issrf - newsletter - 15th ed sept 2014

A p u b l i cat io n o f t h e Ind ian Soc iety for the Study of Reproduct ion and Fert i l i ty

September, 2014Editor-in-Chief: Prof. N. K. Lohiya Editors: Dr. Dheer Singh & Dr. P. K. Mishra

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Message

Dr. Harsh VardhanMinister of Health & Family Welfare, Government of India, New Delhi

Under Millennium Development Goals (MDG 5) we have made extensive efforts to provide universal access to reproductive healthcare but the progress has been uneven and inequitable. With over 1.21 billion people, sexual and reproductive health in our country is regarded a social phenomenon often influenced by contextual factors. Interplay of five main structural determinants: economic status, gender, education, social stratification and age have severely affected reproductive health services imposing large burden on individuals and society. Therefore, plan and policies needs to judiciously target and help decide what needs to be done where, for whom, and when to reach equitable progress toward improved reproductive health for one and - for all. To overcome this burden associated with reproductive health needs, we have two choices: continue on the current path or pursue swift strategies. To stimulate substantial improvement, collaboration among key organizations to spread greater public awareness is an imminent need.

The Indian Society for the Study of Reproduction & Fertility (ISSRF) established in the year 1988 comprising of members representing distinguished scientists, public health executives, program managers and clinicians from the field of reproductive sciences is now entering the 25th year of its inception. A retrospective analysis of society's accomplishments during the course of this journey is necessary but to plan the future discourse will also be highly imperative. ISSRF is uniquely positioned to foster collaborative efforts among academicians, scientists, clinicians, health care workers and policy makers involved in the area of Reproductive Health Research. I am sure; along with public, private and non-profit organizations ISSRF in coming years will plan new strategies that facilitate efforts to achieve “Reproductive Health for All”.

I am happy to note publication of 15th edition of ISSRF Newsletter that is designed to highlight advances in the field of Reproductive Epigenetics, an important area of reproductive health research.

(Harsh Vardhan)

Message

Dr. V. M. KatochSecretary-Department of Health Research & Director General , New Delhi, Indian Council of Medical Research

I am happy to congratulate the Indian Society for the Study of Reproduction & Fertility (ISSRF), in publishing the 15th edition of the ISSRF-Newsletter with focuses on “Epigenetics: an emerging paradigm in reproductive health”.

Emerging knowledge in the area of molecular biology of reproduction has surpassed our understanding of what constitutes heritability and the acquisition of phenotypic traits across generations. While “particulate genetic inheritance” was considered the hallmark for heritability of traits in the past, presently “epigenetic modifications” have been included to the genomic backbone as a primary mechanism in the intricate series of molecular interactions which ultimately coordinates development and cell fate. By large, it has been realized that epigenetic code is highly essential in efficiently regulating gene expression patterns that must occur for a cell to leave its pluripotent state and become fully differentiated to then function in adaptive homeostasis processes of the organism. Therefore, it can be unambiguously stated that one’s epigenetic signatures are the net outcome of genotype, developmental lineage, and gene-environmental interaction.

Communicating the importance of epigenetically-driven change as an influence on reproductive outcomes is likely to have important role to play in helping the academia, move towards a more refined understanding of how these factors influence the patterns of health and disease. With technology for genome-wide interrogation of both chemical modifications to nucleotides and the binding and composition of nucleosomes in place, we will gain knowledge to explain more complex reproductive traits in times to come. I am optimistic that new knowledge of epigenetics will help us in understanding the basis of a variety of human ailments. This is a long journey – plenty of opportunities to identify the basis of adaptations and may be some day also effectively interfere after gaining solid understanding of these phenomena.

I take this opportunity to greet all the readers and wish them good luck.

(V. M. Katoch)

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Message

Prof. N. K. Lohiya

President-Indian Society for the Study of Reproduction and Fertility &

Emeritus Medical Scientist, University of Rajasthan, Jaipur

As we edge ever-closer to the 2015 deadline of the Millennium Development Goals where official negotiations will begin in the U.N.

General Assembly in September, greater attention to strategically focus on sexual and reproductive health and rights are an essential

priority. Sexual health is fundamental to the physical and emotional health and well-being of individuals, couples and families, and to

the social and economic development of communities and countries. Therefore, universal access to comprehensive information

about sexuality, knowledge about the risks they face, their vulnerability to the adverse consequences of sexual activity, their access to

good-quality sexual health care, available contraceptive choices, are the key determinants for promoting sexual health within a range

of country settings. For a country like ours with diverse social and cultural values, it is important for us to re-engage in multi-sectoral

reforms on five crucial domains: laws, policies and human rights; education; society and culture; economics; health systems for

promoting sexual health and wider implementation of our planned strategies. We at ISSRF strongly believe that it is high time to

encourage more comprehensive and multidisciplinary integration of sexual and reproductive health and rights into public health

education. Collective efforts to incorporate sexual and reproductive health and rights in our university teaching curriculum,

formalising research groups that might increase credibility, attract students and facilitate fund raising for research, especially in

collaboration with other inter-sectoral groups (both at local or international level) is required to build a public health workforce that

has the skills to be more responsive to current national reproductive health challenges. Although sexual and reproductive health

issues are viewed often through a critical lens, but now required to be addressed through biomedical and behavioural research as

these would have highly significant health consequences for individuals and communities. If we fail, these in turn, will not only have

serious health burden but social and economic implications as well, for our country at large.

Although our National Health Agencies have made significant contributions to advancing sexual and reproductive health, there is

need for a focused, reinvigorated research agenda. Given the interrelationships among sexual and reproductive health problems,

greater public emphasis to some of the intricate, fundamental and interesting questions and problems is necessary to increase

understanding of and improve provision of needed health services that still persist in our society. The time is long overdue to improve

prevention efforts for limiting size of our population growth. It is clear that more complex and multilevel research, including new and

innovative designs and methodologies at societal and community levels (in terms of policies, programs, norms and values); family,

partner and peer influences; and the individual level (demographic, socioeconomic and psychosocial factors) must be assigned

highest priority. Knowing the magnitude and importance of the problems that might result from all these overlapping issues and

damaging consequences associated with failing to resolve them, it is time for us to address the problems within the field of sexual and

reproductive health through a strong and concerted national effort.

With our strong commitment to 3D’s: discuss, deliberate and disseminate knowledge and information, we have made concerted

efforts to invite your timely attention to several priority areas in reproductive health science and medicine. I am sure this volume of

newsletter that primarily focuses on epigenetics will potentially revolutionize your understanding of the structure and behaviour of

reproductive processes. Selected articles published in this edition might help provide a readily understandable introduction to the

foundations of reproductive epigenetics. As we realize that epigenetic programming of the genome by DNA methylation, histone

modification and chromatin remodelling during gametogenesis and early embryogenesis sets the agenda for developmental origins

of adult diseases, an in-depth understanding of these processes is also of high clinical utility and translational significance. Co-

incidentally, work from the laboratory of Dr. Lanlan Shen from Baylor College of Medicine (USA) published recently in Journal of

Clinical Investigation (July 25, 2014) delineating first straight forward involvement of epigenetic modifications in tumorigenesis

opens up the door for a whole new biological paradigm.

On behalf of the Executive Committee of ISSRF, I congratulate the Editors of this volume: Dr. Dheer Singh, Principal Scientist,

National Dairy Research Institute, Karnal & Dr. Pradyumna Kumar Mishra, Associate Professor, Department of Biological

Sciences, Central University, Sagar for their excellent job.

(N. K. Lohiya)

ISSRF

It's Not All in Our GenesPradyumna Kumar Mishra 4

Epigenome Dynamics in Early Embryo & Germline DevelopmentSonam Mehrotra, Sanjeev Galande 6

Epigenetic Modulation and Reproduction: A Link between Environment and GeneticsJens Vanselow 8

Male Infertility and Epigenetic Modulation

Swetasmita Mishra, Rima Dada 11

Dynamics of DNA Demethylation and Epigenetic Reprogramming in Stem CellsSwayamsiddha Kar, Samir Kumar Patra 15

Assisted Reproductive Technology (ART) and Imprinting Disorders: Epimutation in Gametes as a Functional CauseShaoni Bhattacharjee, Jana Chakrabarti 19

Epigenetics: A Paradigm in Germ Cells Differentiation and In Vivo DevelopmentS. D. Kharche, S. K. Agarwal 22

The Expanding Role of Epigenetics in Reproductive HealthM. Ankolkar, N.H. Balasinor 24

Emerging Role of Epigenetics in Ovarian CancerSeema Sharma 26

Genomic Imprinting in Male Germ-Line Stem CellsPallavi Pushp, Hoon Taek Lee, Mukesh Kumar Gupta 31

Post Translational Histone Modifications in Ovarian Epithelium and their Plausible Implications in Reproductive Plasticity and HealthG. V. Raghuram 33

Polycystic Ovary Syndrome: Plausible Epigenetic Distress and Role of Environmental and Endogenous ModulatorsPooja Sagvekar, Srabani Mukherjee 37

Epigenetic Changes and Human Assisted Reproductive TechnologiesRajvi Mehta 40

MicroRNA in Testis –An OverviewPanneerdoss Subbarayalu, Manjeet K. Rao 43

Epigenetics: A Key Paradigm in Reproductive HealthVarij Nayan, Suneel Kumar Onteru, Dheer Singh 45

Contents

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About the ISSRF

Dr. R. S. Sharma

Secretary-Indian Society for the Study of Reproduction and Fertility &

Sr. Deputy Director General, Indian Council of Medical Research, New Delhi

The Indian Society for the Study of Reproduction and Fertility (ISSRF) was established in the year 1988 with the aim to provide a unique

platform to academicians, scientists, clinicians, public health experts, program managers and policy makers engaged in reproductive health

research to interact and disseminate their findings and also to generate collaborations for optimization of resources and abilities. Over these

years, the society has grown exponentially and is very well represented with 1186 current life memberships. The society encourages active

participation and diverse presence spanning from national to international scale that include almost each national institute, universities,

international agencies and regulatory bodies in events organized under its umbrella/banner. Since inception the society has successfully

organized 43 international/national conferences, including 24 annual meetings of the Society.

Embarking upon an endeavour to reach beyond national limits and exert a global influence, ISSRF began publication of society Newsletter

in July 1999. Since then the society has successfully published 14 Newsletters covering broad range of issues from biology of reproduction

to reproductive health. To offer broad academic coverage and draw maximum attention, the published Newsletters are increasingly

circulated to ISSRF members, national and international funding agencies, institutes and universities.

ISSRF is now entering the 25th year of establishment. We must take cognizance of the historical moment through which we made this

journey despite all thick and thins. Therefore, it is befitting to plan an academic event commemorating this occasion. National Institute for

Research in Reproductive Health, a premier institute under the umbrella of Indian Council of Medical Research (ICMR) is pleased to host the

25th Annual Meeting of the Indian Society for the Study of Reproduction and Fertility (ISSRF) and International Conference on

Reproductive Health at Mumbai during 14-17 February, 2015. The scientific program will include plenary lectures, symposia sessions,

panel discussions, debates, short communications and poster presentations. It will address a broad range of key areas with contributions from

various disciplines including basic, clinical, veterinary, operational and socio-behavioral research in sexual and reproductive health.

As a part of ISSRF mandate, we have also planned a National Seminar on Reproductive Health Awareness at the IIS University, Jaipur

during September 12-13, 2014 and we are delighted to release this newsletter during this mid-term activity of the Society.

A Word from the Editor

It's Not All in Our Genes

Pradyumna Kumar Mishra

School of Biological SciencesCentral [email protected]

That there is a heritable element of susceptibility to chronic human ailments is well established, but there is compelling evidence that few components of such heritability are transmitted through non-genetic factors. Due to an overly complex reproductive process, dissecting into inheritance patterns of these factors though not been easy but little doubt exists that besides the genomic backbone, a range of epigenetic cues affect our genetic programme. The inter-generational transmission of epigenetic marks is believed to operate via four principal means that dramatically differ in their information content: DNA methylation, histone modifications, miRNAs and nucleosomal positioning. Alone or cohesive interaction between these epigenetic signatures, influence the cellular machinery through positive and negative feedback mechanisms. To understand some of the basics of how this mechanism work and activate and deactivate parts of our genetic program not only on day-to-day basis but actually over generations is an important area of reproductive health research.

DNA methylation, the most well characterized epigenetic modification is a heritable covalent modification and binary in nature. Most methylation occurs at the number five carbon of the cytosine pyrimidine ring (5-methyl-cytosine) and represents less than 5 % of all cytosines in our genomes. Genomic methylation patterns are propagated during cell division by DNA methyl transferases (DNMT1, DNMT3A/B). One of the most vital sites of gene regulation by DNA methylation are CpG-enriched regions associated with promoters called "CpG islands”. DNA methylation patterning first established during embryonic development by DNMT3A/B, is preserved in succeeding cell divisions by DNMT1 maintenance. Based on the presence of methylation in the CpG dinucleotide in the complementary template strand, the specificity of DNMT1 for hemi-methylated CpG dinucleotides provides a machinery whereby CpG in the newly synthesized DNA strands are methylated. Along with other enzymes, DNA methylation can orchestrate gene silencing and maintain a repressive chromatin state. Conventionally, DNA methylation is inversely related to both the expression of developmentally regulated genes and the strength of cells. Besides, DNA methylation is also involved in regulating a myriad of cellular processes that

includes chromatin structure and remodelling, X-chromosome inactivation, genomic imprinting and chromosomal stability.

While nucleosomes represent the primary step in the construction of higher-order chromatin structures, histones, the globular proteins undergo post-translational modification and alter regulation of gene expression, DNA replication, recombination, and repair. Histones have a protruding charged 15-38 amino acid N-terminus (“histone tail”) that influences nucleosome assembly into higher order chroma tin structure. In condensed state, chromatin remains in a folded configuration so the nucleosomes are stacked, hence not readily accessible to gene activation. However, covalent modifications such as acetylation, methylation, phosphory la t ion , po ly-ADP r ibosyla t ion and ubiquitination at long tails of H3 and H4 alter histones-DNA interaction and higher order chromatin folding. These post-translational covalent modifications regulate the contact between the octamer core and DNA, and determine DNA accessibility to transcription factor complexes. The capability to accumulate information appears to dwell in the amino-terminal tails of the four core histones which are exposed on the nucleosome surface and are subject to enzyme-catalyzed post-translational modifications of select amino acids, including lysine acetylation, lysine and arginine methylation, serine or threonine phosphorylation, lysine ubiquitination, lysine sumoylation, or glutamine ADP ribosylation. Epigenetic modification of histone tail have key roles in transcriptional regulation, DNA repair, DNA replication, alternative splicing and chromosome condensation. With reference to transcriptional state, human genome can be approximately compartmentalized into actively transcribed euchromatin and transcriptionally inactive heterochromatin. Euchromatin is characterized by towering levels of acetylation and trimethylated H3K4, H3K36 and H3K79. On the other hand, heterochromatin is categorized by low levels of acetylation and elevated levels of H3K9, H3K27 and H4K20 methylation.

miRNAs are single-stranded RNAs of approximately 21-23 nucleotides in length that are transcribed from DNA but not translated into proteins. miRNA genes primarily reside between genes (intergenic) or within introns (intronic) of genes and are transcribed to a primary miRNA (pri-miRNA) mediated by polymerase II or III. The pri-miRNA is processed within the nuclear compartment to a precursor miRNA ( pre-miRNA) by Drosha, a class 2 RNase III enzyme. Subsequently, the transport of pre-miRNAs to the cytoplasm is arbitrated by exportin-5 (EXP-5). In the cytoplasmic region, they are processed further to develop into mature miRNAs by Dicer an RNase III type protein and loaded onto the Argonaute (Argo) protein to generate the effector RNA-induced silencing complex. While majority believes that miRNAs restrain translation, evidence that miRNAs can actually augment translation through alterations in the

Epigenetics: An Emerging Paradigm in Reproductive Health September, 2014 v

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Argo component of the RISC also has been reported. Thus, while miRNAs appear to police translation in an inhibitory fashion, they may also might boost translation in defined biological settings.

DNA packaging in nucleosomes might affect all stages of transcription, thereby regulating gene expression. The precise position of nucleosomes around the transcription start off sites has an essential control on the initiation of transcription. Nucleosome positioning not only decides accessibility of the transcription factors to their target DNA sequence but has also been reported to take part in shaping the methyla t ion landscape . Bes ides transcription regulation, nucleosome occupancy also participates in directing meiotic recombination events. The precise function of nucleosomes is influenced by the incorporation of different histone variants that are incorporated into chromatin independently, outside the S-phase. Often linked with specific histone modifications, nucleosome remodeling machinery is also influenced by DNA methylation. Thus, the interaction among diverse epigenetic partners is evident more often.

While alterations in the epigenomic landscape are required for regular growth and development, they can also be accountable for some disease states. The significance of epigenetics in maintaining typical development and biology is reflected by the observation that many diseases build up when the wrong type of epigenetic marks are introduced or are added at the wrong time or at the wrong place. Disrupting any of the four systems that contribute to epigenetic alterations can cause abnormal activation or silencing of genes. Epigenetic control systems generally involve three types of proteins: “writers”, “readers”, and “erasers.” Writers attach chemical marks, such as methyl groups (to DNA) or acetyl groups (to the histone proteins that DNA wraps around). So-called “readers” bind to these marks, thereby influencing gene expression; erasers remove the marks. The marks are passed down as cells divide, providing a sort of cellular memory to ensure that cell proliferation is effectively regulated. The reversibility of epigenetic marks provides the possibility that the activity of key genes and pathways can be regulated as a therapeutic approach. Recent technological advances are allowing the study on a more genome-wide scale which is facilitating a more “systems biology” based approach to understand disease aetiology. The epigenome has truly arrived as a drug development target as pharmaceutical companies and biotech giants have programmes aimed squarely at proteins that operate in the epigenetic space.

It was often argued that most epigenetic modification, by whatever mechanism, is erased with each new generat ion, dur ing gameto-genesis and af ter fertilization. However, one of the more startling experiments conducted by Skinner published in Science (2005) challenged this belief and suggested that epigenetic changes may endure in at least four subsequent generations. These findings provided a new paradigm for disease aetiology and basic mechanism in evolution not previously appreciated before. Today, epigenetic aberrations have been conjectured to be highly relevant to sexual and reproductive health as these accounts for interactive relationship among genomic landscape, gene-environment interactions and disease phenotype. Novel ins ights in to aetiologies of complex non-Mendelian disease traits have burgeoned interest in the field of reproductive epigenetics. How range of epigenetic mechanisms can differentially influence the male and female germ lines and developmental process is closely monitored. Of late, the subtle and elegant modulation of fidelity of transmissible heritable characteristics through epigenetic

reprogramming has also received wider scientific attention. Developmental activation and deactivation of epigenetic signatures at pre-implantation phase provides putative links between assisted reproductive technologies and imprinting disorders. Occurrence of imprinting errors also disrupts placental growth and development in assisted conception procedures. Besides, epigenetics has potentially helped the livestock industry to find part of the missing causality and heritability of complex economic traits (milk yield and fertility) and animal diseases as well.

With a focus to provide biological nuances and depth on this key determinant of reproductive function, the 15th edition of ISSRF Newsletter brings you leading edge articles on "Epigenetics : an emerging paradigm in reproductive health". We have made best possible efforts to provide you a compendium of contributions from leading experts working in premier institutions of our country on various aspects of reproductive epigenetics. It is our pleasure to introduce this new issue of the ISSRF newsletter. Not only does it bring to readers various contributions linked to the topics of epigenetics, but it also presents some important translational perspectives of reproductive health research, an area that especially excites us. Hope, this new issue of the ISSRF Newsletter will provide you with challenging thoughts and contribute to broadening the debate on how epigenetic factors can play a significant role in our reproductive function and well-being.

“What makes us who we are ? - Begin before birth and it's not all in our genes”

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Epigenome Dynamics in Early Embryo & Germline Development

Sonam Mehrotra, Sanjeev Galande*

Centre of Excellence in Epigenetics Indian Institute of Science Education and ResearchPune *[email protected]

Summary

The fertilization of two highly differentiated haploid cells: the oocyte and the sperm results in the zygote, a cell with complete developmental potential and capable of producing any differentiated cell of the adult organism. The success of the embryonic developmental program is determined by a series of dynamic epigenetic changes that allow configuration of parent-specific epigenetic states in the gametes and support totipotency of the zygote. During early embryogenesis, the cells undergo critical irreversible cell fate decisions. The developmental potential of these cells at different stages is manifested through a combination of multiple epigenetic changes including DNA methylation, modification of histones, incorporation of histone variants, RNA mediated silencing and chromatin modeling. These mechanisms underlie execution of specific functions by different cells carrying identical genetic information through selective activation/repression of specific gene subsets thus creating unique transcription profiles.

Epigenetics Changes in Early Embryonic Development

The series of epigenetic programming and/ or reprogramming events that dominate the early embryonic development culminate in the zygote just after fertilization, with erasure of DNA methylation marks from the paternal pronuclear genome (Figure 1). DNA-methylation marks from 5’ position of cytosine residues from most of the CpG dinucleotides are erased through Tet-catalyzed oxidation followed by decarboxylation or through DNA repair pathways (1, 2). This is followed by another passive wave of DNA demethylation during cleavage /mitotic stages of embryogenesis and finally extensive chromatin modeling to silence pluripotency genes in the inner cell mass and promote differentiation. Chromatin remodeling at this stage is also associated with re-activation of the inactive X chromosome in the female embryos. Subsequently, de novo methylation mediated by DNA cytosine-5’ methyl transferase (DNMT) family enzymes allows reacquisition of methylation signatures to establish expression patterns of essential developmental genes in the embryo (3, 4).

A minority of sequences such as imprinted genes escapes this reprogramming and maintains their parent of origin germ-cell specific epigenetic patterns into adulthood. Genetic imprinting is an epigenetic phenomenon that regulates certain genes such that only one allele is expressed based on the parental origin. This phenomenon may also ensure epigenetic silencing of transposable elements such as

Line1 elements, hence critical in maintaining genomic integrity (5, 6).

The second major reprogramming event occurs in germ cell precursors known as primodial germ cells (PGCs) of the developing embryo. For both male and female gametogenesis involves establishment of cells with germ line specific chromatin signatures which are distinct from the surrounding somatic cells (Fig. 1). These primordial germ cells are direct derivatives of the pluripotent cells of the inner cell mass of the blastocyst that migrate to urogenital ridges during gastrulation. Since these cells consist of essential epigenetic information required for development of the somatic embryo, the reprogramming at this stage involves erasure of all pre-existing epigenetic modifications including those that were maintained during pre-implantation stage. These result in repression of genes involved in somatic differentiation and/or activation of genes involved in maintenance of specific germ cell identity (7). These phenomena therefore establish sex-specific epigenetic profiles and transcriptional networks essential for normal development of the germ-line as well as regulation of epigenetic inheritance mechanisms such as genomic imprinting (8).

The Distinct Epigenomes of the Male and Female Germ Cells

The two mature gametes comprising of maternal and paternal DNA respectively present distinct epigenetic information at the time of fertilization. At this stage the two parental genomes remain physically separate and undergo distinct programs of chromatin remodeling. The maternal DNA contained in the oocyte is bound by histones acquired during oocyte growth comprising of post-translational modifications associated with stalled metaphase II. At fertilization the maternal genome undergoes only few epigenetic changes, these involve euchromatin marks associated with activation of DNA replication and transcription such as H3 and H4 acetylation and argininemethylation. The major difference between the chromatin of oocyte and somatic nuclei is the absence of H1 linker histones in the oocyte which are substituted by a specific H1 variant (9). The role of these H1 histone variants in the oocyte chromatin during early embryogenesis remains to be understood.

In case of the male gamete, the highly compacted paternal DNA residing in the sperm head undergoes extensive remodeling via many decondensation cycles. During spermatogenesis the paternal DNA in the sperm is compacted by replacement of histones with protamines, which are then replaced again by histones when the paternal genome begins to decondense after fertilization. Recent studies suggest that chromatin of mature spermatozoa retain small amounts of histones. Such histone enrichment has been observed on developmentally regulated genes which are important for early development and differentiation as well as ‘priming’ of the zygote (10). The male pronucleus also exhibits high levels histoneacetylation which supports higher transcription from the S-phase in zygote stage and thereafter (11).


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Following fertilization, incorporation of different histone variants also contributes to the differences between the two parental genomes. For example, incorporation of H3.3 histone variant is observed only in the male pronucleus. It is possible that the incorporation of H3.3 variant plays a role in directing or preventing other paternal specific histone modifications. Modification ofH3.3 has also been linked with paternal pericentric heterochromatin and mutations in lysine 27 of H3.3 leads to developmental arrest (12). Incorporation of other histone variants has also been shown to be important for early mammalian development. For instance, the phosphorylation of the C-terminus of SQEY motifs in H2A.X variant occurs in response to DNA double strand breaks in somatic cells and is also enriched duringformation of male pronucleus in Xenopus as well as mouse embryos (13) Although the exact functions of H2A.X and H2A.Z variants are not known in early development, their deletion causes developmental arrest and failure of implantation. During decondensation cycles the male pronuclear genome lacks several modifications such as H3K9me2, H3K9 me3, and H3K27me3 heterochromatin marks which are detectable in the female pronucleus genome (14).

The epigenetic asymmetry between the two parental pronuclei is also detected by differences in the global levels of DNA methylation. Initially both parental nuclei exhibit high levels of DNA methylation. However, specific targeting of DNA demethylation to the male pronucleus may be regulated by asymmetries in the chromatin template and lack of repressive histone modification marks. The specificity of DNA demethylation is also directed by maternally inherited Stella protein (15). Differences in the global DNA methylation levels are detected only until the two-cell stage, which are diluted due to passive DNA methylation loss in the combined embryonic genome. The significance of these epigenetic asymmetries in the parental genomes and their persistence in the early stages of zygotic development and how they affect early embryonic development still remains unclear.

Implications of Epigenetic Inheritance

It has been speculated that alterations in the environment may influence changes in the germ-line epigenome and these modifications may provide mechanisms that allow evolutionary adaptations. Recent studies provide evidence that certain mutations in epigenetic modifiers may lead to paternal inheritance of epigenetic changes. For example, haploinsufficiency of Dnmt1 and Smarca5 in male mice is sufficient for altering gene expression of maternal Avy gene in an otherwise genetically wild-type animal (16). Studies in C.eleganshave also demonstrated involvement of non-coding RNAs such as piRNAs in mechanisms of paternal epigenetic inheritance (17). Studies involving identification RNA sequencing of sperm RNA also suggest the possibility that these RNAscould also contribute to epigenetic states in the early embryo (18).

Additionally, the epigenetic errors during spermatogenesis in humans have been identified which lead to reduced

competency of the sperm and fertility in the males (19). In the females, factors such as maternal nutrition and exposure to certain toxins have also been associated with epigenetic changes in the oocyte that have been linked with neonatal developmental and gestational defects (20). Therefore, even in a genetically normal organism, environmentally induced epimutations in the parent’s germ-line may be transmitted to the offspring and result in phenotypic variation, range of diseases and/or developmental disorders. Therefore, understanding how epigenetic information is established in the parental germ-line and mechanisms underlying epigenetic inheritance is of special interest. This knowledge is crucial in determining the relationship between environmental influences on epigenetic changes and eventually towards the development of the organism.

Figure 1: Summary of Epigenetic Programming and Reprogramming Events During Mammalian Development.

The two mature gametes present genomes in distinct epigenetic states at the time of fertilization. With fertilization, the materal genome (depicted in purple) completes meiosis and undergoes relatively fewer epigenetic changes, involving euchromatic marks. In contrast, the paternal genome (depicted in light blue) undergoes several cycles of decondensation, demethylation and replacement of protamines by histones and histone variants. The formation of the zygote is followed by a wave of demethylation of the genome with every cleavage till the formation of the sixteen-cell stage. The imprinted regions however, escape this demethylation and maintain their parent of origin specific epigenetic states. Following blastocyst formation the diploid genome undergoes global remethylation, which results in erasure of imprinting. Few cells in the inner cell mass acquire epigenetic marks that distinguish these cells from the surrounding somatic cells. These cells migrate to the urogenital ridges in the developing gonads of the embryo, where they undergo paternal and maternal specific epigenetic changes during the development of mature sperm and ovum (21).

References

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2. Hajkova P, Jeffries SJ, Lee C, et al (2010). Genome wide

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20. Stringer JM, Barrand S, Western P (2013). Fine-tuning

evolution: Germ-line epigenetics and inheritance. Reproduction; 146: 37-48.

21. Cantone I, Fisher AG (2013). Epigenetic programming and reprogramming during development. Nature Structural and Molecular Biology; 20: 282-9.

Epigenetic Modulation and Reproduction: A Link Between Environment and Genetics

Jens Vanselow

Leibniz Institute for Farm Animal Biology (FBN)[email protected]

Epigenetic Mechanisms Modulate Gene Expression

The term “epigenetic” was firstly used by C.H. Waddington, actually years before the molecular basis of inheritance has been established. He used this term in the context of “epigenetic landscape” as a conceptual model of how genes might interact with their surroundings to produce a phenotype (1). Currently we understand epigenetics as “…mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence.” (2). However, despite the exploding knowledge of the molecular nature and function of genes as well as their role in heredity the tremendous importance of epigenetic mechanisms for reproduction, development, behavior and health was recognized only recently. Now it is clear that epigenetic mechanisms are crucially involved in regulating the activity of genes (i.e. gene expression) and thus the phenotypic shape. The physical substrates of genes are DNA molecules, which are tightly associated with histone proteins to form chromatin and consolidated within chromosomes. In the most stringent way, epigenetic modulation means the chemical modification of the DNA and associated histone proteins. Histone modifications are posttranslational covalent modifications to histone tailsexhibiting either activating or repressive functions. DNA methylation is the covalent binding of methyl groups to cytosinesin the context of CpG dinucleotides. DNA methylation is typically associated with gene silencing (3). Generally, histone modification is considered as a more transient mechanism, whereas DNA methylation patterns are usually stably inherited across mitotic divisions. Surprisingly, the connection between histone modification and DNA methylation was not discovered earlier than 1998 with the discovery of the methyl-CpG-binding protein MeCP2, which brings together methylated DNA, histone deacetylation and transcriptional silencing (4).

Epigenetic Marks Determine the Cellular Fate and Parental Imprinting of Genes

Developmental epigenetic marks are established during gametogenesis and early embryogenesis (5). Particularly in mammals, during oogenesis and spermatogenesis gender specific differential DNA methylation marks are established

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on a subset of about 150 genes, thus persistently silencing expression of these genes in a parent-specific manner. This mechanism called “genomic imprinting” was discovered in 1984 (6). Genomic imprinting is responsible for the non-equivalence of the maternal and paternal genomes in mammals thus allowing only biparental embryos to develop and survive. During early embryogenesis another wave of genomic methylation does occur, which is responsible for initiating tissue-specific differentiation of previously pluripotent and omnipotent cells. Methylation marks are established, which stably determine the cell fate during subsequent ontogenetic development. Corresponding to the term genomic imprinting this process can be referred to as somatic imprinting. Recently however, it could also be shown that DNA methylation marks can change during ageing, pathological processes like cancer (7) or infectious diseases (8), but also during iterative processes of differentiation like folliculogenesis in adults (9, 10).

Assisted Reproductive Technology can Provoke Epigenetic Alterations

Nowadays, assisted reproductive technology (ART) including artificial insemination (AI), superovulation, in vitro maturation (IVM), in vitro fertilization (IVF), embryo culture and transfer, and intra-cytoplasmic sperm injection (ICSI) is an indispensable component of animal breeding. But also for human reproduction ART is becoming increasingly important. Currently, ART accounts for 1 to 3% of annual births in industrialized countries and continues to expand rapidly (11). Except for an increased incidence of premature births, these technologies are considered safe. However, the timing of two key techniques commonly used during ART, ovarian stimulation and in vitro culture, coincide with important epigenetic programming events that naturally occur during gametogenesis and early embryonic development (12). Accordingly, this gives reason to expect interference with epigenetic alterations.Indeed, experimental evidence published during the past decade has suggested a growing incidence of imprinting disorders in children conceived by ART. Among children with Beckwith–Wiedemann syndrome or Angelman syndrome caused by an imprinting defect several clinical studies have reported an increased frequency of ART conceptions (12). Beckwith–Wiedemann syndrome (BWS) is an overgrowth disorder caused by aberrant genetic and epigenetic regulation at the KCNQ1OT1 and H19 imprinted domains. ART-conceived BWS children commonly experience maternal loss of methylation (LOM) at KCNQ1OT1 and maternal gain of methylation (GOM) at H19. Angelman syndrome (AS) is a neurological disorder that is caused by genetic and epigenetic disturbances at the SNRPN-imprinted domain. ART-conceived AS patients often possess maternal SNRPN LOM (3). Further evidence comes from the observation that a high proportion of these ART-associated imprinting disorders appears to be caused by epimutations that 1) are not commonly found in the general population and 2) a majority of which occur on the maternal allele (12). However, the actual incidence of imprinting

defects in oocytes and embryos from superovulated females appears to be low and stochastic (13, 14).

Also, one has to consider that human embryos produced via ARTs are the product of underlying infertility/subfertility problems. This has led to questions regarding the origin of epigenetic instability, i .e. whether underlying infertility/subfertility compromises epigenetic integrity in gametes/embryos, whether gamete/embryo manipulations cause epigenetic instability, or whether a combination of subfertility and ARTs leads to epigenetic disruption. The relationship between impaired fertility, ARTs, and epigenetic stability is unquestionably complex. However, the possibility exists that ARTs and infertility may disrupt the same biological pathways that lead to epigenetic instability. If this is the case, perturbations induced by infertility/subfertility may be deteriorated by gamete or embryo manipulation, similar to combined ART treatments (3). Other studies suggest that in humans the increase in the incidence of imprinting disorders in individuals born by ART may be due, in some cases, to the use of sperm with intrinsic imprinting mutations. An important consideration is to determine if the implicated association between ART and imprinting disorders is actually related to the procedures or to infertility itself. Several lines of evidence, however, suggest that multiple aspects of the ART process, including the gonadotropin stimulation of folliculogenesis, embryo culture and/or embryo transfer, have the potential to induce epimutations in offspring produced by these methods (15).

In animal breeding it is well known that reproductive techniques, and in particular somatic cell nuclear transfer (SCNT), but also other procedures connected with ART, can cause severe abnormalities like large offspring syndrome (LOS). Interestingly, LOS and BWS show phenotypic and epigenetic similarities (16) thus suggesting similar molecular mechanisms including epigenetic dysregulation of growth associated genes. Data of several studies indicate that imprinting marks are erased during the reprogramming of the somatic cell nuclei during early development, indicating that such epigenetic anomalies may play a key role in mortality and morbidity of cloned animals (17).

Environmental Factors can Induce Transgenerational Epigenetic Alterations

From epidemiological studies it was first discovered in humans that unfavorable nutritional conditions during specific sensitive life periods in particular during the prepubertal slow growth period can increase susceptibility to cardiovascular and metabolic diseases and may significantly influence longevity even during subsequent generations (18, 19, 20, 21). These statistical association studies led to the formulation of the ‘developmental origins of health and disease’ hypothesis, which suggests that many adult-onset diseases can be attributed to altered growth and development during early life (22). However, underlying molecular mechanisms were not revealed by these studies. In contrast, studies in animal models not only demonstrated deleterious, transgenerational effects of toxic substances and endocrine disruptors, but also clearly demonstrated that

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persistent epigenetic alterations were responsible for these effects. Exposure of gestating female rats to the agriculture fungicide vinclozolin during gonadal sex determination revealed in their offspring altered expression profiles of about 400 testicular genes over three generations and demonstrated epigenetic transgenerational inheritance as an important component of the molecular etiology of male infertility (23). Also exposure to dioxin during fetal day 8 to 14 induced prostate disease, ovarian primordial follicle loss and polycystic ovary disease for at least three generation. In this study 50 differentially DNA methylated regions could be identified in specific promoters thus pointing to an altered DNA methylation pattern as a possible cause for dioxin induced transgenerational effects (24). Similar studies were performed with the endocrine disruptor compounds bisphenol-A (BPA), bis(2-ethylhexyl)phthalate (DEHP) and dibutyl phthalate (DBP), which demonstrated epigenetic transgenerational inheritance of adult onset disease and associated DNA methylation epimutations (25). Molecular mechanisms were most convincingly demonstrated in a mouse model showing that maternal exposure to endocrine disruptors BPA or Genistein can stably affect DNA methylation levels within the agouti locus and thus the coat color of offspring. In addition, also metabolic parameters can be negatively affected in these mice (26, 27).

Conclusions

A plethora of studies published during the last decade demonstrated that environmental factors as nutrition, toxins or endocrine disruptors, but also procedures associated with assisted reproduction technologies can essentially affect the formation of the phenotype. Data from animal models indicate that persistent alterations of DNA methylation at specific loci might be responsible. Surprisingly, these epimutations and associated phenotypic alterations can be passed transgenerationally. Thus, the emerging field of environmental epigenomics clearly deserves our greatest attention and research efforts not least because of our responsibility for future generations.

References

1. Wadington CH. The epigenotype. Endeavour. 2042;1:18-20.

2. Russo VEA, Martienssen RA, Riggs AD (1996). Epigenetic mechanisms of gene regulation. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

3. Denomme MM, Mann MR (2012). Genomic imprints as a model for the analysis of epigenetic stability during assisted reproductive technologies. Reproduction;144: 393-409.

4. Nan X, Ng H-H, Johnson CA, et al (1998). Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature; 393: 386-9.

5. Reik W, Walter J (2001). Genomic imprinting: parental influence on the genome. Nat Rev Genet; 2: 21-32.

6. Surani MA, Barton SC, Norris ML (1984). Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis. Nature; 308: 548-50.

7. Kondo Y (2009). Epigenetic cross-talk between DNA methylation and histone modifications in human cancers. Yonsei Med J; 50: 455-63.

8. Vanselow J, Yang W, Herrmann J, et al (2006). DNA-

remethylation around a STAT5-binding enhancer in the alphaS1-casein promoter is associated with abrupt shutdown of alphaS1-casein synthesis during acute mastitis. J Mol Endocrinol; 37: 463-77.

9. Vanselow J, Fürbass R (2010). Epigenetic control of folliculogenesis and luteinization. Animal Reproduction; 7: 134-9.

10. Vanselow J, Spitschak M, Nimz M, et al (2010). DNA Methylation Is Not Involved in Preovulatory Down-Regulation of CYP11A1, HSD3B1, and CYP19A1 in Bovine Follicles But May Play a Role for Permanent Silencing of CYP19A1 in Large Granulosa Lutein Cells. Biol Reprod; 82: 289-98.

11. Eroglu A, Layman LC (2012). Role of ART in imprinting disorders. Semin Reprod Med; 92-104.

12. deWaal E, McCarrey JR (2010). Effects of exogenous endocrine stimulation on epigenetic programming of the female germline genome. Animal Reproduction; 7: 154-64.

13. Sato A, Otsu E, Negishi H, et al (2007). Aberrant DNA methylation of imprinted loci in superovulated oocytes. Hum Reprod; 22: 26-35.

14. Market-Velker BA, Zhang L, Magri LS, et al (2010). Dual effects of superovulation: loss of maternal and paternal imprinted methylation in a dose-dependent manner. Hum Mol Genet; 19: 36-51.

15. deWaal E., Yamazaki Y, Ingale P, et al (2012). Gonadotropin stimulation contributes to an increased incidence of epimutations in ICSI-derived mice. Hum Mol Genet; 21: 4460-72.

16. Chen Z, Robbins KM, Wells KD, et al (2013). Large offspring syndrome: a bovine model for the human loss-of-imprinting overgrowth syndrome Beckwith-Wiedemann. Epigenetics; 591-601.

17. Smith LC, Suzuki J, Jr., Goff AK, et al (2012). Developmental and epigenetic anomalies in cloned cattle. Reprod Domest Anim; 47 Suppl 4: 107-14.

18. Pembrey ME, Bygren LO, Kaati G, et al (2006). Sex-specific, male-line transgenerational responses in humans. Eur J Hum Genet; 14: 159-66.

19. Whitelaw E (2006). Epigenetics: sins of the fathers, and their fathers. Eur J Hum Genet; 14: 131-2.

20. Kaati G, Bygren LO, Pembrey M, et al (2007). Transgenerational response to nutrition, early life circumstances and longevity. Eur J Hum Genet; 15: 784-90.

21. Kaati G, Bygren LO, Edvinsson S (2002). Cardiovascular and diabetes mortality determined by nutrition during parents' and grandparents' slow growth period. Eur J Hum Genet; 10: 682-8.

22. Dorey ES, Pantaleon M, Weir KA, et al (2014). Adverse prenatal environment and kidney development: implications for programing of adult disease. Reproduction; 147: R189-98.

23. Guerrero-Bosagna C, Savenkova M, Haque MM, et al (2013). Environmentally induced epigenetic transgenerational inheritance of altered Sertoli cell transcriptome and epigenome: molecular etiology of male infertility. PLoS One; 8: e59922.

24. Manikkam M, Tracey R, Guerrero-Bosagna C, et al (2012). Dioxin (TCDD) induces epigenetic transgenerational inheritance of adult onset disease and sperm epimutations. PLoS One; 7: e46249.

25. Manikkam M, Tracey R, Guerrero-Bosagna C, et al (2013). Plastics derived endocrine disruptors (BPA, DEHP and DBP) induce epigenetic transgenerational inheritance of obesity, reproductive disease and sperm epimutations. PLoS One; 8: e55387.

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26. Dolinoy DC, Huang D, Jirtle RL (2007). Maternal nutrient supplementation counteracts bisphenol A-induced DNA hypomethylation in early development. Proc Natl Acad Sci U S A; 104: 13056-61.

27. Dolinoy DC, Jirtle RL (2008). Environmental epigenomics in human health and disease. Environ Mol Mutagen; 49: 4-8.

Male Infertility and Epigenetic Modulation

Swetasmita Mishra, Rima Dada*

Department of AnatomyAll India Institutes of Medical SciencesNew Delhi*[email protected]

Epigenetics is the study of heritable changes in gene activity that are not caused by changes in the DNA sequence; it also can be used to describe the study of stable, long-term alterations in the transcriptional potential of a cell that are not necessarily heritable. Examples of mechanisms that produce such changes are DNA methylation and histone modification, each of which alters gene expression without altering the underlying DNA sequence. Gene expression can be controlled through the action of repressor proteins that attach to silencer regions of the DNA. These epigenetic changes may last through cell divisions for the duration of the cell's life, and may also last for multiple generations even though they do not involve changes in the underlying DNA sequence of the organism (1); instead, non-genetic factors cause the organism's genes to behave (or "express themselves") differently. Specific epigenetic processes include paramutation, bookmarking, imprinting, gene silencing, X chromosome inactivation, position effect, reprogramming, transvection, maternal effects, the progress of carcinogenesis, many effects of teratogens, and regulation of histone modifications and heterochromatin.

Chromatin Remodelling

Since DNA methylation and chromatin remodeling play such a central role in many types of epigenetic inheritance, the word "epigenetics" is sometimes used as a synonym for these processes. However, this can be misleading. Chromatin remodeling is not always inherited, and not all epigenetic inheritance involves chromatin remodelling (2).

Because the phenotype of a cell or individual is affected by the genes that are transcribed, heritable transcription states can give rise to epigenetic effects. There are several layers of regulation of gene expression. One way that genes are regulated is through the remodeling of chromatin. Chromatin is the complex of DNA and the histone proteins with which it associates. If the way that DNA is wrapped around the histones changes, gene expression can change as well. Chromatin remodeling is accomplished through two main mechanisms, post translational modification of the histones and methylation of the DNA.

Histone Modifications

Histone proteins are made up of long chains of amino acids. If the amino acids that are in the chain are changed, the shape of the histone might be modified and hence the expression of the genes is changed. These histone proteins carry specific epigenetic marks on them. DNA is not completely unwound during replication. The parental histones are carried into each new copy of the DNA during replication. These histones may act as templates, initiating the surrounding new histones to be shaped in the new manner. By altering the shape of the histones and the epigenetic marks on them these modified histones cause a lineage-specific transcription program which is maintained after cell division. The epigenetic marks on the histone proteins are heritable and play an important role in regulation of spatial and temporal expression of the genes.

Although histone modifications occur throughout the entire sequence, the unstructured N-termini of histones (called histone tails) are particularly highly modified. These modifications include acetylation, methylation, ubiquitylation, phosphorylation, sumoylation, ribosylation and citrullination. Acetylation is the most highly studied of these modifications. For example, acetylation of the K14 and K9 lysines of the tail of histone H3 by histone acetyltransferase enzymes (HATs) is generally related to transcriptional competence. Methylation of lysine 9 of histone H3 has long been associated with constitutively transcriptionally silent chromatin (constitutive heterochromatin). It has been shown that the histone lysine methyltransferase (KMT) is responsible for the methylation activity in histones H3 & H4. This enzyme utilizes a catalytically active site called the SET domain (Suppressor of variegation, Enhancer of zeste, Trithorax). The SET domain is a 130-amino acid sequence involved in modulating gene activities. This domain has been demonstrated to bind to the histone tail and causes the methylation of the histone (3).

Differing histone modifications are likely to function in differing ways; acetylation at one position is likely to function differently from acetylation at another position. Also, multiple modifications may occur at the same time, and these modifications may work together to change the behavior of the nucleosome. Multiple dynamic histone modifications regulate gene transcription in a systematic and reproducible way and this is called the histone code.

DNA Methylation

Methylation of the DNA is the addition of methyl groups to the CpG sites, to convert cytosine to 5-methylcytosine. 5-Methylcytosine performs much like a regular cytosine, pairing with a guanine in double-stranded DNA. However, some areas of the genome are methylated more heavily than others, and highly methylated areas tend to be less transcriptionally active, through a mechanism not fully understood. Methylation of cytosines can also persist from the germ line of one of the parents into the zygote, marking the chromosome as being inherited from one parent or the other (genetic imprinting).

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DNA methylation frequently occurs in repeated sequences, and helps to suppress the expression and mobility of 'transposable elements' (4). As 5-methylcytosine can be spontaneously deaminated (replacing nitrogen by oxygen) to thymidine, CpG sites are frequently mutated and become rare in the genome, except at CpG islands where they remain unmethylated. Epigenetic changes of this type thus have the potential to direct increased frequencies of permanent genetic mutation. DNA methylation patterns are known to be established and modified in response to environmental factors by three independent DNA methyltransferases, DNMT1, DNMT3A, and DNMT3B, (5). DNMT1 is the most abundant methyltransferase in somatic cells localizes to replication foci has a 10–40-fold preference for hemimethylated DNA and interacts with the proliferating cell nuclear antigen (PCNA).

By preferentially modifying hemimethylated DNA, DNMT1 transfers patterns of methylation to a newly synthesized strand after DNA replication, and therefore is often referred to as the ‘maintenance' methyltransferase. DNMT1 is essential for proper embryonic development, imprinting and X-inactivation. Furthermore, in addition to the maintenance and transmission of methylated DNA states, the same principle could work in the maintenance and transmission of histone modifications and even cytoplasmic (structural) heritable states (6). Mechanisms of heritability of histone state are not well understood, however much is known about the mechanism of heritability of DNA methylation state during cell division and differentiation. Heritability of methylation state depends on certain enzymes (such as DNMT1) that have a higher affinity for 5-methylcytosine than for cytosine. If this enzyme reaches a "hemimethylated" portion of DNA (where 5-methylcytosine is in only one of the two DNA strands) the enzyme will methylate the other half.

It has been suggested that chromatin-based transcriptional regulation could be mediated by the effect of small RNAs. Small interfering RNAs can modulate transcriptional gene expression via epigenetic modulation of targeted promoters (7). Various studies on different animal models and humans have established the concept that epigenetic programming/reprogramming are essential for proper spermatogenesis and embryo development. Any defect in the process leading to epigenetic errors may lead to sperm disorders and infertility. This review is mainly focussed on the epigenetics of the sperm genome especially sperm DNA methylation and the role of altered methylation of sperm DNA on fertility.

Sperm Cell Epigenetics

During spermiogenesis the round spermatids with active nuclear activities like transcription and translation are converted into inert flagellated cells with a compact nucleus. DNA compaction is a characteristic feature of male germ cells and a fundamental process required for the transmission of paternal genome to the next generation (8). This specific event of sperm DNA compaction is believed to

convey essential epigenetic information to the developing embryo (9, 10). In the early stages of spermatogenesis the spermatids have a less compact genome in the form of DNA around nucleosomes. Nucleosome is comprised of DNA coiled around an octamer of histones H2A, H2B, H3, and H4. Then as the spermatogenesis process proceeds further there occurs significant compaction of the sperm genome by the replacement of histones by nonhistone proteins. Histones are first replaced by transition proteins (TNP1 and TNP2) and ultimately by protamines (P1 and P2) (11). The protamine bound sperm genome structure is 6-20 times more compact than the histone bound nuclear structure (12). After the protamine bound compact sperm genome structure is reached the sperm cells become transcriptionally and translationally inert. The mature sperm cells contain only the mRNA and small RNAs which was present in the spermatids at the early stages of spermatogenesis. Previously it was believed that there is no involvement of sperm transcript in the embryo development but now several studies report the involvement of sperm genome organisation and paternal transcriptome in early embryo development (9, 10).

Methylation and Male Fertility

The molecular basis of male infertility remains largely unknown (13). A number of candidate gene studies revealed that abnormal sperm DNA methylation patterns are associated with reduced sperm count and function (14, 15, 16, 17, 18 ) as well as outcome of assisted reproductive technologies (ART) (19). Accumulating evidence suggests that the spermatozoa contribute more to the embryo than the paternal genome (20, 21). Epigenetic factors in the sperm cells may regulate the expression of various genes necessary for proper embryogenesis.

Methylation establishment in germ cells is a basic process which is necessary for proper spermatogenesis (22). It is a well studied phenomenon in mouse germ cells. There is a global demethylation remethylation process that takes place during embryogenesis. This process involves erasure of somatic DNA methylation and subsequent establishment of sex-specific de novo DNA methylation (23). Demethylation takes place in the developing embryo between 8.0–13.5 days postcoitum (dpc) and all the methylation marks are removed (24). Then a specific remethylation process begins at 15.5 dpc (25). This methylation remethylation process during embryonic development gives rise to a phenomenon called genomic imprinting.

Genomic Imprinting

It is an inheritance process independent of the classical Mendelian inheritance, an epigenetic phenomenon ensuring the expression of certain genes in a parent-of-origin-specific manner (26).

Diploid organisms possess two copies of the genome in the somatic cells. Each gene is represented by two copies or alleles (one copy inherited from each parent at fertilisation). For majority of autosomal genes expression occurs from both alleles simultaneously. However, in mammals some genes (<1% of genes) are imprinted so that gene expression occurs from only one allele. The expressed allele is

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dependent upon its parental origin. For example, the gene encoding Insulin-like growth factor 2 (IGF2/Igf2) is only expressed from the allele inherited from the father.

In humans, imprinted alleles are silenced such that the genes are either expressed only from the non-imprinted allele inherited from the mother (e.g. H19 or CDKN1C), or in other instances from the non-imprinted allele inherited from the father (e.g. IGF-2). Genomic imprinting can involve DNA methylation and histone modulation in order to achieve monoallelic gene expression without altering the genetic sequence. These epigenetic marks are established in the germline that are maintained through mitotic divisions. Appropriate expression of imprinted genes is important for normal development. There are several diseases pertaining to imprinting defects like Beckwith–Wiedemann syndrome, Silver–Russell syndrome, Angelman syndrome and Prader–Willi syndrome and many more.

Imprinting marks are erased in primordial germ cells (PGCs) and a specific remethylation occurs in spermatogonia and type I spermatocytes, before their entry into meiosis, so that all spermatozoa transmit the correct paternal imprint (27). Only four imprinted loci are methylated in the male germ line Igf2/H19, Rasgrf1, Dlk1-Gtl2, and Zdbf2 (28). DNA methylation is carried out by a family of enzymes called DNA methyltransferases (DNMTs). DNMT1 is responsible for methylation maintenance (29), and DNMT3A, 3B, and 3L specifically allow the methylation process in germ cells. The activities of these methyltransferases are essential for completion of spermatogenesis. It has been demonstrated that Dnmt3a knockout in germ cells causes germ cell apoptosis and ultimately impairs spermatogenesi (30). Knocking out Dnmt3l gene induced meiotic arrest in spermatocytes.

Genomic imprinting is relatively less studied area in human developmental biology. Only two studies have reported methylation marks of imprinted genes in germ cells of adult men. Studies has confirmed the methylation of the H19 in spermatogonia and the demethylated state of MEST/PEG1 in spermatogonia and type I spermatocytes (31). In humans, methylation marks are acquired before entry into meiosis but it is still unknown whether this process occurs at the fetal developmental period, perinatal period or at puberty.

Several studies have investigated the relationship between DNA methylation levels and male fertility in humans. Methylation defects of the sperm DNA has been demonstrated as a cause of infertility. High methylation levels of mature sperm DNA has been reported to be associated with high pregnancy rates (32). Several studies have been conducted to study global methylation patterens with sperm quality and hence infertility. Elevated levels of DNA methylation of imprinted and nonimprinted genes and repetitive elements was reported in poor-quality semen samples (33). Improper erasure of methylation marks was elucidated as a cause of elevated methylation levels in cases of oligoasthenoteratozoospermia (OAT) rather than de novo methylation. They also suggested that not only imprinted genes, but also broad epigenetic defects are involved in causing sperm

defects. Similar kind of study by Aston et al. reported a genome-wide altered DNA methylation pattern in men with altered semen parameters (34) giving a connection of global methylation levels of sperm with fertility status of men.

Marques et al. studied methylation patterns in some important imprinted genes in different sperm populations. Loss of methylation of H19 DMR was found in men with OAT, and also they reported an association of reduced methylation of H19 DMR with decreased sperm count (14). Besides H19 DMR they also found altered methylation levels in MEST DMR in oligozoospermic infertile patients. In a different study loss of methylation in H19 and GTL2 DMRs and altered methylation acquisition at PEG1, LIT1, ZAC, PEG3, and SNRPN loci were reported in cases of oligozoospermia (35). Improper imprinting and methylation in the sperm has been found to be associated with ART failures. Using bisulfite conversion and cloning-sequencing technique it has been observed that imprinting errors in the spermatozoa resulted in spontaneous abortions in ART (35). Boissonnas et al. performed a quantitative analysis of methylation levels at each CpG position included in IGF2 and H19 DMRs (47 different CpGs) in a population of normozoospermic and oligozoospermic men (36). Loss of methylation in IGF2 DMR2 (primary IGF2DMR) and H19DMR was correlated with the severity of oligozoospermia.

Various other groups also demonstrated the association of improper methylation with sperm quality and fertility outcomes (18, 34, 37). Some other groups studied the methylation status of the promoter of important genes involved in sperm formation. The promoters of DAZL (17), CREM (38) and MTHFR (39) genes were studied for methylation and in all of these genes altered methylation patterns were found in the OAT patients. Hypermethylation of the MTHFR promoter was also reported in nonobstructive azoospermia and in idiopathic infertility (40).

By functional analysis it was reported that there is a gain of methylation in spermatogenesis-related genes and a loss of methylation in inflammation- and immune response-related genes (41). By targeting members of the PIWI-associated RNA (piRNA) processing machinery altered DNA methylation patterns has been observed in these genes. Male infer t i l i ty was associated with the promoter hypermethylation-associated silencing of PIWIL2 and TDRD1. It was also shown that methylation errors in these genes resulted in a defective production of piRNAs and a hypomethylation of the LINE-1 repetitive sequence in the patients. This study proves the role of altered DNA methylation in PIWIL2/TDRD1 has a role in the control of gene expression in spermatogenesis and hence may have an important role to play in infertility (42). Chunlin et al detected the testis and epididymis-specific methylated promoters in human cfsDNA. This finding may be of great clinical significance and may be used for diagnosis of male infertility by employing these methylated promoters as noninvasive epigenetic biomarkers (43).

All the studies till date on epigenetics of sperm suggests the

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fact that sperm DNA methylation patterns are essential for normal sperm function, good fertility outcomes and embryo development. However, the underlying causes of these epigenetic faults (methylation errors), and the timing of their occurrence remain poorly investigated. When are these methylation errors acquired? It is to be still answered that whether these methylation errors occur during fetal or early postnatal development? Epigenetic abnormalities are linked to abnormal DNA packaging and configuration during spermiogenesis where histones and protamines play a role in the maintenance of the methylation marks of male gametes (44).Studies are ongoing in our lab to correlate free radical levels and methylation pattern and if oxidative stress apart from causing non specific DNA damage, accelerated telomere shortening also cause methylation errors and thus affect not only the normally imprinted loci but also cause genome wide methylation defects.

Remarks

As discussed in this review epigenetic or methylation errors in both imprinted and non imprinted genes in the sperm DNA are responsible for the sperm defects, the major cause of male infertility and impaired embryonic development which manifests as recurrent spontaneous abortions and congenital malformations. Many studies provide evidence that epigenetic effects are induced by ART procedures which use epigenetically unstable and immature germ cells and are also impacted by the hormonal mileu. There are many confounding factors, such as parental subfertility, age and the interaction between epigenetic responses and genetic variations. All of these factors should be considered in investigations carried out to elucidate the effects of ART on epigenetic marks. More research is needed to evaluate the risk of transmission of epigenetic abnormalities by gametes of infertile men and women and in our lab we are trying to study correlation between oxidative stress and methylation errors. Genetic defects explain only a minor part of male fertility problems (13). The role of epimutations should be investigated. Epigenetics provides the most liable molecular mechanism for gene– environment interactions in the male germ line that may adversely affect semen quality and embryonic development. As is well understood that epigenetics is the result of gene environment interactions. It largely depends on the micro environment in which the gene has to be expressed. In our lab studies are ongoing to assses the alterations in methylation patterns in cases with oxidative stress and understand if epigenetic abnormalities are the underlying cause of infertility, congenital malformations and cancers in cases with oxidative stress. We have initiated a three year study on impact of yoga and meditation on oxidative stress levels, DNA fragmentation index , telomere length and telomerase levels and alterations in methylation levels. Infertility is beleiveed to be an early marker of cancer. We hypothesise that seminal oxidative stress, sperm DNA damage of both nuclear and mitochondrial genome, rapid telomere attrition and epigenetic abnormalities may be the underlying cause of cancer in both the infertile person ans well as the offspring.

Methylation errors in the form of hypermethylation of tumour suppressor genes and hypomethylation of oncogenes may explain for this link. Yoga and meditations are actually therapeutic and cause decline in free radical levels, upregulation in telomerase activity even 10 days post meditation/yoga and study is ongoing for longer periods.Yoga caused a significant decline in levels of inflammatory markers like interleukins and upregulation in the levels b endorphins. Studies should be conducted to investigate the role of environmental factors and lifestyle habits like dietary intake on the sperm epigenome and the consequent effect on the fertility outcome. Since epigenetic changes are reversible, a better understanding of epigenetic dysregulation in the male germ line may help in establishing new strategies to reinstate fertility in male infertility cases by modulating DNA methylation by dietary regulations and lifestyle modifications.

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6. Ogryzko VV (2008). Erwin Schroedinger, Francis Crick and epigenetic stability. Biol Direct; 3: 15.

7. Morris KL (2008). "Epigenetic Regulation of Gene Expression". RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity. Norfolk, England: Caister Academic Press. ISBN 1-904455-25-5

8. Ausió J, Eirín-López JM, Frehlick LJ (2007) . Evolution of vertebrate chromosomal sperm proteins: implications for fertility and sperm competition. Soc Reprod Fertil Suppl; 65: 63-79.

9. Hammoud SS, Nix DA, Zhang H, et al (2009). Distinctive chromatin in human sperm packages genes for embryo development. Nature; 460(7254): 473-8.

10. Arpanahi A, Brinkworth M, Iles D, et al (2009). Endonuclease-sensitive regions of human spermatozoal chromatin are highly enriched in promoter and CTCF binding sequences. Genome Res; 19(8): 1338-49.

11. Oliva R (2006). Protamines and male infertility. Hum Reprod; 12(4): 417-35.

12. Balhorn R (2007). The protamine family of sperm nuclear proteins. Genome Biol; 8(9): 227.

13. Gianotten J, Lombardi MP, Zwinderman AH, et al (2004). Idiopathic impaired spermatogenesis: genetic epidemiology is unlikely to provide a short-cut to better understanding. Hum Reprod; 10(6): 533-9.

14. Marques CJ, Costa P, Vaz B, et al (2008). Abnormal methylation of imprinted genes in human sperm is associated with

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oligozoospermia. Mol Hum Reprod; 14(2): 67-74.

15. Kobayashi H, Hiura H, John RM, et al (2009). DNA methylation errors at imprinted loci after assisted conception originate in the parental sperm. Eur J Hum Genet; 17: 1582–91.

16. Hammoud SS, Purwar J, Pflueger C, et al (2010). Alterations in sperm DNA methylation patterns at imprinted loci in two classes of infertility. Fertil Steril; 94(5): 1728-33.

17. Navarro-Costa P, Nogueira P, Carvalho M, et al (2010). Incorrect DNA methylation of the DAZL promoterCpG island associates with defective human sperm. Hum Reprod; 25(10): 2647-54.

18. Poplinski A, Tüttelmann F, Kanber D, et al (2010). Idiopathic male infertility is strongly associated with aberrant methylation of MEST and IGF2/H19 ICR1. Int J Androl; 33(4): 642-9.

19. El Hajj N, Trapphoff T, Linke M, et al (2011). Limiting dilution bisulfite (pyro)sequencing reveals parent-specific methylation patterns in single early mouse embryos and bovine oocytes. Epigenetics; 6(10): 1176-88.

20. Krawetz SA (2005). Paternal contribution: new insights and future challenges. Nat Rev Gene; 6(8): 633-42.

21. Carrell DT, Hammoud SS (2010). The human sperm epigenome and its potential role in embryonic development. Mol Hum Reprod; 16(1): 37-47.

22. Carrell DT (2010). Biology of spermatogenesis and male infertility. Syst Biol Reprod Med; 56(3): 205-6.

23. Rousseaux S, Caron C, Govin J, et al (2005). Establishment of male-specific epigenetic information. Gene; 345(2): 139-53.

24. Hajkova P, Ancelin K, Waldmann T, et al (2008). Chromatin dynamics during epigenetic reprogramming in the mouse germ line. Nature; 452(7189): 877-81.

25. Li JY, Lees-Murdock DJ, Xu GL, et al (2004). Timing of establishment of paternal methylation imprints in the mouse. Genomics; 84(6): 952-60.

26. Tilghman SM (1999). The sins of the fathers and mothers: genomic imprinting in mammalian development. Cell; 96(2): 185-93.

27. Reik W, Dean W, Walter J (2001). Epigenetic reprogramming in mammalian development. Science; 293(5532): 1089-93.

28. Arnaud P (2010). Genomic imprinting in germ cells: imprints are under control. Reproduction; 140(3): 411-23.

29. Bestor TH (2000). The DNA methyltransferases of mammals. Hum Mol Genet; 9(16): 2395-402.

30. Kaneda M, Sado T, Hata K, et al (2004). Role of de novo DNA methyltransferases in initiation of genomic imprinting and X-chromosome inactivation. Cold Spring Harb Symp Quant Biol; 69: 125-9.

31. Marques CJ, JoãoPinho M, Carvalho F, et al (2011). DNA methylation imprinting marks and DNA methyltransferase expression in human spermatogenic cell stages. Epigenetics; 6(11): 1354-61.

32. Benchaib M, Braun V, Ressnikof D, et al (2005). Influence of global sperm DNA methylation on IVF results. Hum Reprod; 20(3): 768-73.

33. Houshdaran S, Cortessis VK, Siegmund K, et al (2007). Widespread epigenetic abnormalities suggest a broad DNA methylation erasure defect in abnormal human sperm. PLoS One; 2(12): e1289.

34. Aston KI, Punj V, Liu L, et al (2012). Genome-wide sperm deoxyribonucleic acid methylation is altered in some men with abnormal chromatin packaging or poor in vitro fertilization embryogenesis. Fertil Steril; 97(2): 285-92.

35. Kobayashi H, Sato A, Otsu E, et al (2007). Aberrant DNA methylation of imprinted loci in sperm from oligospermic patients. Hum Mol Genet; 16(21): 2542-51

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Dynamics of DNA Demethylation and Epigenetic Reprogramming in Stem Cells

Swayamsiddha Kar

Samir Kumar Patra*

Department of Life ScienceNational Institute of TechnologyRourkela *[email protected]

Introduction

The molecular hallmarks guiding the smooth transformation of a one-celled yet all-powerful, totipotent zygote into a multicellular entity with over 220 specialized unipotent cell types are essentially scripted by synchronization between genetic and epigenetic programs in the stem cells. While genetic content of the stem cells acts as the template for generating varied transcriptional outcomes and remains basically unchanged, the epigenetic choreographers interpret and translate this information into cell-specific functional destinies (1, 2). Stem cells, with their ability to differentiate into any mature cell type and capacity to


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undergo indefinite replicative cycles are considered to be strategically ideal vessels for enactment of the developmental evolution (3). Epigenetic reprogramming of the stem cell transcriptome is the ultimate control mechanism that endows the stem cells with the adaptability, flexibility and versatility to modify their gene expression profile in response to developmental cues and differentiate into any cell type in the adult body (4, 5). This review focuses on the epigenetic determinants participating in reconfiguration of the genome towards cellular differentiation with emphasis on the role of DNA demethylation in this scheme. Exploring the machinery behind epigenomic regulation of stem cell fate will provide better understanding of the intricate system supervising the stem cells as they execute the molecular floor plan of cellular development.

Epigenetic Reprogramming is the Master Architect in Designing Stem Cell Destiny

The mammalian developmental scheme entails the gradual conversion of a small group of genetically homogeneous cells into structurally and functionally heterogeneous organism. As cellular differentiation proceeds towards terminally specialized states, cells with higher potency gradually lose their potential and become committed for specific lineages (6, 7). In terms of transcriptional states, while totipotent and pluripotent stem cells promote the expression of pluripotency-associated factors and transiently restrict the expression of regulatory genes that lead to lineage-speciation; the onset of lineage-orientation witnesses gradually stripping off the restrictive marks on lineage-devoted progenitor and lineage-committed genes, thus allowing them to reassert their identity (8). The decision of the stem cell to either maintain its pluripotency or differentiate into a specialized cell is influenced predominantly by epigenetic events that deliver heritable and stable instructions for the specification of chromatin organization and differential transcriptional outcomes. In fact, the epigenetic signature of any cell is a sensitive indicator of its past and current developmental standing and may ultimately decide its future phenotype and functional character (9). Thus, epigenetic restructuring of the genomic domain is an essentiality during transition of a totipotent zygote into pluripotent stem cells, subsequent channelization towards multipotent lineage progenitors and final acquisition of lineage-specific identity of unipotent somatic cells.

A wide number of epigenetic enforcers converge in the chromatin, modify and manipulate the nuclear architecture to yield varied transcriptional consequences in the stem cells. Of the many agents, the most notable ones are reversible DNA-methylation and covalent post-translational histone modifications. These two modifications work in a stratified manner wherein short-term flexibility imposed by transient histone modifications on the lineage-committed genes during early development is reinforced by induction of long-term or permanent repression of pluripotency genes by DNA-methylation,

nucleosomal rearrangement and higher-order chromatin reorganization at the onset of differentiation (4, 10). Maintenance of such a fine-tuned and efficient arrangement is ensured via co-ordinations among numerous enzymatic players responsible for deposition (writers) and removal (erasers) of the epigenetic marks/tags, as well as various protein complexes that recognize these modifications (readers) and convert them into functional capacity of the cell (11). Alongside these factors, a rigidly coordinated network of four master transcriptional regulators—OCT4, SOX2, NANOG and KLF4 stringently guard stem cell pluripotency and smoothly maneuver transition between differential gene expression states (12, 13). The congregation of numerous epigenetic factors, all working synchronously towards a single goal, guarantees robustness at the level of individual cells and warranties high fidelity of the entire differentiation program.

DNA-Demethylation Plays Godfather Role During Reorganization of Stem Cell Fate

DNA-methylation and demethylation are considered to be significantly more important players participating in the epigenetic scheme behind regulation of development and differentiation (14, 15). Transcriptional plasticity and versatility of the stem cells is achieved as a result of a delicate balance between the processes that add and remove the epigenetic labels especially the methyl tags on the cytosine bases. While DNA methylation integrates the transient restrictive marks previously enforced by histone modifications to permanently turn off unnecessary genes during early embryonic divisions and lineage speciation, DNA-demethylation purges off repressive marks from the genomic database for rapid reactivation of silenced genes leading to assertion of individual cell fate (16, 17). Cellular differentiation and lineage-commitment pathways witness both genome-wide and gene-specific erasure of methyl marks via active and passive mechanisms, thus emphasizing the part played by DNA-demethylation in this scheme (18). Although, DNA-demethylation can’t be held responsible for all the differentiation related changes but a hypomethylated state may facilitate transcriptional changes essential to erase the existing epigenomic memory and generate somatic cell-destiny. In a nutshell, dynamic changes in the pattern of methyl marks both at the global, individual chromosome and gene-specific level by DNA-methylation and demethylation represents a fundamental mechanism in developmental progress.

DNA-demethylation arbitrated erasure of epigenetic barriers appears consistently at every crucial juncture during mammalian development. Starting at the onset of fertilization, DNA-demethylation activates the quiescent transcriptional machinery in the totipotent zygote, facilitates extensive germ-line reprogramming in the primordial germ cells and ultimately steers the pluripotent stem cells into lineage-restricted paths. Just after fertilization and before the initial zygotic divisions, DNA demethylation mediates remodeling of the paternal genomes via both active and passive mechanisms. Before pronuclear fusion and first


cleavage division, the paternal pronucleus experiences genome-wide active demethylation (19, 20). During the progress towards the 16-celled morula, the maternal pronucleus is passively deprived of its methylation load via passive, replication-dependent loss of methylation (21). At the blastocyst stage, the delineation of trophoectoderm (TE) from the inner cell mass (ICM) is also aided by promoter hypomethylation of transcription factor E-74 like Factor 5 (Elf5) (6, 22). Thus, DNA-demethylation stimulates the onset of pluripotency in the zygote leading to expression of lineage-determining transcription factors and segregation into the three embryonic germ layers.

The second critical phase of DNA demethylation mediated epigenetic reorganization is seen during germ-line reprogramming of PGCs. In the first round, the early PGCs acquire the ability to generate pluripotent embryonic germ cells (EGCs) by transcriptional redirection and activation of the germ cell genome from a somatic cell fate (23). In the second round, demethylation in the PGCs results in the expression of a number of genes involved in myriad aspects of germ-line genome activity. For example, activation of Deleted in Azoospermia-Like (Dazl) and Synaptonemal Complex Protein 3 (Sycp3) is necessary for gametogenesis (24), hypomethylation of germ-line genome-defense specific genes such as Testis expressed gene (Tex19.1, Tex19.2), Mili (also called Piwi-like 2) is necessary to suppress any unmasked retrotransposon activity and ensure genome integrity (25) whereas demethylation assisted removal of parent-of-origin-specic imprinting marks including the maternally methylated small nuclear ribonucleoprotein N (Snrpn), Lit1, insulin-like growth factor 2 (Igf2) and the paternally methylated H19 and Ras protein-specific guanine nucleotide-releasing factor 1 (Rasgrf1) leads to imprint erasure (26). Hence, DNA-demethylation induced erasure of epigenetic barriers in the PGCs results in rebirth of the totipotent zygote in the next generation.

Finally, as the pluripotent ESCs gear up for lineage-commitment, DNA-demethylation assists in deciding their final destination. Hypomethylation mediated over-expression of lineage-determining transcription factors, lineage-specific and lineage-restricted genes leads to a satisfying end to the process of organogenesis in the form of terminally differentiated mature cell types (27). Promoter DNA-demethylation helps in hematopoietic speciation in three concurrent steps—firstly, conversion of ESCs into hematopoietic stem cell (HSC) population by hypomethylation of HSC-specific genes such as Runt-Related Transcription Factor 1 (Runx1) and Vasorin (Vasn), followed by over-expression of hematopoietic lineage-specific genes that creates myeloid (Myeloperoxidase (Mpo), β-globin, Platelet Glycoprotein 6 (GP6/GPVI)) and lymphoid (Lymphocyte-Specific Protein Tyrosine Kinase (Lck), ADP-ribosylation factor-Like 4C (ArL4c), Smad7) lineages from the multipotent HSCs and finally, demethylation of hematopoietic-restricted genes in the lineage-specific progenitors to form the mature cells such as

B-cells, T-cells, Natural Killer (NK) cells, myeloid cells, red blood cells, platelets, etc…….(28, 29, 30). Similarly, dissemination of the mesenchymal stem cells (MSCs) into different functional states such as myogenic, osteogenic, chondrogenic or adipogenic lineages (31, 32) and sequential differentiation of neural stem cells (NSCs) into the three major neural lineages-neurons, astrocytes and oligodendrocytes is also mediated by global and gene-specific hypomethylation (33). Thus, DNA-demethylation is a major factor instigating epigenetic manipulation of the pluripotent stem cell niche into functional specialization in the adult.

Histone Modifications and Nuclear Remodeling also Regulate Stem Cell Chromatin Landscape

Post-translational covalent reversible histone modifications are crucial agents directing epigenetic regulation of stem cells. Specific histone modification profiles are responsible for generating differential transcriptional outcome. Depending on the presence and abundance of histone marks, three different states of chromatin arrangement is seen in case of ESCs (34). Firstly in the totipotent zygote and pluripotent ESCs open, active euchromatin is associated with permissive marks such as histone H3 lysine 4 trimethylation (H3K4me3), histone H3 lysine 9 acetylation (H3K9ac) and histone H4 acetylation (H4ac). As differentiation advances towards somatic cells, lineage-control genes are stably silenced by repressive histone marks such as histone H3 lysine 9(H3K9me3) and histone H3 lysine 27 (H3K27me3) trimethylation. The final type of histone mark arrangement is a unique feature known as bivalent domains. Bivalent chromatin is ESCs and multipotent neural progenitors, MSCs and hematopoietic stem and progenitor cells. These bivalent chromatin domains contain active H3K4me3 and repressive H3K27me3 along with non-methylated CpG DNA regions (non-mCpG) and repressive H2AK119Ub1 marks (35, 36). The presence of these domains on key developmentally regulated genes indicates that the lineage-specific gene expression programs in these loci are repressed or poised, yet are ‘primed’ for rapid induction of expression upon receiving differentiation cues (11). Thus, histone modifications act as a gateway for smooth conduct of differentiation traffic along the right direction during lineage-commitment.

Nuclear architecture also undergoes extensive changes such as level of chromatin compaction, its accessibility and positioning within specialized nuclear domains and changes in chromatin organization components – heterochromatin and centromere positioning when ESCs progress along the differentiation axis. As gradual decrease in cellular potency sets in, chromatin landscape witness a drastic metamorphosis, i.e. from an open and globally accessible state in the undifferentiated ESCs permitting stem cell pluripotency to a rigid, transcriptionally restrained state of a lineage-committed cell (34). Also, deviations in relative positioning of epigenetic enforcers and transcription factors between the transcriptionally-restrictive nuclear periphery

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and transcriptionally-permissive nuclear interior promote cellular differentiation. Thus, the different degree of condensation state of chromatin and changes in nucleosomal organization allows various transcriptional profiles to be established during the course of cell commitment (11).

Epigenetic Makeover of Stem Cells via DNA-Demethylation – A Breaking Dawn in Biomedical Research

Molecular reorganization of the epigenetic determinants in stem cells for cellular therapy, tissue repair and regeneration is now considered to be enterprising option in the field of biomedical r e s e a r c h . I n t h i s scenario, stem cell based p h a r m a c o l o g y spearheaded by DNA-demethylation can fulfil the dream of achieving individually-tailored patient-specific remedies. In vitro alteration of MSCs via DNA-demethylation have been considered for cartilage tissue engineering, remodelling, repair, and rejuvenation of bone tissues and gene therapies for local or systemic pathologies (37). In a similar approach, DNA-demethylation induced in vivo differentiation of N S C s i n t o n e u r a l p r e c u r s o r c e l l s i s c u r r e n t l y investigated for futuristic treatment of brain cancer, ischemic spastic paraplegia, chronic spinal cord injury and chronic stroke. Treatment of a number of hematological malignancies can also be envisioned by applying the same principle of DNA-demethylation of lineage-unrelated genes such as that will lead to normal haematopoietic differentiation (38). Along with prognostic and treatment-related benefits, DNA-demethylation arbitrated molecular reorganization of stem cells also offers possibilities for resolving many frustrating problems in the field of human reproductive health. Germ-line reprogramming via global hypomethylation can be employed for solving human infertility problems, treatment of sex-linked genetic disorders and improving the efficiency of mammalian embryo cloning. Addressing the issue of aging by rejuvenating older somatic cells has by far been the proverbial Pandora’s Box. Reversing the course of the aging somatic cells and transforming them into younger ones via global demethylation is now thought to be a likely solution to this dilemma. In a nutshell, epigenomic renovation of stem cells via DNA-demethylation is the ultimate weapon in the area of healthcare management with promising answers

to mysteries in the field of developmental biology.

Concluding Remarks

Epigenetic modifications are increasingly implicated to be participating in cellular homeostasis, preservation of

genome integrity and of course disease progression; hence, it becomes necessary to script an

epigenetic instruction manual highlighting the molecular details of cellular

commitment and differentiation. Elucidation of intricacies of

epigenomic regulatory mechanism will help

actively manipulate cell fate conversion and coax stem cells to a d o p t a d e s i r e d phenotypic outcome.

U n d e r s t a n d i n g t h e d y n a m i c s o f D N A -

demethylation will offer answers to problems faced

during cellular programming events such as induced stem cell pluripotency,

transdifferentiation and nuclear reprogramming of adult stem cells. Exploring and establishing the

founding principles upon which cell lineages are defined and maintained by

hypomethylation facilitated changes will go a long way in shaping the molecular floor plan of cellular development.

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14. Patra SK, Bettuzzi S (2008). Epigenetic DNA-(Cytosine-5-carbon) Modifications: 5-Aza-2’-deoxyctyidine and DNA modification. Biochemistry; 74(6): 613-9.

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16. Patra SK, Patra A, Rizzi F, et al (2008). Demethylation of (cytosine-5-Cmethyl) DNA and regulation of transcription in the epigenetic pathways of cancer development. Cancer Metast Rev 27(2): 315-34.

17. Meissner A, Mikkelsen TS, Gu H, et al (2008). Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature; 454(7205): 766-70.

18. Arney KL, Erhardt S, Drewell RA, et al (2001). Epigenetic reprogramming of the genome–from the germ line to the embryo and back again. Int J Dev Biol; 45(3): 533–40.

19. Mayer W, Niveleau A, Walter J, et al (2000). Demethylation of the zygotic paternal genome. Nature; 403(6769): 501–2.

20. Oswald J, Engemann S, Lane N, et al (2000). Active demethylation of the paternal genome in the mouse zygote. Curr Biol, 10(8): 475–8.

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Assisted Reproductive Technology (ART) and Imprinting Disorders: Epimutation in Gametes as a Functional Cause

Shaoni Bhattacharjee

Jana Chakrabarti*

Department of BiotechnologyPresidency UniversityKolkata*[email protected]

According to the CDC's (Centres for Diseases Control and Prevention) definition (based on 1992 Fertility Clinic Success Rate and Certification Act) 'Assisted Reproductive Technology' (ART) includes all fertility treatments in which both eggs and sperms are handled (1). Generally procedures included in ART treatments are, In vitro Fertilisation (IVF), Intracytoplasmic Sperm Injection (ICSI), Frozen Embryo Transfer (FET), Gamete Intra-Fallopian Transfer (GIFT), Assisted Hatching etc. (2). But It should also be remembered that they do NOT include treatments in which only sperm are handled (i.e., intrauterine - or artificial—insemination) or procedures in which a woman takes medicine only to stimulate egg production without the intention of having eggs retrieved (1).

Assisted reproductive technology (ART) has grown by leaps and bounds in the last few years in India. In a latest survey,

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based on the number of applications received for National ART Registry, Indian Council of Medical Research (ICMR) puts the number of such clinics as 125 in the capital city of India. Officials however believe that the actual figures are around 250-300 (3). In the year 2000, only 5500 cycles of IVF-ICSI were performed in our country, but this number increased to 21,500 in 2006 and in 2011 it became 110,000 (4). However, still ART has some safety problems and risks that need to be described and evaluated so that current clinical policies and laboratory procedures can be revised, if necessary.

The term 'Imprinting' corresponds to a specific epigenetic regulation leading to expression of only one parental allele of a gene. Some imprinted genes exhibit paternal expression whether others exhibit maternal expression. The best-characterized mark of gene imprinting is DNA methylation / unmethylation (5, 6). Usually, methylated DNA sequences are transcriptionally inactive, whereas unmethylated DNA sequences are transcriptionally active (7). There are two mechanisms by which DNA methylation inhibits gene transcription: the first one is the interference of the methyl group with the binding of particular transcription factors to the DNA (8). On the other hand the second one involves the methyl-binding domain proteins mediating transcriptional repression through binding to the DNA (9).

Animals studies on epigenetic reprogramming in the oocyte suggest that imprints are established in growing oocytes during primordial to antral follicle transition (10, 11) and are not completed for some genes until just prior to ovulation. For this reason, maternal imprinting mechanisms could be vulnerable to ovarian stimulation. Because of these concerns, it is important to scrutinize the strength of current evidences regarding the association between ART and imprinting. But the rigorous analysis of the issue becomes complicated by the variability in ART protocols and the rarity of imprinting disorders.

Table 1: Selected Human Disorders Linked to an Imprinting Defect that has been Reported after ART

The word ‘Epimutation’ means a heritable change in gene expression that does not affect the actual base pair sequences of DNA. It is thought that an epimutation in the germ line would produce a phenotype equivalent to that resulting from an inactivating germ line mutation in the same gene.

Evidences accumulated from the literatures published during last few years that gamete progressively acquire epigenetic information, it is possible that immature gametes do not have the full and correct complement of epigenet ic information necessary for normal development. Round spermatids, (largely used in ICSI), are transcriptionally active, and introducing their RNAs into the oocyte via ICSI may lead to altered gene expression, either directly or through RNA interference mechanisms (12). Ejaculated spermatozoa, elongated spermatids and round spermatids all have the correct paternal imprint on chromosome 15q11-13, suggesting that this specific paternal imprint is established in immature testicular spermatids. However, other imprinted regions must be assessed before round spermatid injection may be considered safe (13).

Analysis of epimutations should include ontogenetic specificity of epigenetic processes at the different stages of individual development. Most significant changes of the epigenetic organization of the genome occur during maturation of germ line cells and at early stages of pre- and post implantation development of mammals (14).

Inheritance of genetic alterations is undoubtedly among the central issues of genetics that can be addressed to the inheritance of epimutations over generations. Investigation of inheritance of epimutations can be taken into account at least two circumstances: first, epigenetic genome modifications are reversible and second, there are ontogenetically determined periods of total epigenetic genome reprogramming that provide elimination of aberrant epigenotypes. In this circumstance, a special interest is driven to inheritance of epimutations of imprinted genes, since their phenotypic manifestation depends on sex of the parent that transmitted the epimutation to the offspring.

In most of the cases of mutation in Imprinting Centers (IC) with Angelman Syndrome (AS) and Prader-Willi Syndrome (PWS) are familial. Since in Angelman Syndrome Shortest Region of deletion Overlap (AS-SRO) mutations involve only maternal imprinting, they are transmitted via male germ line without phenotypic expression. Likewise, IC-SRO mutations involving paternal imprinting are not expressed when transmitted through maternal gametogenesis. This explains the unusual mode of inheritance of IC mutations in generations. For instance, mutation in IC-SRO, appearing on chromosome 15 in grandmother, will lead to any phenotypic consequences neither in her nor in her son (Fig.1). However, passing through spermatogenesis, this mutation will cause PWS in her grandchildren. Likewise, to cause AS, the IC mutations must pass through maternal gametogenesis (15).

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Disorders Candidate Chromosomal

Location

BWS (Beckwith-Wiedemann syndrome) 11p15

AS (Angelman Syndrome) 15q11-13

PWS (Prader–Willi syndrome ) 15q11-13

SRS (Silver–Russell syndrome) 7

Isolated hemihyperplasia 11p15

Autism 15q11-13

Bipolar disorder 18p11.2

Schizophrenia 18p11.2

Retinoblastoma 13q

[Source: Carter M. Owen and James H. Segars Jr. Imprinting Disorders and

Assisted Reproductive Technology. Semin Reprod Med.; 2009 September;

27(5): 417-428. Doi: 10.1055/s-0029-1237430.]


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Figure 1: Hypothetic Pedigrees Explaining the Inheritance of Chromosome 15 IC Mutations Fixing Maternal (a) or Paternal (b) Imprinting in Prader–Willi and Angelman Syndromes. When Fixed Paternal Imprinting Passes Through Oogenesis, the Patient has the Maternal Homolog with the Paternal Epigenotype—mat (pat), which Results in the Formation of AS

[Source: N. Lebedev and E. A. Sazhenova. Epimutations of Imprinting Genes in the Human Genome: Classification, Causes, Association with Hereditary Pathology. Russian Journal of Genetics; 2008; Vol. 44, No. 10, pp. 1176–1190. DOI: 10.1134/S1022795408100062]

In a another study, to examine the origin of chromosome 15 with AS-SRO in 18 AS patients, it was found that seven patients inherited the mutation from the grandmothers and eleven patients, from the maternal grandfathers (16). This mode of inheritance might be explained by the appearance of the imprinting defect after the erasure of the parental epigenotypes and possibly even after fertilization. An additional support of the post zygotic appearance of the epimutations is provided by the above-mentioned (Fig-1) somatic mosaicism for IC methylation, revealed in approximately 30% of AS patients. The mechanisms underlying this mosaicism are unclear, but they are likely associated with IC demethylation in some somatic cells.

Moreover, in addition to inheritance of epimutations of imprinted genes, reports on possible transmission to offspring of aberrant epigenetic modifications of non-imprinted genomic loci is also described in two patients with colorectal cancer, who had mosaic hypermethylation of the promoter region of the mismatch repair gene MLH1(17). An epimutation was also present in sperm of one of the patients, indicating a defect in the embryonic germ line and possible transmission to the offspring. The available reports therefore attest to a potential contribution of the conformation (epigenotype) of DNA in determining the functional activity of the gene that can also be inherited, thus causing the

development of diseases in certain cases. This epigenetic inheritance may be characterized by transmission of the epimutation over several generations and possible restoration of the normal epigenetic status in the pedigree. However, it is still unclear whether the repair of resistance of epimutations is determined by sex (spermatogenesis or oogenesis), which can be of special importance in case of imprinted genes, whose activity depends on the sex of the parent having transmitted them.

Therefore, to date the possibility of transmission of primary epimutations and their repair in generations is still under debate. No convincing molecular evidence is available to explain such mechanisms of epigenetic inheritance. Diseases like Autism, Bipolar disorder, Schizophrenia, Retinoblastoma (Table 1) are still in the light of further research to get linked with assisted reproductive technologies. However, in the present discussion, an attempt has been made to mention in short about the diversity of imprinted genes because investigation on their causes, phenotypic effects, and inheritance patterns seems to be a very promising line of research in the modern human genetics, to enhance our understanding of etiological basis of hereditary diseases.

References

1. http://www.cdc.gov/art

2. http://www.rtc.org.au/glossary


3. Chandra N (2013). When you cannot produce a baby, design it! Over 500 babies born each month in Delhi fertility clinics; Mail Today

4. Pratap RK (2011). IVF Baby Boom. Outlook Business; 10.

5. Li E, Beard C, Jaenisch R (1993). Role for DNA methylation in genomic imprinting. Nature; 366: 362–5.

6. Kaneda M, Okano M, Hata K, et al (2004). Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting. Nature; 429: 900–3.

7. Dennis C (2003). Epigenetics and disease: altered states. Nature; 421: 686–8.

8. Iguchi-Ariga SM, Schaffner W (1989). CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation. Genes Dev; 3: 612–9.

9. Swales AK, Spears N (2005). Genomic imprinting and reproduction. Reproduction; 130: 389–99.

10. Hajkova P, Erhardt S, Lane N, et al (2002). Epigenetic reprogramming in mouse primordial germ cells. Mech. Dev; 117(1–2): 15–23.

11. Obata Y, Kaneko-Ishino T, Koide T, et al (1998). Disruption of primary imprinting during oocyte growth leads to the modified expression of imprinted genes during embryo-genesis. Development; 125 (8): 1553–60.

12. Borghol N, Blachere T, Lefevre A (2008). Transcriptional and epigenetic status of protamine 1 and 2 genes following round spermatids injection into mouse oocytes. Genomics; 91: 415 – 22.

13. Huntriss J (2011). Epigenetics and Assisted Reproduction in Kay Elder and Brian Dale (with contribution from Harper J, Huntriss J) ed. Book on In-Vitro Fertilization: Third Edition;; Cambridge University Press; 252-267 [ISBN: 978-0-521-73072-3 (Paperback)]

14. Li E (2002). Modification and Epigenetic Reprogramming in Mammalian Development.;

Nat Rev Genet; 3: 662–73.

15. Lebedev N, Sazhenova EA (2008). Epimutations of Imprinting Genes in the Human Genome: Classification, Causes, Association with Hereditary Pathology. Russian Journal of Genetics; 44(10): 1176–90.

16. Buiting K, Gross S, Lich C, et al (2003). Epimutations in Prader–Willi and Angelman Syndromes: A Molecular Study of 136 Patients with an Imprinting Defect. Am J Hum Genet; 72: 571–7.

17. Suter CM, Martin DI, Ward RL (2004). Germ line Epimutation of MLH1 in Individuals with Multiple Cancers. Nat Genet; 36: 497–501.

Epigenetics: A Paradigm in Germ Cells Differentiation and In Vivo Development

S. D. Kharche*, S. K. Agarwal

PR & SM DivisionCentral Institute for Research on Goats Makhdoom*[email protected]

Epigenetics has evolved very quickly from the study of an obscure collection of diverse phenomena to become one of the most exciting topics in contemporary biology. The traditional view that gene and environment interactions

control disease susceptibility can now be expanded to include epigenetic reprogramming as a key determinant of origins of human disease. It is a rapidly expanding field of study in which the molecular mechanisms of unrelated normal processes, such as paramutation in maize, position effect variegation (PEV) in the fruit fly, and genomic imprinting and X-chromosomal inactivation in mammals, are now recognized as evolutionarily conserved epigenetic processes. Currently, epigenetics is defined as heritable changes in gene expression that do not alter DNA sequence but are mitotically and transgenerationally inheritable. The epigenetic regulation of gene expression is essential for the normal growth, development, and aging of higher organisms (1). Epigenetics also underlies genomic imprinting, programming, and reprogramming in early life and the increased susceptibility to disease in later life. Epigenetic reprogramming is the process by which an organism’s genotype interacts with the environment to produce its phenotype and provides a framework for explaining individual variations and the uniqueness of cells, tissues, or organs despite identical genetic information. The main epigenetic mediators are histone modification, DNA methylation, and non-coding RNAs. They regulate crucial cellular functions such as genome stability, X-chromosome inactivation, gene imprinting, and reprogramming of non-imprinting genes, and work on developmental plasticity such that exposures to endogenous or exogenous factors during critical periods to permanently alter the structure or function of specific organ systems. Aberrant changes in linear DNA sequences result in mutations, deletions, gene fusion, tandem duplications, or gene amplifications causing dysregulation of gene expression that lead to the genesis of disease (2). Recently, however, it has become clear that epigenetic disruption of expression plays an equally important role in the development of disease (3) and this process is more susceptible as compare to environmental modulation.

Germ cells and somatic cells have the identical genome. However, unlike the mortal fate of somatic cells, germ cells have the unique ability to differentiate into gametes that retain totipotency and produce an entire organism upon fertilization (4). The processes by which germ cells differentiate into gametes, and those by which gametes become embryos, involve dramatic cellular differentiation accompanied by drastic changes in gene expression, which are tightly regulated by genetic as well as epigenetic mechanisms (5). Epigenetic regulation refers to heritable changes in gene expression that are not due to changes in primary DNA sequence. It alters chromatin structure by changing the position, organization and composition of nucleosomes, while preserving primary DNA sequence and contributes significantly to “cellular memory”, which maintains a particular cell fate through both mitotic and meiotic cell divisions (6). Epigenetic regulation of the genome involves factors such as histone modifications (i.e. acetylation and methylation) and DNA methylation that directs chromatin structure and gene transcription. Among all known cell types, germ cells are unique due to their

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abilities to produce the next generation of an entire organism upon fertilization. Because of their immortal nature, it is critical for germ cells to not only undergo proper cellular differentiation within one generation, but also retain accurate information to initiate the next generation. Specifically, epigenetic mechanisms may play roles in: (1) ensuring meiosis and terminal differentiation program during gametogenesis; (2) reliably retaining information in gametes for the next generation; (3) erasing improper features in zygotes before initiating a new life cycle, in order to prevent inheritable epimutations.

In many species, gametogenesis is initiated by germ cells, which undergo transit-amplificationbefore committing to meiosis and terminal differentiation. The chromatin structure of germ cells acts as an intrinsic mechanism to maintain their unique self-renewal ability and block differentiation. In addition to chromatin remodeling, dynamic regulation of his tone modifications is also required to maintain germ cells identity. Meiosis is a process unique to germ cells. It is also thought that histone modifying enzymes that remove and generate these histone modifications mustact cooperatively to ensure the changes. Indeed, histone modifying enzymes with similar or distinct activities act in an orchestrated manner in regulating germ cell differentiation. Another dynamic epigenetic regulation during mammalian gametogenesis is the establishment of differential DNA methylation at imprinted genes, which are differentially expressed according to their parental origin and important for embryonic development. In females, genomic imprinting occurs after birth in oocytes arrested at diplotene stage of meiotic prophase I, and the de novo methylation process is complete by the fully-grown oocyte stage (7).

A recent report has revealed that de novo DNAmethylation in mouse male germline occurs mainly in spermatocytes at early meioticprophase, in addition to spermatogonial cells (8). During spermiogenesis in post-meiotic germ cells histones are displaced by the transition nuclear proteins (Tnps) followed by protamines (Prms), allowing for extensive chromatin condensation and DNA packaging in sperm nuclei. This process is regulated by a variety of epigenetic mechanisms, including histone modifications, chromatin remodeling, and histone variants deposition. The DNA methylation pattern of the genome becomes reprogrammed following de-methylation and re-methylation processes after fertilization and during early embryonic development. This epigenetic reprogramming during early embryonic cell differentiation transmits a unique DNA methylation pattern to developing organs in the offspring. An additional epigenetic reprogramming event (i.e. DNA methylation) occurs later in development in the germ line during sex determination. A small subset of imprinted genes is transmitted to subsequent generations through the male or female germ line. Imprinted genes have an allele specific DNA methylation pattern and expression that is maternally or paternally transmitted between generations. Clearly a number of different epigenetic

mechanisms (e.g. histone modifications, chromatin structure and DNA methylation) will be involved in programming the germ line. Alterations in the epigenetic reprogramming of the germ line can promote heritable changes on transcription and disease (9, 10).

Parthenogenesis, which is a successful development of unfertilized eggs, is observed in many vertebrate species and, if it were possible in mammals, would provide a way to produce clones of livestock animals. However, imprinting is a major barrier for parthenogenetic embryo development in mammals because expression levels of the imprinted genes, which include many important developmental genes, are unbalanced in such embryos. However, by modifying imprinted genes by genetic engineering and developmental manipulation, it was possible to derive adult female mice with two maternal genomes and no paternal complement (11, 12). As the method involves genetically engineered animals and highly complex nuclear-transfer technologies, its direct application to livestock seems difficult. Similarly, genomic imprinting and expression of developmental genes in parthenogenetic goat embryos also play an important role during in vivo embryo development. Hence the parthenogenetic goat embryos could not developed in vivo beyond thirty four days following embryo transfer in to surrogate mother (13).

References

1. Feinberg AP (2007). Phenotypic plasticity and the epigenetics of human disease. Nature; 447: 433–40.

2. Garg V (2006). Insights into the genetic basis of congenital heart disease. Cell Mol Life Sci; 63: 1141–8.

3. Dolinoy DC, Weidman JR, Jirtle RL (2007). Epigenetic gene regulation: linking early developmental environment to adult disease. Reprod Toxicol; 23: 297–307.

4. Cinalli RM, Rangan P, Lehmann R. (2008). Germ cells are forever. Cell; 132: 559–62.

5. Kimmins S, Sassone-Corsi P (2005). Chromatin remodelling and epigenetic features of germ cells. Nature; 434: 583–9.

6. Ringrose L, Paro R (2004). Epigenetic regulation of cellular memory by the Polycomb and Trithorax group proteins. Annu Rev Genet; 38: 413–43.

7. Hiura H, Obata Y, Komiyama J, et al (2006). Oocyte growth-dependent progression of maternal imprinting in mice. Genes Cells; 11: 353–61.

8. Oakes CC, La Salle S, Smiraglia DJ, et al (2007). Developmental acquisition of genome-wide DNA methylation occurs prior to meiosis in male germ cells. Dev Biol; 307: 368–79.

9. Tarozzi N, Bizzaro D, Flamigni C, et al (2007). Clinical relevance of sperm DNA damage in assisted reproduction. Reproductive BioMedicine Online; 14: 746–57.

10. Yang J, Yang S, Beaujean N et al (2007). Epigenetic marks in cloned rhesus monkey embryos: comparison with counterparts produced in vitro. Bio Reprod; 76: 36–42.

11. Kono T, Obata Y, Wu Q, et al (2004). Birth of parthenogenetic mice that can develop to adulthood, Nature; 428: 860–4.

12. Kuwahara, M, Wu Q, Takahashi N, et al (2007). High-frequency generation of viable mice from engineered bi-maternal embryos. Nat Biotechnol; 25: 1045–50.

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13. Kharche SD, Goel AK, Jindal SK, et al (2014). Developmental potency of parthenogenetic goat embryos following in vivo transfer in caprahirus. International conference on reproductive health: Issue and strategies under charging climate scenario held on 6-8 February, IVRI, Izatnagar Bareilly.

The Expanding Role of Epigenetics in Reproductive Health

M. Ankolkar, N. H. Balasinor*

Neuroendocrinology DivisionNational Institute for Research inReproductive Health (ICMR)Mumbai*[email protected]

Epigenetics: An Emerging Concept

Ever since the theory of “Genes” and “Mendelian inheritance” has been proposed by legendary personality Gregor Mendel in the early 19th century, and the work of other eminent Nobel laureats, Thomas H. Morgan, Barbara Mclintock; our perception of genetic hereditary and inheritance has been that it is written in the language of DNA and the nucleotide backbone. For decades we have assumed how genetic mutations and changes in the nucleotide sequence can predict and explain the observed phenotypic traits. Although genetics plays a large role in defining the physique and physiology of organisms, it is a combination of genetics with the organisms' experiences (a.k.a. environment) that determines the ultimate phenotype. One of the mechanisms by which this can be achieved is by controlling the expression of genes depending on need. Although, genetic mutations are a known mechanism of evolution in respect to the change in environment, it is a very slow process that occurs over thousands of years and is thus insufficient to explain the continuous adaptation of an organism to its constantly changing environment. Around 1942, a concept was introduced by a noted developmental biologist, Sir Conrad H. Waddington, to explain this phenomenon called "The theory of Epigenesis". He coined the term "Epigenetics" which was defined as the interactions of the genes with the environment that brings the phenotype into being (1). In modern biology, it is defined as the changes in gene expression without affecting the DNA sequence. The concept of epigenetics is capable of explaining the ability of the cells to adapt to the dynamic environment despite having the same genetic constitution. It is brought about by 3 basic mechanisms DNA methylation at the 5th Carbon atom of Cytosine residue at CpG dinucleotides, Histone tail specific residue modifications and heritable RNAs.

Epigenetics is involved in two main aspects of reproductive health, namely, gametogenesis and embryogenesis. One such epigenetic phenomenon is genomic imprinting, whereby expression of some genes or chromosomal regions depends on the parental origin of the allele. Genomic imprinting is important for embryo development; however its establishment is during gametes formation. The parental

specific expression is due to differential marking on the DNA on the two parental alleles by DNA methylation during gametogenesis. In addition to establishment of imprinting marks during gametogenesis, another significant epigenetic modification happens during spermatogenesis, when round spermatids undergo cyto-differentiation to form mature spermatids with condensed chromatin, which is due to the replacement of histones by protamines, thereby modifying the necleosomal chromatin structure into toroidal.

During early embryogenesis in mammals, following the union of the two unipotent gametes, a wave of genome-wide DNA methylation reprogramming takes place. Firstly, the paternal and maternal genome undergoes demethylation leading to totipotent zygote. After implantation, there is global de novo methylation, which is maintained throughout life in the somatic cells. It has been hypothesized that these process of DNA demethylation and remethylation may be affected during assisted reproductive technique, where early embryo development till the blastocyst takes place in-vitro.

Epigenetic Alterations in Assisted Reproductive Techniques

ARTs account for 1-4% of births in the developed countries. During recent years ARTs have been found to be associated with in developmentally associated disorders related to genomic imprinting. One such problem is the overgrowth syndrome called the Beckwith-Wiedemann syndrome (BWS). It occurs in approximately 1 in 13700 births. A case-control study showed that 1 out of 148 healthy children in the control group were born after IVF, whereas 4 out of 37 BWS children were born from IVF procedures (2). BWS has been associated in some cases, with the imbalanced expression of genes in the 11p15.5 region, either due to trisomy in case of chromosomal relocalization or imbalanced expression of the paternally expressed IGF2 gene or H19 gene due to DNA methylation aberration. Similarly, other imprinting disorders like Angelman Syndrome, Prader-willi syndrome have been found to be associated with IVF procedures (3, 4, 5). However, studies linking ART to imprinting disorder is controversial with some studies showing no association and hence systematic multinational long-term follow-up studies are needed (6).

Based on the results from animals and humans studies, epigenetic errors related to ARTs seem to be caused by gamete and embryo manipulations procedures carried out at a critical time when old epigenetic marks are erased, new marks established and maintained during gametogenesis and read during embryogenesis. Manipulations include ovarian stimulation, superovulation and in-vitro preimplantation embryo culture. In-vitro culture in animal models has been found to lead to reduced viability and growth, developmental abnormalities, behavioural changes and loss of imprinting. Gonadotropins used for maturing many oocytes simultaneously could also cause the premature release of oocytes that has not completed the imprinting process leading to errors in imprinting (7). Abnormal methylation patterns in 2-cell embryos from

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superovulated females as compared to non-superovulated females have been reported in rodents (8). In vitro maturation of cultured embryos might induce epigenetic aberrations due to components of the culture medium (9). Alternatively, it is possible that the imprinting error is inherent in the gametes and could be a cause of infertility, which is unmasked with assisted reproduction technology.

Association of Epigenetics with Primary Infertility and Embryonic Death

As mentioned above it is possible that the epigenetic aberrations might already be underlying the very infertility for which people undertake ART. For example, the first study of genomic imprinting marks at H19 gene showed significant DNA hypomethylation in 17% moderate and 30% severe oligozoospermic men. Subsequently, many studies showed that such DNA methylation aberrations are present in other genes like the PEG1, LIT1, ZAC, PEG3 etc. as well, in oligozoospermia (10, 11). Similarly, superovulation of oocytes in infertile women has been found to be associated with DNA hypomethylation in PEG1 gene which, in normal oocytes, is methylated (7). The association of DNA methylation aberration is not only present in oligozoospermia but also in some other reproductive disorders. Study done in our laboratory demonstrated DNA methylation aberrations at imprinted genes in spermatozoa of couples with idiopathic recurrent spontaneous miscarriages (RSM) (12). Also it has been observed in one study that certain genetic polymorphisms at imprinted genes might predispose an individual towards recurrent spontaneous abortions. They observed a higher genotypic frequency of the ApaI polymorphism in the embryo mitogenic IGF2 gene, in idiopathic RSM cases as compared to Control group of proven fertility (13). Similar to Recurrent Spontaneous Miscarriges, but much more frequent are cases of single spontaneous abortions and peri-natal fetal death. In a study of 38 cases of spontaneous miscarriages/ fetal deaths, the paternally imprinted PHLDA2 gene was found to be over expressed in a number of cases in the first 12 weeks of gestation (14). Another study analyzing spontaneous abortions and induced abortions (of 12-24 weeks gestation) observed extreme methylation values in multiple imprinted genes in tissues obtained from such material (15).

Environmental Factors and Epigenetic Aberrations in Adverse Pregnancy Outcome

Another adverse embryonic complication is the teratogenic effects of growing embryo due to either parenteral or in utero alcohol exposure which is associated with a wide range of neurobehavioural and physical abnormalities collectively referred to as Fetal Alcohol Spectrum Disorders (FASD). Ever since its recognition, a lot of research in the causal mechanism has been focusing on myriad of perspectives including genetic, biomolecular, cellular and morphological (16), however, the epigenetic factors are also slowly getting realized. One of the main implications of the epigenetic perspective has realized that FASD is not only limited to in utero exposure

of alcohol but also parenteral consumption of alcohol during the preconception period. Alcohol is known to affect one-carbon-metabolism that controls the supply of the methyl donor (S-adenosyl methionine-SAM) and thus, perhaps, DNA methylation (17). Research has also focused on role of many histone modifications and histone and modifying enzymes like histone acetyl transferases as well as DNA methyltransferase in the pathophysiology of chronic alcohol exposure (18). An increased levels of MIR9 (miR-9) miRNA (a calcium- and voltage-activated potassium (BK) channel were observed after exposure of rat neurons to 20 mM alcohol suggest the role of the epigenetic factor- the small non-coding RNAs being one of the targets of alcohol exposure. In summary, ethanol is a known inhibitor of one-carbon metabolism and DNA methyltransferase, and it interferes with various epigenetic factors, including DNA As a result of the Industrial Revolution, many potentially, though subtly, harmful chemicals are released in the environment and are encountered by humans in day-to-day life. Many of these compounds, like Bisphenol A (BPA- found in plastics), Vinclozolin, Pthalates, etc. have endocrine disrupting properties. Since the growth of the embryo, is very intricately modulated and controlled by hormones, endocrine disruption in pregnant mothers would not only lead to fetal malformations but also affect transgenerationally. Mounting evidence reveals that endocrine disruption causes alterations in the male germ line and leads to transgenerational effects by epigenetic mechanisms (19). Studies in our laboratory on the use of tamoxifen, a Selective Estrogen Receptor Modulator (SERM) revealed significant post implantation implantation loss in female rats upon mating with adult male rats treated with tamoxifen for 60 days, owing to epigenetic aberrations of imprinted genes in the spermatozoa (20).

Conclusion

Reproductive Health, that was once analyzed through wide parameters like fertility, sperm analysis, genetic polymorphisms etc. is being viewed in the light of epigenetics, which promises to give a mechanistic understanding to the dynamics of reproductive physiology. The role of assisted reproduction in mediating epigenetic alterations; though looks possible, still needs to be ascertained by more focused studies in non-confounding model systems. At the same time, another avenue of research has also sprouted which shows that epigenetic alterations could well be associated with the infertility for which people are resorting to ARTs. Similarly, a lot of research in environmental influence and endocrine disruption on reproductive health is coming to the fore. In our experience, although ART procedures could be playing a significant role in altering reproductive epigenetics, there is a lot of scope in elucidating epigenetic variability in infertility cases. Since epigenetic alterations are so sensitive to hormonal imbalance, we presume environmental pollutants could be playing a significant role in fluctuating reproductive outcome transgenerationally and needs to be stressed upon. All in all, epigenetics indeed, is an emerging paradigm and a novel perspective is studying reproductive health.

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methylation, histone modifications, and small ncRNAs (21).

References

1. Speybroeck VL (2002). From epigenesis to epigenetics: the case of C. H. Waddington. Ann N Y Acad Sci; 981: 61-81.

2. Hal l iday J , Oke K, Breheny S, e t a l (2004) . Beckwith–Wiedemann syndrome and IVF: a case control study. Am J Hum Genet; 75: 526–8.

3. Maher ER, Brueton LA, Bowdin SC, et al (2003). Beckwith–Wiedemann syndrome and assisted reproduction technology (ART). J Med Genet; 40: 62–4.

4. Maher ER, Afnan M, Barratt CL (2003a). Epigenetic risks related to assisted reproductive technologies: epigenetics, imprinting, ART and icebergs? Hum Reprod; 18: 2508–11.

5. Allen C, Reardon W (2005). Assisted reproduction technology and defects of genomic imprinting. BJOG; 112: 1589–94.

6. Paoloni-Giacobino A (2007). Epigenetics in reproductive medicine. Pediat Res; 61: 51R-57R.

7. Sato A, Otsu E, Negishi H, et al (2007). Aberrant DNA methylation of imprinted loci in superovulated oocytes. Hum Reprod; 22(1): 26-35.

8. Shi W, Haaf T (2002). Abnormal methylation patterns at the two-cell stage as an indicator of early developmental failure. Mol Reprod Dev; 63: 329-34.

9. El Hajj N, Haaf T (2013). Epigenetic disturbances in in vitro cultured gametes and embryos: implications for human assisted reproduction. Fertil Steril; 99: 632-41.

10. Kobayashi H, Sato A, Otsu E, et al (2007). Aberrant DNA methylation of imprinted loci in sperm from oligospermic patients. Hum Mol Genet; 16: 2542–51.

11. Marques CJ, Costa P, Vaz B, et al (2008). Abnormal methylation of imprinted genes in human sperm are associated with oligozoospermia. Mol Hum Reprod; 14: 67–74.

12. Ankolkar M, Patil A, Warke H, et al (2012). Methylation analysis of idiopathic recurrent spontaneous miscarriage cases reveals aberrant imprinting at H19 ICR in normozoospermic individuals. Fertil Steril; 98: 1186-92.

13. Ostojić S, Pereza N, Volk M, et al (2008). Genetic predisposition to idiopathic recurrent spontaneous abortion: contribution of genetic variations in IGF-2 and H19 imprinted genes. Am J Reprod Immunol; 60(2): 111-7.

14. Dória S, Sousa M, Fernandes S, et al (2010). Gene expression pattern of IGF2, PHLDA2, PEG10 and CDKN1Cimprinted genes in spontaneous miscarriages or fetal deaths. Epigenetics; 5: 444-50.

15. Pliushch G1, Schneider E, Weise D, et al (2010). Extreme methylation values of imprinted genes in human abortions and stillbirths. Am J Pathol; 176(3): 1084-90.

16. Goodlett CR, Horn KH (2001). Mechanisms of alcohol-induced damage to the developing nervous system. Alcohol Res Health; 25: 175-84.

17. Cravo ML, Gloria LM, Selhub J, et al (1996). "Hyperhomocysteinemia in chronic alcoholism: correlation with folate, vitamin B-12, and vitamin B-6 status". American J Clin Nutrit; 63(2): 220–4.

18. Shukla SD, Velazquez J, French SW, et al (2008). Emerging role of epigenetics in the actions of alcohol. Alcohol Clin Exp Res; 32(9): 1525-34.

19. Skinner M (2011). Environmental epigenetic transgenerational inheritance and somatic epigenetic mitotic stability;

Epigenetics 6(7): 838-42.

20. Pathak S, Kedia-Mokashi N, Saxena M, et al (2009). Effect of tamoxifen treatment on global and insulin-like growth factor 2-H19 locus-specific DNA methylation in rat spermatozoa and its association with embryo loss. Fertil Steril Vol 91 (Suppl): 2253-63.

21. Haycock PC (2009). Fetal alcohol spectrum disorders: the epigenetic perspective. Biol Reprod; 81: 607-17.

Emerging Role of Epigenetics in Ovarian Cancer

Seema Sharma

Department of Obstetrics & GynecologyMahatma Gandhi University of Medical Sciences & [email protected]

Introduction

Ovarian cancer ranks as the fifth leading cause of cancer-related deaths among women, and the leading cause of death from gynecological cancer (1). Difficulty to diagnose the disease at an early stage and the persistence of dormant, drug-resistant cancer cells that cause relapse, are the primary reasons for the high mortality rate in ovarian cancer patients (2). Lack of early detection strategies and unfavourable anatomical situation make it difficult to diagnose in early stages. By the time most ovarian cancers are diagnosed, they are already at stage III or IV. Ovarian cancer screening with transvaginal ultrasound and CA125 was evaluated in the Prostate, Lung, Colorectal, and Ovarian (PLCO) trial; and it was revealed that the predictive value of both tests was relatively low (3). Increasing evidence indicates that epigenetic mechanisms may play a major role in the development of ovarian cancer. Administration of therapies that reverse epigenetic “Silencing” of tumor suppressors and other genes involved in drug response cascades could prove useful in the management of drug-resistant ovarian cancer patients. Moreover, when used in combination with conventional chemotherapeutic agents, epigenetic-based therapies may provide a means to resensitize ovarian tumors to the proven cytotoxic activities of conventional chemotherapeutics (4).

Epigenetic Modification in Ovarian Cancer

Altered epigenetic states are intimately associated with ovarian tumorogenesis. Based on morphology, genetics, and site of origination, ovarian cancers of epithelial-cell origin have been categorized into two groups. The type I group are those that are strictly confined to the ovary and are low- grade serous, endometrial, mucinous, and clear-cell type. These tumors are genetically more stable, have few to rare p53 mutations, are easily diagnosed and have a good prognosis. The Type 2 group contains tumors are aggressive and comprise high-grade serous carcinomas, undifferentiated carcinomas and carcinosarcomas. These

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tumors constitute 75% of the ovarian cancers with a 90% death rate and the site of origin stems from tissues other than the ovary. These tumors exhibit genetic instabilities with a higher percentage of p53 mutations (5, 6, 7).

Histones and DNA are primary targets of epigenetic regulation (8). Genes that harbor CpG islands are susceptible to epigenetic modifications, consisting primarily of DNA methylation (9). Both types, histone-based and DNA-based methylation marks, are largely known to inhibit gene expression through transcriptional inhibition. The well-studied acetylation modifications primarily affect the exposure of the DNA to the transcriptome machinery. In some cases, non-histone proteins like p53 when acetylated affect its cellular functions through its DNA binding interactions and stability (10).

Increased methylation of tumor suppressor genes have also been observed in ovarian tumors. Increase in promoter methylation of O-methylguanine DNA methyltransferase (MGMT), paired box 5 (PAX5), Cadherin 13, H-Cadherin (Rose et al.) (CDH13), Wilms tumor 1 (WT1), Thrombospondin 1 (THBS1), and GATA5 have been observed in endometroid ovarian cancer as compared to serous ovarian cancer (11). Tumor suppressors cyclin-dependent kinase inhibitor 2B (CDKN2B), CDH13and RASSF1, a gene that encodes Ras association domain-containing protein 1 have significant hypermethylation and CDKN2B promoter hypermethylation was observed in clear cell carcinomas as compared to other histological types (12). Hypermethylation of BRCA1 has been shown to be frequent in spontaneous breast and ovarian cancers. Demethylation of BRCA1 appears to decrease chemosensitivity of platinum-sensitive cells associated with partial increase of BRCA1. Thus BRCA1 hypermethylation favors treatment sensitivity and has been shown to function independently of PI3K-Akt pathway (13). Methylation analysis of ovarian tumors of genes involved in the Wnt pathway demonstrated that the naked cuticle homolog 1 (NKD1) and disheveled homolog (DVL1) methylation increased risk of disease progression. Hypermethylation of members of SHh pathway, zinc finger protein 1 (ZIC1), results in poor progression free survival (PFS) (14). The relationship between DNA hypermethylation generally favors reduced gene expression. Ovarian cancer cell lines tend to have higher methylation patterns as compared to primary tumors.

Adult stem cells are found in the ovaries and may provide a key link into how tumors arise in the ovary (15). Linking the methylation patterns to these adult stem cells, normal epigenetic marks may undergo a change mediated by environmental cues (external/internal) e.g., parity, inflammation, toward a more tumorigenic phenotype by the suppression or loss of tumor suppressor genes. Hypoacetylation of H3 and H4 in association with GATA4 and 6 transcription factors have been found in a variety of ovarian cancer cells (16).

Micro RNAs (miRNAs) are tightly controlled in normal cells but become highly deregulated in cancer cells. They are single stranded non-coding RNA molecules about 22

nucleotides in length and regulate the levels of gene expression by performing silencer-like type functions, degrading the mRNA to which they bind (17). They bind either to certain sequences within the mRNA or to the 3’-untranslated region of the gene. The post-transcriptional modification of genes by miRNA and the presence of varied miRNA expression levels within solid tumors provides a map of miRNA signatures for specific cancers. Formulating drugs against these miRNAs may provide for a therapeutic approach.

Epigenetic Pathways in Ovarian Cancer

Several pathways and genes are found to be involved in the biogenesis of ovarian cancers. Tumor suppressor proteins play a pivotal role in the cell cycle process and these proteins are highly deregulated in ovarian cancers. Epigenetic changes in p53, p21 and p27 has been most commonly observed in ovarian malignancy.

p53 : Tumor suppressor gene p53 controls the regulation and function of cyclin D1, p16INK4a, p27 and p21 and also predominantly mutated in high grade ovarian cancers. The changes in p53 could be both at the gene and protein level. Point mutations, missense mutations and truncations have been observed, and over expression of the p53 protein has been detected in many of the immunohistochemical studies . The aberrations in p53 result in the accumulation of the altered protein within the cell that has a negative effect on BAX, a transcriptional target of p53 (18). It seems likely that p53 alterations in high grade tumors reduce BAX expression allowing the progression of solid tumors.

The BCL family of apoptotic proteins along with p53 can serve as tools for histotyping (19). The expression of p53-BCL-2 and BCL-2-BAX have been shown to have a strong association with tumor grade and histopathological sub-typing, factors that could be vital for identifying the specific epithelial ovarian carcinoma for adjuvant or combined therapies. Ovarian tumors are initially very responsive to treatment but later become chemoresistant. Possibly the treatment itself may cause a few cells within the tumor mass to harbor mutations in p53 that, in addition to epigenetic silencing of promoter regions of apoptotic favorable genes such as p16 or Rb, may account for the relapse and progression of the tumor.

p21 and p27

Cyclin dependent kinase (CDK) inhibitors are major co-regulatory proteins in the cell cycle along with the p16, p53 and retinoblastoma (Rb) pathways. p21 is a direct transcriptional target of p53 (20). In the presence of wild-type (WT) p53, p21 induction ensues followed by the inhibition of cyclin E/CDK2 preventing the G1-S transition, encouraging the apoptotic phenotype of cells (21) CDK inhibitors p21 and p27 control various phases of the cell cycle based on the cyclin with which they associate. The role of p27 in ovarian cancer is somewhat contradictory. There appears to be no correlation between p21 and p27 expression and in terms of ovarian cancers, as yet these proteins are not especially useful tools for the prognosis of the disease (22).

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Cell proliferation regulatory pathways implicated in the biogense of ovarian cancer are:

• p16INK4a and Rb pathway

• KRAS/MAPK/ERK pathway

• PI3K/PTEN/AKT pathway

• PI3K/PTEN/AKT pathway

• The PI3K/AKT/mTOR pathway

• Hedgehog pathway

• Multiple Drug resistant (MDR) pathway

• Notch Pathway

• Forkhead Box M1pathway (FOXM1)

• Breast cancer type 1/2 susceptibility protein (BRCA1)/(BRCA2) and homologous repair and nucleotide excision repair pathway

In ovarian cancer cells, cell proferation regulator pathway p16INK4a is found mutated or its promoter region is hypermethylated switching off its expression. p16INK4a is considered to be a direct target of Rb expression (23). Some studies have contradicted this finding.

Kirsten rat sarcoma oncogene (KRAS) activation triggers a sequence of events through the RAF/MEK and mitogen activated protein kinases (MAPK) pathways, and in conjunction with mammalian target of rapamycin (mTOR), a target of the protein AK strain thymoma (AKT) pathway, control cell proliferation (24). Point mutations in KRAS provide an advantage for the survival and progression of tumors (25). KRAS mutations have been implicated in the genesis of low-grade ovarian tumors, inducing an overactive proliferative phenotype. In addition to breast cancer associated protein 1 (BRCA1) and 2 (BRCA2) that have familial roles in sporadic germline-based breast and ovarian cancers, KRAS is now considered the third player (26). KRAS expression levels differ based on the histopathological type of ovarian tissue and the expression levels may help determine the various type of ovarian epithelial cancers (27). These signatures have a specific pattern in tandem with the expression of RAF/MAPK components, phosphatase and tensin homolog (PTEN) levels and may help differentiate normal from borderline to early-stage cancers. It is also probable that in late stage ovarian cancers, the earlier mutations in KRAS can stimulate and induce the over-activation of NF-κB in cancer stem cells (CSCs) that survive chemotherapy.

K R A S i n d u c e s m i t o g e n a c t i v a t e d p r o t e i n kinase/extracellular signal-regulated kinases (MAPK/ERK) through Serine thrconine protein kinase (BRAF). Stimulation of KRAS via GTPase activity activates BRAF. The stimulation can be mediated by cytokines, growth factors or proto-oncogenes. The downstream target of BRAF, MEK stimulates ERK when activated. ERK targets transcription factors that are involved in cell proliferation such as Myc (28).

Phosphatidylinositide-3 kinase (PI3K) and phosphatase and tensin homolog (PTEN) proteins influence multiple

pathways within the cell that have many effects on the cellular phenotype through protein AK strain thymoma (AKT), also known as protein kinase B (PKB). These genes are crucially required to transcend the input from external stimuli (growth factors) and convey them to AKT. Phosphorylation of key amino acid residues in these proteins triggers the downstream activation processes. PTEN is a tumor suppressor gene and either mutations in the gene or loss of function of the protein is observed in many cancers, including ovarian cancers (29). Loss of function in this gene is mediated through epigenetic silencing and its role in ovarian cancer needs to be further assessed (30).

PI3K is a heterodimeric protein comprised of a regulatory subunit, p85, and a catalytic subunit, p110 (31). The PI3K/AKT/mTOR pathway is involved in type I and type II ovarian cancers. Single mutations of a member of the pathway coupled with a mutation with members of another pathway promote ovarian hyperplasia, and double mutations within the same pathway are necessary for ovarian tumorogenesis. Mutations in parallel pathways that are involved in cross-talk are found to be mutated in ovarian carcinomas. The integrated genomic analysis study of ovarian carcinomas showed that at least 45% of the cases contained mutations in the PI3K/RAS signaling pathway, where PTEN deletions (7%); mutations (<1%), PIK3CA amplifications (18%); mutations (<1%), AKT isoform amplifications AKT 1 and AKT 2 (3 and 6 % respectively), were observed in conjunction with KRAS amplification (11%) (32).

Hedgehog is a signaling pathway that controls development and is expressed during embryonal development (33). The expression of Hh in adult ovarian tissue has not been observed. The signaling mechanism is likely to be activated within the stem cell population in the ovarian tissue that is necessary for the repair of the ovarian surface epithelium (OSE). Cancers arising from OSE have an epithelial phenotype. They are thought to arise through mutated Hh signaling and produce spheroid like structures with cancer stem cell-like properties (34).

ATP-binding cassette (ABC) transporters are implicated in multidrug resistance (MDR) of many tumors. Ovarian cancers by far appear to build up resistance to many treatments and ABC upregulation is thought to play a major role in this process (35). Changes in ABC transporters appear to stem from treatment rather than in situ tumor development. Abnormal expression of the transcription factor Gli1, a downstream target of Hh signaling, has been shown to induce MDR resistance in a subset of ovarian cancers and that the promoter regions of ABCB1 and ABCG2 genes contain Gli1 binding specific consensus sequence (35).

Notch pathway is regulated by ligands such as Jagged1, 2 and Delta-like 1, 3, 4 (36). Elements of the Notch pathway are expressed in EOCs (37). Notch pathway signaling appears to be fundamentally important to cell survival, motility and development of vasculature (38). The chemoresistance observed to platinum-based therapy of

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ovarian cancers stems from the expansion of cancer stem cells that have Notch activated (Notch 3) (39).

FOXM1 is a transcription factor that regulates genes that control the cell cycle and thus proliferation and tumor progression (40) and its role in angiogenesis has also been observed (41). Serous ovarian cancers express high levels of the protein that correlates with tumor progression (42). FOXM1 is also involved in the induction of breast cancer type 2 susceptibility protein (BRCA2) a downstream target that regulates DNA repair (42). FOXM1 is involved in cell migration/invasion in ovarian cancers via ERK that acts upstream of FOXM1 (42) and regulates cell proliferation through a number of elements in the pathway.

BRCA1 and BRCA 2 are tumor suppressors that readily associate with p53 to serve apoptotic functions. Most of the mutations seen in ovarian tumors are somatic mutations. Mutations in BRCA1 and BRCA2 are of heritable germline type. Only 10% of the ovarian tumors that arise are hereditable, which involve theBRCA1/2 mutations (43) The remaining 90% arise through somatic mutations affecting proteins such as BRCA1/2 or p53 (44) BRCA1 expression is favored over BRCA2 expression in terms of staging the cancer. p53 accumulation is more apparent with BRCA1 mutations in late stage or stage III ovarian cancers (45).

BRCA proteins are associated with DNA repair and exist as a complex with other repair proteins (46). Hereditable ovarian cancer involving BRCA requires two-hits for tumor formation. A single BRCA1 affected allele may not be sufficient to promote tumor formation. However, DNA stability is affected as DNA repair is affected that can encourage tumor formation through the loss of function of the second allele or mutations in genes governing the repair pathway. The paradoxical role of BRCA in cancers is apparent (47). Hereditable and somatic mutations in BRCA are responsible for breast and ovarian cancer formation, and yet, cells that carry WT BRCA with other pathway anomalies are less sensitive to treatment or develop chemoresistance. The observations are controversial as some studies point out that the BRCA status (proficient versus absent) of the cells do not have a significant correlation to treatment outcome, whereas others have shown that the absence of BRCA1 enhances the sensitivity to treatment by agents that induce DNA damage, including ionizing radiations (48) showed that the presence of BRCA1 mutations was associated with better survival outcomes as compared to those patients that carried WT BRCA1.

Epigenetic Mechanisms in Chemoresistance

Current therapies against ovarian cancer involve cisplatin treatment (49) Patients that are initially responsive become resistant to the treatment. Platinum-based cisplatin therapy involves the induction of DNA damage through inter and intrastrand crosslinking between purines (50) The 1, 3 and the 1, 2-intrastrand crosslinks are excised and removed through nucleotide excision repair (NER) with the former lesions being easier to remove due to less distortion of the helix (51). The 1, 3-interstrand lesion are complex and require homologous recombination where double stranded

breaks (DSB) are involved (52) The NER system is robust in terms of lesion recognition and requires a host of various NER components. The up-regulation of these elements by DNA-induced damage could account for the gain of resistance to treatment. In terms of DSBs, Rad51 recombinase is required and acts in conjunction with BRCA2 (53). Therefore, loss of function of BRCA2 or mutations that silence the expression of the protein favors sensitivity to treatment and thus women with BRCA1/2 mutations have a better diagnosis and are more responsive to platinum-based therapy. The direct correlation between the expression of NER genes or its members and resistance to therapy does not always hold true. The data obtained from a study analyzing NER efficiency and cisplatin resistance showed that altering the HRR pathway, but not NER member expression, could enhance the sensitivity of cisplatin-resistant tumors to platinum-based agents (54). These observations have been corroborated by the Spellman et al. study that showed that of the samples tested 23% carried mutations/epigenetic altered states in BRCA1, while 11% carried mutations in BRCA2 (32) and that 51% of the cases had altered HRR pathway that involved BRCA2 and Rad51C (32).

Epigenetic and Diagnosis of Ovarian Cancer

So far only two biomarkers of protein origin (CA125 and HE4) have been approved by the FDA for monitoring ovarian cancer (55). However, benign gynecological and medical conditions such as endometriosis, congestive heart failure, and cirrhosis can also have elevated CA125 levels, and elevated serum HE4 level was only related to the advanced stage of epithelial ovarian cancer. More specific and early detection biomarkers are surely needed. As one of the basic elements of epigenetic mechanisms, DNA methylation has been recognized as a potential ideal biomarker due to its stability compared with RNA and protein, sensitivity of detection by PCR, the possibility of localization to a specific gene region and potential of development as a screening method specific for cancer detection (56).

Epigenetic mechanisms as main players in cancer development are emerging as attractive targets for characterizing reliable biomarkers of ovarian cancer. Hypermethylation of a number of TSG promoters has been detected in the serum or peritoneal fluid of a group of ovarian cancer patients in stage I. These TSGs include RASSF1A, BRCA1, APC, CDKN2A, and DAPK (57) Especially for DAPK methylation, there is a tight association of the methylation status between DNA isolated from the peripheral blood and primary tumor (58).

Genes that are specifically methylated in ovarian cancer still wait to be discovered that have the potential to distinguish ovarian cancer from other cancers and to therefore serve diagnostic purposes. Epigenomics studies consisting of methylomic analysis may hold the key in this regard. However, multicenter-conducted studies and well-controlled clinical trials will eventually be needed to further validate these biomarkers.

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Conclusion

Ovarian cancers are lethal diseases as they slip detection and are far advanced when detected. Research in ovarian cancer is just in infancy in terms of understanding the pathways deregulated in the disease. Clearly, there does not appear to be a strong association between deregulated patterns and the gene specific expression and subcellular correlation patterns. Utilizing more quantitative technologies such as microarray systems, western blots, real-time PCR, or whole-genome sequence analysis might provide insight into the etiology and pathology of the disease. Identifying and utilizing markers of DNA methylation patterns and miRNA expression patterns governing gene expression in ovarian cancer and its subtypes may make treatments and therapies more customized.

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Genomic Imprinting in Male Germ-line Stem Cells

2Pallavi Pushp, Hoon Taek Lee1Mukesh Kumar Gupta *

1Department of Biotechnology & Medical EngineeringNIT, Rourkela 2Bio-Organ Research Center, Konkuk University Seoul, South Korea*[email protected]

Male infertility is a common condition in human and its etiology remains unknown in a high proportion of cases. It can be empirically treated with intracytoplasmic sperm injection (ICSI) as long as at least one sperm per oocyte is available. In cases wherein sperms are not available for ICSI, testes-derived male germ-line stem (GS) cells has been envisaged as future therapeutic module for restoration of male fertility (1, 2). It has particular application in patients expecting to undergo radiotherapy or chemotherapies, for instance, after cancer therapies, which often disrupt

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spermatogenesis. Prior to the start of chemotherapy, GS cells may be isolated from testicular biopsy samples and cryopreserved for restoring the fertility by GS cell transplantation upon successful completion of the cancer therapy (Figure). This approach is especially useful in pediatric patients, whose sperms are not available for preservation. GS cells can also be used for in vitro spermatogenesis to bypass the pre- or post-meiotic barriers to sprematogenic process and restoration of fertility (Figure 1). Recent studies have further revealed that, under appropriate culture conditions, GS cells can acquire pluripotency to become multipotent GS (mGS; from neonatal testes) or multipotent adult GS (maGS; from adult testes) cells, which has the capacity to differentiate into the cells of three germ layers and therefore, has applications in regenerative medicine (3, 4). The mGS/maGS cells originate from the cultured GS cells themselves and therefore, at any particular time point, in vitro cultured GS cells may contain some contaminating mGS/maGS cells which may produce teratoma instead of initiating spermatogenesis upon testicular transplantation. Consequently, a molecular marker that can distinguish GS cells from mGS/maGS cells is essential for their application in clinics. Recent studies have demonstrated that, the androgenetic genomic imprinting in mouse GS cells also changes to embryonic stem (ES) cell-like pattern in mGS/maGS cells. Thus, genomic imprinting may be used as molecular signature to distinguish GS and maGS cells.

Genomic imprinting refers to mono-allelic expression of genes in a parent-of-origin manner. In recent years, abnormal genomic imprinting in germline cells has been identified as a possible cause of impaired spermatogenesis in some men diagnosed with idiopathic infertility. During germ cell maturation and gametogenesis, male germline cells undergo widespread erasure of somatic-like patterns of DNA methylation followed by establishment of sex-specific patterns by de novo DNA methylation. Incomplete programming of DNA methylation in the male germline ce l l s may therefore , resu l t in compromised spermatogenesis. An association between altered DNA methylation of imprinted genes in sperm and male factor infertility has also been established (5, 6). Further, DNA methylation patterns in spermatogonia and male GS cells was generally found to be stable in culture and were highly reproducible (7, 8). Thus, analysis of DNA methylation may serve as a valuable diagnostic tool in clinical andrology. We observed that mouse maGS cells were epigenetically stable for DNA methylation at imprinted Igf2-H19 gene cluster during in vitro culture and differentiation (8) but re-acquired GS cell-like growth and differentiation characteristics with altered DNA methylation pattern when they were re-cultured in the GS-like conditions (9). During reprogramming of GS and maGS cells, presence of leukemia inhibitory factor (LIF) favored the decrease in androgenetic DNA methylation of paternally imprinted genes (Igf2, H19) while glial cell line-derived neurotrophic factor (GDNF)

prohibited the reprogramming of paternal imprinted genes and favored the increase in DNA methylation of maternally imprinted genes (Peg1). Neither LIF nor GDNF affected the DNA methylation of non-imprinted Oct4, Nanog and Stra8 genes (9). It was further observed that, the DNA methylation of mouse GS and maGS cells varied with the degree of reprogramming in different cell lines and hence, could be a marker of reprogramming in GS cells (3, 4, 8, 9, 10).

Besides imprinted genes, we also analyzed the imprinted microRNAs (miRNA) in mouse GS and maGS cells. Impaired biogenesis of miRNAs was found to disrupt spermatogenesis in male mice and lead to infertility (11). On the other hand, miRNAs such as miR-122-transfected human induced pluripotent stem (iPS) cells formed spermatozoa-like cells (12). Several miRNAs (e.g. miR-15b, miR-16, miR-19a, miR-19b, miR-22, miR-34b, miR-34b*, miR-34c-5p, miR-122, miR-383, miR-449a, miR-1973, Let-7 etc.) have now been shown to be differentially expressed in asthenozoospermic and oligoasthenozoospermic males compared with normozoospermic males (13, 14). Imprinted miRNAs represent a family of miRNAs that are mono-allelically expressed in a parent-of-origin manner and acts in trans, generally outside the genomic region from where they are encoded. Genes encoding the imprinted miRNA are mainly clustered in two chromosomal domains [PWS-AS (also called Snurf-Snrpn) cluster and Dkl1-Gtl2 cluster] in mouse although few singleton imprinted miRNA are also present at several genomic regions.

Besides, almost all well-characterized imprinted genes clusters such as Igf2-H19, Peg10, Copg2, Rasgfr1, Gnas-Nespas, Kcnq1 and Igf2r-Air also encode one or more imprinted miRNAs whose expression is restricted in a parent-of-origin manner and is controlled by DNA methylation at imprinted control regions (ICR) of the respective gene cluster. These imprinted miRNAs show distinct temporal- and tissue-specific expression patterns in different tissues, including ES cells, and control a wide range of developmental and physiological pathways, including stem cell pluripotency and differentiation. We observed that the expression pattern of imprinted miRNAs in mouse sperm was distinct from those of ES or somatic cells (10). Sperm showed significantly higher expression of imprinted and paternally expressed miRNAs (miR-296-3p, miR-296-5p, miR-483) and lower expression of imprinted and maternally expressed miRNAs (miR-127, miR-127-5p) than those observed with somatic cells. Similar to sperm, the expression of imprinted and paternally expressed miRNAs (miR-296-3p, miR-296-5p, miR-483) were consistently higher while those of imprinted and maternally expressed miRNA (miR-127, miR-127-5p) were consistently lower in GS cells than in control ES cells. This suggests that, expression pattern of imprinted miRNA in mouse GS cells is likely androgenetic and therefore, may possibly form an epigenetic signature on testes-derived GS cells. DNA methylation analyses of ICRs, that control the expression of all imprinted miRNAs in respective gene clusters (Gnas-Nespas DMR, Igf2-H19 ICR and Dlk1-Dio3 IG-DMR),

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confirmed that imprinted miRNAs were androgenetic in GS cells. On the other hand, DNA methylation of imprinted miRNA genes in maGS cells resembled those of ES cells but the expression pattern of the imprinted miRNAs was intermediate between those of GS and ES cells. These data confirm the conversion of androgenetic GS cells to multipotent maGS cells but also suggest that the acquisition of ES cell-like characteristics in maGS cells was likely incomplete or partial. Indeed, several studies have shown that genomic imprinting in maGS cells stand in between GS and ES cells (4, 9, 15).

Taken together, studies suggest the genomic imprinting and expression of imprinted miRNAs are androgenetic in GS cells but changes to ES cell-like pattern upon their conversion to maGS cells. Differential genomic imprinting may thus, serve as epigenetic signature or molecular marker to distinguish GS cells from maGS or ES cells. Since maGS cells originate from GS cells during their extended in vitro culture, these markers may have implication in clinical settings to distinguish GS cell colonies from maGS cell colonies and thereby, minimize the likelihood of teratoma formation by contaminating maGS cells generated from the GS cells.

References

1. Conrad S, Renninger M, Hennenlotter J, et al (2008). Generation of pluripotent stem cells from adult human testis. Nature; 456(7220): 344-9.

2. Ko K, Arauzo-Bravo MJ, Tapia N, et al (2010). Human adult germline stem cells in question. Nature; 465(7301): E1; discussion E3.

3. Guan K, Nayernia K, Maier LS, et al (2006). Pluripotency of spermatogonial stem cells from adult mouse testis. Nature; 440(7088): 1199-203.

4. Kanatsu-Shinohara M, Inoue K, Lee J, et al (2004). Generation of pluripotent stem cells from neonatal mouse testis. Cell; 119(7): 1001-12.

5. Hammoud SS, Purwar J, Pflueger C, et al (2010). Alterations in sperm DNA methylation patterns at imprinted loci in two classes of infertility. Fertil Steril; 94(5): 1728-33.

6. Klaver R, Tuttelmann F, Bleiziffer A, et al (2013). DNA methylation in spermatozoa as a prospective marker in andrology. Andrology; 1(5): 731-40.

7. Cortessis VK, Siegmund K, Houshdaran S, et al (2011). Repeated assessment by high-throughput assay demonstrates

that sperm DNA methylation levels are highly reproducible. Fertil Steril; 96(6): 1325-30.

8. Oh SH, Jung YH, Gupta MK, et al (2009). H19 gene is epigenetically stable in mouse multipotent germline stem cells. Mol Cells; 27(6): 635-40.

9. Jung YH, Gupta MK, Oh SH, et al (2010). Glial cell line-derived neurotrophic factor alters the growth characteristics and genomic imprinting of mouse multipotent adult germline stem cells. Exp Cell Res; 316(5): 747-61.

10. Shin JY, Gupta MK, Jung YH, et al (2011). Differential genomic imprinting and expression of imprinted microRNAs in testes-derived male germ-line stem cells in mouse. PLoS One; 6(7): e22481.

11. Wu Q, Song R, Ortogero N, et al (2012). The RNase III enzyme DROSHA is essential for microRNA production and spermatogenesis. J Biol Chem; 287(30): 25173-90.

12. Liu T, Huang Y, Liu J, et al (2013). MicroRNA-122 influences the development of sperm abnormalities from human induced pluripotent stem cells by regulating TNP2 expression. Stem Cells Dev; 22(12): 1839-50.

13. Abu-Halima M, Hammadeh M, Schmitt J, et al (2013). Altered microRNA expression profiles of human spermatozoa in patients with different spermatogenic impairments. Fertil Steril; 99(5):1249-55 e1216.

14. Gupta MK, Lee HT, editors (2012). Differences between multipotent adult germline stem cells and germline stem cells for microRNA: Springer, USA. pp 113-29.

15. Ko K, Tapia N, Wu G, et al (2009). Induction of pluripotency in adult unipotent germline stem cells. Cell Stem Cell; 5(1): 87-96.

Post Translational Histone Modifications in Ovarian Epithelium and their Plausible Implications in Reproductive Plasticity and Health

G. V. Raghuram

Clinical Research CentreAdvanced Centre for Treatment, Researchand Education in CancerNavi [email protected]

While the early research in past century has defined “particulate genetic inheritance” as a primary mechanism for the heritability of traits, more recent work in past decades in lower eukaryotes and early mammalian species have speculated “epigenetic” (or “upon the genome”) modifications to this genetic framework, as a primary mechanism, for the varied molecular events regulating the reproductive development (1). Ovarian surface epithelium (OSE) is a single layer of modified squamous or cuboidal mesothelial cells covering the surface of the ovary separated from underlying ovarian stromal tissue by a basal lamina of collagenous connective tissue. The epithelium is physiologically involved in follicular rupture and the subsequent repair of the follicle wall during reproductive

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age. The cyclic ovulation process predisposes ovarian surface epithelium to genetic alterations if left unrepaired (2). Given the dynamic nature of the ovarian follicle, presents it as a best model to study the coordinated activation and inactivation of genes related to cell growth and differentiation during ovulations and the surrounding ovarian epithelium. The gene expression is not only cycle specific but also reflects the differentiation status of the follicular granulose and theca cells of the ovary (3). Though, significant progress has been made in ascertaining factors that promote the transcription of ovarian genes mediating gonadotropin action and steroidogenesis; yet clear understanding is required about the appropriate time at which these specific genes are turned on and off and also how these factors influence the chromatin remodeling needs to be delineated. In this regard, an attempt has been made in this brief monograph on post translational histone modifications in ovarian epithelium and their plausible implications in reproductive plasticity and health.

Last decade, has seen surge in studies focusing epigenetic regulation of ovarian genes through histone modifications on a preliminarily aspect (4). Nucleosome represents the basic repeating unit of chromatin consisting of two copies each of four histone (H2A, H2B, H3, and H4) proteins. Histone H1 is outside the core bound to linker DNA. The core histones have protruding charged amino-terminal tails which undergo various post-translational covalent modifications including methylation, phosphorylation, acetylation, ubiquitnation, sumoylation, ADP ribosylation and citrullination (5). While, some of these modifications promote transcription, others repress it. Open chromatin modifications promoting transcription recruit chromatin remodeling ATPases enzymes eventually leading to nucleosome repositioning and increased access to DNA (6). In addition, transcription factors such as HNF3 and GATA4, upon mutation, open the compacted chromatin themselves (7).

Over the past decade, various studies have substantiated the role of the epigenetic alterations during mammalian germ cell development through candidate regulatory factors. Among all the histone modifications, the most studied ones include the acetylation of lysine (K) residues, the methylation of arginine (R) and K residues, the phosphorylation of serine (S) and threonine (T) residues, and the ubiquitination of K residues.

Histone Acetylation and Deacetylation

Acetylation involves the addition of acetyl groups to lysines residues of the histone tails in neutralizes positive charges in presence of histone acetyltransferases (HATs) resulting in alterations to the chromatin fiber and increase accessibility to the DNA and facilitate transcription. In the tail region, acetylation occurs at lysines at amino acid position 9, 14, 18, and 23 within H3 and at positions 5, 8, 12, and 16 within H4 (8, 9). The coactivators p300 and CREB-binding protein (CBP) are the histone acetyltransferases which acetylate H3 and H4 tails and recruit additional acetyltransferases such as p300/CBP-associated factor (pCAF) to specific gene

promoters by transcription factors associated with the DNA (10).

During folliculogenesis, unique epigenetic and transcriptional changes occur in germ cells, including the differentiating and migrating PGCs, oogonia, and the early mitotic-/meiotic-arrested primary oocytes. Seki et al (11) in their studies showed that H3K9 acetylation (H3K9ac) modifications associated with transcriptionally active chromatin were transiently increased starkly upon their entry into the genital ridge and further global chromatin analysis found that H3K9ac levels in PGC precursors were indistinguishable from those of the PGC cells’ somatic neighbours. While, with increasing age and in fully grown oocytes, the histone of the perinucleolar region is highly acetylated (H3K9ac, H3K18ac, H4K5ac, and H4K12ac) than in the growing oocytes suggestive of hyperactive chromatin pattern. Evaluation of histone acetylation within the steroidogenic acute regulatory protein (STAR) gene promoter region (whose protein product regulates the rate-limiting step in steroidogenesis and active during gonadotrophin surge) in the ovary, have shown hyperacetylated H3 in luteinized granulosa cells exhibited a 32-to 206-fold increase in acetylated H3 associated with the STAR promoter (27 h post-hCG) when compared with non-luteinized granulosa cells under induced ovulatory scenarios (12).

Morever, just as histone acetylation is important for facilitating active gene transcription, deacetylation catalyzed by histone deacetylases (HDACs) too is important for turning off genes and maintaining some genes in a repressed state. Mammalian histone deacetylases fall into three classes (I–III) based on their homology to yeast HDACs. Classes I and II HDACs require trichostatin A (TSA) and Class III HDACs require NAD+ as a cofactor. HDACs form the multiprotein complexes recruited to the chromatin by DNA binding proteins (13). However, the precise functions of many of these HDACs in ovary function are still unknown and specifically in ovarian follicle formation are beginning to be understood. Speculation has been that during genome reprogramming in germ cells acetylated lysines should be deacetylated to erase cell memory by histone deacetylation to create undifferentiated or totipotent zygotes of the next generation. Preliminary studies have shown that during the oocyte growth phase, histone H3 was deacetylated at K9, K14, and K18 as the chromosomes condensed when the fully grown oocytes began maturing with subsequently disappearing completely at the time of germinal vesicle (GV) breakdown (14).

Histone Methylation

Histones are methylated to different degrees of mono-, di-, or trimethylation of K residues leading to gene repression or activation, whereas R residues can bemono- or dimethylated methylation (15). Methylation is controlled in a reversible fashion by methyltransferases and demethyltransferases, often associating in complexes. With reference to follicle formation in primordial germ cells (PGCs), the upregulation of H3K27me3 is complemented by the erasure of H3K9me2

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to maintain a proper repressive chromatin state of the genome during their migration from E7.5 to E10.5, stages. However, first sign of chromatin changes in gonadal PGCs at E11.5 was a rapid loss of linker histone H1 concomitant with loss of H3K9me3 and H3K27me3, as well as loss of the repressive H4/H2AR3me2. At about E12.5, the H3K9me3 marks reappeared, and some other chromatin changes also reverted to the original state of development until at least E10.5 periods examined (16). During follicle maturation, histone methylation modifications play important roles in the regulation of chromatin structure and gene expression, especially for oocyte meiotic maturation. H3 methylation at lysines 4 (K4) and 9 (K9) (H3K9me1 and H3K9me2 methylated) by euchromatic histone-lysine N-methyltransferase 2 (EHMT2) are essential for early meiotic progression. It has been found that H3K9me3 appeared in growing oocytes from early antral follicles, increased thereafter during the growth phase, and was maintained during oocyte meiotic maturation and activation (14, 17). However, a different sensitivity of histone methylation including H3K4, H3K4me, H3K4me2, and H3K4me3 in granulosa cells of follicles was found at different developmental stages. Furthermore, oocytes from primary to antral-stage follicles were positive for H3K4 mono-, di-, and trimethylation with altered patterns throughout oocyte development (18).

Histone Phosphorylation

Phosphorylation of histone tails entails a negative charge altering chromatin accessibility locally. During mitosis and meiosis, phosphorylation of serines 10 and 28 and threonine 11 of histone H3 is associated with chromatin condensation, yet serine 10 (S10) phosphorylation has more recently been implicated in transcriptional activation (19). During follicle formation, high levels of phosphorylated histone variant H2A.Z was detected in early E10.5 PGCs, but most of the signals were lost by E11.5eE12.5. However, levels remained relatively constant in the neighbouring somatic cells (16).

During follicle maturation, Histone H3 phosphorylation is altered with changes in chromatin condensation during mouse oocyte meiosis with both phH3/ser10 and phH3/ser28 presented in GVBD, meiotic I (MI), or meiotic I (MII) oocytes (20). Gu et al. (21) showed a stage-dependent dynamics of phosphorylated H3 (phH3/ser10) and phH3/ser28 during porcine oocyte meiotic maturation with no apparent phH3/ser28 signals in GV-, late-GV-, or early-GVBD-stage oocytes; but high levels were observed in pre-MI-, MI-, and MII-stage oocytes. Furthermore, mechanisms of histone phosphorylation during mammalian follicle development (especially its relationship with granulosa-cell-mediated reproductive hormones) remain unknown.

Histone Ubiquitination

Ubiquitination and deubiquitination of proteins are reciprocal events involved in many cellular processes, including the cell cycle. Monoubiquitination of histone H2B at lysine 120 (H2Bub1) has been shown to have key roles in transcription, the DNA damage response and stem cell differentiation (22). Histone variants and histone

ubiquitination may be other regulatory factors during follicle development (23) showed that ubiquitinated histone H2A is associated with transcriptional silencing of large chromatin regions during meiosis in females (24). This suggests that orderly and proper epigenetic reprogramming in premigratory and migratory germ cells might be necessary for the production of gametes with an appropriate epigenotype to support subsequent normal development.

Histone Modifications and Ovarian Cancer

In lieu of the fact that the ovarian surface epithelium is exposed to stressful agents generated during periovulatory processes, any genetically altered proliferative ovulatory wound-repair responses could give rise to a transformed phenotype. Greater than 95% of ovarian cancers originate in the epithelial cells on the ovary by perturbed ovulation (25). Recent there has been a consensus on significance of epigenetic alterations such as global genomic hypo/hyper-methylation along with changes in the ‘histone-code’ in the transformation and carcinogenesis of ovarian epithelial cells (26).

Ovarian cancer manifests silently, usually revealing no clear symptoms until disease advances to a malignant metastatic stage. Histone modifications have been known to facilitate dysregulated gene expression in such malignant disease. Recent studies have shown that alike in normal embryonic stem cells, both normal and transformed neoplastic ovarian cells/cancer stem cells maintain genetic flexibility or ‘bivalent epigenetic signature’ by co-occupancy of opposing histone marks of transcription activating and/or repressive modifications (epigenetic silencing). It has been shown that the tight junction proteins claudin-3 (CLDN3) and claudin-4 (CLDN3) are frequently overexpressed in ovarian cancer through concurrent hyper-acetylation of histone H3 (H3K9Ac) and H4 (H4K16Ac) and promoter DNA hypomethylation (27). Also, over expression of the HDACs I, II and III have been demonstrated to result in overall aberrant histone deacetylation in ovarian cancers which in turn correlated with high-grade tumors and poor prognosis of the disease (28). Besides, other important several genes the such as GATA family of differentiation-associated transcription factors and the cell cycle inhibitor p21 appeared to be regulated solely by histone modifications (29, 30). Convincing results gained through preclinical and clinical studies using HDAC inhibitors have shown potential for the treatment of ovarian cancers through decreased cancer cell growth, apoptosis and increased cell differentiation. However, further studies are required towards increased survival and better prognosis (31).

Regarding histone methylation, the best characterized histone modification in ovarian cancer is the repression of bivalent mark" of H3K27me3/H3K4me2 in aggressive form of disease. However, recent studies have revealed a multivalent histone methylation marks binary H3K9me3/H3K27me3, trivalent H3K4me2/ H3K9me3/ H3K27me3 and tetravalent H3Ac/H3K4me2/ H3K9me3/ H3K27me3 marks. These have further substantiated the effect of the local microenvironment on the epigenetic

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plasticity in response to the presence two different cellular conditions of three-dimensional multicellular aggregates (spheroids) and two-dimensional monolayers ascites and peritoneal mesothelia, in ovarian malignancies (26, 32). Recently, overexpression of methyltransferase Enhancer of Zeste Homolog 2 (EZH2), a catalytic subunit of polycomb repressive complex 2 (PRC2), has been shown to generate histone H3 (H3K27me3) mark contributing to the proliferative ovarian tumor environment (33). In addition, under stress induced premature senescent conditions, induction of hypermethylation of H3K9me3 through increased senescent heterochromatin foci (SAHF) formation has been noted in ovarian epithelial cells our recent study (34).

Histone phosphorylation has been implicated in the DNA damage response given their fine control of chromatin configuration that determines access by transcription factors and DNA repair proteins. In this regard, ovarian cancer is undoubtedly a tumor driven by aberrant DNA damage signaling. Incidentally, high levels of phosphorylated histone H2AX isoform (γ-H2AX) along with histone H3.3 phosphorylation on serine 10 has been observed in high grade ovarian tumors leading to cell death (35). Therefore the potential exists to improve the way this pathway is targeted with current therapies by a greater understanding of the chromatin landscape.

Among the other histone modifications monoubiquitination of histones H2A/H2B are clear instances of alternative roles for ubiquitin in transcription and DNA repair through controlled by ubiquitin ligases such as the RING finger proteins RNF20 and RNF40 and deubiquitinases (DUBs). Loss of global levels of H2Bub1 has been reported in ovarian cancers. Histone small ubiquitin-like modifiers (SUMO) through sumoylation in response to genotoxic stress, and co-localizes at sites of DNA damage with SUMO1, SUMO2/3 and the SUMO-conjugating enzyme Ubc9. However, exact mechanistic insights into these forms of histone marks are still unknown.

Recently, another form of histone modification, histone citrullination, has been shown to occur. It is catalyzed by an enzyme called peptidylarginine deiminase 4 (PAD4, also called PADI4), which converts both histone arginine (Arg) and mono-methyl arginine residues to citrulline. PAD6 is found in ovaries, which needs to investgated further for functional significance. Preliminary studies have found that histone citrullination counteracts the effect of histone arginine methylation and functions as a repressive marker to turn off gene expression (36). However, whether citrullination is the cause or the consequence of the pathological alterations such as tumorigenesis needs to be investigated.

Taking together, the study of histone modifications and their impact on ovarian epigenetic plasticity and function are worth investigating as these processes aim to regulate the transcription activity of genes and foster to establish a new expression system. The orderly and proper epigenetic reprogramming in ovary occupies prime importance as germ

cells might be necessary for the production of gametes with an appropriate epigenotype to support subsequent normal ovarian development. Future investigations on the histone post translational epigenetic modifications along with new epigenetic modifiers could act as surrogate biomarkers of disease risk that could potentially facilitate tailored treatment and further refine preventive measures for ovarian epithelial cancers.

References

1. Segars JH, Aagaard-Tillery KM (2009). Epigenetics in reproduction. Semin Reprod Med; 27: 349-50.

2. Papadaki L, Beilby JOW (1971). The fine structure of the surface epithelium of the human ovary. J Cell Sci; 8: 445–65.

3. Richards JS (2001). Perspective: the ovarian follicle—a perspective in 2001. Endocrinol; 142: 2184–93.

4. Aagaard-Tillery KM, Suter MA, Harris A, et al (2010). Epigenetics and reproduction and the developmental origins of health and disease. Anim Reprod; 7: 103-16.

5. Fischle W, Wang Y, Allis CD (2003). Histone and chromatin cross-talk. Curr Opin Cell Biol; 15: 172–83.

6. Eberharter A, Becker PB (2004). ATP-dependent nucleosome remodelling: factors and functions. J Cell Sci; 117: 3707–11.

7. LaVoie HA (2005). Epigenetic control of ovarian function: the emerging role of histone modifications. Mol Cell Endocrinol; 243: 12-18.

8. Strahl BD, Allis CD (2000). The language of covalent histone modifications. Nature; 403: 41–45.

9. Eberharter A, Becker PB (2002). Histone acetylation: a switch between repressive and permissive chromatin. Second in review series on chromatin dynamics. EMBO Rep; 3: 224–9.

10. Wang Y, Fischle W, Cheung W, et al (2004). Beyond the double helix: writing and reading the histone code. Novartis Found Symp; 259: 3–17.

11. Seki Y, Yamaji M, Yabuta Y, et al (2007). Cellular dynamics associated with the genome-wide epigenetic reprogramming in migrating primordial germ cells in mice. Developmen;. 134: 2627-38.

12. Christenson LK, Stouffer RL, Strauss JF 3rd (2001). Quantitative analysis of the hormone-induced hyperacetylation of histone H3 associated with the steroidogenic acute regulatory protein gene promoter. J Biol Chem; 276: 27392–9.

13. Sengupta N, Seto E (2004). Regulation of histone deacetylase activities. J Cell Biochem; 93: 57–67.

14. Bui HT, Van Thuan N, Kishigami S, et al (2007). Regulation of chromatin and chromosome morphology by histone H3 modifications in pig oocytes. Reproduction; 133: 371-82.

15. Biermann K, Steger K (2007). Epigenetics in male germ cells. J Andro;l 28: 466-80.

16. Hajkova P, Ancelin K, Waldmann T, et al (2008). Chromatin dynamics during epigenetic reprogramming in the mouse germ line. Nature; 452: 877-81.

17. Tachibana M, Nozaki M, Takeda N, et al (2007). Functional dynamics of H3K9 methylation during meiotic prophase progression. EMBO J; 26: 3346-59.

18. Seneda MM, Godmann M, Murphy BD, et al (2008). Developmental regulation of histone H3 methylation at lysine 4 in the porcine ovary. Reproduction; 135: 829-38.

19. Nowak SJ, Corces VG (2004). Phosphorylation of histone H3: a balancing act between chromosome condensation and

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transcriptional activation. Trends Genet; 20: 214-20.

20. Swain JE, Ding J, Brautigan DL, et al (2007). Proper chromatin condensation and maintenance of histone H3 phosphorylation during mouse oocyte meiosis requires protein phosphatase activity. Biol Reprod; 76: 628-38.

21. Gu L, Wang Q, Wang CM (2008). Distribution and expression of phosphorylated histone H3 during porcine oocyte maturation. Mol Reprod Dev; 75:143-9.

22. Cole AJ, Clifton-Bligh RJ, Marsh DJ (2014). Ubiquitination and cancer: Histone H2B monoubiquitination - roles to play in human malignancy. Endocr Relat Cancer; ERC-14-0185.

23. Santenard A, Torres-Padilla ME (2009). Epigenetic reprogramming in mammalian reproduction: contribution from histone variants. Epigenetics; 4: 80-84.

24. Baarends WM, Wassenaar E, van der Laan R, et al (2005). Silencing of unpaired chromatin and histone H2A ubiquitination in mammalian meiosis. Mol Cell Biol; 25: 1041-53.

25. Murdoch WJ, Martinchick JF (2004). Oxidative damage to DNA of ovarian surface epithelial cells affected by ovulation: carcinogenic implication and chemoprevention. Exp Biol Med; 229: 546-52.

26. Bapat SA (2010). Modulation of gene expression in ovarian cancer by active and repressive histone marks. Epigenomics; 2: 39-51.

27. Bapat SA, Jin V, Berry N, et al (2010). Multivalent epigenetic marks confer microenvironment - responsive epigenetic plasticity to ovarian cancer cells. Epigenetics; 5: 716-29.

28. Weichert W, Denkert C, Noske A, et al (2008). Expression of class I histone deacetylases indicates poor prognosis in endometrioid subtypes of ovarian and endometrial carcinomas. Neoplasia; 10:1021–27.

29. Caslini C, Capo-chichi CD, Roland IH (2006). Histone modifications silence the GATA transcription factor genes in ovarian cancer. Oncogene; 25: 5446–61.

30. Strait KA, Dabbas B, Hammond EH, et al (2002). Cell cycle blockade and differentiation of ovarian cancer cells by the histone deacetylase inhibitor trichostatin-A are associated with changes in p21, Rb, and Id proteins. Mol Cancer Ther; 1: 1181–90.

31. Takai N, Narahara H (2010). Histone deacetylase inhibitor therapy in epithelial ovarian cancer. J Oncol; 458431.

32. Kwon MJ, Kim SS, Choi YL, et al (2010). Derepression of CLDN3 and CLDN4 during ovarian tumorigenesis is associated with loss of repressive histone modifications. Carcinogenesis; 31: 974-83.

33. Li H, Zhang R (2013). Role of EZH2 in Epithelial Ovarian Cancer: From Biological Insights to Therapeutic Target. Front Oncol; 3: 47.

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Polycystic Ovary Syndrome: Plausible Epigenetic Distress and Role of Environmental and Endogenous Modulators

Pooja Sagvekar*, Srabani Mukherjee

Molecular Endocrinology DepartmentNational Institute for Research in Reproductive Health (ICMR)Mumbai*[email protected]

Polycystic ovary syndrome (PCOS) is a multifactorial, chronic disease state of a yet unknown etiology affecting 6-10% of women of childbearing age (1). It is one of the leading causes of anovulatory infertility and subfertility demarcated by an array of ovarian, hormonal and metabolic derangements and characterized by chronic menstrual irregularities, folliclular cysts on ultra-sonography, hyperandrogenemia, hyperinsulinemia and obesity. PCOS pathophysiology can be outlined starting from hypothalamo-pituitary-ovarian axis, where persistent, elevated GnRH release leads to increased LH secretion that exerts gonadotropic effects through its receptors on theca and granulosa cells. In PCOS, immature follicles get prematurely sensitized to LH and facilitate excess ovarian steroidogenesis. Elevated steroid production coupled with aromatization defects downstream in these pathways favor increased synthesis of androgens over estrogens, thus contributing to hyperandrogenemia. Another important characteristic of PCOS is insulin resistance (IR), in which peripheral muscles and fat tissues exhibit low tolerance to insulin and glucose, further promoting compensatory hyperinsulinemia. Excess insulin acts synergistically with LH to augment ovarian steroidogenesis and aggravates the intensity of androgenic stress. Further, ovarian androgens coupled with adrenal androgens result in clinical manisfestation of hyperandrogenism (Textbook Ref, 2). Also obesity, a commonly observed trait that stems from inherent or de-novo abnormalities in lipid metabolism is an added risk factor in development of PCOS, though lean phenotypes are also prevalent. Therefore, PCOS women develop co-morbidities like hyperglycaemia, type-2 diabetes mellitus (T2DM), hypertension, cardio-vascular disorders (CVDs), coronary heart diseases (CADs) and metabolic syndrome (MS) that predispose them to life threatening health risks.

Although PCOS bears a well-established genetic component, it holds equal ground for epigenetic mechanisms that bring about phenotypic plasticity, fetal reprogramming, late-life onset of disease and such phenomena that persist beyond the understanding of gene sequence abnormalities. Recent information on heritability of epi-mutations over several generations of progeny (transgenerational epigenetic inheritance), has raised concerns among scientists in reproductive health circles (3).

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Events such as early/late-life exposure to endocrine disruptors (ECDs) and environmental pollutants, unfavorable diet and lifestyle malpractices or even altered circadian or diurnal rhythmicity are known to affect this inheritance mechanism. Several such factors have been linked with PCOS etiology in the form of circumstantial as well as direct evidences on epigenetic modifications in PCOS (Table 1).

Table 1: Epigenetic Studies in PCOS Women and PCO Models. (Conclusive Inference Marked in Blue)

However, the lack of solid evidence pertaining to these changes has necessitated high throughput investigations on this subject. This article therefore, highlights the significance of epigenetic distress in PCOS as an outcome of endogenous remodeling events in response to environmental contaminants and unfavorable life styles.

Epigenetic programming is achieved by an intricate control over mechanisms that bring about addition or removal of chemical groups in the chromatin i.e DNA methylation and post-translational histone modifications without altering the genetic code (4). These alterations may interfere with basic chromatin structure and functions and result in global epigenetic changes (eg: DNA hypo and hypermethylation in cancer) and/or differential expression changes in target genes and alterations in non-coding RNAs (ncRNAs). Such fluctuations may manifest as easily palpable, rare phenotypic anomalies like the Angelman syndrome (a gene imprinting disorder) or subtle and common variations like obesity, caused by metabolic perturbations (5, 6).They also confer tissue specificity by controlling the transcriptional “switch” responsible for gene expression or repression, and are therefore associated with cell specific or tissue associated derangements contributing to disease states.Of these epigenetic modifications, DNA methylation changes are less labile, relatively easy to track and are hence more frequently investigated than the rest. Thus, epigenetic studies are crucial in understanding the obscure but significant phenomena related to pathophysiology of chronic diseases. PCOS is one such disorder that has recently gained attention owing to the extent of reproductive dysfunction in women.

Role of Inadvertent Exposure to Hormones, EDCs/drugs and other Environmental Pollutants in PCOS

Epigenetic modification studies provide a nexus to translate the emerging effects of environmental exposures to reproductive morbidities in both men and women and the extent of incurred damage is determined by both, frequency and intensity of a given exposure. In PCOS, a significant portion of its current understanding on reproductive anomalies comes from data extrapolated from experimental animal models owing to the limitations discussed earlier. Untimely or excess exposure of female fetus to uterine androgens in rodent and rhesus monkey models of PCO has improved our understanding on fetal origins of this disease. These prenatally androgenized females developed PCOS like features in adulthood, which may be explained by epigenetic priming processes (7, 8). Similarly, fetal estrogen excess studies in PCO and non-PCO animal models demonstrate development of PCOS features like hyperandrogenism, irregular menstrual cycles and impaired fertility. Interestingly, estrogen deficiency in fetus also promoted hyperandrogenic, multicystic ovarian phenotypes, thus emphasizing that optimal estrogen levels are necessary for normal reproductive functioning (9). Also, studies on hormone receptor genes eg: X-chromosome inactivation in androgen receptor (AR) (Laisk et al., 2010), hypomethylation in LH receptor (LHCGR) (Table.1) in PCOS and other genes eg: Lamin A/C (LMNA) hypermethylation 10) in insulin resistant PCOS phenotypes have encouraged additional studies on role of hormone induced epigenetic dysregulation. These findings are in support of Barker’s hypothesis of developmental origins of health and disease (DOHaD) in PCOS (11).

EDCs predominating the external environment are additional culprits in epigenetic programming and can be classified as phyto-estrogens, heavy metal toxins, fungicides, pesticides, pharmaceutical and industrial wastes, plasticizers, flame-retardants and hydrocarbon mixtures. Their actions are exerted by modifying estrogen or androgen dependent steroidogenic pathways with most endocrine damage incited by estrogenic mimetics that act via estrogen receptors (ESR). Bisphenol-A (BPA), a plasticizer with such actions is widely used in the industrial manufacture of plastic wares, dental resins and food-can lining has been reported to be high in PCOS (12). High circulatory BPA levels are proposed to interfere with insulin signalling mechanisms of glucose metabolism thereby causing IR (13, 14). They are also associated with other PCOS features ofincreased testosterone synthesis by theca-interstitial cells, lowering of hepatic androgen-related hydroxylases that stall testosterone degradation and competence with endogenous androgens that bind to sex hormone-binding globulin (SHBG). Diethylstilbestrol (DES) is yet another EDC that was exploited for its ability to mimic estrogen signaling and was prescribed heavily during 1950s-60s to achieve pregnancy (9) Third generation studies have revealed reproductive anomalies in sons and daughters of women exposed to DES in utero, more-so in daughters who showed excess GnRH secretion, hyperandrogenism

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and infertility due to menstrual irregularities that are suggestive of PCOS (9). However till date, information on whether epigenetic mechanisms play a role in EDC induced PCOS features in women is not available.

There are several more undetectable environmental components namely volatile agents from exhaust fumes, hydrocarbon emissions, electromagnetic and radio waves or even unknown constituents that have more pronounced impact during prenatal stages of organ development (16). This is because key epigenetic programming in fetal stages occurs in two phases: (i) in pre-implantation embryo bearing three germ layers (affecting overall organ development) (ii) in primordial germ cells (PGCs) during their migration from extra-gonadal sites to the genital ridge (affecting germline reprogramming and subsequent progeny) (16). Although, exposure hazards during adulthood are reported to be equally debilitating, they are reversible and therefore manageable.

Effect of Dietary Indulgence

Methylation at 5-carbon (C5) position of cytosine bases in DNA is brought about by DNA methyl transferases (DNMTs) while methylation of lysine and arginine residues in the histone tails is a function of histone methyl t r a n s f e r a s e s ( H M Ts ) a n d p r o t e i n a r g i n i n e methyltransferases (PRMTs) respectively. Both these processes are effectively facilitated by S-Adenosyl methionine (SAM), an active derivative of the essential amino acid methionine that acts as a methyl donor for most of the biochemical reactions. These SAM-dependent methyltransferases release methyl groups by conversion of SAM to S-adenosylhomocysteine (SAH), which acts as a feedback inhibitor for SAM dependent methyltransferases. Thus, an excess of methionine in the system favours hypermethylation in chromtatin while high amounts of homocysteine are conducive to hypomethylated states (17). Systemic depletion of SAM and a concurrent rise in SAH levels has been attributed to less intake of folate and choline rich diet. Limited availability of folate restricts the synthesis of its active counterpart, 5’-methyl tetrahydrofolate, thus depleting SAM and resulting in decreased cytosine methylation in DNA (18). In PCOS women, high levels of homocysteine have been reported earlier, which may be associated with low intake of folate supplements and resultant defects in DNA methylation machinery (19). Studies on PCOS associated co-morbidities namely CAD and MS carried out in leucocytes and visceral adipose tissue respectively also indicate towards global DNA hypomethylation in PCOS (20, 21). Adherence to protein restricted diet and supplementation of folate co-factors, choline and vitamin B12 has experimentally proven to facilitate restoration of methylation loss by increasing the abundance of SAM (22, 23). Therefore, women with PCOS can be recommended for consumption of food with low cholesterol, low glycaemic index (GI) and high folate content.

Another important dietary risk factor for PCOS is ‘advanced glycation end-products’ (AGEs), which are reactive

metabolites of non-enzymatic glucose-protein reactions produced in response to hyperglycemia, oxidative stress or exogenously ingested carbohydrate-rich food processed at high-temperatures. They contribute to preeminent risks of diabetes and cardiovascular disease in the general population. PCOS women are reported to have high concentrations of AGEs in their sera, which positively correlate with elevated serum androgen levels (24). Also, women with insulin resistance demonstrate heightened serum AGEs and their respective receptors (RAGEs) on ovarian theca and granulosa cells (25), suggesting a putative direct action of AGEs in PCOS.

ART Induced Endocrine Milieu as an Additional Epigenetic Stressor

Diagnosis of infertility in couples and the surging trend in delay of maternal age at first childbirth, especially in metropolitan sectors, has provided impetus to taking support of assisted reproductive technologies (ART) in child conception. PCOS, as aforesaid maximally contributes to anovulatory infertility and thereby oocyte defects in women who thereby resort to in-vitro fertilization (IVF) programs for conception. Infertility, as we know, has been already ascertained as a consequence of erroneous epigenetic programming in quality-compromised oocytes or sperms, thereby, making PCOS women more susceptibility to a whole new set of ART induced epigenetic alterations as well. IVF encompasses protocols like controlled ovarian hyper-stimulation (COH) for oocyte retrieval and in-vitro oocyte maturation (IVM) that precede embryo-transfer (ET) and intra-cytoplasmic sperm injection (ICSI).With every proceeding step, the spermatozoa and ovarian cells, especially granulosa cells and oocytes get exposed to external environmental agents including the culture media, laboratory pathogens, temperature fluctuations and so on. These fluctuations also contribute to errors in epigenetic mechanisms that are manifested in both early developmental stages and/or adulthood (26, 27).

These initiatives were based on observations that upto 4% of the ART-borne infants presented with severely abnormal phenotypes that matched with Beckwith–Weidemann syndrome while 1 in 15,000 newborns presented with Angelman syndrome (28). Extensive methylation analyses of regulatory regions in several imprinted genes have identified susceptible targets that develop such phenotypes. H19, Ubiquitin-protein ligase E3A (UBE3A) and insulin-like growth factor-2 (IGF2) are some of these imprinted gene targets (29). However, there are no reports thus far, explaining the outcome of ART in PCOS affected women, thereby keeping this aspect open for investigation.

To conclude, self-imposed dietary choices, life-style vagaries and undesired exposure to combinations of exogenous and endogenous agents discussed thus far, can lead to development of PCOS. However, additional tendencies like alcohol consumption, smoking, substance abuse, lack of physical exercise and prolonged late-night work shifts, contact with bacteria, viruses etc. cannot be ignored as the confounding factors while investigating

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epigenetic effects in such life-style related disease states. With eminent progress in epigenetic studies, we therefore speculate that the enigma behind complex disorders like PCOS can be resolved and would lead to the use of appropriate epigenetic therapy in alleviation of their health risks in future times.

References

1. Bu Z, Dai W, Guo Y, et al (2013). Overweight and obesity adversely affect outcomes of assisted reproductive technologies in polycystic ovary syndrome patients. Int J Clin Exp Med; 6(10): 991–5.

2. Dunaif D, Chang R, Franks S, et al (2008). Polycystic Ovary Syndrome: Current controversies, from the ovary to the pancreas, Contemporary Endocrinology, ISBN: 978-1-58829-831-7 (Print)

3. Nilsson E, Larsen G, Manikkam M, et al (2012). Environmentally induced epigenetic transgenerational inheritance of ovarian disease. PloS One, 7(5), e36129.

4. Zama A, Uzumcu M (2010). Epigenetic effects of endocrine-disrupting chemicals on female reproduction: An ovarian perspective. Front Neuroendocrinol; 31(4): 420–39.

5. Lalande M, Calciano MA (2007). Molecular epigenetics of Angelman syndrome. Cellular and Molecular Life Sciences CMLS; 64(7-8): 947–60.

6. Oken E, Gillman MW, Fetal MWG (2003). Fetal Origins of Obesity. Obes Res; 11(4): 496-506.

7. Abbott DH, Barnett DK, Bruns CM, et al (2005). Androgen excess fetal programming of female reproduction: a developmental aetiology for polycystic ovary syndrome? Human Reproduction Update; 11(4): 357–74.

8. Li Z, Huang H (2008). Epigenetic abnormality: a possible mechanism underlying the fetal origin of polycystic ovary syndrome. Med Hypotheses;70(3): 638-42.

9. Abbott DH, Padmanabhan V, Dumesic DA (2006). Contributions of androgen and estrogen to fetal programming of ovarian dysfunction. Reprod Biol Endocrinol; RB&E, 4, 17.

10. Ting W, Yanyan Q, Jian H, et al (2013). The relationship between insulin resistance and CpG island methylation of LMNA gene in polycystic ovary syndrome. Cell Biochem Biophy; 67(3): 1041–7.

11. De Boo HA, Harding JE (2006). The developmental origins of adult disease (Barker) hypothesis. Aust New Zealand J Obst Gynaeco; 46(1): 4–14.

12. Kandaraki E, Chatzigeorgiou A, Livadas S, et al (2011). Endocrine disruptors and polycystic ovary syndrome (PCOS): Elevated serum levels of bisphenol A in women with PCOS. J Clin Endocrino Metabol; 96(3): E480–4.

13. Alonso-Magdalena P, Morimoto S, Ripoll C, et al (2005). The estrogenic effect of bisphenol a disrupts pancreatic β-cell function in vivo and induces insulin resistance. Environ Health Perspect; 114(1): 106–12.

14. Lin Y, Sun X, Qiu L, et al (2013). Exposure to bisphenol A induces dysfunction of insulin secretion and apoptosis through the damage of mitochondria in rat insulinoma (INS-1) cells. Cell Death Disease; 4: e460.

15. Reik W, Dean W, Walter J (2001). Epigenetic reprogramming in mammalian development. Science; 293(5532): 1089–93.

16. Herceg Z (2007). Epigenetics and cancer: towards an evaluation of the impact of environmental and dietary factors. Mutagenesis; 22(2): 91–103.

17. Duthie SJ (1999). Folic acid deficiency and cancer: mechanisms of DNA instability. Brit Medi Bull; 55(3): 578–92.

18. Gupta S, Fedor J, Biedenharn K, et al (2013). Lifestyle factors and oxidative stress in female infertility: is there an evidence base to support the linkage? Exp Rev Obstet Gynecol; 8(6): 607–24.

19. Turcot V, Tchernof A, Deshaies Y, et al (2012). LINE-1 methylation in visceral adipose tissue of severely obese individuals is associated with metabolic syndrome status and related phenotypes. Clin Epigenet; 4 (1): 1–8.

20. Wei L, Liu S, Su Z, et al (2013). LINE-1 Hypomethylation is Associated with the Risk of Coronary Heart Disease in Chinese Population. Arq Bras Cardiol; (37): 1–8.

21. Crider KS, Yang TP, Berry RJ, et al (2012). Folate and DNA Methylation: A Review of Molecular Mechanisms and the Evidence for Folate’s Role. Adv Nutr; (14): 21–38.

22. Niculescu MD, Zeisel SH (2002). Trans-HHS Workshop: Diet, DNA Methylation Processes and Health Diet, Methyl Donors and DNA Methylation: Interactions between Dietary, Folate, Methionine and Choline. J Nutr; Supplement: 2333S–2335S.

23. Diamanti-kandarakis E (2005). Increased levels of serum advanced glycation end-products in women with polycystic ovary syndrome. Clin Endocrinol; 62: 37–43.

24. Maria E, Costa F, Spritzer PM, et al (2014). Effects of endocrine disruptors in the development of the female reproductive tract. Arq Bras Endocrinol Metab; 58 (2): 153–161.

25. Batcheller A, Cardozo E, Maguire M, et al (2011). Are there subtle genome-wide epigenetic alterations in normal offspring conceived by assisted reproductive technologies? Fertil Steril; 96: 1306–11.

26. Manipalviratn S, DeCherney A, Segars J (2009). Imprinting disorders and assisted reproductive technology. Fertil Steril; 91: 305–15.

27. Ludwig M, Katalinic A, Gross S, et al (2005). Increased prevalence of imprinting defects in patients with Angelman syndrome born to subfertile couples. J Med Gen; 42(4): 289–91.

28. Godfrey KM, Lillycrop KA, Burdge GC, et al (2007). Epigenetic Mechanisms and the Mismatch Concept of the Developmental Origins of Health and Disease. Pediatr Res; 61(5): 31–6.

Epigenetic Changes and Human Assisted Reproductive Technologies

Rajvi Mehta

Trivector Embryo Support [email protected]

Introduction

Assisted Reproductive Technologies [ART] are now an established modality for treating infertility. The International Committee for the Monitoring of Assisted Reproductive Technology (ICMART) has reported that approximately 350000 ART cycles are performed worldwide per year; 5 million babies in the world have been born through ART till 2013; of which nearly 25% have been born in the last 6 years.

As ART involves many ‘non-natural’ procedures such as 40

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ovarian stimulation, physical manipulation of the sperms and oocytes; embryo culture and cryopreservation – there has always been a concern on whether these procedures would affect the health of the children born through ART. In a meta-analysis, Bower and Hansen (1) reported on an increase in low birth weight as well as small for gestational age babies born after ART as compared with those born after natural conception. There is a 2.6-fold increased risk of low birth weight in term IVF infants, with a greater risk of prematurity such that 0.4% of all very low birth weight infants are conceived by IVF. This observation was only for singleton pregnancies (2, 3) ruling out the possibility of the low birth babies being due to multiple gestations. Although, there does not appear to be any significant difference in the physical growth or hormonal parameters between ART and naturally conceived children.

The question that now arises is whether the low for gestation age and low birth weight of babies conceived through ART would affect the future development of these children? Barker showed a connection between birth weight and adult disease expressed later in life (4). He proposed the fetal origins hypothesis of adult disease. According to this hypothesis the origins of diseases in adults begin in utero. This hypothesis has gained credibility through epidemiologic studies performed in Europe and the United States (5). If the onset of diseases in adults originates in utero then could the onset of abnormalities in utero or low gestational age be a result of early embryonic developments? With the extensive manipulations on the gametes and embryos in ART, there is a concern on whether these could be responsible for the low gestational age or low birth weight of the babies born through ART.

Abnormalities in Children Born After ART

Birth defects: Studies have been carried out comparing the prevalence of birth defects in ART babies especially comparing IVF with babies born following ICSI.

As ICSI is more invasive than IVF, there is a concern on whether the procedure would have a detrimental effect on the embryos. Bonduelle et al. in a multi-centric study compared 540 ICSI and 437 IVF-conceived 5-year-old children from five European countries and found that major malformations were more frequent in the ICSI group, in particular in ICSI boys, beyond the neonatal period with the majority of these increased defects due to an excess in urogenital malformations (6). However, in other recent large studies and meta-analyses, no significant differences were found between ICSI and IVF children (7, 8).

In one of the largest meta-analysis, Wen et al. reviewed 46 studies and a total of 124468 children born following IVF/ICSI. They reported on a pooled risk estimation of 1.37 (95% CI 1.26–1.48) following IVF-ICSI which was not statistically different when the data of IVF was compared to that of children born through ICSI. Thus it appears that there is no increase in birth defects following ICSI (9).

Long Term Development

The growth patterns of ART-conceived children have

attracted the attention of many researchers. The vast majority of these studies have not found any differences in the postnatal growth until 12 years of age between ART and naturally conceived children (7, 10, 11). However, some studies do suggest that ART children are taller. Miles et al. (12) found that IVF/ICSI-conceived children aged 5–6 years were significantly taller than naturally conceived controls following adjustment for age and parental height (12). If these observations are corroborated by other studies than one needs to determine whether such an outcome is specifically due to ART manipulations?

Increased Vascular Disorders

Healthy children conceived through ART seem to have an elevated risk of suffering from cardiovascular diseases in the future (13, 14, 15). Ceelen et al. (13) observed that 8 to 18 year olds conceived following ART had higher blood pressure and fasting glucose levels compared with age and gender-matched controls (13). Scherrer et al. (15) compared systemic and pulmonary vascular function in 65 healthy ART-conceived children and 57 naturally conceived controls and reported that ART-conceived children were apparently normal but may have had generalized vascular dysfunction (15). They found that flow-mediated dilation of the brachial artery was 25% smaller ((6.7±1.6)% versus (8.6±1.7)%), carotid-femoral pulse-wave velocity was significantly faster, carotid intima-media thickness was significantly greater, and the systolic pulmonary artery pressure at high altitude was 30% higher in ART-conceived children than in controls.

What is the cause of the vascular abnormalities remains unclear. It is hypothesized that these could be due to epigenetic changes or genomic imprinting disorders.

Methylation of Genomes and Gene Silencing

With the understanding of epigenetics in the 1950s, it was realized that genetic expression can be altered in response to the environment. The mechanism by which genetic expression is altered as well as its role in fetal development is now being understood. The alteration in the DNA methylation pattern can cause gene silencing and alter gene function without any changes in the gene itself (16). The delivery of a methyl group to the gene spurs a tight recoiling of the DNA. The contraction makes it harder, or impossible, for transcription factors to interact with the gene leading to ‘silencing’ of the gene Methylation of the CpG dinucleotide causes gene silencing. 70% to 80% of the genome are methylated during development and when some of the CpG islands become methylated then the associated gene gets silenced (17, 18).

Epigenetic Changes During Gametogenesis and Embryogenesis

During mammalian development, there are various phases of epigenetic modification.

• During gametogenesis, there is genome-wide demethylation followed by remethylation before fertilization.

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• Early embryogenesis is then characterized by a second genome-wide demethylation event (19) following which methylation is re-established early in embryonic life following implantation.

These post fertilization demethylation and remethylation phases are likely to play a role in the removal of acquired epigenetic modifications, particularly those acquired during gametogenesis (20, 21).

Genes that undergo genomic or parental imprinting are among the most well-understood examples of epigenetic transcriptional modification. A subset of approximately 80 genes in humans display mono-allelic expression, i.e. expression only occurs from a single parent. Genomic imprinting-induced silencing of one parental allele results in mono-allelic expression from either the paternal or maternal copy of a gene. The imprint control regions for genomic imprinting usually contain a differentially methylated CpG island in which one parental allele is methylated and the other unmethylated.

Epigenetic Changes in Response to In Vitro Culture

As the human embryos ‘created’ by ART are exposed to a varied environment, there have been concerns on whether they are subject to epigenetic alterations. There is a concern that exposure to an exogenous environment could potentially lead to epigenetic alterations and subsequently immediate and long-term impacts on the fetus and the health of the offspring. Although, gene expression studies on human embryos are rare due to obvious ethical considerations, development of children born following ART is being monitored to determine whether there are any differences in children born after ART as compared with those born after natural conception; And, changes if any can be attributed to epigenetic alterations. Extrapolations are also being made from animal experimentation.

Data from animal studies clearly show that nutrition during embryonic development does lead to changes in DNA methylation patterns because mammalian one carbon metabolism, which is the source of all methyl groups for all biologic methylation reactions, is very dependent upon dietary methyl donors and cofactors (22). If the dietary methyl donors influence embryonic development then it is hypothesized that the composition of the culture medium in ART would alter methylation patterns and thereby expression of imprinted genes (23, 24).

Mouse embryos cultured in Whitten's media showed loss in methylation of the H19 differentially methylated region that was not seen in embryos cultured in Bigger’s KSOM medium which contained amino acids (25). When mouse embryos were cultured in fetal cord serum, which for a long time had been a protein supplement even in human embryo culture media had reduced viability, reduced body weight, decreased expression of H19 and IGF2, and increased methylation of the H19 imprinting control region when compared with controls (26). All these studies on mice ART clearly indicate that epigenetic changes do occur when embryos are cultured in vitro and these alterations do affect

the development of the embryos and development of embryos into fetuses and live births.

Studies in mice demonstrate that ART alters the methylation pattern of genes involved in vascular function and induces vascular dysfunction, a problem that can be prevented by modification of the culture media used for ART (27).

Abnormalities in ART Conceived Children Specifically due to Epigenetic Alterations

As crucial imprinting events coincide with the specific ART procedures raising serious concerns on the epigenetic changes due to ART. Since 2002, several reports have raised concerns that children conceived by ART are at an increased risk of having imprinting disorders, especially some rare and severe imprinting-related diseases, such as Beckwith-Wiedemann syndrome (BWS), Angelman syndrome (AS), and retinoblastoma (28) . However, these disorders are very rare and therefore it is difficult to determine whether their occurrence is related to ART, However, some studies have suggested that the subfertile condition of the parents may also be responsible for imprinting disorders and not essentially the in vitro culture conditions (29-30). However, other studies show no correlation between ART and genomic imprinting disorders, including BWS and AS (31). Some epigenetic modifications appear to be dynamic throughout life and could contribute to adult-onset diseases Thus, ART could lead to subtle abnormalities in children that could present later in life.

Katari et al. found a modest change in the methylation level of CpG sites in about 700 genes from the cord blood samples from ART-conceived children (32). Many of these altered genes have been implicated in chronic metabolic disorders, such as obesity and type II diabetes. When the peripheral blood cord blood of 18 children conceived through ART were analysed, it found hypomethylation at specific regions in 3 of 18 clinically normal children conceived by ART. (33). Nevertheless, many other studies do not corroborate these observation on aberrant methylation patterns in children conceived following ART (34, 35).

Conclusions

Most children conceived following any of the ART are healthy. There are some risks on poorer perinatal outcome, low birth weight and small for gestational age in ART conceived children. However, it is unclear whether these risks are an outcome of epigenetic changes induced by the in vitro culture conditions or the infertility per se. Larger studies with long-term follow-up are needed to answer these questions. The current data on epigenetic alterations in ART conceived children are controversial which in turn could be de to the epigenetic changes being reversible!

References

1. Bower C, Hansen M (2005). Assisted reproductive technologies and birth outcomes: overview of recent systematic reviews. Reprod Fertil Dev; 17: 329–33.

2. Wennerholm UB, Bergh C (2004). Outcome of IVF pregnancies. Fetal Matern Med Rev; 15: 27–57.

3. Schieve LA, Meikle SF, Ferre C, et al (2002). Low and very

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low birth weight in infants conceived with use of assisted reproductive technology. N Engl J Med; 346: 731–7.

4. Barker DJ (1990). The fetal and infant origins of adult disease. BMJ; 301: 1111.

5. Curhan GC, Willett WC, Rimm EB, et al (1996). Birth weight and adult hypertension, diabetes mellitus, and obesity in US men. Circulation; 94: 3246–50.

6. Bonduelle M, Wennerholm UB, Loft A, et al (2005). A multi-centre cohort study of the physical health of 5-year-old children conceived after intracytoplasmic sperm injection, in vitro fertilization and natural conception. Hum Reprod; 20(2): 413–9.

7. Sutcliffe AG, Saunders K, McLachlan R, et al (2003). A retrospective case-control study of developmental and other outcomes in a cohort of Australian children conceived by intracytoplasmic sperm injection compared with a similar group in the United Kingdom. Fertil Steril; 79(3): 512–6.

8. Tararbit K, Houyel L, Bonnet D, et al (2011). Risk of congenital heart defects associated with assisted reproductive technologies: a population-based evaluation. Eur Heart J; 32(4): 500–8.

9. Wen J, Jiang J, Ding C, et al (2012). Birth defects in children conceived by in vitro fertilization and intracytoplasmic sperm injection: a meta-analysis. Fertil Steril; 97: 1331–7.

10. Knoester M, Helmerhorst FM, Vandenbroucke JP, et al (2008). Perinatal outcome, health, growth, and medical care utilization of 5- to 8-year-old intracytoplasmic sperm injection singletons. Fertil Steril; 89(5): 1133–46.

11. Woldringh GH, Hendriks JC, van Klingeren J, et al (2011). Weight of in vitro fertilization and intracytoplasmic sperm injection singletons in early childhood. Fertil Steril; 95(8): 2775–7.

12. Miles HL, Hofman PL, Peek J, et al (2007). In vitro fertilization improves childhood growth and metabolism. J Clin Endocrinol Metab; 92(9): 3441–5.

13. Ceelen M, van Weissenbruch MM, Vermeiden JP, et al (2008). Cardiometabolic differences in children born after in vitro fertilization: follow-up study. J Clin Endocrinol Metab; 93(5): 1682–8.

14. Wikstrand MH, Niklasson A, Strömland K, et al (2008). Abnormal vessel morphology in boys born after intracytoplasmic sperm injection. Acta Paediatr; 97(11): 1512–7.

15. Scherrer U, Rimoldi SF, Rexhaj E, et al (2012). Systemic and pulmonary vascular dysfunction in children conceived by assisted reproductive technologies. Circulation; 125(15): 1890–6.

16. Waddington C (1957). The Study of the Genes. Allen and Unwin, London.

17. Larsen F, Gundersen G, Lopez R, et al (1992). CpG islands as gene markers in the human genome. Genomics; 13: 1095–107.

18. Bird AP (1986). CpG-rich islands and the function of DNA methylation. Nature; 321: 209–13.

19. Santos F, Hendrich B, Reik W, et al (2002). Dynamic reprogramming of DNA methylation in the early mouse embryo. Dev Biol; 241:172–82.

20. Pickard B, Dean W, Engemann S, et al (2001). Epigenetic targeting in the mouse zygote marks DNA for later methylation: a mechanism for maternal effects in development. Mech Dev; 103: 35–47.

21. Reik W, Dean W, Walter J (2001). Epigenetic reprogramming in mammalian development. Science; 293: 1089–93.

22. Van den Veyver IB (2002). Genetic effects of methylation diets.

Annu Rev Nutr; 22: 255–82.

23. Niemann H, Wrenzycki C (2000). Alterations of expression of developmentally important genes in preimplantation bovine embryos by in vitro culture conditions: implications for subsequent development. Theriogenology; 53: 21–34.

24. Eppig JJ, O'Brien MJ (1998). Comparison of preimplantation developmental competence after mouse oocyte growth and development in vitro and in vivo. Theriogenology; 49: 415–22.

25. Doherty AS, Mann MR, Tremblay KD, et al (2000). Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo. Biol Reprod; 62: 1526–35.

26. Khosla S, Dean W, Brown D, et al (2001). Culture of preimplantation mouse embryos affects fetal development and the expression of imprinted genes. Biol Reprod; 64: 918–26.

27. Rexhaj E, Giacobino A, Nicod P, et al (2010). Mice generated by assisted reproductive technologies, a model organism for the study of epigenetic mechanisms of vascular dysfunction in vivo [abstract]. Circulation; 122: A19117.

28. Laprise SL (2009). Implications of epigenetics and genomic imprinting in assisted reproductive technologies. Mol Reprod Dev; 76(11): 1006–18.

29. Horsthemke B, Ludwig M (2005). Assisted reproduction: the epigenetic perspective. Hum Reprod Update; 11(5): 473–82.

30. Ludwig M, Katalinic A, Gro S, et al (2005). Increased prevalence of imprinting defects in patients with Angelman syndrome born to subfertile couples. J Med Genet; 42(4): 289–91.

31. Bowdin S, Allen C, Kirby G, et al (2007). A survey of assisted reproductive technology births and imprinting disorders. Hum Reprod; 22(12): 3237–40.

32. Katari S, Turan N, Bibikova M, et al (2009). DNA methylation and gene expression differences in children conceived in vitro or in vivo. Hum Mol Genet; 18(20): 3769–78.

33. Gomes MV, Huber J, Ferriani RA, et al (2009). Abnormal methylation at the KvDMR1 imprinting control region in clinically normal children conceived by assisted reproductive technologies. Mol Hum Reprod; 15(8): 471–7.

34. Li L, Wang L, Le F, et al (2011). Evaluation of DNA methylation status at differentially methylated regions in IVF-conceived newborn twins. Fertil Steril; 95(6): 1975–9.

35. Tierling S, Souren NY, Gries J, et al (2010). Assisted reproductive technologies do not enhance the variability of DNA methylation imprints in human. J Med Genet; 47(6): 371–6.

MicroRNA in Testis –An overview

Panneerdoss Subbarayalu

Manjeet K. Rao*

University of Texas Health Science Center at San AntonioSan Antonio, Texas *[email protected]

MicroRNA (miRNA) are naturally occurring small non-coding RNAs that have withstood years of evolutionary pressure to be abundantly expressed in animals and humans. miRNAs are endogenously expressed molecule that are present either in the intron where they are transcribed as a part of the genes or in the intergenic region where they are

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transcribed by the independent transcriptional complex. miRNAs have emerged as important regulators of gene expression as they bind to 3' untranslated region (UTR) of the target gene and silence their expression at the transcriptional and/or translational level. Studies in several organisms have revealed that miRNAs play important roles during normal development and differentiation in various tissues (1, 2).Recent studies have established an equally important roles for miRNAs in regulating testicular development and spermatogenesis. One of the first evidence that miRNAs may be key players in testicular physiology came from Kotaja et al. (3) when they reported that miRNA processing machinery is localized in the chromatoid bodies of the male germ cells and likely play an important role during germ cell differentiation. Another exciting study came from Dr. Wei Yan's group claiming that many X-linked miRNAs escape meiotic sex chromosome inactivation during spermatogenesis (4). Subsequent studies reported in vivo model that showed importance of miRNA during spermatogenesis. In these in vivo models, miRNA processing enzyme dicer was specifically deleted in the primordial germ cells resulting in spermatogenic arrest likely due to compromised spermatogonial proliferation and differentiation (5). Though insightful, these studies could not specifically address the importance of miRNAs in post-meiotic germ cell development, as Dicer conditional knockout in early germ cell development produced few if any Dicer-deleted germ cells that entered meiosis (5). To address these issues, we generated post-meiotic germ cell-specific Dicer knockout mouse model. Our results revealed that loss of miRNA expression in haploid germ cells resulted in impaired spermatid differentiation and compromised fertility. Specifically, we showed that loss of miRNA-dependent control of actin-associated protein Arpc5 resulted indefective chromatin compaction in differentiating post-meiotic germ cells (6). This was an important finding as sperm chromatin compaction state is an important independent prognostic characteristic associated with human male fertility.

MicroRNA Biogenesis

The majority of miRNA genes located in the non-protein coding intronic region which is transcribed by RNA polymerase II/III to produce long primary miRNA (pri-miRNA), which is approximately 400 bp, that includes several hairpin loop structures. This pri-miRNA transcripts are processed in the nucleus by a microprocessor complex enzyme Drosha III, which generates ~70 bp precursor miRNA (pre-miRNA). The Pre-miRNA is then transported to cytoplasm by nuclear membrane exportin5. Cytoplasmic RNase III enzyme Dicer cleaves this pre-miRNA duplex into two separate strands. One strand called guide strand which ultimately target the 3'UTR of gene and induce mRNA degradation or translational suppression. Another strand called passenger strand (*) which gets degraded shortly.

miRNAs are also highly expressed in the Sertoli cells and like germ cells miRNAs are reported to play an equally important role in the Sertoli cells. For example, Sertoli cell-specific knockout of miRNA processing enzyme Dicer during embryonic stages were shown to result in testicular dysgenesis due to alteration in Sertoli cell architecture and infertility (7). Importantly, genes including Gdnf, Kitl, Man2a2, and Serpina5 that are known to be critical for spermatogenesis were reported to be significantly down-regulated in mice lacking Dicer in the Sertoli cells. These results clearly underscored the importance of miRNAs in the developing Sertoli cells. Little was known about the role of miRNAs in adult Sertoli cell until recently when we showed that miRNAs may control androgen-mediated events in the adult Sertoli cells. We showed that androgen directly regulates the expression of several miRNAs in adult Sertoli cells (8). Importantly, we found that several of these miRNAs are preferentially expressed in the testis and target genes that are highly expressed in the Sertoli cells. Examples of such genes included Foxd1, a forkhead/winged-helix transcription factor important in Sertoli cell metabolism (9), and desmocollin-1 (Dsc1), a desmosomal cadherin that plays a crucial role in establishing cell-cell adhesion and desmosome formation in epithelial cells (10). These findings suggested that miRNAs may regulate androgen-mediated events either directly, by targeting genes in the androgen receptor-responsive Sertoli cells, or indirectly, by regulating germ cell-specific genes.

Conclusion and Perspective

Emerging evidence suggests that miRNAs are important players in male fertility. A concerted effort to identify role of individual miRNAs during spermatogenesis will pave the way for establishing whether miRNAs can serve as important prognostic indicators and/or therapeutic regimens to diagnose/treat male infertility.

References

1. Bernstein E, Kim SY, Carmell MA, et al (2003). Dicer is essential for mouse development. Nat Genet; 35: 215-7.

2. Kanellopoulou C, Muljo SA, Kung AL, et al (2005). Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing. Genes Dev; 15: 489-501.

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3. Kotaja N, Bhattacharyya SN, Jaskiewicz L, et al (2006). The chromatoid body of male germ cells: similarity with processing bodies and presence of Dicer andmicroRNA pathway components. Proc Natl Acad Sci U S A; 103: 2647-52.

4. Song R, Ro S, Michaels JD, et al (2009). Many X-linked microRNAs escape meiotic sex chromosome inactivation. Nat Genet; 41: 488-93.

5. Hayashi K, Chuva de Sousa Lopes SM, Kaneda M, et al (2008). MicroRNA biogenesis is required for mouse primordial germ cell development and spermatogenesis. PLoS One; 5: e1738.

6. Chang YF, Lee-Chang JS, Imam JS, et al (2012). Interaction between microRNAs and actin-associated protein Arpc5 regulates translational suppression during male germ cell differentiation. Proc Natl AcadSci U S A;109: 5750-5.

7. Papaioannou MD, Pitetti JL, Ro S, et al (2009). Sertoli cell Dicer is essential for spermatogenesis in mice. Dev Biol; 326: 250–9.

8. Panneerdoss S, Chang YF, Buddavarapu KC, et al (2012). Androgen-responsive microRNAs in mouse Sertoli cells. PLoS One; 7: e41146.

9. Dahle MK, Gronning LM, Cederberg A, et al (2002). Mechanisms of FOXC2- and FOXD1-mediated regulation of the RI alpha subunit of cAMP-dependent protein kinase include release of transcriptional repression and activation by protein kinase B alpha and cAMP. J BiolChem; 277: 22902–8.

10. King IA, O'Brien TJ, Buxton RS (1996). Expression of the “skin-type” desmosomal cadherin DSC1 is closely linked to the keratinization of epithelial tissues during mouse development. J Invest Dermatol; 107: 531–8.

End Note from the Editor

Epigenetics: A Key Paradigm in Reproductive Health

Varij Nayan, Suneel Kumar Onteru

Dheer Singh*

National Dairy Research Institute (NDRI) Indian Council of Agricultural Research (ICAR) Karnal*[email protected]

Several evidences have reiterated that reproductive health is a consequence of interplay between genetic, biochemical, physiological, pathological, nutritional, environment and management factors. Hormones, steroids, growth factors and even the follicular microenvironment (1) have greater roles in reproductive outcome. Transcription factors regulated by specific hormone-activated signalling pathways obviously dictate the activation of target genes of reproductive events. Recently, the reproductive events also involve epigenetic changes that impact gene expression and thus cellular and physiological functions. It is imperative now that epigenetic regulatory machinery may contribute to the fertility and reproductive health. Perturbations to these epigenetic mechanisms may further delineate the intrinsic causes of infertility or state of fertility. External factors including environment and food are represented as examples of the nutriment and nurture-epigenetic-

phenotype relationship. Lately, there is increased realization that “the environment modulates the organism” (2), and is gradually taking shape as one of the basic tenet of modern biological science.

Definition, Mechanisms and Reproductive Perspective

Historically, Conrad Waddington (1905–1975) was given credit for introducing the term “epigenetics” as equivalent to experimental embryology. It was assumed as the branch of biology that studies the causal interactions between genes and their products, and which bring the phenotype into being (3). Epigenetics was ascribed as a developmental program, where genes determine the individual’s phenotype by considering the internal and external environmental cues (4, 5). In spite of the word epigenetic being originally derived from an older terminology “epigenesis” - referring to an embryological concept, it literally gives the sense of being “beyond or above genetics” (6). However, for molecular biologists, a more suitable definition of epigenetics is “the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence” (7).

These changes are effected by several molecular mechanisms now commonly known as epigenetic mechanisms that include DNA methylation, histone modi f i ca t ions (me thy la t ion , ace ty la t ion and phosphorylation) and chromatin remodelling such as altering the position of nucleosomes, all of which bring about changes in chromatin structure and function. The important role of non-coding RNAs (ncRNA) like miRNA as an additional epigenetic mechanism in this process is also emerging. All these diverse groups of successive epigenetic modifications ensure the creation of a healthy being and as a consequence, the reproductive health. Important epigenetic reprogramming events occur during germ cell development and early embryogenesis in mammals.

Figure 1: A General Overview of Dynamics of Epigenetic Reprogramming Events During DNA Methylation in Mammalian Female Developmental Time Line in Mammalian Female [Adapted from (8)]

Any disruption during early embryogenesis or germ cell development has the potential to alter epigenetic reprogramming. This is evident from the presence of large offspring syndrome (LOS) in animals during in vitro embryo

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production (IVEP). Thus any venture of IVEP and assisted reproductive technology (ART) should take into account of epigenetic reprogramming considerations.

Epigenetics vis-à-vis Nutriment, Nurture and Developmental Origins of Health and Disease

It is worthwhile to realize that epigenetics goes beyond DNA sequences. Different nutritional and environmental factors are known to influence developmental plasticity and thus phenotypes in a wide variety of animals, from yeast, insects to humans (9, 10). Mounting evidence has re-affirmed the importance of early environmental conditions for adult health and disease. Utilizing this idea, about twenty two years ago, Hales and Barker (11) proposed the ‘thrifty phenotype’ hypothesis. It was argued that nutritional conditions during uterine development have effects much later in life, and influence the occurrence of adult metabolism and diseases. They proposed that, under poor nutritional conditions, the foetal environment could modify the development of the embryo to prepare the offspring for a future environment with low resources during adult life (a thrifty phenotype). Later on, many epidemiological and animal studies supported the thrifty phenotype theory and gradually, it has evolved into a more general theory known as the ‘developmental origins of health and disease’ (DOHaD), which proposes that a wide range of environmental conditions during embryonic development and early life determine susceptibility to disease during adult life (10)

Figure 2: Hales and Barker's 'thrifty phenotype' Hypothesis

It is now recognised that nutrients have the ability to interact and modulate molecular mechanisms underlying an organism’s physiological functions and has pressed for a revolution in the eld of nutrition (12). One of the SCFAs, butyrate apart from participating in metabolism as nutrients, also functions as histone deacetylase (HDAC) inhibitor and histone hyperacetylation (13, 14, 15). HDAC is one of the important epigenetic regulators. Liu et al (16) reported that transient exposure to NaBu after GVBD improves meiotic competence, but not later on, through histone acetylation during oocyte maturation. It was also found that butyrate induced histone acetylation regulates miRNA expression, and also, miRNAs may interfere with butyrate induced modulation of gene expression and cellular functions, which may be due to the cross-talk of miRNA and histone

acetylation (17). In mice, epigenetic transgenerational inheritance of ovarian disease states was induced by environmental compounds and it is suggestive of a role of environmental epigenetics in ovarian disease etiology (18). All these results are encouraging and provide us a great opportunity for exploring and understanding of the role of environment, management conditions, feed/dietary components in changing epigenetic landscape and definitely will have impact on integrated-omics research in humans and animals alike.

Translational Significance from our Experiences

Recognizing the recent advances in epigenetics, we worked on its potential in regulation of female fertility. Specifically, recent works concerning DNA methylation and chromatin modification of the buffalo CYP19 gene, a key fertility gene, during folliculogenesis and luteinization confirmed this. Our laboratory reported a T/C single nucleotide polymorphism (SNP) in a regulatory region at the exon 2 of CYP19 gene (19). The C allele adjacent to the putative TATA binding element could be a putative methylation site. Methylation analysis of 5 CpG dinucleotides of placenta-specific promoter I.1 and proximal promoter of the CYP19 gene (PII) showed hypo-methylation in early and term placenta and hyper-methylation in mid-placenta for PI.1, whereas PII was found to be hypomethylated in all (20). We also conducted methylation analysis of the CpG dinucleotides of two major promoters of CYP19 gene in granulosa cells and luteinized tissue (21). Methylation analysis of five CpG dinucleotides of ovary specific proximal promoter II showed hyper-methylation in corpus luteum while hypo-methylation in large follicle. It was also implied that a mechanism other than differential methylation is involved in the change in Cyp19 gene expression, where the expression of Cyp19 was found lower in retained foetal membrane cases and higher in normal ones and both the major promoters were hypomethylated in the two tissues. All these support that DNA methylation provides a heritable, stable and critical component of epigenetic regulation. While conducting analysis of histone modifications using ChIP assay, our group also revealed that the distal promoter (PI.1) of CYP19 gene is more enriched with acetylated histone H3 in corpus luteum than in the large follicle.

Concluding Remarks

In summary, reproduction involves a complex series of endocrinological, biochemical and molecular events during folliculogenesis and further developmental stages. Fertility, encompassing the successful fertilization of quality oocytes and sustained development of viable embryo, set the stage for successful pregnancy. We have a wealth of information regarding mammalian reproduction but many fundamental and emerging questions related to it need further insight and research. The human and animal phenotypes are summation of gene-environment-nutrition interactions. The genetic and epigenetic health of female gamete and its further development is thus regulated and affected by epigenetic modifiers such as prenatal nutrition, endocrine disruptors, in vivo maternal environment and also in vitro culture

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Views and opinions published in ISSRF Newsletter are not necessarily endorsed by the Indian Society for the Study

of Reproduction and Fertility or the institutions and organizations to which the respective authors belong.

DISCLAIMER

The ISSRF President, Executive and Life Members heartily express their appreciation and gratefulness for valuable contributions being made by the authors and the editors in the publication of this newsletter.

A WORD OF GRATITUDE

ACKNOWLEDGMENTS

The scientific inputs and assistance provided by Dr. A. S. Ansari & Mr. Deepak Jaiswal in compilation of the newsletter are gratefully acknowledged.

conditions. The epigenetic modifications influence viability of female gamete and further embryo development. This is also important in the success of assisted reproductive technologies (ARTs), embryo transfer programs and recent initiatives of stem cell therapy and cloning in some animals. A comprehensive holistic policy and plans comprising the management, environmental factors, nutrition, and in vitro embryo production for fertility augmentation and ARTs should be taken care of with epigenetic perspectives on horizon.

References

1. Fortune JE, Rivera GM, Yang MY (2004). Follicular development: the role of the follicular microenvironment in selection of the dominant follicle. Anim Reprod Sci; 82-83: 109-26.

2. de Magalhães JP, Wuttke D, Wood SH, et al (2012). Genome-environment interactions that modulate aging: powerful targets for drug discovery. Pharmacol Rev; 64: 88-101.

3. Waddington CH (1942). The epigenotype. Endeavour; 1: 18-20.

4. Jablonka E, Lamb MJ (2002). The changing concept of epigenetics. Ann. NY Acad Sci; 981: 82-96.

5. Holliday R (2006). Epigenetics: a historical overview. Epigenetics; 1: 76-80.

6. Speybroeck LV (2002). From epigenesis to epigenetics: the case of C.H.Waddington. Ann NY Acad Sci; 981: 61–81.

7. Riggs AD, Martienssen RA, Russo VEA (1996). Introduction. In Epigenetic mechanisms of gene regulation (Ed. Russo, VEA. et al.), (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) pp 1.

8. Nayan V, Onteru SK, Singh, D (2014). Epigenetics: A Promising Paradigm for Controlling Fertility in Dairy Animals. In: Contemporary Topics in Life Sciences (Ed. Mathur, PP), (Narendra Publishing House, New Delhi) pp.53-74.

9. Sadhu MJ, Guan Q, Li F, et al (2013). Nutritional control of epigenetic processes in yeast and human cells. Genetics; 195: 831-44.

10. Feil R, Fraga MF (2012). Epigenetics and the environment: emerging patterns and implications. Nat Rev Genet; 13: 97-109.

11. Hales CN, Barker DJ (1992). Type 2 (non-insulin-dependent) diabetes mellitus: the thrifty phenotype hypothesis. Diabetologia; 35: 595–601.

12. Mutch DM, Wahli W, Williamson G (2005). Nutrigenomics and nutrigenetics: the emerging faces of nutrition. FASEB J; 19: 1602-16.

13. Davie JR (2003). Inhibition of histone deacetylase activity by butyrate. J Nutr; 133: 2485S-93S.

14. Myzak MC, Ho E, Dashwood RH (2006). Dietary agents as histone deacetylase inhibitors. Mol Carcinog; 45: 443-6.

15. Steliou K, Boosalis MS, Perrine SP, et al (2012). Butyrate histone deacetylase inhibitors. Biores Open Access; 4: 192-8.

16. Liu L, Song G, Gao F, et al (2012). Transient exposure to sodium butyrate after germinal vesicle breakdown improves meiosis but not developmental competence in pig oocytes. Cell Biol Int; 36: 483-90.

17. Li CJ, Li RW, Elsasser TH (2010). MicroRNA (miRNA) expression is regulated by butyrate-induced epigenetic modulation of gene expression in bovine cells. Genet. Epigenet; 3: 23-32.

18. Nilsson E, Larsen G, Manikkam M, et al (2012). Environmentally induced epigenetic transgenerational inheritance of ovarian disease. PLoS ONE 7 e36129.

19. Onteru SK, Sharma D, Singh D et al (2008). CYP19 (cytochrome P450 aromatase) gene polymorphism in murrah buffalo heifers of different fertility performance. Res Vet Sci; 87: 427-37.

20. Ghai S, Monga R, Mohanty TK, et al (2010). Tissue-specific promoter methylation coincides with Cyp19 gene expression in buffalo (Bubalus bubalis) placenta of different stages of gestation. Gen Comp Endocrinol; 169: 182-9.

21. Monga R, Ghai S, Datta TK et al (2011). Tissue-specific promoter methylation and histone modification regulate CYP19 gene expression during folliculogenesis and luteinization in buffalo ovary. Gen Comp Endocrinol; 173: 205-15.

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Dr. Dheer Singh received Ph.D. degree from National

Dairy Research Institute (NDRI) Deemed University

at Karnal (Haryana) in India. After his doctoral degree

he was selected as Agricultural Research Scientist

(ARS) and joined Animal Biochemistry Division at

NDRI as scientist and subsequently promoted to

Principal Scientist. Awarded BOYSCAST (Better

opportunity for young scientist in chosen area of

science and technology) fellowship from Department

of Science and Technology (DST), Ministry of

Science and Technology, Govt. of India to pursue

higher study abroad and worked in Department of Cell

and Developmental Biology and Anatomy at School of

Medicine, University of South Carolina, USA in the area of transcriptional regulation of

eukaryotic genes. He was a visiting scientist to School of Medicine, Stanford University, USA

and identified novel endocrine genes using DNA microarray, computational tools and RNAi in

functional analysis of genes in granulosa cells. He is Life Member of prominent professional

national scientific societies namely, SBC, India; ISSRF; ISCB etc. Apart from several educational

fellowships such as ICMR Junior research fellowship (JRF), NDRI-JRF and SRF etc, he is th threcipient of 9 & 11 AAAP/CAPI Outstanding Research Award of Asian Australian Animal

Production Society and the prestigious Labhsetwar Award-2013 of the Indian Society for the

Study of Reproduction & Fertility. Handled more than 15 research projects funded by various

funding agencies, including Indo-German joint collaborative project (DST-DFG & DST-DAAD)

on epigenetic regulation of genes and published more than 40 research papers in peer reviewed

International Journals. His current research interests include: 1) Identification and analysis of the

regulation of selected trait-affecting (e.g. fertility) genes; 2) Application of RNA interference

tools for livestock functional genomics; 3) Epigenetic regulation of gene expression; and 4)

Development of nano-biosensor.

Dr. Dheer Singh Principal ScientistAnimal Biochemistry Division National Dairy Research Institute Karnal 132 001, Indiae-mail: [email protected]

Dr. Pradyumna Kumar Mishra is currently working

as Associate Professor in School of Biological Sciences

at Central University - Sagar. Earlier, he served as

Scientist E, Division of Translational Research, Tata

Memorial Centre, Mumbai and Head of Research Wing,

BMHRC, Indian Council of Medical Research, Bhopal.

Dr. Mishra is a recipient of prestigious Fulbright-Nehru

Fellowship (USA) and is a Visiting Faculty Associate to

Weizmann Institute of Science, Israel and Osaka

University, Japan. He is a recipient of Young Investigator

Award from Department of Biotechnology, New Delhi.

For his seminal contribution in developing Dendritic

Cell based therapeutic vaccines, he received JEM Young

Scientist Award in 2008 at Kobe, Japan. His laboratory has received extra-mural funding support

from DBT, DST and CSIR. He has validated a number of biomarkers for human pathologies and has

developed two quantitative PCR based technologies for rapid identification and characterization of

occult hepatitis C virus and latent tuberculosis infection. To his credit, he has one US and one WIPO

patent. He has authored more than 80 peer-reviewed original research articles (cumulative impact

index 245.97) with more than 1225 citations and his publication h-index is 21. Dr. Mishra is

Associate Editor-in-Chief of WJG and serves in editorial board of three highly impact international

journals. He has guided 7 students for their Ph.D. programme and more than 40 M. Phil. & PG

dissertations. Dr. Mishra is a Post-graduate & Doctoral Committee Member of 2 central, 11 state

universities and 4 national institutes. He is life-member of eight scholarly societies and his primary

research focus is molecular and translational medicine. Of late, his laboratory has been engaged in

delineating the epigenetic dimension of oxygen radical injury in spermatogonia which is considered

an inextricable component responsible for genome integrity maintenance in testicular milieu.

Dr. Pradyumna Kumar MishraAssociate Professor (Sr. Grade) School of Biological Sciences Central University Sagar 470 003, Indiae-mail: [email protected]

About Our Editors

President

Dr. N. K. Lohiya, Jaipur

Vice-Presidents

Dr. Suneeta Mittal, New Delhi

Dr. Sudha Salhan, New Delhi

Secretary

Dr. R. S. Sharma, New Delhi

Joint Secretary

Dr. A. H. Bandivadekar, Mumbai

Treasurer

Dr. A. S. Ansari, Jaipur

Members

Dr. Nomita Chandhiok, New Delhi

Dr. S. G. Dastidar, Kolkata

Dr. Sujata Kar, Bhubaneswar

Dr. S. S. Majumdar, New Delhi

Dr. P. B. Seshagiri, Bangalore

Dr. G. Taru Sharma, Bareilly

Dr. Dheer Singh, Karnal

Co-Opted Members

Dr. M. M. Misro, New Delhi

Dr. Roya Rozati, Hyderabad

Ex-Officio Members

Immediate Past President

Dr. C. P. Puri, Mumbai

Immediate Past Secretary

Dr. Smita Mahale, Mumbai

ISSRF Executive

48

Valuable comments of the

readers will serve as the

source of inspiration & also

help us to improve upon future

newsletters of the Society.

49

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An International Conference on Reproductive Health: Issues and Strategies under Changing Climate Scenario and the 24th Annual Meeting of the Indian Society for the Study of Reproduction and Fertility (RH-ISCS-ISSRF 2014) has been organized by Dr. G. Taru Sharma, Director, CAFT, Principal Scientist and Head Division of Physiology & Climatology at the Indian Veterinary Research Institute (IVRI), Izatnagar (UP) under the auspices of the ISSRF during February 6-8, 2014. The conference was held in conjunction with the celebration of 125 years of establishment of IVRI with a glorious history of serving the country in animal health and production.

The conference focused on the issues and strategies related to reproductive health at a juncture when scientists across the globe are concerned about the adverse effects of climate change on earth. Optimized nutrient requirement plays a vital role in reproduction and as per predictions of the Inter-Governmental Panel on Climate Change (IPCC) global earth

otemperature is likely to increase by 1.8 to 4.0 C by the end of this century, and this may have an adverse effect on food production. Reproduction is optimized within a narrow range of environmental conditions and this process is compromised due to change in climate, because nutrients are diverted to maintain homeostasis, which is a priority over reproduction.

Existence and continuity of all living organisms is ensured only through the process of reproduction. Various reproductive events involving male-female gametes, fertilization, embryo development, implantation, pregnancy and birth of the young ones are beautifully orchestrated processes. Yet the strings of these physiological processes are delicately balanced to maintain good reproductive health and require a perfect harmony, else it leads to disturbances. Therefore, it is imperative that biologists study the effect of climatic changes on reproductive health and develop suitable strategies to mitigate its adverse impact.

The inaugural session of the conference was held at 9.00 a.m. on February 6, 2014 at Central Auditorium, Administrative Block, IVRI, Izatnagar. The conference was inaugurated by the Chief Guest Dr. V.P. Kamboj, Former Director CDRI & Chairman, Biotech Consortium India Ltd., New Delhi. Dr. G. Taru Sharma, Organizing Secretary gave a conference briefing and offered the welcome address. Prof. N.K. Lohiya, President-ISSRF and Prof. Gaya Prasad, ADG (AH),

thICAR and Former Director, IVRI addressed the inaugural function. The proceedings of the conference and 14 issue of the ISSRF Newsletter on Challenges of reproductive health due to influence of changing environment and lifestyle edited by Dr. Savita Yadav, AIIMS New Delhi were formally released on the occasion. The presidential address was delivered by Dr. R.K. Singh, Director & Vice-Chancellor, IVRI. During inaugural function, Lifetime Achievement Award was conferred to Dr. V.P. Kamboj. Dr. G. Singh, Sr. Scientist proposed a vote of thanks. More than 200 delegates and 60 invited speakers from different parts of India and overseas attended this conference.

International Conference on Reproductive Health:thIssues and Strategies under Changing Climate Scenario and the 24 Annual Meeting of the ISSRF

(RH-ISCS-ISSRF 2014)

February 6-8, 2014, IVRI, Izatnagar

Inaugural session was followed by the keynote addresses of Dr. M.L. Madan and Dr. S.M. Totey.

Scientific program included keynote addresses, invited talks, oration lectures, senior and young scientist's oral presentations and poster presentations. Conference had a total of sixteen sessions; six award sessions, two oral and one young scientists session, seven scientific sessions namely, Gametogenesis, Epigenetics and reprogramming, Climate-endocrine fertility relationship, Reproductive health, Stem cell biology and regenerative therapy, Upstream reproductive techniques and Implantation biology besides three poster sessions of one hour each. Tradition of ISSRF

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was followed by inviting reproductive biologists from different organizations to interact and intensify network to address the issues related to reproductive health in the context of ONE WORLD ONE HEALTH. A parallel session was avoided, so as to encourage a useful discussion and exchange of ideas with and amidst the scientific resource persons. Noted scientists from India and abroad in the field of reproductive health delivered their talks.

Amongst the senior colleagues, invited for the meeting were Dr. Vijayaraghavan Srinivasan, Kent State University, Kent, Ohio, USA; Dr. Dieter Schams, Technische Universität München, Freising, Germany; Dr. Pradeep Kumar G., Rajiv Gandhi Centre for Biotechnology, Trivandrum; Dr. Dheer Singh, National Dairy Research Institute, Karnal; Dr. Savita Yadav, All India Institute of Medical Sciences, New Delhi; Dr. Polani Seshagiri, Indian Institute of Science, Bangalore; Dr. S. Shivaji, CCMB/LVP Eye Institute, Hyderabad; Dr. Surendra Sharma, Women and Infants Hospital, Brown University, Providence, RI, USA; Dr. S.N. Kabir, Indian Institute of Chemical Biology, Kolkata; Dr. Ashutosh Halder, All India Institute of Medical Sciences, New Delhi; Dr. Rajvi Mehta, Trivector Embryology Support Academy, Mumbai; Dr. C.V. Rao, Florida International University, Miami, Florida, USA; Dr. Bhanu Prakash Telugu, University of Maryland, College Park, USA; Dr. Ramesh Bhonde, Manipal Institute of Regenerative Medicine, Bangalore; Dr. Taruna Madan, National Institute for Research in Reproductive Health, Mumbai; Dr Riaz Ahmad, Faculty of Veterinary Sciences, SKUAST, Srinagar. There were sixteen oral presentations by younger colleagues during the meeting.

Young scientists oral session was organized to encourage the junior scientists, total seven presentations were scheduled in the session. All the presentations were appreciated. However, the panel of judges recommended three best presentations, and the awardees were presented a certificate, plaque and a cash award of Rs. Three thousands each during valedictory function.

Several awards of ISSRF were given away during this 3-day conference. Labhsetwar award was shared by Dr. Malini Laloraya and Dr. A. S. Ansari. Dr. S.D. Kholkute received Prof. N. R. Moudgal Memorial Oration Award. Dr. S. Majumdar received Founder President Dr. T.C. Anand Kumar Memorial Oration Award. Prof. N. R. Moudgal Young Scientist Award was given to Ms. Swapna Desai. Dr. Jaysree Sengupta received Prof. L.S. Ramaswami Memorial Oration Award. Prof. G.P. Talwar Gold Medals went to Dr. S. Mondal for middle career scientist, Dr. Pankaj Suman and Dr. Shikha Saini for young scientist awards. All the awardees delivered their exciting award lectures.

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Day one banquet with the cultural evening was organized at the beautiful lawns of Executive Club, Bareilly. An elegant Sufi dance performed by the artists to give the fragrance of Northern India, was appreciated by everyone. Cultural evening witnessed the spark from the delegates too, specially Dr. S.D. Kholkute, Dr. Subeer Majumdar, Dr. Surendra Sharma, Dr. A. H. Bandivadekar and Dr. Sandeep Goel, who made it very memorable with their lively and energetic participation. Day two banquet was enjoyed by all at Hotel Awadh Plaza with good mood and cheer.

The Executive Committee Meeting of the ISSRF was held on 7th February, 2014 and that of General Body Meeting on 8th February, 2014. Prof. N.K. Lohiya, President – ISSRF presided over the meetings and the Secretary Dr. R.S. Sharma presented minutes of the ISSRF EC & GB meetings held at the RGCB, Thiruvananthapuram, and mid-term EC meeting held at NIRRH, Mumbai along with a brief about the activities of past one year of the Society. The General Body appreciated joining of large number of scientists as life member of the Society leading to 1176 members on roll. Both, the ISSRF Executive Committee and General Body complimented organizers of the International Conference on Reproductive Health: Issues and Strategies under Changing Climate Scenario and the 24th Annual Meeting of the ISSRF for very well planned scientific programme with participations of leading scientists from different parts of the country and abroad.

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The valedictory function was the last activity of the conference. Padmabhushan Prof. G.P. Talwar, Director, Talwar Research Foundation, New Delhi was the Chief Guest of the function. Dr. R.K. Singh, Director & Vice-Chancellor, IVRI, presided over the function. Prof. N.K. Lohiya gave the opening remarks followed by a report of the conference by Dr. G. Taru Sharma. Prof. G.P. Talwar gave a talk on Multiple Facets of Human Chorionic Gonadotropin. It was followed by the award ceremony. The meeting concluded with the vote of thanks offered by the Organizing Secretary.

Best poster presentation awards were also given by the Society. A total of 152 abstracts were invited for the poster presentations, spread over all three days. A four member jury selected three best posters. The best poster awards were given to following young scientists: Dr. Indrashis Bhattacharya, National Institute of Immunology, New Delhi, Mr. Eswara Murali S. and Ms. Sarbani Saha, CSIR-Indian Institute of Chemical Biology, Kolkata.

The ISSRF has instituted following awards. The applications/nominations for various awards should to

addressed to the President, Indian Society for the Study of Reproduction & Fertility (ISSRF), Centre for Advanced

Studies, Department of Zoology, University of Rajasthan, Jaipur – 302 004. The last date of receipt is November

30, every year.

• Labhsetwar Award

• Founder President Dr. T. C. Anand Kumar Memorial Oration

• Prof. G. P. Talwar Young Scientist Award / Prof. G. P. Talwar Gold Medal for Middle Career Scientists

• Prof. L. S. Ramaswami Memorial Oration

• Prof. N. R. Moudgal Memorial Oration / Prof. N. R. Moudgal Young Scientist Awards

• The Lifetime Achievement Award & The Fellowship Award

Kindly visit ISSRF website for further details. www.issrf.org

CALL FOR ISSRF AWARDS

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RECENT ACCOMPLISHMENTS OF LIFE MEMBERS OF THE SOCIETYDr. Anil Suri is well recognized eminent scientist in the field of Cancer Biology. Dr. Suri is the Fellow of National Academy of Sciences and a Fellow of National Academy of Medical Sciences (India). His outstanding cancer research has resulted in discovery of unique molecules which are important for early detection and diagnosis and can be implicated as immunotherapeutic targets for reproductive tract cancers. Dr. Suri was recently conferred upon a

prestigious oration award which carries Citation and Silver plate for 6th RNC thMemorial Oration Lecture award for the year 2014-2015 on 11 August, 2014 at

Post Graduate Institute of Medical Education and Research, Department of Experimental Medicine and Biotechnology, Chandigarh. Dr. Suri delivered award lecture on “BENCH-TO-BEDSIDE DEVELOPMENT OF CANCER THERAPY”.

Compliments & hearty congratulations, on behalf of the ISSRF, for taking over as Director of the National Institute for Research in Reproductive Health, Mumbai - a premier institute of the country.

Dr. Smita D. Mahale

February 14-17, 2015

th25 Annual Meeting of the Indian Society forthe Study of Reproduction and Fertility

andInternational Conference on Reproductive Health

"ISSRF Silver Jubilee Celebrations"

Dr. Jayanti Mania-Pramanik & Dr. Nafisa H. BalasinorScientist 'E'National Institute for Research in Reproductive Health(Indian Council of Medical Research)Jehangir Merwanji StreetParel, Mumbai - 400 012, IndiaTel.: +91-22-24192000/2025/2039Fax: +91-22-24139412E-mail : [email protected] : www.nirrh.res.in & www.issrf.org

stDeadline for submission of Abstract : 31 October, 2014thDeadline for early Registration : 30 November, 2014

IMPORTANT DATES

Organizing Secretaries

THEMES

Infertility

Stem Cells

Fertility Regulation

Reproductive Cancer

RTI/STI including HIV

Reproductive Toxicology

Reproductive Endocrinology

Reproductive Ageing, Maternal and Child Health

Lifestyle, Stress, Occupation and Reproductive Health

Environment, Endocrine Disruptor and Reproductive Health

NEWSLETTER FOR THE ISSRF-2015 EVENTIt is proposed to bring out the 16th special issue of the Newsletter to commemorate Silver Jubilee celebrations of our Society to be released during ISSRF 2015 event at the NIRRH, Mumbai. The newsletter will cover the following:

• attainment of specific objectives relating to reproductive health as per the national scenario

• highlights the priorities for setting up an agenda beyond millennium development goals

• showcase contributions of Indian Scientists in arena of reproductive health research that translated from bench to bedside.

• provides a perspective of ISSRF - journey of 25 years and still counting; and importantly

• need to reinvigorate national strategies to achieve 'Reproductive Health for All.'

The thematic focus may be "Perspectives of Reproductive Health Research on a Post - 2015 Development Framework".

For further information please contact the ISSRF secretariat.

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• Official peer reviewed journal of Indian Society for the Study of Reproduc�on and Fer�lity (ISSRF).

• Aims to publish juried original scien�fic ar�cles in clinical and laboratory research relevant to reproduc�ve and developmental biology, andrology, immunology, gene�cs, contracep�on, menopause and infer�lity.

• Target audience comprises obstetricians, gynecologists, reproduc�ve endocrinologists, physiologists, pathologists, urologists and scien�sts working on human reproduc�on issues.

• Accepts original ar�cles, reviews, short communica�ons, opinions, commentaries, news and le�er to the editors.

Na�onal Editorial BoardProf. N. K. Lohiya, JaipurDr. Subeer S. Majumdar, New Delhi Dr. Rakesh K. Tyagi, New DelhiProf. P. B. Seshagiri, Bangalore Dr. Smita Mahale, Mumbai Dr. R. S. Sharma, New Delhi Dr. G. Taru Sharma, BareillyDr. Dheer Singh, Karnal Prof. K. Muralidhar, New Delhi Dr. Pradeep Kumar G., Thiruvananthapuram Dr. Sa�sh Kumar Gupta, New Delhi Prof. Sujoy K. Guha, Kharagpur

EDITORIAL BOARDChief EditorsProf. Debabrata Ghosh, New DelhiProf. Jayasree Sengupta, New Delhi

Interna�onal Editorial BoardProf. David Kennaway, Australia Prof. Berthold Huppertz, AustriaProf. Carlos Simon, USAProf. Denny Sakkas, USAProf. Thomas D'Hooghe, BelgiumProf. Shigeru Saito, JapanProf. C. V. Rao, USA

ISSRF Secretariat: Centre for Advanced Studies, Department of Zoology, University of Rajasthan, Jaipur - 302 004, IndiaTelephone No.: +91-141-2701809 • Fax No.: +91-141-2701809 • E-mail: [email protected] • Website: www.issrf.org

Design & Print at Popular Printers, Jaipur • +91-141-2606883

issrf - newsletter - 15th ed sept 2014

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