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Oocyte differentiation is genetically dissociable from meiosis in mice GREGORIY A DOKSHIN, ANDREW E BALTUS, JOHN J EPPIG & DAVID C PAGE NATURE GENETICS (2013) 45: 877
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INTRODUCTION • Oogenesis • Meiotic prophase • Oocyte differentiation • Stra8 deficiency
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INTRODUCTION
What is oogenesis? • Creation of a fully-functional egg from an ovarian germ cell • Involves both cellular and chromosomal events
OOGENESIS SPERMATOGENESIS
oocyte sperm
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INTRODUCTION
OOGENESIS SPERMATOGENESIS
oocyte sperm
Responsibilities of an egg: • Can undergo fertilization and give rise to offspring • Contributes one and only one copy of each chromosome to the
developing embryo • Drive follicle formation
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INTRODUCTION
Gonadal primordium
PGC
Prospermatogonia
Testis
Oocyte
PGC
Ovary
meiosis
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INTRODUCTION
Meiosis in germ cells 2c diploid
4c diploid
premeiotic S phase
2c haploid
1c haploid
prophase
Ist meiotic division
2nd meiotic division
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INTRODUCTION
2c diploid
4c diploid
premeiotic S phase
2c haploid
1c haploid
prophase
Ist meiotic division
2nd meiotic division
In the fetus
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INTRODUCTION
2c diploid
4c diploid
premeiotic S phase
2c haploid
1c haploid
prophase
Ist meiotic division
2nd meiotic division
ARRESTS
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INTRODUCTION
Oocyte Differentiation • During fetal development: • Cells arrest in meiotic prophase I and differentiate into
primordial oocytes
• Following puberty: • Primordial oocytes become fully developed oocytes
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INTRODUCTION
Previous research • In vitro development of oocyte-like cells • Underwent second meiotic division prior to fertilization
What does it mean? • Potentially difficult to coordinate meiosis and oocyte
differentiation because they may be regulated independently
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OBJECTIVES
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OBJECTIVES
Dokshin et al. • Previously characterized Stra8 deficient ovarian germ cells in
mixed genetic background
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Gonadal primordium
PGC
Prospermatogonia
Retinoic acid Msx1/Msx2
Stra8 Testis
Oocyte
PGC
Ovary
meiosis
Role of Stra8
OBJECTIVES
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OBJECTIVES
• Does Stra8 deficiency effect oocyte growth and differentiation, in addition to blocking meiotic initiation? • Confirm Stra8 deficiency using inbred genetic background
• Ultimately, understand the different pathways involved in
oogenesis • Implications for derivation of oocytes in vitro
HYPOTHESIS: If Stra8 (and thus meiotic prophase) is required for oocyte growth and differentiation, then these processes will not occur Stra8 deficient mice
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RESULTS • Data • Methods
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RESULTS
Meiotic defect in Stra8 deficient inbred mice • Assembly of the synaptonemal complex • Localization of meiotic cohesin REC8 • Formation of DNA double-strand breaks
Confirmed that Stra8 is required for meiotic prophase.
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RESULTS
Meiotic defect in Stra8 deficient inbred mice • Assembly of the synaptonemal complex
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RESULTS
Meiotic defect in Stra8 deficient inbred mice • Localization of meiotic cohesin REC8
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RESULTS
Meiotic defect in Stra8 deficient inbred mice • Formation of DNA double-strand breaks
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RESULTS
Fetal and postnatal loss of germ cells • At birth, ovaries had fewer germ cells compared to wild type • By 6 to 8 weeks of age, no germ cells remaining
• Dokshin et al. examined the cells that survived during the early stages
• Investigated the oogenic potential of these cells
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RESULTS
Fetal and postnatal loss of germ cells • Some germ cells survived in the early stages
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RESULTS
Stra8 deficient oocyte-like cells HYPOTHESIS: If Stra8 (and thus meiotic prophase) is required for oocyte growth and differentiation, then these processes will not occur Stra8 deficient mice How to test? • Look at ovarian histology in wild type and Stra8 deficient mice
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RESULTS
Stra8 deficient oocyte-like cells • Compare to wild-type using histological techniques
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RESULTS
Stra8 deficient oocyte-like cells • Compare ultrastructural features using electron microscopy Cytoplasmic processes (dashed arrow), vacuolated mitochondria (M), multivesicular bodies (MVB), lattice structures (L), cortical granules (CG), cumulus granulosa cells (CCP), zona pellucida (ZP).
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RESULTS
Stra8 deficient cells have not entered meiotic prophase • Confirm that these cells did not manage to enter meiotic
prophase
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RESULTS
Stra8 deficient cells have not entered meiotic prophase • Confirm that these cells did not manage to enter meiotic
prophase
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RESULTS
Stra8 deficient cells have not entered meiotic prophase • Confirm that these cells did not manage to enter meiotic
prophase
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RESULTS
Follicle formation and ovulation • Immunohistochemical staining of ovary sections
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RESULTS
Follicle formation and ovulation • Superovulation induced by hormonal stimulation • Expanded cumuli oophori in both groups
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RESULTS
Asymmetric division to produce polar bodies • Do breakdown of the nuclear envelope, chromosome
condensation and asymmetric division occur in Stra8 -/- ?
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RESULTS
Asymmetric division to produce polar bodies • Breakdown of the nuclear envelope (geminal vesicle) • Seen in “most” oocyte-like cells
• Asymmetric division and formation of polar bodies • Wild type: 28 out of 35 • Stra8 deficient: 11 out of 42
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RESULTS
Premeiotic chromosome replication has not occurred • Immunoflourescent labeling for DNA, spindle and centromeres
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RESULTS
Premeiotic chromosome replication has not occurred
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RESULTS
Stra8 deficient cells can form two cell embryos • Cannot produce live offspring, but early steps of embryogenesis
can be observed
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RESULTS
Recap of Methods Mice • Stra8 deficient mice backcrossed with inbred genetic background
(C57BL/6) – females used for testing
Histology • Fixed and sectioned ovaries (minimum 4 samples for each
genotype) Electron Microscopy • Used to observe ultrastructural characteristics of oocytes
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RESULTS
Recap of Methods Immunocytochemistry • For observation of ovarian germ cell spreads
Immunohistochemistry • Antibodies: mouse yH2A.X, goat hVASA, rat ZP2, rabbit
NOBOX, goat FOXL2
Superovulation • Stimulated with pregnant mare serum gonadotropin & human
chorionic gonadotropin • Cumulus-oocyte complexes were dissected out
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RESULTS
Recap of Methods Oocyte maturation • Stimulated with pregnant mare serum gonadotropin • Ovaries dissected
Microscopy • Z stacks at 0.5 micrometer spacing
In vitro Fertilizatoin • Cumulus-oocyte complexes were incubated with sperm & MEM-
alpha and 0.3% BSA for 6 hours
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DISCUSSION
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DISCUSSION
Meiosis and Differentiation – potential for different pathways • Oocyte growth and differentiation appears dissociable from
meiotic initiation and prophase I
• Meiosis remains absolutely critical for later steps of embryogenesis
• However, Stra8 deficient oocyte-like cells are fertilization-competent and develop despite lack of meiotic initiation
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DISCUSSION
Meiosis and Differentiation – potential for different pathways
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DISCUSSION
Meiosis and Differentiation – potential for different pathways • Stra8 mutants could prove useful in the future for studying this
proposed independent pathway • Look at ovarian germ cell differentiation when meiosis does
not occur
• Meiotic initiation and oocyte differentiation occur concurrently but are regulated independently • These pathways contribute independently to the formation
of a fully grown egg
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DISCUSSION
Meiosis and Differentiation – potential for different pathways • Despite not entering meiotic prophase, these cells maintained an
ovarian cell fate • Cell fate and differentiation do not appear linked to meiotic
initiation • Meiotic prophase should not be considered a sign that cells
have committed to a fate
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DISCUSSION
Opportunities for future study • Are these processes dissociable in other organisms as well?
Vertebrates? Invertebrates?
• What genes are expressed in Stra8 deficient ovarian germ cells? How do they drive ovary development?
• Why do Stra8 deficient ovarian germ cells die prematurely and in such great numbers?
• Implications for fertility and in vitro oogenesis
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STRENGTHS
• Improving and confirming a previous study more thoroughly
• Helps answer an important question regarding current hypotheses concerning mouse germ cells
• Discovery that other researchers can build on
• Important topic relevant to many individuals • Potential implications for in vitro fertilization advancements
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STRENGTHS
• Each figure shows a direct comparison between Stra8 mutant and the wild type or Stra8 heterozygote
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STRENGTHS
• Detailed investigation of oocyte growth and development
• Made observations at the subcellular level all the way to examination of entire tissues
• Variety of techniques used, in vitro & in vivo
• Concise and well organized paper • Provides a complete and easy to follow introduction to the
topic • Additional detail given in supplementary materials
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WEAKNESSES
• Imprecise terminology used throughout – subjective meaning • “properly” • “most” • “similar”
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WEAKNESSES
• All figures are qualitative data – very little (if any) quantitative data is given
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WEAKNESSES
• Significance of differences between wild type and Stra8 mutants is not quantified
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WEAKNESSES
• Some techniques like superovulation are unlikely to accurately represent what happens in vivo
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WEAKNESSES
• Contradiction to argue that oocyte differentiation is entirely dissociable from meiosis • Lack of meiosis resulted in failure to produce polar bodies in
many cells later on – this is a type of interdependence
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WEAKNESSES
• All findings are based on a small subset of ovarian germ cells (in Stra8 deficient females) that survived postnatally • Difficult to draw meaningful conclusions from a small set • Later stages cannot be observed
• Optimistic to say Stra8 mutants can be used as a tool considering most germ cells die • Stra8 likely has many essential functions
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Oocyte differentiation is genetically dissociable from meiosis in mice GREGORIY A DOKSHIN, ANDREW E BALTUS, JOHN J EPPIG & DAVID C PAGE NATURE GENETICS (2013) 45: 877