Comparative Biology of Oocyte Aging

Pierre Comizzoli, D.V.M., Ph.D.

Smithsonian Conservation Biology Institute

National Zoological Park, Washington DC

Disclosure

I have nothing to disclose

Contribution of Reproductive Science and Gamete

Biology to Conservation Biology

Value of basic/comparative studies

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gesta

gens (

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→ Scholarly knowledge and conservation actions in situ and ex situ

(enhancing natural mating, maintaining genetic diversity)

→ Development of Assisted Reproductive Techniques and

Genome Resource Banking

(overcoming mating difficulties, preserving fertility, maintaining genetic diversity)

Diversity and Complexity

of Reproduction and Gamete Biology

Only few species well described among 5,400 mammal species

As many mechanistic differences in reproduction as there are species

Diversity in fertility issues (aging, teratospermia, sensitivity to stress…)

Species-specificities in hormonal stimulations and in vitro culture conditions

Difficult to compare human and animal reproductive aging (different lifespan)

No ‘menopause’ in animals but age-related loss of fecundity

Diverse Reproductive Physiologies

Seasonal

Breeder

Estrous Cycle

(days)

Estrus

(days) Ovulation

Gestation

(days)

Black-footed

cat No 5 – 29 2 – 9 Ind. 63 – 71

Bobcat Yes 44 5 – 10 Ind. 50 – 70

Cheetah No 7 – 23 2 – 6 Ind. 90 – 98

Clouded

leopard Yes 25 – 30 3 – 6 Spont. 85 – 93

Domestic cat Varies 14 – 21 3 – 7 Both 64 – 67

Asian

elephant No 100 – 110 2 – 4 Spont. 640 – 660

Eld’s deer Yes 20 – 24 1 – 2 Spont. 230 – 240

Giant panda Yes 7 – 10 1 Spont. 80 – 180

Values of Comparative Folliculogenesis

and Oocyte Biology

Animal models are essential to improve our understanding of aging

mechanisms and develop mitigation strategies

Limitations of existing laboratory models because of size, anatomy, and

physiology (including lifespan)

Differences in ovarian anatomy and histology

Rodents Bovids Suidae Felids Human

Oocyte diam.

(µm) 80 110 125 110 110

Germ. vesicle

diam. (µm) 30 35 35 40 40

In vitro

maturation <24 hr ~24 hr ~44 hr ~28 hr ~24 hr

Differences in oocyte size (minimal), nucleo-cytoplasmic ratio, lipid content

Differences in folliculogenesis (timing, follicle waves, polyovulations)

Differences in oocyte competence related to the follicular size

Differences in GV competence related to the follicular size

Values of Comparative Folliculogenesis

and Oocyte Biology

Reproductive Aging:

Complex and Multifactorial Mechanisms

Mainly described in human

Aging of organism affecting folliculogenesis and ovulation

(hormone level changes)

Egg quantity and quality significantly declines with reproductive age (>35 yr)

Increase in miscarriages, infertility, and birth defects

Changes in reproductive tract affecting conception, embryo development,

implantation, and pregnancy

Accumulations/exposures during reproductive life:

Irreparable damage, long arrest at the GV stage, increased oxidative stress

during folliculogenesis

Oocyte Aging:

Complex and Multifactorial Mechanisms Too!

Changes in:

GV chromatin configuration and integrity

GV epigenetics/transcriptomics

GV proteomics

Cytoplasm (mitochondria number and function,

protein metabolism)

Zona pellucida

Connections with cumulus cells

Ovarian environment (fibrosis)

As a result:

Defects in meiotic maturation (chromosome segregation, aneuploidy)

Defects in fertilization, embryo development, implantation, pregnancy

Need systematic approaches with proper models for each aspect

Chromatin configuration and competence in the cat model

A B C D

E F G H

Chromatin Configuration and Integrity

~8% of oocytes with abnormal configuration and DNA damage at any age

~10% in adult ungulates vs. 25% in old individuals (past 14 yr)

Germinal Vesicle Epigenetics

Aging mouse oocyte - Decrease in expression of

histone deacetylases (HDAC) and DNA methyl-transferases (DNMT)

Genome-wide DNA methylation is lower

Histones are more acetylated

Key histone methylations are altered

Need for alternate models (closer to human in size and timing)

Germinal Vesicle Epigenetics

Distribution of Histone Deacetylase 2 during folliculogenesis and transcriptional

silencing in the cat model

Translocation occurs earlier in older individuals (>12 year)

Manipulation of Epigenetics in Germinal Vesicles

Reversible and global de-acetylation to mitigate aging

Control

0.5 mM resveratrol

1.0 mM resveratrol

1.5 mM resveratrol

Primary regulations of histone methylations are modified in old cats (>12 year)

Germinal Vesicle Epigenetics

Primary regulations of histone methylations during folliculogenesis in

the cat model

Changes in Ovarian Environment

Squirrel monkey

Cheetah

Cattle

Aging Study in Cheetahs (Acinonyx jubatus)

A Counterexample

Drop of fertility in older females

No pregnancies after natural breeding in old females

Normal ovarian cycles

Good ovarian response to exogenous gonadotropins

But no conception after intra-uterine artificial insemination

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eCG

hCG

Oocyte

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Aging Study in Cheetahs (Acinonyx jubatus)

Percentages of fertilization and embryo development were not different

between young and old females

Oocyte quality is not affected by the age of the female

(including microtubules, mitochondrial functions…)

Aging Study in Cheetahs (Acinonyx jubatus)

Comparison of ovarian anatomy

Oocyte aspiration and IVF

Increase of uterine wall thickness in old females

High prevalence of cystic endometrial hyperplasia

Aging Study in Cheetahs (Acinonyx jubatus)

Age Effect in Eld’s Deer (Rucervus eldii thamin)

In vitro maturation

In vitro fertilization and culture

for 7 days in deer SOF medium

Age Effect in Eld’s Deer (Rucervus eldii thamin)

Less embryo development and no pregnancy with donors >10 yr old

Heat stress > aging - Older females are more sensitive

Adapted hormone treatments to stimulate folliculogenesis in old donors

Have induced ovulator less oocyte aging issues?

(higher production rate of oocytes)

Ungulates seems to be more prone to effect of age

Understanding oocyte aging through methods used for mitigations

(optimization and development of new tools):

Adapted hormone stimulation in aged patients (deer)

Resveratrol exposure in cat oocytes

Germinal vesicle transfer

(cytoplasmic aging > nuclear aging)

Lessons Learned

Values of comparative studies to advance knowledge on oocyte aging

and ‘shed a new light’ on human fertility studies

Difficult to compare oocyte aging in animal species

(no menopause, uterine pathology or heat stress are prevalent)

We know very little about oocyte aging in animal species

Elephants could be an excellent model but no knowledge on the oocyte

Systems biology will help to better understand and mitigate oocyte aging

Take-Home Messages