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 deciency !

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!

INTRODUCTION!

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! !
OOGENESIS! SPERMATOGENESIS!

oocyte!

sperm!

INTRODUCTION!

Gonadal primordium!

Testis! PGC! meiosis! Prospermatogonia! Oocyte!

PGC!

Ovary!

INTRODUCTION!

Meiosis in germ cells!

premeiotic S phase! prophase!

2c diploid!

4c diploid!

Ist meiotic division!

2c haploid!
2nd meiotic division!

1c haploid!

INTRODUCTION!

In the fetus!
premeiotic S phase! prophase!

2c diploid!

4c diploid!

Ist meiotic division!

2c haploid!
2nd meiotic division!

1c haploid!

INTRODUCTION!

2c diploid!
premeiotic S phase! prophase! ARRESTS!

4c diploid!

Ist meiotic division!

2c haploid!
2nd meiotic division!

1c haploid!

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! !

INTRODUCTION!

Previous research! ! ! In vitro development of oocyte-like cells! ! Underwent second meiotic division prior to fertilization! ! ! What does it mean? ! ! ! Potentially difcult to coordinate meiosis and oocyte differentiation because they may be regulated independently!

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OBJECTIVES!

OBJECTIVES!

Dokshin et al. ! ! ! Previously characterized Stra8 decient ovarian germ cells in mixed genetic background ! !

OBJECTIVES!

Role of Stra8!
Gonadal primordium! Retinoic acid! Msx1/Msx2! Stra8! PGC! meiosis! Prospermatogonia! Oocyte!

Testis! PGC!

Ovary!

OBJECTIVES!

! Does Stra8 deciency effect oocyte growth and differentiation, in addition to blocking meiotic initiation?! ! Conrm Stra8 deciency 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 decient mice !

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RESULTS!
! Data! ! Methods!

RESULTS!

Meiotic defect in Stra8 decient inbred mice! ! ! Assembly of the synaptonemal complex! ! Localization of meiotic cohesin REC8 ! ! Formation of DNA double-strand breaks! Conrmed that Stra8 is required for meiotic prophase. !

RESULTS!

Meiotic defect in Stra8 decient inbred mice ! ! Assembly of the synaptonemal complex! ! !

RESULTS!

Meiotic defect in Stra8 decient inbred mice ! ! Localization of meiotic cohesin REC8 !

RESULTS!

Meiotic defect in Stra8 decient inbred mice ! ! Formation of DNA double-strand breaks!

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!

RESULTS!

Fetal and postnatal loss of germ cells! ! Some germ cells survived in the early stages!

RESULTS!

Stra8 decient oocyte-like cells! ! HYPOTHESIS: If Stra8 (and thus meiotic prophase) is required for oocyte growth and differentiation, then these processes will not occur Stra8 decient mice ! ! How to test?! ! ! Look at ovarian histology in wild type and Stra8 decient mice!

RESULTS!

Stra8 decient oocyte-like cells! ! Compare to wild-type using histological techniques ! !

RESULTS!

Stra8 decient 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). ! !

RESULTS!

Stra8 decient cells have not entered meiotic prophase! ! Conrm that these cells did not manage to enter meiotic prophase ! !

RESULTS!

Stra8 decient cells have not entered meiotic prophase! ! Conrm that these cells did not manage to enter meiotic prophase ! !

RESULTS!

Stra8 decient cells have not entered meiotic prophase! ! Conrm that these cells did not manage to enter meiotic prophase ! !

RESULTS!

Follicle formation and ovulation! ! Immunohistochemical staining of ovary sections ! ! !

RESULTS!

Follicle formation and ovulation! ! Superovulation induced by hormonal stimulation! ! Expanded cumuli oophori in both groups ! ! !

RESULTS!

Asymmetric division to produce polar bodies! ! Do breakdown of the nuclear envelope, chromosome condensation and asymmetric division occur in Stra8 -/- ?! !

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 decient: !11 out of 42!

RESULTS!

Premeiotic chromosome replication has not occurred! ! Immunoourescent labeling for DNA, spindle and centromeres! !

RESULTS!

Premeiotic chromosome replication has not occurred!

RESULTS!

Stra8 decient cells can form two cell embryos! ! Cannot produce live offspring, but early steps of embryogenesis can be observed !

RESULTS!

Recap of Methods! ! Mice! ! Stra8 decient 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 ! !

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 ! !

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 & MEMalpha and 0.3% BSA for 6 hours ! !

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DISCUSSION!

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 decient oocyte-like cells are fertilizationcompetent and develop despite lack of meiotic initiation !

DISCUSSION!

Meiosis and Differentiation potential for different pathways ! !

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 ! !

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 ! !

DISCUSSION!

Opportunities for future study! ! ! Are these processes dissociable in other organisms as well? Vertebrates? Invertebrates?! ! What genes are expressed in Stra8 decient ovarian germ cells? How do they drive ovary development?! ! Why do Stra8 decient ovarian germ cells die prematurely and in such great numbers?! ! Implications for fertility and in vitro oogenesis ! !

STRENGTHS!

! Improving and conrming 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!

STRENGTHS!

! Each gure shows a direct comparison between Stra8 mutant and the wild type or Stra8 heterozygote !

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 !

! ! ! ! ! ! ! ! ! ! ! ! WEAKNESSES!
! Imprecise terminology used throughout subjective meaning ! ! properly! ! most! ! similar!

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! All gures are qualitative data very little (if any) quantitative data is given !

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! Signicance of differences between wild type and Stra8 mutants is not quantied !

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! Some techniques like superovulation are unlikely to accurately represent what happens in vivo ! !

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! 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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! All ndings are based on a small subset of ovarian germ cells (in Stra8 decient females) that survived postnatally! ! Difcult 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 !

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 ! !