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  

                                         

INTRODUCTION •  Oogenesis •  Meiotic prophase •  Oocyte differentiation •  Stra8 deficiency

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

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

INTRODUCTION

Gonadal primordium

PGC

Prospermatogonia

Testis

Oocyte

PGC

Ovary

meiosis

INTRODUCTION

Meiosis in germ cells 2c diploid

4c diploid

premeiotic S phase

2c haploid

1c haploid

prophase

Ist meiotic division

2nd meiotic division

INTRODUCTION

2c diploid

4c diploid

premeiotic S phase

2c haploid

1c haploid

prophase

Ist meiotic division

2nd meiotic division

In the fetus

INTRODUCTION

2c diploid

4c diploid

premeiotic S phase

2c haploid

1c haploid

prophase

Ist meiotic division

2nd meiotic division

ARRESTS

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 difficult to coordinate meiosis and oocyte

differentiation because they may be regulated independently

                                         

OBJECTIVES

OBJECTIVES

Dokshin et al. •  Previously characterized Stra8 deficient ovarian germ cells in

mixed genetic background

Gonadal primordium

PGC

Prospermatogonia

Retinoic acid Msx1/Msx2

Stra8 Testis

Oocyte

PGC

Ovary

meiosis

Role of Stra8

OBJECTIVES

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

                                         

RESULTS •  Data •  Methods

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.

RESULTS

Meiotic defect in Stra8 deficient inbred mice •  Assembly of the synaptonemal complex

RESULTS

Meiotic defect in Stra8 deficient inbred mice •  Localization of meiotic cohesin REC8

RESULTS

Meiotic defect in Stra8 deficient 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 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

RESULTS

Stra8 deficient oocyte-like cells •  Compare to wild-type using histological techniques

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).

RESULTS

Stra8 deficient cells have not entered meiotic prophase •  Confirm that these cells did not manage to enter meiotic

prophase

RESULTS

Stra8 deficient cells have not entered meiotic prophase •  Confirm that these cells did not manage to enter meiotic

prophase

RESULTS

Stra8 deficient cells have not entered meiotic prophase •  Confirm 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 deficient: 11 out of 42

RESULTS

Premeiotic chromosome replication has not occurred •  Immunoflourescent labeling for DNA, spindle and centromeres

RESULTS

Premeiotic chromosome replication has not occurred

RESULTS

Stra8 deficient cells can form two cell embryos •  Cannot produce live offspring, but early steps of embryogenesis

can be observed

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

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 & MEM-

alpha and 0.3% BSA for 6 hours

                                         

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 deficient oocyte-like cells are fertilization-competent 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 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

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

STRENGTHS

•  Each figure 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”

WEAKNESSES

•  All figures are qualitative data – very little (if any) quantitative data is given

WEAKNESSES

•  Significance of differences between wild type and Stra8 mutants is not quantified

WEAKNESSES

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

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

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

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