1. BIOL30001 Reproductive Physiology
Germ Cells - Danielle Hickford
BIOL30001 Reproductive Physiology
Germ cells
Is this still the correct course code??
Danielle Hickford

2. Early observations on sperm and eggs…
BIOL30001 Reproductive Physiology
Germ Cells - Danielle Hickford
Early observations on sperm and eggs…
Animalcules- “little animals”
homunculus “little man”

3. BIOL30001 Reproductive Physiology
Germ Cells - Danielle Hickford
Germ cells
the gametes (eggs and sperm)
primordial germ cells (PGCs) are precursors to the gametes
transmit genetic information from one generation to the next
GC VS PGCs – check definition

4. BIOL30001 Reproductive Physiology
Germ Cells - Danielle Hickford
Life cycle of germ cells
up-regulation of pluripotency genes
1. specification
down-regulation of somatic genes
5. fertilisation
proliferation (mitosis)
2. migration to the genital ridges (future gonads)
4. gametogenesis (ova or spermatozoa)
proliferation
(mitosis)
XX inactivation- after entry into embryo, during migration to the gonads. Reactivation during somatic gonadal differentiation
Epigenetic imprint erasure –during somatic gonadal differentiation
XY epigenetic reprogramming – 2 days after erasure (after mitotic arrest). XX reprogramming just after birth
erasure of epigenetic imprints
epigenetic
re-programming
3. sexual differentiation
(mitotic arrest or meiosis)

5. Mechanisms of PGC specification
Determinative (preformisitic)
depends on inheritance of germ plasm
Regulative (epigenetic)
germ cell fate specified by cell-cell interactions and signalling

6. Determinative (preformistic) PGC specification
BIOL30001 Reproductive Physiology
Germ Cells - Danielle Hickford
Determinative (preformistic) PGC specification
insects, nematodes, fish, birds & frogs
inheritance of germ plasm – cytoplasm rich in specialised RNA binding proteins, RNA and mitochondria
germ plasm contains inhibitors of transcription and translation
germ cells specified very early in development
Germ plasm in many species contain inhibitors of transcription and translation. Egs include vasa, nanos (?)
Drosophila: Gcl (germ cell-less)- transcription inhibitor. Also Polar granule component (trans. Inihib). Nanos in a translational inhibitor.
Vasa – essential for initiating GC diff and meiosis
Germ plasm during cleavage in the zebrafish
Yoon et al., 1997

7. BIOL30001 Reproductive Physiology
Germ Cells - Danielle Hickford
Determinative PGC specification- zebrafish
(4 vasa-positive cells)
(~25 vasa-positive cells)
At 1000 cell stage, ~4 positive cells
At 6 hr vasa +ve cells.
8h-75% epiboly
16 hr- 14 somites
= vasa-positive germ plasm
Modified from IlluScientia

8. Regulative PGC specification
mammals, urodeles (eg, salamanders)
depends on signals from adjacent cell populations
ancestral form of germ cell specification?
BMP4, BMP8b (bone morphogenetic protein)
BMP2
Alkaline phosphatase positive PGCs in a E7.5 mouse embryo
(Ginsburg et al., 1990)
PGCs in a 3 week old human embryo
(Essential Reproduction, Johnson, 2013)

9. Germ cells VS somatic cells
Pluripotent cell lineages
Somatic cell lineages
Essential Reproduction, Johnson, 2013

10. Key processes of PGC specification
BIOL30001 Reproductive Physiology
Germ Cells - Danielle Hickford
Key processes of PGC specification
pluripotency genes
(eg, Sox2, Nanog)
Prdm1
germ cell-specific genes (eg, Stella, Nanos3)
Prdm14
somatic mesodermal genes (eg, Hox genes, Brachyury)
Prdm1
Check order of these processes.
Extensive epigenetic remodelling
Prdm14

11. Germ cell proliferation
E6.25 mouse: ~6 PGCs. By E13.5, ~25,000 germ cells
proliferation requires numerous growth factors & proteins
autocrine or paracrine signals (SCF/c-kit, FGFs, LIF)
Numbers of human ovarian germ cells during development
Essential Reproduction, Johnson, 2013

12. BIOL30001 Reproductive Physiology
PGC migration
Germ Cells - Danielle Hickford
migratory route guided by ECM
chemoattractive & repulsive signals are also involved
complex migratory route - many different stages
Migration from mesoderm into endoderm. 2. Migration along endoderm (ie, along gut) migration into dorsal mesentary migration to genital ridges
Richardson and Lehman review
Essential Reproduction, Johnson, 2013

13. BIOL30001 Reproductive Physiology
Germ Cells - Danielle Hickford
gonads
mesonephros
gut
body wall
dorsal mesentery
PGC migration in a wallaby fetus

14. BIOL30001 Reproductive Physiology
Germ Cells - Danielle Hickford
gonads
mesonephros
gut
body wall
dorsal mesentery
PGC migration in a wallaby fetus
CXCR4 on GC, SDF1a (in genital ridges) –

15. PGC migration in a wallaby fetus
gonad
dorsal mesentery
PGCs
mesonephros
PGC migration in a wallaby fetus

16. BIOL30001 Reproductive Physiology
Germ Cells - Danielle Hickford
PGC migration in a wallaby fetus
CXCR4
SDF1
ckit
SCF
Guidance by ECM – integrins and E-cadherin on PGCs, ECM in fetus

17. Germ cell sexual differentiation
BIOL30001 Reproductive Physiology
Germ cell sexual differentiation
Germ Cells - Danielle Hickford
first step is meiosis
in mice: at E female germ cells enter meiosis, males enter mitotic arrest
germ cell differentiation depends on the somatic environment initially, then on the chromosomal component of the germ cells
Condense further
Premature entry into meiosis - not enough germ cells
unique to germ cells
exchange of genetic material (via recombination)
creates haploid gametes

18. Meiosis unique to germ cells exchange of genetic material
production of haploid gametes
Essential Reproduction, Johnson, 2013

19. Control of entry into meiosis
Cyp26b1 RA
Stra8
meiosis
VitA RA
Stra8
meiosis
VitA
Mesonephros
Ovary
Testis
RA = retinoic acid

20. Inequivalence of information from eggs and sperm
gametes carry the same genetic information but some of it is differentially modified between the sexes (= epigenetic modification)
From: Principles of Development. Wolpert/Tickle

21. Epigenetic modifications
heritable changes to DNA or chromatin structure but not to DNA sequence

22. Mechanisms of epigenetic modifications
Histone modifications
DNA modifications
Essential Reproduction, Johnson, 2013

23. Epigenetic modifications
main epigenetic modification in germ cells is DNA methylation
genomic imprinting – expression of a gene in a parent-of origin specific manner
Male germ cells
Female germ cells
Imprinted genes
17 maternally methylated
4 paternally methylated
Average level of DNA methylation
~40%
~89%

24. Epigenetic control of germ cell development
BIOL30001 Reproductive Physiology
Germ Cells - Danielle Hickford
Epigenetic control of germ cell development
Epigenetic reprogramming of germ cells is required for:
- correct gene expression
- X chromosome inactivation/reactivation
- progression of meiosis
- gametogenesis
Control of gene expression - - repression of somatic genes (Prdm1), onset of other germ cell specific genes at the correct time (eg, vasa, Scp3, Dazl, after arrival at genital ridge)

25. Modification of imprint status
BIOL30001 Reproductive Physiology
Germ Cells - Danielle Hickford
Modification of imprint status
main epigenetic modification in PGCs is DNA methylation
epigenetic erasure of imprinted loci before and as germ cells arrive at genital ridge
new imprint status established after sexual differentiation:
- females: after birth, during prophase I
- males: during mitotic arrest
Males – 3 imprinted loci, females, >10
new imprint status (via DNA methylation)
demethylation (epigenetic erasure)

26. Modification of PGC imprint status
Removal of DNA methylation by TET (ten-eleven translocation) proteins
Re-establishment of DNA methylation by de novo DNA methyltransferases (DNMTs)
TET2 protein in Day 25 pp wallaby testis
DNMT3A protein in Day 40 pp wallaby testis

27. Epigenetic control of gametogenesis
BIOL30001 Reproductive Physiology
Epigenetic control of gametogenesis
Germ Cells - Danielle Hickford
spermatids: histones replaced by protamines
oocytes contribute factors for post-fertilisation reprogramming (transcription factors and epigenetic modifiers)
Protamines- small proteins that allow DNA to be condensed further
Factors for re-programming in oocyte – can be cytoplasmic and/or nuclear. Not yet identified
Protamines are small, arginine-rich, nuclear proteins that replace histones late in the haploid phase of spermatogenesis and are believed essential for sperm head condensation and DNA stabilization. They may allow for denser packaging of DNA in spermatozoon than histones, but they must be decompressed before the genetic data can be used for protein synthesis. However, in humans and maybe other primates, 10-15% of the sperm's genome is packaged by histones thought to bind genes that are essential for early embryonic development

28. Life cycle of germ cells
up-regulation of pluripotency genes
1. specification
down-regulation of somatic genes
5. fertilisation
proliferation (mitosis)
2. migration to the genital ridges (future gonads)
4. gametogenesis (ova or spermatozoa)
proliferation
(mitosis)
erasure of epigenetic imprints
epigenetic
re-programming
3. sexual differentiation
(mitotic arrest or meiosis)

29. BIOL30001 Reproductive Physiology
Germ Cells - Danielle Hickford
Biol30001 – Germ cell lecture Reading and references
General reviews:
Primordial germ cells in mice. Cold Spring Harb. Persperct. Biol. 4:11 (2012) Saitou & Yamaji
Mouse germ cell development: From specification to sex determination. Molec. & Cell Endocrin. 323: 76–93 (2010) Ewen & Koopman
Germ cell migration:
Mechanisms guiding PGC migration: strategies from different organisms. Nat. Rev. Molec. Biol. 11:37-49 (2010) Richardson & Lehmann
Germ cell genetics:
Genetics of germ cell development. Nat. Rev. Genet. 13: (2012) Lesch & Page
Germ cell epigenetics:
De novo DNA methylation: a germ cell perspective. Trends in Genet 28: (2012) Smallwood & Kelsey