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Early observations on sperm and eggs…
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
1. specification
2. migration to the genital ridges (future gonads)
4. gametogenesis (ova or spermatozoa)
5. fertilisation
3. sexual differentiation (mitotic arrest or meiosis)
up-regulation of pluripotency genes
down-regulation of somatic genes
erasure of epigenetic imprints
proliferation (mitosis)
proliferation (mitosis)
epigenetic re-programming
Life cycle of germ cells
Mechanisms of PGC specification
Determinative (preformisitic)• depends on inheritance of
germ plasm
Regulative (epigenetic)• germ cell fate specified by cell-cell interactions and signalling
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 during cleavage in the zebrafishYoon et al., 1997
Modified from IlluScientia
Determinative PGC specification- zebrafish
(4 vasa-positive cells)
(~25 vasa-positive cells)
= vasa-positive germ plasm
Regulative PGC specification• mammals, urodeles (eg, salamanders)
• depends on signals from adjacent cell populations• ancestral form of germ cell specification?
Alkaline phosphatase positive PGCs in a E7.5 mouse embryo(Ginsburg et al., 1990)
PGCs in a 3 week old human embryo
BMP4, BMP8b (bone morphogenetic protein)
BMP2
(Essential Reproduction, Johnson, 2013)
Germ cells VS somatic cells
Essential Reproduction, Johnson, 2013
Pluripotent cell lineages
Somatic cell lineages
Key processes of PGC specification
pluripotency genes (eg, Sox2, Nanog)
somatic mesodermal genes (eg, Hox genes, Brachyury)
germ cell-specific genes (eg, Stella, Nanos3)
Extensive epigenetic remodelling
Prdm1
Prdm1
Prdm14
Prdm14
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
PGC migration
• migratory route guided by ECM• chemoattractive & repulsive signals are also involved
Essential Reproduction, Johnson, 2013
gonads
mesonephros
gut
body wall
dorsal mesentery
PGC migration in a wallaby fetus
gonads
mesonephros
gut
body wall
dorsal mesentery
PGC migration in a wallaby fetus
gonad
gonad
dorsal mesentery
PGCs
mesonephros
PGC migration in a wallaby fetus
PGC migration in a wallaby fetus
CXCR4SDF1ckit SCF
Germ cell sexual differentiation• first step is meiosis• in mice: at E13.5-14.5 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
• unique to germ cells• exchange of genetic
material• production of haploid
gametes
Meiosis
Essential Reproduction, Johnson, 2013
RA Stra8
meiosis
RA
VitA
Cyp26b1RA
Stra8
meiosis
RA
VitA
Mesonephros Ovary Testis
Control of entry into meiosis
RA = retinoic acid
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
Epigenetic modifications
• heritable changes to DNA or chromatin structure but not to DNA sequence
Mechanisms of epigenetic modifications
DNA modifications
Histone modifications
Essential Reproduction, Johnson, 2013
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%
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
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
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
Epigenetic control of gametogenesis
• spermatids: histones replaced by protamines • oocytes contribute factors for post-fertilisation
reprogramming (transcription factors and epigenetic modifiers)
1. specification
2. migration to the genital ridges (future gonads)
4. gametogenesis (ova or spermatozoa)
5. fertilisation
3. sexual differentiation (mitotic arrest or meiosis)
up-regulation of pluripotency genes
down-regulation of somatic genes
erasure of epigenetic imprints
proliferation (mitosis)
proliferation (mitosis)
epigenetic re-programming
Life cycle of germ cells
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. 13:781-
794 (2012) Lesch & PageGerm cell epigenetics: • De novo DNA methylation: a germ cell perspective. Trends in
Genet 28: 33-42 (2012) Smallwood & Kelsey
