biol30001 reproductive physiology germ cells danielle hickford hickford@unimelb.edu.au

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BIOL30001Reproductive Physiology

Germ cells

Danielle Hickford

hickford@unimelb.edu.au

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

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