BIOL30001 Reproductive Physiology Germ Cells - Danielle Hickford BIOL30001 Reproductive Physiology Germ cells Is this still the correct course code?? Danielle Hickford hickford@unimelb.edu.au

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”

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

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)

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 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

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- 16-25 vasa +ve cells. 8h-75% epiboly 16 hr- 14 somites = vasa-positive germ plasm Modified from IlluScientia

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)

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

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

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

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). 3. migration into dorsal mesentary 4. migration to genital ridges Richardson and Lehman review Essential Reproduction, Johnson, 2013

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

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) –

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

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

Germ cell sexual differentiation BIOL30001 Reproductive Physiology Germ cell sexual differentiation Germ Cells - Danielle Hickford 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 Condense further Premature entry into meiosis - not enough germ cells unique to germ cells exchange of genetic material (via recombination) creates haploid gametes

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

Control of entry into meiosis Cyp26b1 RA Stra8 meiosis VitA RA Stra8 meiosis VitA Mesonephros Ovary Testis 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 Histone modifications DNA 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 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)

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)

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 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

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)

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. 13:781-794 (2012) Lesch & Page Germ cell epigenetics: De novo DNA methylation: a germ cell perspective. Trends in Genet 28: 33-42 (2012) Smallwood & Kelsey