1-Germ cell development, gametogenesis and fertilisation

Developmental biology - understanding the mechanisms that control embryo development 

• Understanding which genes are turned on when which are turned off & how its regulated 

Differentiation 

Gene expression - genes that are actively transcribing mRNA 

Genetic equivalence - genetic material in almost all cells is the same  - exception is immune cells that have more variation in their DNA for generating antibodies 

Evidence = Sir John Burden 1960s - nucleus of a frog's oocyte replaced with nucleus from gut cell - tadpoles can still be produced 

• Different distinct cell phenotypes occur because only a proportion of the genes are expressed (eg. neurons, pancreatic) 

Somatic cells: 

• Cells of the body 

• Limited life  

Germ line cells:

• Cells for reproduction 

• Immortal 

• Female - oocytes 

• Male - sperm

GERM LINE

• Oocyte and sperm cells generated from primordial germ cells 

Oogenesis 

• Meiosis 

• Haploid DNA content 

• Large gamete 

Spermatogenesis 

• Meiosis 

• Small motile gamete 

• Haploid DNA 

Mammalian development 

• Most development knowledge comes from studying mice - easy to manipulate 

Monotremes - lay eggs 

Marsupials - shorter gestation, immature young

Placentals - placentas, more mature when born than marsupials 

Mouse embryo development: 

• Human embryos don't undergo the elongation - they remain as a flat disc 

Why don't all the epiblast cells become primordial germ cells?

• Cell signalling 

• Bone morphogenetic protein 4 (BMP4) - signalling molecules secreted from extraembryonic tissue 

• BMP4 acts on cells in the epiblast that its most closely in contact with - it triggers the cells to become PGCs

• High levels of BMP4 are needed for th

BMP4 receptors:

• Transmembrane proteins 

• Extracellular part is where the BPM4 binds 

• Binding causes phosphorylation in cytoplasmic part - leads to cascade - new genes in the nucleus are turned on 

How do we know this? 

Cells from the top of the epiblast were transferred and transplanted to the region closest to the extraembryonic tissues

These cells are exposed to high enough levels of BMP4 to become PGCs 

• Shows that fate of the epiblast cells depends on their position in the embryo

• Differentiation of PGCs is controlled by the environment 

Migration of PGCs: 

Mechanism of their migration not fully understood 

• They’re generated in the epiblast on day 6-7

• Day 8 - migration into the gut 

• Day 11 - enter the genital ridges (tissue that gives rise to ovaries and testes) 

6PGCs at the start - around 5000 when they enter the genital ridges 

Differentiation into sperm or oocytes?

• PGCs in female genital ridge are committed to an oocyte fate and vice versa 

• XY PGCs in a foetal ovary will develop as oocytes 

• XX PGCs in foetal testis will develop as sperm 

Anne McLaren 

• Worked on PGC differentiation 

• - suggested that retinoic acid is important (metabolite of vitamin A) 

• IVF - showed the possibility to fertilise mouse oocytes in vitro to generate viable embryos

Retinoic acid signalling: 

Retinoic acid: 

• Metabolite of vitamin A 

• Lipophilic - easily passes over the cell membrane 

• Important in stages of embryonic development by regulating gene expression 

• Potent 

• Present in female and male foetal gonads 

• Needed for PGCs to become oocytes 

• Male gonad cells express enzyme cytochrome P450 which degrades RA - inhibits oocyte differentiation 

• If XY PGCs are removed from the male gonad and placed in female gonad:

RA levels in environment increase, RA induces PGCs into oocytes 

• If XX PGCs are placed in male gonad - presence of cytochrome P450 

RA degraded - PGCs differentiate into sperm 

→ sperm                                     

→ oocyte 

• Absence of retinoic acid - staining shows retinoic acid binding proteins on the outer area of the cell 

• Presence of retinoic acid - rapid translocation of the protein into the nucleus 

SPERMATOGENESIS 

• Production of mature sperm cells from PGCs 

• Continuous and prolific 

• Occurs in seminiferous tubules of the testes

1. In male gonad, PGCs give rise to stem cells called spermatogonial sperm cells  (SSCs) 

2. SSCs generate more SSCs and differentiate to form primary spermatocytes 

3. Primary spermatocytes undergo meiosis to become secondary spermatocytes 

4. Secondary spermatocytes give rise to spermatids 

• SSC proliferation and differentiation needs to be tightly controlled 

• Too MUCH proliferation could lead to formation of a tumour 

• Too LITTLE proliferation and too MUCH differentiation could deplete the SSC population - leading to male infertility 

Function of sperm components: 

Acrosome - derived from golgi, contains enzymes that digest proteins and sugars; these enzymes are required to lyse the outer coverings of egg

Nucleus - contains haploid number of chromosomes 

Midpiece - contains mitochondria that produce the energy (ATP) required for motility 

Flagellum - required for propulsion, motor portion of the flagellum is the axoneme 

OOGENESIS 

• Development of mature oocytes from PGCs 

• Occurs in ovaries

• Begins in the embryo with the differentiation with the PGCs - to stem cells called oogonia 

• Oogonia multiply by mitosis and begin meiosis - stops at prophase 1 - primary oocytes 

• Primary oocytes remain arrested in prophase 1 until puberty 

• Each month follicle stimulating hormones trigger division of some of the primary oocytes 

• First mitotic division produces uneven sized cells - one is secondary oocyte, one is polar body 

• Secondary oocyte begins second mitotic division but is arrested in metaphase 2 

• If fertilised by sperm - complete second division - gives rise to 2nd polar body 

• Once meiosis 2 is complete - nucleus of ovum fuses with sperm nucleus - forms a zygote  

Structure of oocyte:

Nucleus - nucleus of mature oocyte is arrested in 2nd metaphase 

Zona pellucida - thich extracellular matrix that binds sperm 

Cumulus - layer of ovarian follicular cells surrounding oocyte, layer adjacent to zona called corona radiata 

Cytoplasm - contains proteins, ribosomes, tRNA, mRNA 

FERTILISATION 

1. Attraction and activation of sperm by contents of female reproductive tract 

• Different regions of tract secrete molecules that attract and affect sperm motility 

• In some mammals, sperm becomes hyperactivated in the oviduct 

• Ovarian follicle may secrete chemo-attractants that attract sperm towards oocyte 

2. Binding of sperm to zona pellucida 

• Binding is species specific eg. mouse sperm only bind to oocyte that contain ZP3 glycoprotein on their surface 

3. Release of enzyme from acrosome to lyse hole in zona 

• Eg. in mice - acrosomal reaction triggered by cross linking of proteins on sperm surface to ZP3 

• Enzymes released to make hole in zona so sperm can reach plasma membrane of the ovum 

• Passage of sperm through zona 

• Plasma membranes of sperm and ovum fuse 

• Sperm nucleus enters

4. Fusion of sperm and oocyte pronuclei 

• Sperm entry - female pronucleus stimulated to complete its 2nd meiotic division 

• Chromatin of male pronucleus uncoils

• Each pronucleus migrates towards the other 

• Two nuclear envelopes break down 

• Chromosomes orientate themselves on the mitotic spindle - creating a zygote 

→ female and male pronuclei are not equivalent 

• Some genes are imprinted and only expressed from either maternal or paternal chromosome 

• Hydatiform mole - only has male chromosomes - mass of placenta like cells - embryo doesn't develop - can give rise to tumours 

• Parthenogenetic embryos - only has female chromosomes - sometimes has organs - chaotic development and embryo becomes grossly disorganised 

Prevention of polyspermy: 

• After fusion - cortical reaction occurs

• Egg releases enzymes that harden zona pellucida so no more sperm can penetrate

• Mice - enzymes modify ZP3 so oocyte can't bind to sperm