Sex Determination, Germ Cells and Fertilization

Sex determination, Germ cells and Fertilization

Determination of the sexual phenotype

(Principles of Development, Wolpert, 2002)

Mammals

Sex-determing gene on the Y-chromosome

Y- chromosome and maleness:

XXY (Klinefelter syndrome) : ♂, infertile, small testes

XO (Turner syndrome): ♀, w/o egg

XY (SRY lost)  ♀

XY (SRY crossing over)  ♀(Fig. 12.2)

XX (SRY transgenic mice)  ♂, w/o sperm  infertile

SRY (sex-determining of the Y chromosome) = sex- determining factor

SRY  testis development

Gonadal hormones and sex determination (Fig.12.3)

Testes  Muellerian - inhibiting substance ︳♀ development

Testes  testosterone  ♂ development

Testes  testosterone  receptor (--) throughout the body  ♀ development

XX ♀ + testosterone  ♂ development

prospective testes removed during embryonic stage  ♀

depending on number of X chromosomes

Drosophila:

XY: ♂

XXY: ♀

Sex-lethal gene  transformer double sex (Fig.12.6, Fig.12.7)

2 × numerator gene on X-chromosome  Pe promoter of sex-lethal gene

depending on number of X chromosomes (Fig. 12.9)

C. elegans:

X-inactivation: dosage compensation (Fig.12.7), Barr body in mammals,

XX active: early cleavage

XX inactivation: after uterus implantation  1) paternal X inactivation: extra-embryonic tissue  2) gastrulation: random X inactivation throughout life

XX reactivation: germ cell developing

XY, XX, XXY, XXXY: 1 X active

Xist gene: non-coding RNA, produced from inactive X

Xist gene, introduced into other chromosome  inactive,

Methylation of DNA  inactivation

The determination and development of germ cells

The germ line (From chapter 19 of Developmental Biology, 6th ed., Gilbert, 2000)

The precursors of germ line = primordial germ cells

The determination of germ cells

Germ plasm (cytoplasmic components)

1) Nematodes: (Fig. 12.14) P granules

roundworm (Parascaris aequorum) Fig. 19.1,

chromosome diminution in somatic cells, 2 intact chromosomes in germ line

C. elegans: P4 blastomere, P-granules: contain several transcriptional inhibitors, RNA binding proteins, homologoues of Drosophila Vasa and Nanos proteins.

2) Insects: (Drosophila): (Fig. 12.13, 12.15) pole plasm

pole cells, pole plasm (mRNA, mitochondria, fibrils, polar granules),

mRNA: from nuse cells

germ cell-less (gcl mRNA) Fig.19.2

oskar: localization of the protein or RNAs (e.g.nanos) necessary for germ cell formation

nanos: pole cell migration, preventing mitosis & transcription

vasa: RNA binding protein

mitochondrial ribosomal RNA (mtrRNA)

polar granule component (Pgc) :non-translatable RNA, migration of pole cells

3) Amphibians (Fig.19.4):

Germ plasm + yolk = islands, after the roatation, releasing from yolk  together migrating to the vegetal pole (Fig. 19.4~5) genes homologous to nanos (Xcat2) and vasa localized to vegetal region  inhibit transcription and translation  preventing it from differentiating into anything else.

Germ cell migration + proliferation:

1) Amphibians:

Pole plasm in vegetal pole  floor of the blastocoel  posterior region of the larval gut  dorsal side of the gut  dorsal mesentery (the tissue  mesodermal organs)  genital ridges

Fibronectin along the pathway: substrate for PGC migration

filopodium

2) Mammals:

-No obvious germ plasm,

-Day 7, embryonic mesoderm, posterior to the primitive streak, 8 PGCs

 yolk sac  allantois (尿囊臍帶) hindgut genital ridges (day11),

-migration mechanism: unknown,

Fibronectin along the pathway: substrate for PGC migration

Integrin receptor on PGC

Genital ridges  TGF-β-like protein: attracting mouse PGCs

Oct4(Fig. 19.7): expression in early-cleavage blastomere nuclei in inner cell mass  gastrulation,  posterior epiblast cells  primordial germ cells

-extending filopodia  penetrating cell monolayers  migrating

day 12, 2500-5000PGCs in the gonads

- Cell migration pathway  stem cell factor (White)  binding to stem cell membrane (receptor: Steel) proliferation

3) Birds (Fig.19.10): Germinal crescent (in hypoblast at the anterior border of the area pellucida)

4) Drosophila(Fig.19.12):

Sex determination: cell signals and genetic constitution

Mouse: migrating female and male germ cells are indistinguishable, sex is determined after residing in gonad (Fig. 12.10)

XX germ cells  ovary  eggs

XY germ cells  testis  sperms

XY germ cells  ovary  no reproductive eggs

XX germ cells  testis  no reproductive sperm

Imprinting: (Fig. 12.19, Fig. 12.20)

CG island, C - methylation

Androgenic, gynogenetic

Demethylation: during early germ cell development?

Methylation: during germ cell differentiation

Fertilization: (Fig. 12.22)

Structure of sperm: head (acrosome + nucleus), neck (mitochondria), tail (axoneme)

Haploid nucleus: streamlined, DNA tightly compressed by H1t, which will be replaced by protamine during sperm maturation

Acrosomal vesicle: a sac of enzymes, derived from Glogi apparatus,

Globular actin: (sea urchin) between nucleus and the acromosmal vesicle

Flagellum: axoneme, microtubule doublet (A,B), protofilaments (α,βtubulin subunites) (Fig. 4.3)

Dynein protein: a ATPase, dynein-deficient  sterile

Histone 1 (H1): stabilizes the flagella and microtubules

Differentiation of of sperms: testis seminferous tubules lumen  store in epididymis, acquire mobility  ejaculation: able to move

Structure of the Egg

Oocyte: developing egg, before it is haploid

Ovum: mature egg

Remarkable cytoplasm: proteins (for energy and amino acids), ribosomes & tRNA, messenger RNA, morphogenetic factors, protective chemicals (UV filters, DNA repair enzymes, distasteful for predators, antibodies)

cortex: contains cortical granules and actin,

cortical granules contains digestive enzymes, mucopolysaccharides hyalin

digestive enzymes, mucopolysaccharides  prevent polyspermy

hyalin + adhesive glycoproteins (surround early embryo)  support for cleavage-stage blastomeres

plasma membrane

vitelline envelope ( ≒zona pellucida of mammals)

jelly layer ( ~ cumulus cells = ovarian follicular cells, corona radiata = innermost cells of cumulus or the follicular cells immediately adjacent to the zona pellucida)

Recognition of egg and sperm: Action at a distance,

Sea urchin:

Sperm attraction: (chemotaxis)

egg jelly  chemotactic factor (resact)  sperm chemotaxis,

Resact: species-specific, timing of release (sperm receptor) immediately after 2nd meiotic division

Sperm Activation:

1) activation of flagellum by resact: Resact  sperm transmembrane receptor  conformation change  guanylate cyclase activity of inner receptor  cAMP ↑ dynein (ATPase)  tail beating↑

2) Acrosome reaction by egg jelly: relative species-specific,  jelly  fusion of acrosomal membrane and sperm cell membrane i) release of contents (enzyme) of the acrosomal vesicle (exocytosis) (Fig.4.9)  ii) globular actin  actin microfilaments,  iii) bindin

Mammals:

Human: 200 reach ampullary region / 280 x 106 ejaculated sperms

Translocation by the muscular activity of the uterus. (借力使力)

Sperm motility is minor to transportation. Within 30 min, they reach oviduct.

Capacitation: (適化，馴化，臨陣磨鎗！？) new ejaculated sperm (fertilization X)

Sperm residing in reproductive tract (or media) fertilization OK

Media: Ca ions, bicarbonate, serum albumin.

Molecular changes:

i) lipid composition: cholestrol removed by albumin and lipid transfer protein

ii) particular proteins or carbohydrates lost

iii) cAMP-dependent pathway activated: adenylyl cyclase  cAMP PKA (cAMP dependent protein kinase)  tyrosine kinase •P  activation of proteins for zona pellucida binding and exocytosis of the acrosomal vesicle

iv) membrane potential ↓(-30mV-50 mV)

Hyperactivation: swimming ↑(上點油？)

Different regions of female reproductive tract  different specific molecules for sperm motility

Chemotaxis: ovum, ovarian follicles  chemotactic substances

The race is not always to the swiftest.

Recognition of egg and sperm: Contact of gametes

Species-specific Recognition in Sea Urchins

Bindin (species-specific) on sperm after acrosomal reaction  species-specific receptor on vitelline envelope of egg

Garmete binding and recognition in Mammals:

Primary binding:

Mammalian sperm vs zona ≒ sperm of sea urchins vs vitelline membrane

Zona protein:

ZP3: secreted by oocyte: i) for sperm binding, 2) initiating acrosome reaction (Fig.4.16)

Sperm-zona adhesion proteins (on sperms): (Fig.4.16)

Galactose-binding protein (56K or SP56)  • galactosidase of ZP3  open Ca++ chanels on sperm membrane

Galatosyltransferase  • N-acetylglucosamine on zona,  G protein activation  acrosomal reaction

Zona receptor kinase (95K, ZRK)  • ? on zona = RTK (receptor tyrosine kinase)  acrosomal reaction

Secondary binding: lysis of zona

Acrosome-reacted sperm binds to ZP2 (for ZP1, 2, 3 see Fig. 4.18)

Egg cortical granules release contents  protease  alters ZP2 ] further secondary binding

Sperm-ZP2 binding protein = ?

Proacrosin  acrosin (protease)  digesting zona

Gamete fusion

Sea urchins: (Fig. 4.19), microvilli, fertilization cone, actin polymerization  microfilaments: necessary fo cell division, formation of microvilli, membrane fusion., fusogenic protein, bindin, lysin  dissolve vitelline envelope

Mammalian sperm, fertilin  • α6β1 integrin on egg plasma  union of two membranes  sperm nucleus, mitochondria, centriole, and flagellum can enter the egg

Prevention of polyspermy

Aberrant development of dispermic sea urchin egg (Fig.4.21)

Membrane potential

♀ ♂

barrier overcome

hyaluronic acid on cumulus cells hyaluronidase on sperms

zona pellucida (glycoproteins): acrosomal reaction:

ZP3 receptor (species specific) galatosyltransferase (binding)

-N-acetylglucosamidase (break )

acrosin (protease)

cytoplasm fusion

integrin-like receptor fertilin

releasing cortical granules (Fig. 12.24) prevention of polyspermy

calcium wave calmodulin dependent protein kinase II  MPF (maturation - promoting factor, cyclin)  cyclin degradation  meiosis  pronuclei fusion  mitosis

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