Sex determination, Germ cells and Fertilization

Sex determination, Germ cells and Fertilization
Determination of the sexual phenotype
(Principles of Development, Wolpert, 2002)
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  ♀
XX ♀ + testosterone  ♂ development
prospective testes removed during embryonic stage  ♀
depending on number of X chromosomes
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
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,
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
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
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
-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
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,β
tubulin 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
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
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
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
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
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
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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