Sex determination, Germ cells and Fertilization

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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: ♀
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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
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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
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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
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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,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 &
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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 
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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.
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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 = ?
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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
hyaluronic acid on cumulus cells
overcome
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
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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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