gametogenesis, fertilisation & preimplantation development Flashcards
testis cell types and functions
leydig: testosterone production
sertoli: production of activin, inhibin, AMH and androgen binding proteins, regulate internal environment of seminiferous tubules
izumo1
binds to folate receptor 4 (Juno) essential for sperm egg fusion
path of PGC migration
from dorsal body wall to genital ridges
regulation of PGC migration
chemotaxis: stem cell factor binds to and activates C-kit (PGCs have C-kit receptor) failure of migration -> teratomas
how does the SRY cause sexual differentiation and testes cell types
SRY (on long arm of Y chromosome) -> SOX9 ->
- FGF9 -> differentiation of Sertoli from coloemic epithelium (threshold of Sertoli cells needed for male differentiation; proliferation determines size of male gonad)
- steroidogenic factor 1 -> Leydig
- PDGF -> interstitial cells (occurs later than other 2)
development of male ductal system
Wolffian ducts -> male ductal system
AMH -> degeneration of Mullerian ducts by apoptosis
testosterone/DHT important for formation of epididymis, vas deferens, seminal vesicles
formation of seminiferous cords
Sertoli cells cluster and surround germ cells
perimyoid cells surround these cords
differentiation of perimyoid cells
Dhh (desert hedgehog) produced by Sertoli cells binds to PTCH1 (patched 1)
DAX-1 receptor also important
difference between oogonia and oocytes
PGCs are oogonia once they have migrated into the genital ridge
oogonia differentiate into oocytes which can no longer proliferate
formation of primordial follicles
pregranulosa cells (origin unknown) surround oocytes
inner oocytes (medulla) form follicles first
role of Sertoli cells
production of AMH (until puberty), inhibin, oestrogen and androgen binding proteins
regulates internal environment of seminiferous tubule
FSH -> proliferation
mouse study demonstrating potential therapy for male infertility
cells from testes of other mouse that express Oct-4 (stem cell potential) injected into infertile mouse restored spermatogenesis
spermatogenesis (5)
2017 SAQ
- Primordial germ cells
- Spermatogoonia: undergo mitosis
- Type A: undifferentiated, sits on basement membrane
- Type B differentiate to…
- Spermatocyte: undergo meiosis (can no longer undergo mitosis)
- Primary (meiosis I)
- Secondary (meiosis II)
- Spermatids (round)
- Mature spermatozoa (elongated) develops flagellum, forms acrosome, released from Sertoli cell
functions of epididymis (4)
- concentration of sperm
- functional maturation
- storage
- removal of decapacitated sperm
segments of epididymis and functions (4)
- initial segment: water reabsorption via sodium channels
- caput: synthesis of compounds in epididymal fluid
- corpus
- cauda: synthesis of compounds in epididymal fluid
function of epididymal proteins
mask membrane proteins on sperm, released when exposed to female reproductive tract environment, allowing binding to zona pellucida of oocyte
loss of cytoplasmic droplet in sperm function? when does it occur?
during transit from caput to corpus
ejaculate w/o cytoplasic droplets leads to decreased fertility and proportion of spermatozoa
how do sperm overcome acidic pH (<5) of vagina
seminal fluid pH is ~7 and coagulates
effects of oestrogen on cervical mucus
oestrogen (during ovulation) -> increased hydration of cervical mucus -> easier for sperm to penetrate
role of progesterone in fertilisation
progesterone (released by cumulus cells of oocyte) → ↑calcium permeability of membrane → calcium influx into spermatozoa →
- capacitation of spermatozoa
- acrosome reaction: persistent Ca entry via TRPC2 channel
- oocyte activation: calcium oscillations caused by a sperm-specific PLCζ released into oocyte following gamete fusion
characteristics of capacitated sperm
- hyperactivated motility
- change in surface properties
- ability to undergo acrosome reaction
stages of follicular development (9)
- activation of follicle growth (regulation unknown)
- primordial follicle: single layer of flattened pre-granulosa cells surrounding
- transitional follicle: granulosa cells at one pole become cuboidal
- primary follicle: one layer of cuboidal granulosa cells
- secondary follicle: two layers + theca cells surrounding + zona pellucida
- pre-antral follicle: multilayered, oocyte growth
- antral follicle: antrum (fluid-filled cavity prevents necrosis of centre of follicle)
- graafian follicle: matured and ready for ovulation
- corpus luteum: structure left after ovulation
theca and granulosa cell hormone secretion
LH -> theca cells -> produce androgens
FSH -> granulosa cells convert androgens to oestrogens
regulation of folliculogenesis (4)
hormones not necessary for early follicle development
FIGL-alpha: essential for formation of follicles
GDF-9: essential for granulosa cell proliferation (multilayering), theca formation
connexin-43: gap junction protein, communication between granulosa cells, KO -> arrest at primary stage
cx-37: communication between oocyte and granulosa cells, KO -> loss of antral follicle formation
selection of dominant follicle
dominant follicle most sensitive to FSH, produces most E2 which inhibits FSH production
LH receptors so that can respond to LH surge
what effects does the LH surge have (6)
- decreased cAMP -> resumption of meiosis
- cumulus expansion: hyaluranon produces attracts water -> gel-like complex more easily picked up by fimbriae of Fallopian tube
- ovulation
- luteinsation: corpus luteum formation, switch from granulosa cell oestrogen production to progesterone
- withdrawal of granulosa cell transzonal processes from oocyte
- decreased gap junction communication
luteolysis
- functional: decreased progesterone production -> development of new follicles
- structural: removal of corpus luteum
- corpus luteum -> corpus albicans: scar of collagen, eventually resorbed
factors required for entry into meiosis (3)
prev SAQ
Intrinsic factor DAZL: PGC → meiosis competent germ cell
Extrinsic factor retinoic acid produced by mesonephros
Females: RA important for expression of Stra8, which is required for premeiotic DNA replication → oogonium
Males: Cyp26b1 in testis degrades retinoic acid, preventing Stra8 expression → spermatogonium
functions of meiosis
- maintain chromosome numbers by producing haploid gamete
- increased genetic variation by: random assortment of parental chromosomes and recombination of genetic material
stages of meiosis until LH surge
inner oocytes (medulla) enter meiosis first
DNA synthesis -> 2 chromosomes connected by cohesins
- leptotene: chromosomes condense
- zygotene: distance pairing (homologues lie side by side)
- pachytene: paired homologues synapse and form bivalent, recombination w formation of chiasmata
- diplotene: synaptonemal complexes removed -> homologues held together by chiasmata only
meiotic arrest until LH surge
c-Kit receptor
- ligand, produced where?
- location
- function
- KO -> ?
- Kit-L produced by granulosa cells
- binds to c-Kit receptor on oocyte
- triggers oocyte growth and theca cell recruitment
- KO -> loss of fertility (mice)
functions of gap junctions between granulosa cells and oocyte
avascular environment within basal lamina
- allows passage of small molecules e.g. cAMP
- coordination of luteinzation, atresia, etc.
- transport of growth factors, metabolic precursors e.g. amino acids, nucleotides
interactions between oocytes and granulosa cells
oocytes -> GDF9 -> granulosa cell proliferation
granulosa cells -> Kit-L -> oocyte growth
how is diplotene meiotic arrest maintained (cellularly)
cGMP from granulosa cells -> oocyte via gap junction
cGMP inhibits PDE31 (phosphodiesterase) which usually degrades cAMP -> maintenace of meiotic arrest by cAMP
function of anaphase promoting complex (APC)
APC degrades securin, releasing separase, which cleaves Rec8 (cohesin between long arms of chromosomes) -> separation of homologues -> cell cycle progress
cyclin B/Cdk1 complex (MPF: maturation-promoting factor) degrades APC → meiotic arrest
cytostatic factors e.g. mos and emi2 help to inhibit APC
stages of meiosis after LH surge
resumption of meiosis following LH surge
metaphase I: homologues align at metaphase plate and attach to spindle
anaphase I: separation of homologues between poles
telophase I: asymmetrical cell division -> extrusion of 1st polar body arrest at metaphase II
uncapacitated sperm store
at isthmus of fallopian tube
only few sperm become capacitated and migrate towards oocyte (~100)
zona pellucida glycoproteins in humans and mice + functions
ZP1, 2, 3 in mice / ZP1 - 4 in humans
ZP3: primary sperm receptor, binds to head of sperm and interacts w TRPC2 channels -> calcium influx, induces acrosome reaction, provide species specificity
ZP2: secondary sperm receptor (binds acrosome reacted sperm)
ZP1, 2, 4 involved in induction of acrosome reaction
what happens after sperm bind to zona pellucida
acrosome reaction: irreversible exocytosis -> hydrolytic enzymes break down zona pellucida
acrosomal process grows and penetrates further through coating and egg plasma membrane
egg and sperm membranes fuse
cortical granule exocytosis
zona reaction: enzymes from granules
A protease cleaves ZP2
B hexosaminidase cleaves ZP3 -> removes sperm binding properties
cross-linking of tyrosine kinase residues on ZPs -> zona hardening
how does calcium oocyte activation occur
sperm binds to oocyte and releases PLC-zeta which generates InsP3 which binds to receptors on ER -> calcium efflux (oscillations)
resumption of meiosis II following fertilisation
anaphase II: chromatids separate
telophase II: one set of chromatids expelled in polar body
protective functions of zona pellucida during preimplantation development
- prevents sticking of embryo to oviduct walls and each other
- protects attack against leukocytes (embryos w/o zona disintegrate in oviducts)
- maintains normal cleavage pattern
processes during first cleavage
- pronucleus formation and migration together (but not fusion), duplication of DNA
- mitosis metaphase: pronuclear membranes break down, alignment of chromosomes on metaphase plate
- mitosis anaphase: separate of chromatids, formation of cleavage furrow
important of E-cadherin in preimplantation development
calcium dependent compaction
at 16-cell stage in humans
models for differentation of cell types in blastocyst (2)
- inside-outside model: outer cells -> trophoectoderm / inner cells -> inner cell mass
- polarisation model: conservative division -> 2 polar cells / differentiation division (when less space) -> 1 polar 1 apolar
polar cells -> trophoectoderm / apolar cells -> inner call mass
functions of trophoectoderm (3)
- pumps fluid into cavity
- transport of metabolites between maternal tissues and ICM
- proliferative source of cells for placental TE
when does activation of the embryonic genome occur
between 4 and 8 cell stage
when does most embryo growth arrest occur
between 4 and 8 cell stages (activation of embryonic genome)
leading cause of miscarriage (%)
aneuploidy (35%)
differences in timing of meiosis in males vs females
F: entry into meiosis during fetal development, meiotic arrest during childhood, LH surge triggers resumption
M: entry into meiosis after puberty
aneuploidy: true non-disjunction
no loss of cohesins -> both homologues go to one pole (monosomy/trisomy)
‘achiasmate’ non-disjunction
no formation of chiasmata between homologues -> separate and connected to different microtubules -> chance to both can go to same pole (trisomy/monosomy)
small chromosomes (e.g. 21 -> Down’s syndrome) more likely to be achiasmate
aneuploidy in meiosis I (3)
- true non-disjunction
- ‘achiasmate’ non-disjunction
- premature separation of sister chromatids
mitotic non-disjunction
both chromatids go towards one pole (failure of loss of cohesins from centromere)
earlier in cleavage -> more cells affected
aneuploidy in meiosis II
meiotic non-disjunction: failure of loss of cohesins from centromere -> both chromatids to one pole
post-zygotic non-disjunction
aneuploidy that occurs during cleavage stages -> mosaicism (mixtures of diploid, monosomic and trisomic cells)
fate of abnormal cells unknown: ?apoptosis, ?slower cleavage so that normal cells predominate
anaphase lag
chromosome falls off spindle
free-floating in cytoplasm -> formation of micronucleus
examples of different origins (paternal/maternal, meiosis I/II etc.) of trisomys
- trisomy 16 100% maternal, meiosis I
- XXY mostly sperm, meiosis II
causes of increased aneuploidy w age
- relaxed selection hypothesis: uterus less able to recognise abnormal pregnancy (now thought to not be the case)
- ‘two hit’ hypothesis: reduced recombination -> predisposition to non-disjunction, which increases w age due to oxidative stress, decreased microcirculation around dominant follicle, defective granulosa cells
- limited pool hypothesis: antral follicle no declines w age
- local factors hypothesis: degeneration of cohesins -> decreased cohesion between chromatids, increased FSH, prolonged exposure to environmental toxins
key meiotic processes which predispose to aneuploidy when disrupted
- recombination
- cohesion between chromatids (cohesin)
- spindle
what is CatSper? function?
sperm specific Ca channel in flagellum
Controls sperm chemotaxis and guides it to oocyte
