10. Fertilisation and early development Flashcards
What are the mechanisms for sperm selection?
From the ejaculate only ~100 sperms reach Fallopian tube - mechanisms for selection of the fittest:
- acidic pH + leukocytes in vagina
- cervical mucus eliminates sperm with poor motility
- basic pH in Fallopian tubes
- cumulus mass around the egg - requires penetration, HA interaction, chemotaxis
What are the parts of oviduct and what are their roles?
Oviduct parts:
- Utero-tubal junction (UTJ) - sperm selection - number reduction (different mechanisms between species)
- Isthmus (Is) - stores sperm while preserving its vaibility
-** Ampulla (Am)** - hold oocyte, site of fertilisation
Explain sperm selection in UTJ
Sperm selection in UTJ - in mice:
- ADAM3 protein must be recognised on sperm by receptors - allowed to pass - possibly interacts with uterine lining
Ensures selection for:
- liveliness
- motility
- normal morphology
- uncapacitated
- intact acrosome
What are the stages fo fertilisation process?
Fertilisation stages:
1) sperm prepapration: capacitation + acrosome reaction
2) sperm binding and fusion: IZUMO-JUNO binding, microvilli + pinocytosis fusion
3) cortical reaction
Explain what is the sperm structure
Explain the first step of sperm preparation in fertilisation
Sperm preparation step 1 = capacitation - testing sperm’s capacity:
- takes place in female reproductive tract
- physiological changes: plasma membrane re-organisation - loss of cholesterol, phospholipids, surface glycoproteins
- molecular changes: increase in ROS generation, Ca2+ influx, tyrosine phosphorylation
-> after capacitation sperm becomes less stable but higher motility + ability to respond to chemoattractants (ex: progesterone) => sperm hyperactivation
How is sperm capacitated in IVF?
What are the stages of sperm capacitation?
Sperm capacitation stages:
1) Insemination
2) Initial capacitation in vagina + cervix
3) Formation of transient sperm reservoir in isthmic region
4) Hyperactivation
5) Cumulus mass penetration
6) Zona pelucida penetration
Explain sperm hyperactivation
Hyperactivation - sperm are held at transient reservoir in isthmic region - endocrine signalling (progesterone) triggers hyperactivation - sperm break away from oviduct epithelium reservoir - swim to penetrate cumulus complex
Explain the second step of sperm preparation in fertilisation
Sperm preparation step 2 = acrosome activation - fusion of sperm plasma membrane + outer acrosomal membrane (OAM) -> creates pores - enzymes released - new surface antigens of sperm head exposed - Izumo1 - for oocyte (oolema) binding
Only capacitated sperm can undergo acrosomal reaction
Compare mouse vs human zona pellucida
Zona pellucida (ZP) - outer layer of the egg
Mouse: more densely packed mesh
Human: less tightly packed - larger pores
What are the functions of ZP?
ZP functions:
- mediates species-specific interaction of oocyte + sperm - antigens must match
- prevents polyspermy
- protects preimplantation embryo from reabsorption when it is moving towards uterus (before hatching)
What proteins are involved in sperm binding to oocyte?
Sperm binds to oolemma of the oocyte:
- IZUMO1 protein (sperm) + JUNO receptor (oocyte)
- CD9 and CD81 proteins in oolemma - stabilise IZUMO1-JUNO interaction - essential in fertilisation - CD9 and CD81 KO are sterile
Explain what is oolemma?
Oocyte’s plasma membrane - beneath ZP
Is sperm-oolemma binding a single protein interaction?
No, recently even more interacting proteins were discovered beyond IZUMO1, JUNO, CD9 and CD81
Explain the sperm-oocyte fusion process
1) Oocyte contain microvilli on the surface - engulf the bound sperm at its equatorial segment
2) Sperm anterior is engulfed by pinocytosis - stops moving
3) Fertilisation cone formed - swelling - at fusion point
4) Sperm head passes into oocyte cytoplasm - two gametes have fused
Explain the cortical reaction
Cortical reaction function - prevent polyspermy - hardens ZP:
1) Sperm engulfed into cytoplasm sperm-oocyte fusion - no other sperm must enter ->
2) Cortical granules in oocyte fuse with its oolemma releasing enzymes into perivitelline space - proteases, peroxidases, polysaccharides and Zn => hardens ZP - no other sperm enters
What are all the post-fertilisation blocks which reduce the chance of polyspermy?
Blocks which reduce polypspermy post-fertilisation:
- Zinc shield - Zn released in cortical granules
- Membrane block - oolemma blocks fusion with another sperm by releasing JUNO vesicles - receptors bind to IZUMO1 deactivating them
- ZP blocks by changing configuration - hardening - change in ZP1, 2, 3 protein configuration
Explain how is the membrane block created
Membrane block - JUNO receptors released from oolemma in vesicles - allocated in perivitelline space - re-organisation of microvilli distribution (needed in sperm-oolemma binding) + 2-fold decrease in CD9 (essential for stabilising IZUMO1-JUNO interaction)
Explain how is the ZP block is created
ZP block - ovastacin cleaves ZP2 protein ->
- hardens ZP structure
- oligosaccharides removed from ZP3 (essential in species-specific recognition for sperm-oolemma fusion)
- adjacent ZP molecules crosslinked
Before fertilisation fetuin-B inhibits ovastacin function - maintains ZP permeability - premature ZP hardening - cause of infertility
What is a common cause of miscarriage?
Embryo polyploidy- lethal - vast majority of polyploidy formed at clevage stage due to mismanagement of centrosomes -> miscarriage
Some species tolerate polyploidy - ex: birds - after multiple sperm have fertilised the egg - can choose the fittest pronucleus and remove the rest
Explain how is oocyte activated for early embryogenesis?
Oocyte activated for early development by calcium influx:
Oocyte is activated by an activating factor from sperm - phospholipase C zeta 1 -> stimulates Ca2+ release from oocyte intracellular stores - refill - empty again -> cycles - these Ca pulse create intracellular Ca spikes (oscillations) - cycles stop when PN forms in oocyte
Explain what is dependent on intracellular calcium spikes
Intracellular calcium spikes in oocytes regulate:
- cortical reaction
- resumption of meiosis in oocytes
- 2nd polar body formation
- translation of maternally deposited mRNAs
Explain what are intracellular calcium spikes
Oocyte is activated by an activating factor from sperm - phospholipase C zeta 1 -> stimulates Ca2+ release from oocyte intracellular stores - refill - empty again -> cycles - these Ca pulse create intracellular Ca spikes (oscillations) - cycles stop when PN forms in oocyte
Explain the events which lead to zygote formation
How and why is sperm DNA differently packed compared to oocyte DNA?
Sperm DNA is more densely packed - additional proteins: transition protein 2 + protamines in packaging - to protect sperm DNA from ROS damage in female reproductive tract
Explain how pronucleus is formed
In mice: male and female pronuclei move towards each other - to the centre - in the oocyte - until overlap (actually not overlap but on top of each other in 3D)
Wht controls when first mitotic division occurs?
When does the embryo loose its totipotency and become pluripotent?
Totipotent -> pluripotent after compaction when goes from 8->16 cell stage before cavitation
What process occur in preimplantation development? What stage is reached before embryo implants?
Explain the process of embryo cavitation
Cavity forms - fluid-filled
When does maternal-zygotic transition occur?
Maternal - zygotic transition - when zygotic genome is activated and started to be transcribed
What are the mechanisms driving cavitation?
Cavitation - fluid accummulation:
- TE undergoes epithelialisation - tight junctions formed
- Na+/K+ pumps in TE establish ion gradient
- ion gradient drives water uptake into the embryo cavity through aquaporins in TE
What are the primary cell lineages formed in the embryo
Which blastocyst lineage contributes to what tissues in the embryo?
Lecture key points
