Fertilisation Flashcards
Methods of achieving fertility differ between organisms…
- nutrient acquisition (materials of the egg must be used to drive the cell cycles)
- differences in number and size of eggs
- necessitates sex trade-offs within the breeding system
- different parental response and behaviours
Describe Futus rhizoid fertility
attach their offspring to rocks to photosynthesise.
Describe Drosophila fertility
larvae
Describe frog fertility
- tadpoles
- rely on metamorphosis, and differing metabolism and different life stages
Describe chicken fertility
use an albuminous egg
Describe mouse fertility
- such as Mus musculus
- placental fertility system
Describe sea urchin fertilisation
- external
- easier to visualise
- fourteen peptides released by the egg, creating a chemotactic gradient up which the small, flagellate and numerous sperm can navigate in its ambient marine environment
- egg releases carbohydrate signals to initiate the calcium ion mediated exocytosis of the sperm’s acrosomal vesicle
- sperm releases necessary enzymes for extracellular envelope penetration
- stimulates the actin-dependent formation of the acrosome for delivery of the male nucleus
- sperm and egg membrane fusion creates the fertilisation membrane
extracellular envelope
also termed the zona pellucida
How is species-specific recognition achieved in sea urchin fertilisation?
binding proteins on the acrosomal process
Describe fusion in mammals
- sperm binds to the zona pellucida, so that its acrosome is anchored between cumulus cells of the cumulus cell layer
- Izumo1 protein must bind to the egg receptor Juno
- allows the acrosome to react and mature, and penetrate through the zona pellucida to the perivitelline space
- Izumo-RFP protein localises at the acrosomal membrane
- attachment of the opened acrosome to a cortical granule then allows fusion of the plasma membranes, and the deposition of the sperm nucleus and contents into the egg cytoplasm
polyspermy
the fusion of multiple sperms
‘block’ to polyspermy has two mechanisms
the fast and the slow.
Describe the fast block to polyspermy
- achieved via a fertilisation-mediated membrane depolarisation event
- small local calcium influx through calcium channels in the internal plasma membrane initiates a calcium wave
- changes potential from -70 to +20mV
- membrane gradually repolarises.
Describe one model for the fast block to polyspermy
- enzyme-activated phosphorylation of PI to PIP to the PIP2 substrate in the plasma membrane
- phospholipid-
- activates PKC
- hydrolyses IP3, releasing it into the cytoplasm
- phosphorylation of the InsP3 stimulates other signalling events
- allows calcium release from the ER at the IP3 receptor, which can go to PKC
PI
- phosphatidyl inositol
- 2 acyl chains, glycerol phosphate and sugar,
DAG
- diacylglycerol
- highly hydrophobic
- usually acts as a landing site for phosphoprylating protein kinases
PKC
protein kinase calcium
InsP3
small, charged, diffuse and hydrophilic
Describe the self-stimulation of calcium release in the fast block to polyspermy
- further calcium on the cytoplasmic side primes the next IP3 receptors
- stimulates further calcium release
- non-equilbrium feedforward loop
Describe ‘store operated calcium entry’
- replace calcium so that it is not lost in the ER
- cellular calcium influx at the STIMI, coupled to the plasma membrane at the ORA1 membrane receptor
- allows calcium to transfer to the SERCA protein calcium pump embedded in the ER membrane
How can the fast block to polyspermy be visualised?
- rapid confocal imaging
- Medaka fish eggs through luminescence after microinjection of aequorin
aequorin
- calcium ion photoprotein
- isolated from Aequoria victoria
Describe aequorin imagery of the fast block to polyspermy
- multiple calcium waves spreading through the egg
- triggered by a calcium ionophore
Describe the visualisation of PIP2 hydrolysis
- fluorescent tagging via microinjection of PIP2 with a chimera of GFP-PH of eggs of Lytechnius pictus
PH
pleckstrin homology domain of PLC
Describe the egg-receptor mediated polyspermy block model
sperm derived factor fertilin would activate an integrin egg receptor via a tyrosine kinase, thus activating PLC.
Describe the sperm oocyte-activating factor-mediated polyspermy block model
- IP3 is directly released by the sperm derived PLCz isoform, via the PIP2 already present in the cytoplasmic oocyte vesicles
- results in the activation of an src-tyrosene kinases that activate the egg PLC
- IP3 also releases calcium ions from the endoplasmic reticulum, via IP3R
Describe the slow block to polyspermy
- cortical granule fusion, mediated by the calcium wave
- CICR
- InsP3 enhances sensitivity of the InsP3-R, and cADPribose to calcium
- elevated cytosolic calcium imitates further calcium release from either ER channel, in a non-equilibrium feedforward loop
- cortical granule and vesicles can be exocytosed (gramnosecretion), resulting in calcium-protein and particle mediated-fusion
- fertilisation envelope layer can form
Describe calcium ‘puffs’ or ‘sparks’
calcium propagation via clusters of channels across the egg
CICR
- calcium-induced calcium-release
- regenerative process where local calcium release spreads into a calcium wave or tide
- activates a plethora of downstream targets
cADPribose
natural ligand of the Ryanodine-receptor
Describe the fertilisation envelope layer
- polymer of mucopolysaccharides, cross-linked vitelline proteins and hyalin
- hardens the zona pellucida, protecting the egg and preventing further sperm entry
- the slow black to polyspermy
How can the slow block to polyspermy be visualised?
using fluorescent reporters
Summarise the polyspermy block
- sperm fusion results in membrane depolarisation: the fast block to polyspermy
- tyrosine kinase activation, which activates PLC
- release of IP3 and DAG
- CICR
- activation of NAD+ kinase, producing NADPH, and exocytosis of the cortical granule
- formation of the hyaline layer: slow block to polyspermy
What moves an egg from quiescence?
- CICR acts in combination with DAG, which activates PKC - results in sodium and potassium ion exchange for an increase in pH
- stimulation of protein synthesis and DNA replication to activate the egg from quiescence and begin division
Describe the first division of the embryo
- male pro-nucleus must migrate to meet the female pro-nucleus
- four molecules of calcium can bind co-operatively to the four EF-hand domains of calmodulin
- CaM-kinase II activated by a transient calcium signal localised around the spindle
- autophosphoryaltion
- calcium can be released to recycle in its binding to calmodulin, leaving the CaM-kinase II calcium independent, but remaining active, at a 50-80% activity rate
- Cam-kinase II can then go on to phosphorylate many other molecules
Describe male pro-nuclear migration
- indirect process of flow-mediated migration via motor drag on the microtubules
- generation of cortical cytoplasmic flow sweeps the two nuceli together
- visualised under mathematical modelling
Describe the zygotic microtubules
- derived from the astromicrotubules
- extend from the centrosome, forming spindles at the site of sperm entry
- reconstitution of the mitosis apparatus
Describe calmodulin
4 domains separated by an alpha helix each
Describe co-operative binding of calmodulin
- induces major conformational change and preferential localisation at the mitotic apparatus during division
- rapid transition to full binding on a single binding event (sigmoidal binding response)
Describe the activation of CaM-kinase II
- binding of calcium ions to calmodulin into a complex
- exposure of its receptor protein for activation
- allows allosteric binding onto the inhibitory domain of CaM-kinase I
- winds catalytic domain inwards and blocked by an inhibitory molecule
Describe the autophosphorylation of CaM-kinase II
- produces ADP from ATP
- phosphate binds to the allosteric inhibitory domain and allows the catalytic domain to unwind, and inhibitory molecule to dissociate
- full activation
Describe the inactivation of CaM-kinase II
- protein phosophotase can dephosphorylate the calcium independent CaM-kinase II
- produces a molecule of inorganic phopahte
- compound becomes inactive once more
Describe the downstream effects of CaM-kinase II activation by calcium
Activation of this CaM-kinase II by calcium results in the inactivation of cytostatic factor, which thus stimulates centrosome duplication, and activates anaphase promoting factor.
