Fertilisation Flashcards
1
Q
Methods of achieving fertility differ between organisms…
A
- 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
2
Q
Describe Futus rhizoid fertility
A
attach their offspring to rocks to photosynthesise.
3
Q
Describe Drosophila fertility
A
larvae
4
Q
Describe frog fertility
A
- tadpoles
- rely on metamorphosis, and differing metabolism and different life stages
5
Q
Describe chicken fertility
A
use an albuminous egg
6
Q
Describe mouse fertility
A
- such as Mus musculus
- placental fertility system
7
Q
Describe sea urchin fertilisation
A
- 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
8
Q
extracellular envelope
A
also termed the zona pellucida
9
Q
How is species-specific recognition achieved in sea urchin fertilisation?
A
binding proteins on the acrosomal process
10
Q
Describe fusion in mammals
A
- 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
11
Q
polyspermy
A
the fusion of multiple sperms
12
Q
‘block’ to polyspermy has two mechanisms
A
the fast and the slow.
13
Q
Describe the fast block to polyspermy
A
- 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.
14
Q
Describe one model for the fast block to polyspermy
A
- 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
15
Q
PI
A
- phosphatidyl inositol
- 2 acyl chains, glycerol phosphate and sugar,
16
Q
DAG
A
- diacylglycerol
- highly hydrophobic
- usually acts as a landing site for phosphoprylating protein kinases
17
Q
PKC
A
protein kinase calcium
18
Q
InsP3
A
small, charged, diffuse and hydrophilic
19
Q
Describe the self-stimulation of calcium release in the fast block to polyspermy
A
- further calcium on the cytoplasmic side primes the next IP3 receptors
- stimulates further calcium release
- non-equilbrium feedforward loop
20
Q
Describe ‘store operated calcium entry’
A
- 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
21
Q
How can the fast block to polyspermy be visualised?
A
- rapid confocal imaging
- Medaka fish eggs through luminescence after microinjection of aequorin
22
Q
aequorin
A
- calcium ion photoprotein
- isolated from Aequoria victoria
23
Q
Describe aequorin imagery of the fast block to polyspermy
A
- multiple calcium waves spreading through the egg
- triggered by a calcium ionophore
24
Q
Describe the visualisation of PIP2 hydrolysis
A
- fluorescent tagging via microinjection of PIP2 with a chimera of GFP-PH of eggs of Lytechnius pictus
25
PH
pleckstrin homology domain of PLC
26
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.
27
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
28
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
29
Describe calcium 'puffs' or 'sparks'
calcium propagation via clusters of channels across the egg
30
CICR
- calcium-induced calcium-release
- regenerative process where local calcium release spreads into a calcium wave or tide
- activates a plethora of downstream targets
31
cADPribose
natural ligand of the Ryanodine-receptor
32
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
33
How can the slow block to polyspermy be visualised?
using fluorescent reporters
34
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
35
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
36
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
37
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
38
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
39
Describe calmodulin
4 domains separated by an alpha helix each
40
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)
41
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
42
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
43
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
44
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.
