Fertilisation

Question 1

Methods of achieving fertility differ between organisms…

Answer 1

• 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

Question 2

Describe Futus rhizoid fertility

Answer 2

attach their offspring to rocks to photosynthesise.

Question 3

Describe Drosophila fertility

Answer 3

larvae

Question 4

Describe frog fertility

Answer 4

• tadpoles
• rely on metamorphosis, and differing metabolism and different life stages

Question 5

Describe chicken fertility

Answer 5

use an albuminous egg

Question 6

Describe mouse fertility

Answer 6

• such as Mus musculus
• placental fertility system

Question 7

Describe sea urchin fertilisation

Answer 7

• 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

Question 8

extracellular envelope

Answer 8

also termed the zona pellucida

Question 9

How is species-specific recognition achieved in sea urchin fertilisation?

Answer 9

binding proteins on the acrosomal process

Question 10

Describe fusion in mammals

Answer 10

• 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

Question 11

polyspermy

Answer 11

the fusion of multiple sperms

Question 12

‘block’ to polyspermy has two mechanisms

Answer 12

the fast and the slow.

Question 13

Describe the fast block to polyspermy

Answer 13

• 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.

Question 14

Describe one model for the fast block to polyspermy

Answer 14

• 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

Question 15

PI

Answer 15

• phosphatidyl inositol
• 2 acyl chains, glycerol phosphate and sugar,

Question 16

DAG

Answer 16

• diacylglycerol
• highly hydrophobic
• usually acts as a landing site for phosphoprylating protein kinases

Question 17

PKC

Answer 17

protein kinase calcium

Question 18

InsP3

Answer 18

small, charged, diffuse and hydrophilic

Question 19

Describe the self-stimulation of calcium release in the fast block to polyspermy

Answer 19

• further calcium on the cytoplasmic side primes the next IP3 receptors
• stimulates further calcium release
• non-equilbrium feedforward loop

Question 20

Describe ‘store operated calcium entry’

Answer 20

• 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

Question 21

How can the fast block to polyspermy be visualised?

Answer 21

• rapid confocal imaging
• Medaka fish eggs through luminescence after microinjection of aequorin

Question 22

aequorin

Answer 22

• calcium ion photoprotein
• isolated from Aequoria victoria

Question 23

Describe aequorin imagery of the fast block to polyspermy

Answer 23

• multiple calcium waves spreading through the egg
• triggered by a calcium ionophore

Question 24

Describe the visualisation of PIP2 hydrolysis

Answer 24

• fluorescent tagging via microinjection of PIP2 with a chimera of GFP-PH of eggs of Lytechnius pictus

Question 25

PH

Answer 25

pleckstrin homology domain of PLC

Question 26

Describe the egg-receptor mediated polyspermy block model

Answer 26

sperm derived factor fertilin would activate an integrin egg receptor via a tyrosine kinase, thus activating PLC.

Question 27

Describe the sperm oocyte-activating factor-mediated polyspermy block model

Answer 27

- 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

Question 28

Describe the slow block to polyspermy

Answer 28

- 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

Question 29

Describe calcium 'puffs' or 'sparks'

Answer 29

calcium propagation via clusters of channels across the egg

Question 30

CICR

Answer 30

- calcium-induced calcium-release - regenerative process where local calcium release spreads into a calcium wave or tide - activates a plethora of downstream targets

Question 31

cADPribose

Answer 31

natural ligand of the Ryanodine-receptor

Question 32

Describe the fertilisation envelope layer

Answer 32

- 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

Question 33

How can the slow block to polyspermy be visualised?

Answer 33

using fluorescent reporters

Question 34

Summarise the polyspermy block

Answer 34

- 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

Question 35

What moves an egg from quiescence?

Answer 35

- 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

Question 36

Describe the first division of the embryo

Answer 36

- 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

Question 37

Describe male pro-nuclear migration

Answer 37

- 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

Question 38

Describe the zygotic microtubules

Answer 38

- derived from the astromicrotubules - extend from the centrosome, forming spindles at the site of sperm entry - reconstitution of the mitosis apparatus

Question 39

Describe calmodulin

Answer 39

4 domains separated by an alpha helix each

Question 40

Describe co-operative binding of calmodulin

Answer 40

- 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)

Question 41

Describe the activation of CaM-kinase II

Answer 41

- 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

Question 42

Describe the autophosphorylation of CaM-kinase II

Answer 42

- 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

Question 43

Describe the inactivation of CaM-kinase II

Answer 43

- protein phosophotase can dephosphorylate the calcium independent CaM-kinase II - produces a molecule of inorganic phopahte - compound becomes inactive once more

Question 44

Describe the downstream effects of CaM-kinase II activation by calcium

Answer 44

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.