Lecture notes from Oxford University Biology Year 1: Building a Phenotype Trinity Term. Includes lectures on fertilisation, the cell cycle, mitosis, plant development & plant hormones, animal signal transduction from neurones and hormones, animal development, stem cells, regeneration, and immunolog...
Fertilisation:
Context:
Major transitions in evolution:
• Origin of sex (asexual -> sexual)
• Transition to Multicellularity
• Cell differentiation = Division of Labour – separation of germline
from soma
There are still examples of organisms undergoing these transitions e.g.
different species of algae (Chlamydomonas to Volvox).
Resource allocation per egg:
How much should be invested into an egg if you want it to become a
breeding adult? Must consider number of eggs, size of eggs, parental
survival, optimal clutch size, and animal behaviour?
I.e. why is a chicken egg bigger than frogspawn?
Insects do not invest enough into a full adult, so there is a larva adapted
for eating to make up these resources.
Mechanisms:
Fertilisation & development in sea urchins:
Like many marine organisms, fertilisation occurs externally. How do they
find each other?...
1. Chemotaxis of sperm to egg in response to 14 amino acid resact
peptide.
2. Egg carbohydrate signals initiate Ca2+-mediated exocytosis of
acrosomal vesicle.
3. Releases enzymes needed to penetrate the extracellular envelope.
4. Stimulates actin-dependent formation of acrosomal process.
5. Species specific recognition through binding protein on acrosomal
process.
6. Fusion between sperm and egg membranes.
Difficult to study fertilisation, as sperm v small in comparison to egg and
fertilisation only takes a few seconds therefore must be lucky to see it on
microscope.
Similar principles in Fucus (brown algae) and mammals (shows v similar
across multicellular tree of life).
Visualisation of mammalian sperm-egg fusion:
Can be tagged with fluorescent proteins. 2 proteins on sperm. Still v
difficult to visualise.
,Fast block to polyspermy via membrane depolarisation:
Fertilisation leads to membrane depolarisation.
Rapid confocal imaging shows calcium entry through plasma membrane
calcium channels as a cause or consequence of membrane depolarisation.
It is possible that influx of calcium is involved in depolarisation, since it is
electrical then this happens very quickly.
Calcium waves in Medaka fish eggs triggered by fertilisation:
- Calcium imaged by luminescence after microinjection of the Ca2+-
photoprotein, aequorin.
- Fertilisation wave (of calcium) spreads through egg – this is far
slower than the depolarisation of the plasma membrane.
- Multiple waves triggered by application of a calcium ionophore.
So far, if you block Ca then the process stops, if you add it, it helps, and
some form of calcium change should take place.
Sperm competition and the fast block to polyspermy:
Only one sperm can fertilise the egg.
1939: Ernest Just – Egg & embryos as excitable systems:
Pretty much got the process right with shit microscopes and no
technology – purely by observation.
What triggers the fertilisation Ca2+ wave?
Initiation of Ca2+ elevation by phosphatidyl inositol signalling:
PI embedded in membrane. It is phosphorylated to make PIP, and then
again to make PIP2.
PIP2 is the substrate for PLC. It will cleave the molecule leaving DAG in the
membrane and InsP3. InsP3 is v hydrophilic and v soluble, do can diffuse
from the cell. DAG stays in the membrane and is the landing site for PKC
(protein kinase C). InsP3 binds to IP3. IP3 lets calcium into the cytoplasm.
One of the roles of the Ca2+ is to activate PKC. Now PKC can
phosphorylate things in the cell.
There must be a mechanism to put calcium back into the ER to reset the
system and let it happen again.
To do this, there is a store-operated calcium entry – there are various
signalling components that activate an influx of calcium from outside, that
recharges the store. It does this using a pump to being calcium from the
cytoplasm back into the ER. This is also seen in muscles to restore the
calcium store, crops up in many signalling pathways.
How do we know hydrolysis of PIP2 takes place?
We use imaging of PIP2 with GFP-PH. We know it is attached to the
membrane. We can visualise how it is removed from the membrane and
then goes into the cytoplasm during fertilisation.
,Alternative models for egg activation:
n Egg-receptor mediated: Binding of a sperm derived factor (fertilin)
activates an egg receptor, possibly an integrin.
n Sperm oocyte-activating factor (SOAF):
- Direct release of a second messenger (IP3)
- Activation of a src-tyrosine kinases that activates egg PLC.
- Introduction of sperm PLC (zeta) isoform (different version of PLC)
Experimentally v difficult to study as very small and very quick.
Model 2: Activation by sperm derived PLC zeta:
• PLC Zeta from the sperm releases IP3 from PIP2 present in
cytoplasmic oocyte vesicles.
• IP3 releases Ca2+ from the ER via IP3R
How does the calcium wave propogate?
Activation of a cluster of channels generates a ‘puff’ or ‘spark’ of calcium.
Spikes of calcium called different things in different cells. Local releases of
calcium are thought to be due to clusters of calcium release channels.
This alone will not generate a wave – we need it to cause a positive
feedback and cause more calcium release of other channels.
Calcium-induced calcium-release (CICR):
1. InsP3 enhances the sensitivity of the InsP3-R to calcium.
2. Elevated cytosolic calcium can initiate further calcium release from
either channel (CICR).
3. cADPribose (cADPR) enhances the sensitivity of the Ryanodine-
receptor (Ry-R) to calcium.
CICR can lead to a regenerative wave of
calcium.
- They behave as independent vesicles,
however in practise the lumen is
connected. So, are they separate
organelles or together? So why do they
not act as one and just release all
calcium? Will find out later.
Coalescence of sparks stimulates a regenerative calcium wave.
(May also be called a calcium tide as once up can stay there for a while.)
Calcium waves and cortical granule secretion:
n Fast block to polyspermy via rapid membrane depolarisation from -
70 mV to +20mV from Na+ or Ca2+ influx.
, n CICR Ca2+-wave causes slow block to polyspermy via cortical granule
fusion.
n Forms the fertilization envelope (mucopolysaccharides, cross-linked
vitelline proteins and hyalin) – no sperm can get through this.
Summary so far:
Initiation of the first division:
Movement of the male pro-nucleus is mediated by motor drag on MTs:
Female and male nucleus appear to move towards each other – but how
do they actually find each other?
At the start of cell division, only one of the nuclei makes microtubules – is
this involved in attracting the two? Biophysical model shows yes.
How does calcium control activation and early division?
Calmodulin has 4 calcium binding EF-hand domains.
Co-operative interactions confer a sigmoidal response.
Calmodulin undergoes a major conformational change on binding calcium.
Ca2+-calmodulin is preferentially localised to the mitotic apparatus during
division. It is localised to the spindle – controls something about the cell
division.
Activation of CaM-kinase II:
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