Fertilization 2
Reference: S. Gilbert Developmental Biology 7th Edition, Sinauer Associates Chapter 7
or as indicated)
We briefly reviewed the sperm acrosome reaction, including the role of calcium release
in triggering exocytocis. We looked at the two figures of AR in sea urchin sperm and
hamster sperm, and mentioned the roles of actin (in the acrosomal process) and bindin in
sperm–egg interaction.
Two more problems in fertilization
Prevention of polyspermy and activation of egg metabolism
Mechanism for the prevention of polyspermy do differ among various metazoans
But typically there is a fast, electrical block and a slow mechanical block, at least among
those that fertilize externally.
7.21 what happens when a dispermic sea urchin egg is created
Electrical block, resting membrane potential is about –70 mV this is due to lower conc. of
+ (and high conc. of – charged macromolecular) ions inside the cell relative to outside
Sea urchin eggs are low in Na+ and high in K+ relative to water (recall from 261 text K+
are counter ions to macromolecular anions so aren’t really free +)
When fertilization occurs the resting membrane potential shifts rapidly to about +20 V
due to influx of sodium (recall Na +K+ ATPase works to keep Na+ out and K+ in, in the
normal condition) Sperm cannot fuse to eggs with a + membrane potential (figure 7.22)
Lower Na+ outside (7.22) increases polyspermy, artificially clamp membrane potential
electrically (Laurinda Jaffe) negative increase polyspermy.
Why does the electrical block work? Perhaps sperm carry a voltage sensitive “fusogenic”
protein. Frogs have an electrical block, most mammals probably do not.
Slow block to polyspermy involves the calcium dependent release of contents from the
egg’s cortical granules (see E.E. Just on your Vade Mecum CD ), the so-called “cortical
reaction”
There are about 15, 000 cortical granules in the cortex region of the sea urchin egg, a
region of cytoplasm just below the plasma membrane with its closely opposed vitelline
envelope (membrane) Exocytosis of the cortical granules (figure 7.23 + 7.24) releases a
serine protease that snips the fibrous proteins of the vitelline envelope (VE) off their
plasma membrane anchors, and removes the bindin receptors (bindin again is the sperm
receptor for egg in sea urchins) Also released from the cortical granules are osmotically
active mucopolysaccharides that effectively draw water into the space between the PM
and the VE this elevates the VE into what is now called the fertilzation envelope (FE)
(see also Vade Mecum-sea urchin). Hardening of the FE is due to the action of a
peroxidase. This enzyme cross links the fibrous proteins of the VE (now the FE)
Hyalin and other cortical granule proteins released into the space between the PM and the
FE serve to hold the embryo together.
In mammals the cortical reaction is less obvious but has the same function of blocking
polyspermy. The ZP is modified so that no more sperm can bind. Carbohydrates, esp Nacetylglucosamine are removed from ZP3 (recall competition experiment earlier in the
chapter that showed that sugars on ZP3 are required for sperm binding) Zp2 is
proteolyzed so that it also loses the ability to bind sperm.
The cortical reaction requires calcium (as does the sperm acrosome reaction) If you use a
fluorescent dye that reacts (that is increases fl with increasing calcium conc. such as
Fura-2 or aequorin) you can watch a wave of calcium release that begins at the point of
sperm entry and spreads across the egg (fig 7.25, takes 30 sec) This happens without
Ca2+ in the sea water and can be artificially triggered (as can the sperm acrosome
reaction) by ionophore A23187. So Ca2+ must already be in the egg. In sea urchins and
vertebrates the calcium required is stored in the endoplasmic reticulum surrounding the
cortical granules (Fig 7.26)
Activation of Egg Metabolism
Calcium is responsible for re-initiation of protein synthesis and re-entry of the egg into
the cell cycle (recall meiotic, and in some species mitotic arrest prior to sperm entry)
In mammal, there are actually several waves of calcium, and metabolic events are trigged
at different times in response to these (some events may require the passage of multiple
calcium waves in order to go to completion.
Rise in intracellular pH and rapid increase (burst) in O2 consumption follow elevation of
the sea urchin FE, with other events falling in order like dominos in a line.
7.1 table events in sea urchin fert., pretty rapid but still it is 5-10 minutes per
Fig 7.27 model for activation of various egg metabolic events. We went over this and
figure 7.28 from the sidelights in detail.
NAD+ kinase resp for NADP+ production, NADPH is a coenzyme for lipid
biosynthesis. Need lots of lipids during cleavage! NADPH also used in the production
of peroxides from O2 that are needed to harden the FE
Later responses in egg activation
Ca and pH rise thought to stimulate DNA synthesis (need a lot of that in cleavage too)
and protein synthesis. In many species, but prob not mammals protein synthesis (fig 7.30
sea urchin) uses stored RNAs placed in the egg maternal (by the various means we’ve
already discussed) Can initiate protein synthesis in sea urchins by adding ammonium ions
to raise cytoplasmic pH. What happens? Maternal RNAs may be held in place in an
untranslatable form by ‘masking’ proteins or RNAs, these get degraded and translation
ensues.
Fusion of genetic material
A relatively late event. In some species (see Parascaris/ascaris) the female pronucleus
is not done with meiosis yet! The male pronucleus decondenses and the two migrate
towards one another. The sperm centriole is really important because it makes the
microtubules that drive the migration ( we looked at figure 7.31 sea urchin) This takes an
hour.
In mammals the process of the fusion of the genetic material takes over 12 hours and
looks very different. The egg has to complete meiosis. The two pronuclei complete
DNA synthesis separately. The sperm centrioles produces the asters, microtubule move
the two pronuclei together, but do not fuse (fig 7.32) the chromosomes condense
separately (bipolar nuclear array, prophase) the sperm and egg chromosomes finally
come together and line up on the spindle during prometaphase of the first mitosis. True
diploid nucleus is only seen in the two cell stage.
The male and female pronuclei are not equivalent in terms of directing development. In
mammals at least, see table 7.2 in the sidelight , transplantation experiments show that
both a paternally and a maternally derived pronucleus are needed to correctly control
development.
Rearrangement of the egg cytoplasm occurs in some species. It is especially pronounced
in the frog =cortical rotation (for next time)