Inhibition of the fast and slow blocks to polyspermy

Objective : to test the effects of inhibiting the fast and slow blocks to polyspermy on sea urchin development.

Background
Sea urchin eggs release components of the egg jelly that attract sperm and induce the acrosome reaction (Gilbert, 2000). In the laboratory, a single egg many be covered with dozens or even hundreds of sperm burrowing through the egg jelly.

Once a sperm contacts the viltelline membrane, it binds to a receptor protein that triggers the events of fertilization. Eventually, the plasma membranes fuse, and the sperm nucleus and centriole enter the egg. However, if a single egg is fertilized by multiple sperm (polyspermy), additional chromosomes and centrioles are brought insideThis can cause defects at the first cleavage division as the chromosomes are segregated randomly between multiple spindles (Just, 1919, Gilbert, 2000).

Two mechanisms have evolved to reduce the occurrence of polyspermy. Sperm binding initially triggers an influx of Na+ ions leading to depolarization, which transiently blocks binding of additional sperm (known as the fast block to polyspermy). This depolarization can be blocked by the addition of nicotine (Jaffe, 1980). In addition, sperm binding also stimulates the release of Ca++ ions from the endoplasmic reticulum, leading to the fusion of a series of exocytic vesicles (the cortical granules) with the plasma membrane. This releases their contents, including a trypsin-like protease, which destroys the sperm binding sites on the vitelline membrane and detaches it from the plasma membrane (Vacquier, et al, 1973). Water rushes into the space, causing the vitelline membrane to rise up and become the fertilization envelope. This produces a more permanent block to polyspermy (the slow block ).

Procedure

1a) To inhibit the fast block to polyspermy, resuspend eggs in ASW or in nicotine (Sigma
Chemical Company) at 0.25 mM, 0.5 mM and 1 mM, (dilute 1.25 ul, 2.5 ul and 5 ul of a
40% solution of nicotine free base in 10 ml ASW and use immediately). Fertilize eggs
with diluted sperm as described in the basic protocol . Be sure to allow sperm to activate
for at least 5 minutes before adding to eggs.

1b) To inhibit the slow block to polyspermy, resuspend eggs in ASW or in soybean trypsin
inhibitor (SBTI, Sigma Chemical Company,) at 0.4 mg/ml (1/10 volume of 4 mg/ml stock).
Fertilize eggs with diluted sperm as described in the basic protocol. Be sure to allow
sperm to activate for at least 5 minutes before adding to eggs.

2. Examine eggs after 10 minutes to score for the presence (or absence) of the fertilization
envelope and periodically to observe the events associated with the first cleavage
division.