The mammalian membrane block to polyspermy: Contributions of the sperm, the egg actin cytoskeleton, the membrane protein CD9 and post-ovulatory aging

This dissertation research examines four areas related to how the mammalian egg plasma membrane undergoes changes that prevent penetration by more than one sperm, a process called the membrane block to polyspermy. First, the effect of egg actin disruption on the establishment of the membrane block to polyspermy was examined. Previous studies in the lab revealed that disrupting the egg actin cytoskeleton with cytochalasin D leads to increased polyspermy during in vitro fertilization (IVF) of ZP-free mouse eggs. However, in our studies the effect is specific to cytochalasin D, since other actin-disrupting drugs do not cause an increased incidence of polyspermy. Second, we examined how sperm contents introduced upon fertilization could contribute to membrane block establishment. There are reports that eggs activated by parthenogenetic stimuli or fertilized by intracytoplasmic sperm injection (ICSI) do not establish the membrane block. Our studies found that neither fertilization-like intracellular calcium release, sperm-induced cortical remodeling nor sperm adhesion to the egg plasma membrane are sufficient to induce the membrane block, but suggest that sperm-egg membrane fusion is a crucial step in the establishment of the membrane block. Third, membrane function in eggs collected at later times after ovulation was studied. There are conflicting reports regarding membrane receptivity to sperm in post-ovulatory aged eggs collected after natural mating and artificial insemination. Our IVF studies of post-ovulatory aged eggs provide evidence for two different aged egg membrane abnormalities. Sperm are able to penetrate a subset of fertilized ZP-free aged eggs, indicative of an impaired membrane block, while another subset of ZP-free aged eggs are slow to fertilize or resistant to fertilization. These data provide new insights into the decreased reproductive success observed with delayed conception. Fourth, changes to the surface expression of CD9, an egg membrane protein that is essential for fertilization was studied. It is possible that the membrane block involves modification or down-regulation of molecules involved in fertilization. Our indirect immunofluorescence studies indicate there is a qualitative change in anti-CD9 fluorescence. This dissertation research extends our current understanding of the cellular mechanism and the molecular changes that contribute to the mammalian membrane block to polyspermy.