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Involvement of Calcium Signaling and the Actin Cytoskeleton in the Membrane Block to Polyspermy in Mouse Eggs¹

^a Department of Biochemistry and Molecular Biology, Division of Reproductive Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205 ^b Center for Research on Reproduction and Women's Health, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

This study examines the effects of actin microfilament-disrupting drugs on events of fertilization, with emphasis on gamete membrane interactions. Mouse eggs, freed of their zonae pellucidae, were treated with drugs that perturb the actin cytoskeleton by different mechanisms (cytochalasin B, cytochalasin D, jasplakinolide, latrunculin B) and then inseminated. Cytochalasin B, jasplakinolide, and latrunculin B treatments resulted in a decrease in the percentage of eggs fertilized and the average number of sperm fused per egg. However, cytochalasin D treatment resulted in an increase in the average number of sperm fused per egg and the percentage of polyspermic eggs. This increase in polyspermy occurred despite the observation that cytochalasin D treatment caused a decrease in sperm-egg binding and did not affect spontaneous acrosome reactions or sperm motility. This suggested that cytochalasin D-treated eggs had an impaired ability to establish a block to polyspermy at the level of the plasma membrane. The effect of cytochalasin D on the block to polyspermy was not due to a general disruption of egg activation because sperm-induced calcium oscillations and cortical granule exocytosis were similar in cytochalasin D-treated and control eggs. However, buffering of intracellular calcium levels with the calcium chelator BAPTA-AM resulted in an increase in polyspermy. Together, these data suggest that a postfertilization decrease in egg membrane receptivity to sperm requires functions of the egg actin cytoskeleton that are disrupted by cytochalasin D. Furthermore, egg activation-associated increased intracellular calcium levels are necessary but not sufficient to affect postfertilization membrane dynamics that contribute to a membrane block to polyspermy.

¹ This work was supported by grants from the American Society for Reproductive Medicine and Organon, Inc. (J.P.E.), from the American Society for Reproductive Medicine and Serono, Inc., and the Burroughs-Wellcome Fund (C.J.W.), and from the National Institutes of Health (HD 37696 to J.P.E.). G.B.W. was supported by training grants from the National Institutes of Health (HD 07276 and CA 09110). Early stages of this work were supported by HD 22732 (to Richard M. Schultz and Gregory S. Kopf).

² Correspondence: Janice P. Evans, Department of Biochemistry and Molecular Biology, Johns Hopkins University, Bloomberg School of Public Health, Room 3606, 615 N. Wolfe Street, Johns Hopkins University, Baltimore, MD 21205. FAX: 410 614 2356; jpevans{at}jhsph.edu

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