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Journal of Andrology, Vol 3, Issue 6 373-381, Copyright © 1982 by The American Society of Andrology

Fertilization of Mouse Eggs In Vitro

Time Resolution of the Reactions Preceding Penetration of the Zona Pellucida

HARVEY M. FLORMAN 1, PATRICIA M. SALING 2, AND BAYARD T. STOREY 3

1 Department of Obstetrics and Gynecology University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
2 Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
3 Laboratories for Cell Biology, Department of Anatomy, University of North Carolina School of Medicine, Chapel Hill, North Carolina

3-Quinuclidinyl benzilate (QNB) is a potent inhibitor of fertilization of mouse eggs in vitro: it acts by blocking penetration of the zonae by sperm. The time course of this inhibition was examined by addition of 50 µM QNB to the in vitro system for fertilization of cumulusfree mouse eggs at increasing times post-insemination. QNB, added prior to and at intervals up to 20 minutes post-insemination, inhibited fertilization by more than 90%. When added at longer times, QNB inhibition decreased; at 60 minutes post-insemination, added QNB had no effect on fertilization. The time course of onset of insensitivity to QNB inhibition was compared to the time courses of other reactions identified as precursors to fertilization in the mouse system. The most rapid reaction is Ca2+-mediated binding of sperm to zonae, which is reversible by EGTA; it has no time lag and is complete by 15 minutes post-insemination. The second reaction is onset of insensitivity of this binding to reversal by EGTA; it has a lag time of 5 minutes post-insemination and all bound sperm become insensitive 20 minutes post-insemination. The acrosome reaction occurring in sperm bound to zonae has a lag time of 25 minutes, and is not completed in all bound sperm until 240 minutes post-insemination. The time course of loss of QNB inhibition lies between the second reaction and the acrosome reaction. These studies allow the time resolution of four sequential reactions, which lead to fertilization of mouse eggs in vitro and occur prior to penetration of the zona pellucida. These are: EGTA-reversible, Ca2+-mediated binding of sperm to zona; onset of insensitivity to EGTA reversal of binding; onset of insensitivity to QNB inhibition of zona penetration; and onset of the acrosome reaction in sperm bound to zonae.

     Key words: in vitro fertilization, penetration of zona pellucida, 3-quinuclidinyl benzilate, acrosome reaction, sperm binding to zonae, mouse sperm, mouse eggs, inhibition of zona binding, inhibition of zona penetration

Accepted on June 21, 1982




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P. Wassarman
The biology and chemistry of fertilization
Science, January 30, 1987; 235(4788): 553 - 560.
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Copyright © 1982 by The American Society of Andrology.