Zona-intact Eggs Research Articles

Mouse gamete interactions: The zona pellucida is the site of the acrosome reaction leading to fertilization in vitro

The question of whether the acrosome reaction, which leads to fertilization, occurs in intact sperm bound to the zona pellucida of the egg or in intact sperm before contact with the egg, was addressed by assessing the effect of 3-quinuclidinyl benzilate (QNB) on the two types of acrosome reaction. QNB is a specific inhibitor of the fertilization of zona-intact mouse eggs by mouse sperm. Mouse spermatozoa in suspension underwent acrosome reactions at a low rate, which could be accelerated by addition of 5 μ M divalent cation ionophore A23187; the occurrence of such acrosome reactions was not inhibited by QNB. The rate at which acrosome reactions occurred in sperm bound to the zona pellucida of cumulus-free eggs, bound to isolated zonae, or exposed to acid-solubilized zona components, was greatly accelerated relative to that observed in the absence of zonae. These acrosome reactions were strongly inhibited by QNB at concentrations which inhibit the fertilization of zona-intact mouse eggs in vitro. These data suggest that the zona pellucida can induce acrosome reactions in mouse spermatozoa and that these acrosome reactions are the ones which lead to the fertilization of zona-intact eggs. In contrast, the acrosome rection in sperm which are not in contact with the zona is not associated with fertilization of zona-intact eggs.

Fertile life of acrosome-reacted guinea pig spermatozoa.

To study the fertile life of acrosome-reacted guinea pig spermatozoa, spermatozoa were induced to undergo a synchronous acrosome reaction after which vigorously motile acrosome-reacted spermatozoa were isolated from acrosome-intact ones. Acrosome-reacted spermatozoa thus prepared were incubated in vitro for various periods of time before they were mixed with zona-intact and zona-free eggs. Such spermatozoa retained their ability to fertilize zona-intact eggs for at least 2 hours after the acrosome reaction; thereafter they rapidly lost this ability. There was a close correlation between the decline and loss of the ability to penetrate the zona and the loss of hyperactivated motility by spermatozoa, indicating the importance of vigorous motility in passing through the zona. The ability of acrosome-reacted spermatozoa to fertilize zona-free eggs was maintained for many hours even after the spermatozoa had completely lost their capacity to fertilize zona-intact eggs. As long as acrosome-reacted spermatozoa are "alive," they seem to retain the capacity to fuse with the egg plasma membrane regardless of the quality of their motility.

Involvement of trypsin-like activity in binding of mouse spermatozoa to zonae pellucidae.

Work from a number of laboratories has shown that fertilization is blocked in the presence of protease inhibitors, although the specific site of inhibition has not been identified. The present experiments were designed to discriminate between sperm binding to zonae pellucidae as opposed to sperm penetration through zonae, so as to assess the effect of protease inhibitors on these two distinct events. Exposure of capacitated mouse spermatozoa to a variety of protease inhibitors directed against trypsin blocked sperm binding to zonae in a concentration-dependent manner. A chymotrypsin-directed inhibitor was not capable of blocking sperm binding to zonae. The trypsin inhibitors did not affect sperm penetration though zonae nor gamete membrane fusion if the sperm had established a firm association with the zona surface before addition of the inhibitors. Previous incubation of zona-intact eggs with the inhibitors did not lead to a reduction in sperm binding, indicating that the activity affected by the inhibitors is borne by spermatozoa. Interaction between spermatozoa and the zona surface appeared to be the specific locus of inhibition; sperm binding to zona-free eggs (i.e., binding to the egg plasma membrane) was unaltered by the trypsin inhibitors. These results suggest a reevaluation of the function of proteases in fertilization focusing on their role in initial sperm contact with the zona pellucida.

Premature cortical granule loss does not prevent sperm penetration of mouse eggs

The role of cortical granules in the mouse egg's plasmalemma block to polyspermy was investigated by examining the effect of premature granule loss on egg fertility. Granule loss, quantitated by transmission electron microscopic examination, was induced in zona-free eggs by exposure to the divalent cation ionophore, A23187, or by mechanical removal of zonae. Egg exposure to ionophore led to the loss of approximately 50% of the egg's complement of granules in the absence of nuclear activation (parthenogenesis), while complete cortical granule loss accompanied the parthenogenetic activation seen in a limited population of mechanically stimulated eggs. Aged eggs underwent nuclear activation without a dramatic reduction in granule complements. The fertility of treated zona-free eggs was identical to that of controls, as measured by the percentage of eggs penetrated and by the mean number of sperm recovered per egg. Moreover, both ionophore-treated and aged eggs subsequently underwent a normal sperm-induced block response. Exposure of zona-intact eggs to ionophore was also without effect on egg fertility. These results indicate that cortical granules are not involved in the plasmalemma block to polyspermy in the mouse.

Induction of zonal and egg plasma membrane blocks to sperm penetration in mouse eggs with cortical granule exudate.

The contents of cortical granules (cortical granule exudate = CGE) were recovered from zona-free mouse eggs inseminated in vitro with capacitated epididymal spermatozoa. Preincubation of cumulus-free, zona-intact eggs in CGE led to reduced penetration levels upon insemination. This reduction was CGE concentration- and time-dependent and was susceptible to inhibition by soybean trypsin inhibitor and by p-aminobenzamidine. Control experiments eliminated active contributions from the sperm suspension used to elicit granule release. CGE was also active in reducing penetration of zona-free eggs. These results indicate that cortical granule contents are capable of modulating sperm penetration of mouse eggs at both the zona pellucida and at the egg plasma membrane.