Previously, we reported that ZP3, one of three different glycoproteins present in the mouse egg's zona pellucida, serves as a sperm receptor. Furthermore, small glycopeptides derived from egg ZP3 retain full sperm receptor activity, suggesting a role for carbohydrate, rather than polypeptide chain in receptor function. Here, we report that removal of O-linked oligosaccharides from ZP3 destroys its sperm receptor activity, whereas removal of N-linked oligosaccharides has no effect. A specific size class of O-linked oligosaccharides, recovered following mild alkaline hydrolysis and reduction of ZP3, is shown to possess sperm receptor activity and to bind to sperm. The results presented strongly suggest that mouse sperm bind to eggs via O-linked oligosaccharides present on ZP3.
The sperm acrosome reaction is a Ca 2؉ -dependent exocytotic event that is triggered by adhesion to the mammalian egg's zona pellucida. Previous studies using ionselective f luorescent probes suggested a role of voltagesensitive Ca 2؉ channels in acrosome reactions. Here, wholecell patch clamp techniques are used to demonstrate the expression of functional T-type Ca 2؉ channels during mouse spermatogenesis. The germ cell T current is inhibited by antagonists of T-type channels (pimozide and amiloride) as well as by antagonists whose major site of action is the somatic cell L-type Ca 2؉ channel (1,4-dihydropyridines, arylalkylamines, benzothiazapines), as has also been reported for certain somatic cell T currents. In sperm, inhibition of T channels during gamete interaction inhibits zona pellucidadependent Ca 2؉ elevations, as demonstrated by ion-selective f luorescent probes, and also inhibits acrosome reactions. These studies directly link sperm T-type Ca 2؉ channels to fertilization. In addition, the kinetics of channel inhibition by 1,4-dihydropyridines suggests a mechanism for the reported contraceptive effects of those compounds in human males.
Sperm adhesion to egg zonae pellucidae initiates sperm acrosome reactions, an exocytotic event that is an early step during fertilization. Previously, it was suggested that zona pellucida-evoked Ca 2؉ entry into sperm through low voltage-activated Ca 2؉ channels is an essential step in acrosome reactions, based on the inhibitory effects of Ca 2؉ channel antagonists. However, analysis of this channel is limited by the inability to apply electrophysiological methods directly to sperm. In this report, optical methods of determining membrane potential and internal Ca 2؉ levels were used to demonstrate that (i) contact with zonae pellucidae activates a transient Ca 2؉ response in sperm that has a time course and antagonist sensitivity anticipated of low voltage-activated Ca 2؉ channels; (ii) these channels are unavailable for opening in uncapacitated sperm because of voltage-dependent, steady state inactivation; (iii) membrane hyperpolarization during sperm capacitation is sufficient to recruit channels into a closed state, from which they are available for opening during fertilization; and (iv) channel conductance state may be a factor in determines the efficacy with which channel antagonists inhibit fertilization. This study provides evidence for the activation of sperm Ca 2؉ channels during gamete adhesion and offers a mechanism that may account for aspects of the regulation of sperm fertility during capacitation through the control of channel availability. Finally, these results suggest that channel conductance state may be a central feature in the design of channel antagonists that inhibit sperm function.
Membrane potential (VM) was investigated in mouse and bovine sperm populations. VM was determined from the fluorescence emission of the lipophilic anion, bis(1,3-diethylthiobarbituric acid)trimethine oxonol (DiSBAC2(3)), and from the lipophilic cation, 3,3'-dipropylthiodicarbocyanine iodide (DiSC3(5)). Fluorescent signals were corrected for contributions of mitochondrial potentials and apparent VM values were obtained by calibrations in sperm selectively permeabilized with valinomycin or with gramicidin D. The calculated VM values of uncapacitated mouse and bovine sperm were approximately -35 and -30 mV, respectively. In contrast, capacitated populations of mouse and bovine sperm have VM values of -50 to -60 mV. Membrane hyperpolarization is due in part to an enhanced K+ permeability. The development of zona pellucida-activated signal transducing mechanisms during capacitation is dependent upon hyperpolarization. It is suggested that VM alterations regulate the activation state of sperm, thereby suppressing premature acrosome reactions in uncapacitated sperm and permitting capacitated sperm to respond to zona pellucida stimuli.
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