Activation of mouse sperm T-type Ca2+ channels by adhesion to the egg zona pellucida

The sperm acrosome reaction is a Ca2+-dependent exocytotic event that is triggered by adhesion to the mammalian egg’s zona pellucida. Previous studies using ion-selective fluorescent probes suggested a role of voltage-sensitive Ca2+ channels in acrosome reactions. Here, whole-cell patch clamp techniques are used to demonstrate the expression of functional T-type Ca2+ 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 Ca2+ 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 pellucida-dependent Ca2+ elevations, as demonstrated by ion-selective fluorescent probes, and also inhibits acrosome reactions. These studies directly link sperm T-type Ca2+ 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.

Calcium-triggered acrosomal exocytosis in human spermatozoa requires the coordinated activation of Rab3A and N-ethylmaleimide-sensitive factor

The acrosome reaction of spermatozoa is a complex, calcium-dependent, regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. However, very little is known about the molecules that mediate and regulate this unique fusion process. Here, we show that N-ethylmaleimide-sensitive factor (NSF), a protein essential for most fusion events, is present in the acrosome of several mammalian spermatozoa. Moreover, we demonstrate that calcium-dependent exocytosis of permeabilized sperm requires active NSF. Previously, we have shown that the addition of the active (GTP-bound) form of the small GTPase Rab3A triggers exocytosis in permeabilized spermatozoa. In the present report we show that Rab3A is necessary for calcium-dependent exocytosis. The activation of Rab3A protects NSF from N-ethylmaleimide inhibition and precludes the exchange of the endogenous protein with recombinant dominant negative mutants of NSF. Furthermore, Rab3A activation of acrosomal exocytosis requires active NSF. Our results suggest that, upon calcium stimulation, Rab3A switches to its active GTP-bound form, triggering the formation of a protein complex in which NSF is protected. This process is suggested to be an essential part of the molecular mechanism of membrane fusion leading to the release of the acrosomal contents.

Sperm capacitation in humans is transient and correlates with chemotactic responsiveness to follicular factors.

In humans, only a small fraction (2-12%) of a sperm population can respond by chemoattraction to follicular factors. This recent finding led to the hypothesis that chemotaxis provides a mechanism for selective recruitment of functionally mature spermatozoa (i.e., of capacitated spermatozoa, which possess the potential to undergo the acrosome reaction and fertilize the egg). This study aimed to examine this possibility. Capacitated spermatozoa were identified by their ability to undergo the acrosome reaction upon stimulation with phorbol 12-myristate 13-acetate. Under capacitating conditions, only a small portion (2-14%) of the spermatozoa were found to be capacitated. The spermatozoa were then separated according to their chemotactic activity, which resulted in a subpopulation enriched with chemotactically responsive spermatozoa and a subpopulation depleted of such spermatozoa. The level of capacitated spermatozoa in the former was approximately 13-fold higher than that in the latter. The capacitated state was temporary (50 min < life span < 240 min), and it was synchronous with the chemotactic activity. A continuous process of replacement of capacitated/chemotactic spermatozoa within a sperm population was observed. Spermatozoa that had stopped being capacitated did not become capacitated again, which indicates that the capacitated state is acquired only once in a sperm's lifetime. A total sperm population depleted of capacitated spermatozoa stopped being chemotactic. When capacitated spermatozoa reappeared, chemotactic activity was restored. These observations suggest that spermatozoa acquire their chemotactic responsiveness as part of the capacitation process and lose this responsiveness when the capacitated state is terminated. We suggest that the role of sperm chemotaxis in sperm-egg interaction in vivo may indeed be selective recruitment of capacitated spermatozoa for fertilizing the egg.