Kinetics of human sperm acrosomal exocytosis.

The acrosome reaction is a unique event in the lifespan of sperm characterized by the exocytosis of the acrosomal content and the release of hybrid vesicles formed by patches of the outer acrosomal membrane and the plasma membrane. This unique regulated exocytosis is mediated by essentially the same membrane fusion machinery present in neuroendocrine cells. However, whereas secretion in neuroendocrine cells occurs in less than a second, the acrosome reaction is normally assessed after several minutes of incubation with inducers. In this report, we measured the kinetics of human sperm exocytosis triggered by two stimuli (calcium ionophore and progesterone) by using electron microscopy and three different approaches based on the incorporation of fluorescent Pisum sativum agglutinin into the acrosome upon opening of fusion pores connecting the extracellular medium with the acrosomal lumen. The results with the different methods are consistent with a slow kinetics (t½ = 14 min). We also manipulated the system to measure different steps of the process. We observed that cytosolic calcium increased with a relatively fast kinetics (t½ = 0.1 min). In contrast, the swelling of the acrosomal granule that precedes exocytosis was a slow process (t½ = 13 min). When swelling was completed, the fusion pore opening was fast (t½ = 0.2 min). The results indicate that acrosomal swelling is the slowest step and it determines the kinetics of the acrosome reaction. After the swelling is completed, the efflux of calcium from intracellular stores triggers fusion pores opening and the release of hybrid vesicles in seconds.

Cholesterol content regulates acrosomal exocytosis by enhancing Rab3A plasma membrane association.

The acrosome is an exocytic granule that overlies the spermatozoan nucleus. In response to different stimuli, it undergoes calcium-regulated exocytosis. Freshly ejaculated mammalian sperm are not immediately capable of undergoing acrosome reaction. The acquisition of this ability is called capacitation and involves a series of still not well-characterized changes in the sperm physiology. Plasma membrane cholesterol removal is one of the sperm modifications that are associated with capacitation. However, how sterols affect acrosomal exocytosis is unknown. Here, we show that short incubations with cyclodextrin, a cholesterol removal agent, just before stimulation promote acrosomal exocytosis. Moreover, the effect was also observed in permeabilized cells stimulated with calcium, indicating that cholesterol plays a direct role in the calcium-dependent exocytosis associated with acrosome reaction. Using a photo-inhibitable calcium chelator, we show that cholesterol affects an early event of the exocytic cascade rather than the lipid bilayers mixing. Functional data indicate that one target for the cholesterol effect is Rab3A. The sterol content does not affect the Rab3A activation-deactivation cycle but regulates its membrane anchoring. Western blot analysis and immunoelectron microscopy confirmed that cholesterol efflux facilitates Rab3A association to sperm plasma membrane. Our data indicate that the cholesterol efflux occurring during capacitation optimizes the conditions for the productive assembly of the fusion machinery required for acrosome reaction.

alpha-SNAP and NSF are required in a priming step during the human sperm acrosome reaction.

The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli it undergoes a special type of Ca2+-dependent exocytosis termed the acrosome reaction (AR), which is an absolute prerequisite for fertilization. Aided by a streptolysin-O permeabilization protocol developed in our laboratory, we have previously demonstrated requirements for Rab3A, N-ethylmaleimide-sensitive factor (NSF), several soluble NSF-attachment protein receptor (SNARE) proteins, and synaptotagmin VI in the human sperm AR. Here, we show that alpha-soluble NSF-attachment protein (alpha-SNAP), a protein essential for most fusion events through its interaction with NSF and the SNARE complex, exhibits a direct role in the AR. First, the presence of alpha-SNAP is demonstrated by the Western blot of human sperm protein extracts. Immunostaining experiments reveal an acrosomal localization for this protein. Second, the Ca2+ and Rab3A-triggered ARs are inhibited by anti-alpha-SNAP antibodies. Third, bacterially expressed alpha-SNAP abolishes exocytosis in a fashion that depends on its interaction with NSF. Fourth, we show a requirement for alpha-SNAP/NSF in a prefusion step early in the exocytotic pathway, after the tethering of the acrosome to the plasma membrane and before the efflux of intra-acrosomal Ca2+. These results suggest a key role for alpha-SNAP/NSF in the AR, and strengthen our understanding of the molecular players involved in the vesicle-to-plasma membrane fusion taking place during exocytosis.