CONICET | Buscador de Institutos y Recursos Humanos

The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to stimuli, it undergoes a special type of calcium-dependent exocytosis termed the acrosome reaction, which is an absolute prerequisite for fertilization. Freshly ejaculated mammalian sperm are not immediately capable of undergoing the acrosome reaction. The acquisition of this ability is called capacitation and involves a series of changes in sperm physiology that are not yet well characterized. Plasma membrane cholesterol removal is one of the sperm modifications associated with capacitation. However, how sterols affect acrosomal exocytosis is unknown. By using streptolysin O-permeabilized human sperm and a photosensitive calcium chelator, we have shown that cholesterol concentration affects an early event of the exocytic cascade rather than mixing of the lipid bilayers. Functional data indicate that one target for the cholesterol effect is Rab3A. The sterol content does not affect the Rab3A activation-deactivation 1 cycle but regulates Rah3A´s membrane anchoring. Western blot analysis and immunoelectron microscopy confirmed that cholesterol efflux facilitates Rab3A association with the sperm plasma membrane. To assess whether similar results could be obtained using nonpermeabilized perm, we added an arginine-rich peptide to the N-terminus of Rab3A. ´The presence of this polybasic peptide promotes the diffusion of proteins through the plasma membrane. Polyarginine-Rab3A entered into nonpermeabilized sperm and triggered exocytosis. Similar to what we have observed in permeabilized sperm, the action of polyarginine-Rab3A was modulated by the cholesterol content of membranes. Our data indicate that the cholesterol efflux occurring during capacitation optimizes conditions for the productive assembly of the fusion machinery required for the acrosome reaction.

enviar mensaje