Membrane dynamics during the acrosome reaction in human sperm

POCOGNONI CA; SOSA CM; MAYORGA, L.S.; DE BLAS GA

La Serena

Workshop; EMBO Workshop: Actualizations in membrane trafficking in health and disease; 2016

Only sperm that have completed the acrosome reaction can successfully fuse with the oocyte. Therefore, the acrosomal reaction is an essential secretory process that must occur at the appropriate time and location for a productive sperm-egg interaction. The acrosome reaction relies on the same highly conserved molecules that drive intracellular membrane fusion and exocytosis in all other cells; however, the change in membrane topology during secretion is unique. Hundreds of fusion pores are opened connecting the acrosomal lumen with the external medium leading to the secretion of the acrosomal content and the release of hybrid vesicles formed by patches of the outer acrosomal and plasma membranes. What defines the zones of interaction and fusion between the essentially flat acrosomal membrane and the plasma membrane is unknown. We have shown that the acrosomal granule swells upon stimulation and that inward invaginations of the limiting membrane delineate ring-shaped membrane microdomains that contact the plasma membrane. We have postulated that the opening and expansion of fusion pores along these rings trigger acrosomal exocytosis. Our observations indicate that swelling is a slow and asynchronic process which is uncoupled from the initial increase of cytsosolic calcium. In contrast, fusion pore opening is much faster and requires the release of calcium from the acrosome. The invaginations of the acrosomal membrane topologically resemble the deformations of the endosomal membrane leading to the assembly of luminal vesicles in multivesicular bodies. In fact, intraacrosomal vesicles are also form during acrosomal exocytosis. We have reported that perturbing the function of members of Endosomal Sorting Complex Required for Transport (ESCRT) with antibodies or recombinant proteins inhibited acrosomal exocytosis in permeabilized cells. These observations indicate that ESCRT-mediated processes are essential for acrosomal secretion, implicating these multifunctional complexes in an exocytic event crucial for sperm-egg interactions. ABSTRACTThe 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, 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.