Role of cAMP in membrane fusion during acrosome reaction of human sperm

LUCCHESI O; BUSTOS MA; RUETE C; QUEVEDO MF; TOMES CN

Workshop; EMBO Workshop: Current advances in membrane trafficking: Implications for polarity and diseases; 2014

Exocytosis of sperm?s single secretory granule or acrosome (AR) is a highly regulated event essential for fertilization. The AR begins with an influx of calcium from the extracellular milieu and continues with the synthesis of cAMP and the activation of its target Epac. At this point, the cascade bifurcates into a Rab3-GTP-driven limb that assembles the fusion machinery and a Rap- GTP-driven limb that mobilizes internal calcium. Once the acrosome is docked to the plasma membrane and the acrosomal calcium is movilized, the acrosome fuses to the plasma membrane and the acrosomal content is released. Because sperm are transcriptionally and translationally inactive, overexpression or silencing experiments cannot be performed. An alternative method is the use of membrane-permeant proteins. We have designed a permeable version of the C-terminal domain of human PKA-RI beta. This protein functions as a cAMP sponge that captures cytoplasmic cAMP, depleting sperm of this second messenger. We analyzed the effect of cAMP shortage in various sperm events. By TEM we proved that cAMP is essential for the acrosomal swelling caused by AR inducers. When we incubated cells with cAMP sponge and challenged with progesterone or A23187, AR levels declined drastically. On sperm permeabilized with streptolysin O and the acrosome loaded with the fluorescent calcium sensor Fluo3-AM, cAMP sponge impaired intraacrosomal calcium release elicited by external calcium. Finally, we designed a method that combines protein-protein interaction with indirect immunofluorescense to detect small G proteins in their active state. Rab3-GTP, Rab27-GTP and Rap1-GTP levels decreased on sperm incubated with the cAMP sponge and challenged with progesterone. We conclude that cAMP has a fundamental role on acrosomal swelling, internal calcium reservoir emptying and granule content release; suggesting that cytosolic levels of this second messenger are critical to regulate membranes fusion and exocytosis.