Both progesterone and the calcium ionophore
Both progesterone and the calcium ionophore A23187 rely on the synthesis of cAMP by sAC to elicit exocytosis in human sperm (Fig. 2E-H and , ). We had reported earlier that digestion of endogenous cAMP with recombinant cAMP-specific phosphodiesterase 4D  or inhibition of its synthesis with the sAC blocker KH7  prevents calcium from accomplishing the AR in SLO-permeabilized sperm. A comparison with results obtained with sAC −/− mouse sperm are discussed in a previous publication . It has been proposed that sAC synthesizes cAMP in the tail and that this cAMP binds PKA to elicit the protein phosphorylation events that correlate with capacitation in mouse  and human  sperm. A transmembrane AC would be responsible for the synthesis of cAMP in the mouse sperm head. Blocking this activity with SQ22536 does not prevent the AR triggered by A23187 . Unfortunately, KH7 was not tested in the mouse studies, and therefore whether the apparent discrepancies with the human are due to different models or to some other reason cannot be ascertained with the information currently available. In any case, what we know beyond any doubt is that sperm exocytosis elicited by all inducers tested, notably progesterone, requires cAMP. Cyclic AMP modulates exocytosis in secretory pcpt receptor by PKA-dependent and/or PKA-independent mechanisms, the latter are mediated by Epacs. In human sperm, calcium-induced AR is mediated by cAMP/Epac and independent of PKA . Raps (Rap1a, b and Rap2a, b, c) are small molecular weight GTPases of the Ras family. Raps serve as substrates for Epac\'s enzymatic activity. The presence and localization of Epac and Rap isoforms in mammalian sperm has been recently reviewed . A large body of evidence shows that the signaling module cAMP/Epac/Rap governs many biological responses in a variety of cells. Yet its relevance in regulated exocytosis has only been scrutinized in a handful of studies: Epac2A/Rap1 are required for cAMP/Epac-dependent potentiation of insulin secretion ,  and intracellular calcium mobilization . Epac1/Rap1 are required for pancreatic amylase  and non-amyloidogenic-soluble form of the amyloid precursor protein α  release. Epac1/Rap2 contribute to chlorine secretion in human intestinal epithelial cells . Epac/Rap1 control cAMP-mediated exocytosis of Weibel–Palade bodies in endothelial cells  and the secretion of hepatic FGF21 elicited by glucagon . The AR elicited by 8-pCPT-2′-O-Me-cAMP, recombinant Rab3A-GTP-γ-S, and calcium requires endogenous, active Rap1. The amount of Rap1-GTP pulled down from cells challenged with 8-pCPT-2′-O-Me-cAMP in human (50μM, 15min, ), mouse (500μM, 15min, ), and boar (50μM, 2h, ) sperm is substantially higher than that from untreated controls. Active Rap1 localizes to the acrosomal region upon challenging SLO-permeabilized sperm with calcium . Here we show that progesterone and A23187 augment the percentage of non-permeabilized sperm cells exhibiting acrosomal Rap1-GTP (Fig. 3A, B, E). Activation requires cAMP (Fig. 3B) but not PKA (Fig. 3E). The stimulation of Epac with 8-pCPT-2′-O-Me-cAMP is sufficient to achieve Rap1 activation in a high percentage of sperm (Fig. 3E). Recombinant R-Rab3A-GTP-γ-S promotes the exchange of GDP for GTP on Rap1 (Fig. S1) and the AR (Fig. 2D) in a cAMP-dependent manner. In the last few years, it became appreciated that Rap1 proteins serve in interconnected signaling networks , . The only references to a possible connectivity between Rabs3 or 27 with Rap1 are indirect insofar as they describe the interactions between Rap1GAP2 and the Rab27 effectors synaptotagmin-like protein 1  and 2 . Here we presented direct evidence of the connection between Rab3A and Rap1 during secretion. The pathway appears to be unidirectional because Rap1 is not required to exchange GDP for GTP on Rab3 . External calcium is dispensable for the AR elicited by cAMP , recombinant Rab3A , , Arf6 , and diacylglycerol , but internal calcium is not. Nevertheless, intracellular calcium mobilization alone does not trigger secretion. In sperm with their plasma membrane permeabilized with SLO, cAMP, but not PKA, is required to mobilize calcium from the granule (Fig. 4, Fig. 5). One important caveat to the interpretation of findings obtained using PKA inhibitors is that they can only establish PKA-independence but cannot definitively establish a role for Epac. Here we present direct evidence that cAMP/Epac/Rap1 mobilize calcium from the acrosome because the stimulation of sperm\'s Epac with 8-pCPT-2′-O-Me-cAMP suffices to elicit intra-acrosomal calcium efflux shortly after the application (Fig. 5B). Anti-Epac (Fig. 5C) and anti-Rap1 (Fig. 7B) antibodies antagonize this effect. Intravesicular calcium mobilization exhibits a lag time that is not due to the kinetics of cAMP diffusion. First, we took great care in mixing the drugs with the bathing solution rapidly to accomplish homogeneous concentration. Second, cells were permeabilized with SLO so that access to the cytosolic compartment was not slowed by diffusion across the plasma membrane. Thus, the delay must reflect the latency of signaling events downstream of Epac. Unlike excitable cells, sperm do not have their secretory granules in close proximity to the plasma membrane primed for immediate exocytosis. Instead, the initiation of exocytosis must accomplish the activation of the fusion machinery and concomitant approximation of the acrosome to the plasma membrane achieved via swelling of the acrosomal contents followed by docking. Swelling is cAMP dependent (Fig. 2E-H) and rate limiting .