Abstract
Epac, a guanine nucleotide exchange factor for the small GTPase Rap, binds to and is activated by the second messenger cAMP. In sperm, there are a number of signaling pathways required to achieve egg-fertilizing ability that depend upon an intracellular rise of cAMP. Most of these processes were thought to be mediated by cAMP-dependent protein kinases. Here we report a new dependence for the cAMP-induced acrosome reaction involving Epac. The acrosome reaction is a specialized type of regulated exocytosis leading to a massive fusion between the outer acrosomal and the plasma membranes of sperm cells. Ca2+ is the archetypical trigger of regulated exocytosis, and we show here that its effects on acrosomal release are fully mediated by cAMP. Ca2+ failed to trigger acrosomal exocytosis when intracellular cAMP was depleted by an exogenously added phosphodiesterase or when Epac was sequestered by specific blocking antibodies. The nondiscriminating dibutyryl-cAMP and the Epac-selective 8-(p-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate analogues triggered the acrosome reaction in the effective absence of extracellular Ca2+. This indicates that cAMP, via Epac activation, has the ability to drive the whole cascade of events necessary to bring exocytosis to completion, including tethering and docking of the acrosome to the plasma membrane, priming of the fusion machinery, mobilization of intravesicular Ca2+, and ultimately, bilayer mixing and fusion. cAMP-elicited exocytosis was sensitive to anti-alpha-SNAP, anti-NSF, and anti-Rab3A antibodies, to intra-acrosomal Ca2+ chelators, and to botulinum toxins but was resistant to cAMP-dependent protein kinase blockers. These experiments thus identify Epac in human sperm and evince its indispensable role downstream of Ca2+ in exocytosis.
Highlights
Of amylase from parotid and pancreatic acinar cells [4]
Reagents that Mimic cAMP Trigger acrosome reaction (AR) in streptolysin O (SLO)-permeabilized Sperm—If the opening of Ca2ϩ channels elicited by protein kinase (PKA) [11] was the only cAMP-dependent signaling pathway during the AR, it would be expected that reagents that augment the intracellular concentration of cAMP would not have an effect in cells where the plasma membrane is permeabilized by SLO, bypassing any requirement for ion channel opening
When SLO-permeabilized human sperm were incubated with 1 mM Bt2cAMP they underwent acrosomal exocytosis of a magnitude similar to that achieved by 10 M Ca2ϩ (Fig. 1A)
Summary
PKA has been described in spermatozoa from several mammalian species It has been implicated in sperm motility and capacitation through still undefined signaling cascades that culminate in enhanced protein tyrosine phosphorylation. These channels are directly opened by either cAMP of cGMP and are permeable to Ca2ϩ ions They form heterooligomeric complexes composed of at least two distinct subunits (␣ and ) [31]. We demonstrate that the activation of Epac alone by a specific cAMP analogue is sufficient to achieve maximum exocytosis levels in intact and SLO-permeabilized cells. This exocytosis relies on the bona fide machinery required for fusion in all secretory cells. Epac functions in a relatively early step during the exocytosis cascade, prior to tethering by Rab, priming by NSF/␣-SNAP, docking by SNAREs, and intra-acrosomal Ca2ϩ release
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