The Intraacrosomal Calcium Pool Plays a Direct Role in Acrosomal Exocytosis

Abstract

The acrosome reaction is a unique type of regulated exocytosis. The single secretory granule of the sperm fuses at multiple points with the overlying plasma membrane. In the past few years we have characterized several aspects of this process using streptolysin O-permeabilized human spermatozoa. Here we show that Rab3A triggers acrosomal exocytosis in the virtual absence of calcium in the cytosolic compartment. Interestingly, exocytosis is blocked when calcium is depleted from intracellular stores. By using a membrane-permeant fluorescent calcium probe, we observed that the acrosome actually behaves as a calcium store. Depleting calcium from this compartment by using a light-sensitive chelator prevents secretion promoted by Rab3A. UV inactivation of the chelator restores exocytosis. Rab3A-triggered exocytosis is blocked by calcium pump and inositol 1,4,5-trisphosphate (IP 3 )-sensitive calcium channel inhibitors. Calcium measurements inside and outside the acrosome showed that Rab3A promotes a calcium efflux from the granule. Interestingly, release of calcium through IP 3 -sensitive calcium channels was necessary even when exocytosis was initiated by increasing free calcium in the extraacrosomal compartment in both permeabilized and intact spermatozoa. Our results show that a calcium efflux from the acrosome through IP 3 -sensitive channels is necessary downstream Rab3A activation during the membrane fusion process leading to acrosomal exocytosis.The acrosome reaction is an exocytotic process induced physiologically by the activation of sperm receptors by ligands in the zona pellucida of the oocyte. This interaction initiates a complex transduction mechanism leading to multiple fusions between the outer acrosomal membrane and the overlying plasmalemma resulting in the release of the acrosomal content and exposure of the molecules present on the inner acrosomal membrane.As in other regulated secretory events, calcium plays a central role in acrosome reaction (1). Recent results from different laboratories suggest that a first transient cytosolic calcium increase (probably mediated by T-type calcium channels (2)) leads to a second sustained increase of cytosolic calcium that is necessary for the acrosome reaction (3-9). Although the connection between the two events is not completely clear, the current hypothesis is that the first calcium increase causes the activation of a phospholipase C (PLC).1 There are several isoforms of PLC in the spermatozoa (10, 11). PLC␦4, in particular, has been implicated in the early events of the acrosome reaction (12). Active PLC would produce inositol 1,4,5-trisphosphate (IP 3 ) that would open IP 3 -sensitive calcium channels in the membrane of intracellular stores. The emptying of these stores would trigger the opening of store-operated calcium (SOC) channels in the plasma membrane causing a second and sustained calcium increase that would trigger acrosomal exocytosis (1, 7). Although direct proof for the nature of the calcium stores involved in this mechanism is lacking, severa...

Rab27 and Rab3 sequentially regulate human sperm dense-core granule exocytosis

“…In the SLO-permeabilized human sperm model, NP-EGTA-AM crosses the plasma and outer acrosomal membranes, accumulates inside the acrosome, and prevents the AR triggered by all inducers by sequestering intra-acrosomal calcium for as long as the system is kept in the dark. UV photolysis of NP-EGTA-AM rapidly replenishes the acrosomal calcium pool, resuming exocytosis (36,39,41). In NP-EGTA-AM-loaded permeabilized sperm, anti-Rab27 and Slac2-b blocked exocytosis when added before, but not after, initiation of the AR by calcium (Fig.…”

“…The acrosome behaves as an internal store of releasable calcium (41)(42)(43)(44); efflux from this reservoir through inositol 1,4,5 triphosphate (IP 3 )-sensitive channels is required for the AR initiated by all inducers (32,33,41,45). We had previously demonstrated that Rab3A acts in the AR before the release of calcium from the intracellular store (32).…”

ADP Ribosylation Factor 6 (ARF6) Promotes Acrosomal Exocytosis by Modulating Lipid Turnover and Rab3A Activation

“…The acrosome behaves as an internal store of releasable calcium (19,47,53,54); efflux from this reservoir through IP 3 -sensitive channels is required for the AR initiated by all inducers (22,28,33,55). Results presented in Figs.…”

The Multi‐PDZ domain protein MUPP1 as a lipid raft‐associated scaffolding protein controlling the acrosome reaction in mammalian spermatozoa

“…influx is not sufficient to promote acrosomal exocytosis, but that acrosomal Ca 2þ release is also necessary (De Blas et al, 2002;Herrick et al, 2005). Although the exact role played by acrosomal Ca 2þ awaits further clarification, it is accepted that its release into the confined space between the outer acrosomal and the plasma membrane is fundamental for the ultimate membrane fusion event (Ramalho-Santos et al, 2002).…”

“…The second sustained increase in cytosolic Ca 2þ is controlled by inositol 1,4,5-trisphosphate receptor (IP 3 R) channels localized in the outer membrane of the acrosomal vesicle (Walensky and Snyder, 1995). The latter acts as an intracellular Ca 2þ store (De Blas et al, 2002;Herrick et al, 2005), the emptying of which activates storeoperated Ca 2þ channels, potentially composed of members of the transient receptor potential (TRP) family of cation channels (Jungnickel et al, 2001;Darszon et al, 2005). Remarkably, mimicking the influx of Ca 2þ mediated by store-operated Ca 2þ channels by incubating permeabilized spermatozoa in high external Ca 2þ , led to the intriguing observation that the sustained increase of intracellular Ca 2þ resulting from Ca 2þ…”