Sperm factor induces intracellular free calcium oscillations by stimulating the phosphoinositide pathway

Abstract

Injection of a porcine cytosolic sperm factor (SF) or of a porcine testicular extract into mammalian eggs triggers oscillations of intracellular free calcium ([Ca2+]i) similar to those initiated by fertilization. To elucidate whether SF activates the phosphoinositide (PI) pathway, mouse eggs or SF were incubated with U73122, an inhibitor of events leading to phospholipase C (PLC) activation and/or of PLC itself. In both cases, U73122 blocked the ability of SF to induce [Ca2+]i oscillations, although it did not inhibit Ca2+ release caused by injection of inositol 1,4, 5-triphosphate (IP3). The inactive analogue, U73343, had no effect on SF-induced Ca2+ responses. To determine at the single cell level whether SF triggers IP3 production concomitantly with a [Ca2+]i rise, SF was injected into Xenopus oocytes and IP3 concentration was determined using a biological detector cell combined with capillary electrophoresis. Injection of SF induced a significant increase in [Ca2+]i and IP3 production in these oocytes. Using ammonium sulfate precipitation, chromatographic fractionation, and Western blotting, we determined whether PLCγ1, PLCγ2, or PLCδ4 and/or its splice variants, which are present in sperm and testis, are responsible for the Ca2+ activity in the extracts. Our results revealed that active fractions do not contain PLCγ1, PLCγ2, or PLCδ4 and/or its splice variants, which were present in inactive fractions. We also tested whether IP3 could be the sensitizing stimulus of the Ca2+-induced Ca2+ release mechanism, which is an important feature of fertilized and SF-injected eggs. Eggs injected with adenophostin A, an IP3 receptor agonist, showed enhanced Ca2+ responses to CaCl2 injections. Thus, SF, and probably sperm, induces [Ca2+]i rises by persistently stimulating IP3 production, which in turn results in long-lasting sensitization of Ca2+-induced Ca2+release. Whether SF is itself a PLC or whether it acts upstream of the egg's PLCs remains to be elucidated.