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

Injection of a porcine cytosolic sperm factor (SF) or of a porcine testicul ar extract into mammalian eggs triggers oscillations of intracellular free calcium ([Ca2+](i)) similar to those initiated by fertilization. To elucida te whether SF activates the phosphoinositide (PI) pathway, mouse eggs or SF were incubated with U73122, an inhibitor of events leading to phospholipas e C (PLC) activation and/or of PLC itself. In both cases, U73122 blocked th e ability of SF to induce [Ca2+](i) oscillations, although it did not inhib it Ca2+ release caused by injection of inositol 1,4,5-triphosphate (IP3). T he inactive analogue, U73343, had no effect on SE-induced Ca2+ responses. T o determine at the single cell level whether SF triggers IP3 production con comitantly 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 incre ase in [Ca2+](i) and IP3 production in these oocytes. Using ammonium sulfat e precipitation, chromatographic fractionation, and Western blotting, we de termined whether PLC gamma1, PLC gamma2, or PLC delta4 and/or its splice va riants, which are present in sperm and testis, are responsible for the Ca2 activity in the extracts. Our results revealed that active fractions do no t contain PLC gamma1, PLC gamma2, or PLC delta4 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, wh ich is an important feature of fertilized and SF-injected eggs. Eggs inject ed with adenophostin A, an IP3 receptor agonist, showed enhanced Ca2+ respo nses to CaCl2 injections. Thus, SF, and probably sperm, induces [Ca2+](i) r ises by persistently stimulating IF, production, which in turn results in l ong-lasting sensitization of Ca2+-induced Ca2+ release. Whether SF is itsel f a PLC or whether it acts upstream of the egg's PLCs remains to be elucida ted.