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Sperm Factor Induces Intracellular Free Calcium Oscillations by Stimulating the Phosphoinositide Pathway¹

^a Molecular and Cellular Biology Program and Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts 01003 ^b Department of Physiology and Biophysics, University of California, Irvine, California 92697-4560 ^c Department of Biochemistry, University of Tokyo, Tokyo 108-8039, Japan

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 PLC1, PLC2, or PLC4 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 PLC1, PLC2, or PLC4 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.

FOOTNOTES

First decision: 24 October 2000.

¹ Supported by grants from the USDA (2371) to R.A.F. and the NIH (GM 57015) to N.L.A.

² Correspondence. FAX: 413 545 6326; rfissore{at}vasci.umass.edu

³ These authors contributed equally.

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