Functional, biochemical, and chromatographic characterization of the complete [Ca2+]i oscillation-inducing activity of porcine sperm

Abstract

AbstractA cytosolic sperm protein(s), referred to as sperm factor (SF), is delivered into eggs by the sperm during mammalian fertilization to induce repetitive increases in the intracellular concentration of free Ca2+ ([Ca2+]i) that are referred to as [Ca2+]i oscillations. [Ca2+]i oscillations are essential for egg activation and early embryonic development. Recent evidence shows that the novel sperm-specific phospholipase C (PLC), PLCζ, may be the long sought after [Ca2+]i oscillation-inducing SF. Here, we demonstrate the complete extraction of SF from porcine sperm and show that regardless of the method of extraction a single molecule/complex appears to be responsible for the [Ca2+]i oscillation-inducing activity of these extracts. Consistent with this notion, all sperm fractions that induced [Ca2+]i oscillations, including FPLC-purified fractions, exhibited high in vitro PLC activity at basal Ca2+ levels (0.1–5 μM), a hallmark of PLCζ. Notably, we detected immunoreactive 72-kDa PLCζ in an inactive fraction, and several fractions capable of inducing oscillations were devoid of 72-kDa PLCζ. Nonetheless, in the latter fractions, proteolytic fragments, presumably corresponding to cleaved forms of PLCζ, were detected by immunoblotting. Therefore, our findings corroborate the hypothesis that a sperm-specific PLC is the main component of the [Ca2+]i oscillation-inducing activity of sperm but provide evidence that the presence of 72-kDa PLCζ does not precisely correspond with the Ca2+ releasing activity of porcine sperm fractions