Functional, biochemical, and chromatographic characterization of the complete [Ca 2+] i oscillation-inducing activity of porcine sperm

Abstract

A 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 Ca 2+ ([Ca 2+] i) that are referred to as [Ca 2+] i oscillations. [Ca 2+] 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 [Ca 2+] 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 [Ca 2+] i oscillation-inducing activity of these extracts. Consistent with this notion, all sperm fractions that induced [Ca 2+] i oscillations, including FPLC-purified fractions, exhibited high in vitro PLC activity at basal Ca 2+ 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 [Ca 2+] i oscillation-inducing activity of sperm but provide evidence that the presence of 72-kDa PLCζ does not precisely correspond with the Ca 2+ releasing activity of porcine sperm fractions.