Kurokawa Manabu, Sato Ken-Ichi, Wu Hua, He Changli, Malcuit Christopher, Black Samuel J, Fukami Kiyoko, Fissore Rafael A
Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA.
Dev Biol. 2005 Sep 15;285(2):376-92. doi: 10.1016/j.ydbio.2005.06.029.
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 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), PLCzeta, 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 microM), a hallmark of PLCzeta. Notably, we detected immunoreactive 72-kDa PLCzeta in an inactive fraction, and several fractions capable of inducing oscillations were devoid of 72-kDa PLCzeta. Nonetheless, in the latter fractions, proteolytic fragments, presumably corresponding to cleaved forms of PLCzeta, 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 PLCzeta does not precisely correspond with the Ca2+ releasing activity of porcine sperm fractions.
一种胞质精子蛋白，称为精子因子（SF），在哺乳动物受精过程中由精子传递到卵子中，诱导细胞内游离Ca2+浓度（[Ca2+]i）重复升高，即所谓的[Ca2+]i振荡。[Ca2+]i振荡对于卵子激活和早期胚胎发育至关重要。最近的证据表明，新型精子特异性磷脂酶C（PLC），即PLCζ，可能是长期以来寻找的诱导[Ca2+]i振荡的SF。在此，我们展示了从猪精子中完全提取SF，并表明无论提取方法如何，似乎单个分子/复合物负责这些提取物的诱导[Ca2+]i振荡的活性。与此概念一致，所有诱导[Ca2+]i振荡的精子组分，包括FPLC纯化的组分，在基础Ca2+水平（0.1 - 5 microM）下均表现出高体外PLC活性，这是PLCζ的一个标志。值得注意的是，我们在一个无活性组分中检测到免疫反应性72 kDa的PLCζ，而几个能够诱导振荡的组分中没有72 kDa的PLCζ。尽管如此，在后者的组分中，通过免疫印迹检测到蛋白水解片段，推测对应于PLCζ的裂解形式。因此，我们的发现证实了精子特异性PLC是精子诱导[Ca2+]i振荡活性的主要成分这一假设，但提供了证据表明72 kDa PLCζ的存在与猪精子组分的Ca2+释放活性并不精确对应。
