Functional, biochemical, and chromatographic characterization of the complete [Ca2+]i oscillation-inducing activity of porcine sperm
Functional, biochemical, and chromatographic characterization of the complete [Ca2+]i oscillation-inducing activity of porcine sperm
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DOI：
10.1016/j.ydbio.2005.06.029
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发表时间：
2005-09-15
影响因子：
2.7
通讯作者：
Fissore, RA
中科院分区：
文献类型：
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作者：
Kurokawa, M;Sato, K;Fissore, RA
一种细胞溶质精子蛋白，称为精子因子（SF），在哺乳动物受精过程中由精子递送到卵子中，以诱导细胞内游离Ca 2+浓度（[Ca 2 +]（i））的重复增加，称为[Ca 2 +]（i）振荡。[Ca2（i）振荡对卵的激活和早期胚胎发育至关重要。最近的证据表明，新的精子特异性磷脂酶C（PLC），PLC zeta，可能是长期追求后[Ca 2 +]（i）振荡诱导SF。在这里，我们证明了从猪精子中完全提取SF，并表明无论提取方法如何，单个分子/复合物似乎是这些提取物的[Ca 2 +]（i）振荡诱导活性的原因。与这一观点一致，所有诱导[Ca 2 +]（i）振荡的精子组分，包括FPLC纯化组分，在基础Ca 2+水平（0.1-5 μ M）下表现出高的体外PLC活性，这是PLC zeta的标志。值得注意的是，我们检测到免疫反应性72-kDa PLC zeta在一个非活性馏分，和几个馏分能够诱导振荡没有72-kDa PLC zeta。尽管如此，在后一种组分中，通过免疫印迹检测到蛋白水解片段，推测其对应于PLQ的切割形式。因此，我们的研究结果证实了这一假设，即精子特异性PLC是精子的[Ca 2 +]i振荡诱导活性的主要成分，但提供的证据表明，72 kDa PLC zeta的存在并不完全对应于猪精子组分的Ca 2+释放活性。(c)2005年爱思唯尔公司All rights reserved.
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), PLC zeta, 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 mu M), a hallmark of PLC zeta. Notably, we detected immunoreactive 72-kDa PLC zeta in an inactive fraction, and several fractions capable of inducing oscillations were devoid of 72-kDa PLC zeta. Nonetheless, in the latter fractions, proteolytie fragments, presumably corresponding to cleaved forms of PLQ, 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 PLC zeta does not precisely correspond with the Ca2+ releasing activity of porcine sperm fractions. (c) 2005 Elsevier Inc. All rights reserved.