Wu H, He C L, Fissore R A
Program in Molecular and Cellular Biology, University of Massachusetts, Amherst 01003, USA.
Mol Reprod Dev. 1997 Feb;46(2):176-89. doi: 10.1002/(SICI)1098-2795(199702)46:2<176::AID-MRD8>3.0.CO;2-N.
Fertilization in mammals is associated with the generation of intracellular calcium ([Ca2+]i) oscillations. The site of, or mechanism(s) utilized by, the sperm to initiate and maintain these Ca2+ responses is not known. In this study, we tested the hypothesis that a factor from the sperm is capable, upon release into the oocyte's cytosol, of initiating oscillations. A sperm factor, prepared from porcine semen, was injected into mouse oocytes and bovine eggs that had been loaded with fura-2 dextran, a fluorescent Ca2+ indicator. The resulting Ca2+ responses were monitored and compared to those characteristic of each species. Our results show that injection of sperm factor triggered long-lasting [Ca2+]i oscillations, and that the observed patterns were species-specific. In mouse oocytes, sperm factor-induced [Ca2+]i rises exhibited high frequency, whereas in bovine eggs, Ca2+ responses were separated by long intervals. Further characterization of the sperm factor revealed that it was predominantly present in sperm preparations, that it contained a protein moiety, and that it was unlikely to be a protease. The intracellular Ca2+ channels/receptors through which the sperm factor-mediated Ca2+ release was investigated by using heparin, a competitive inhibitor of the inositol 1,4,5 trisphosphate receptor (InsP3R), and ryanodine, which binds the ryanodine receptor (RyR). The sperm factor appeared to stimulate InsP3R, at least in mouse oocytes, because sperm factor-induced oscillations were delayed or blocked in all oocytes by injection of heparin. RyR may be involved in the modulation of these oscillations, since addition of ryanodine modified Ca2+ responses to the sperm factor. The present results support the hypothesis that a factor from the sperm is involved in the generation of fertilization-associated [Ca2+]i oscillations.
哺乳动物的受精过程与细胞内钙（[Ca2+]i）振荡的产生有关。精子启动和维持这些钙反应所利用的位点或机制尚不清楚。在本研究中，我们测试了这样一个假设，即精子中的一种因子在释放到卵母细胞胞质溶胶后能够引发振荡。将从猪精液中制备的一种精子因子注射到已加载荧光钙指示剂fura-2葡聚糖的小鼠卵母细胞和牛卵中。监测由此产生的钙反应，并与每个物种的特征反应进行比较。我们的结果表明，注射精子因子引发了持久的[Ca2+]i振荡，并且观察到的模式具有物种特异性。在小鼠卵母细胞中，精子因子诱导的[Ca2+]i升高表现为高频，而在牛卵中，钙反应间隔较长。对精子因子的进一步表征表明，它主要存在于精子制剂中，含有蛋白质部分，并且不太可能是蛋白酶。通过使用肝素（肌醇1,4,5-三磷酸受体（InsP3R）的竞争性抑制剂）和ryanodine（与ryanodine受体（RyR）结合）来研究精子因子介导的钙释放所通过的细胞内钙通道/受体。精子因子似乎至少在小鼠卵母细胞中刺激了InsP3R，因为注射肝素后，所有卵母细胞中精子因子诱导的振荡都被延迟或阻断。RyR可能参与了这些振荡的调节，因为添加ryanodine改变了对精子因子的钙反应。目前的结果支持了这样一个假设，即精子中的一种因子参与了受精相关的[Ca2+]i振荡的产生。
