Identification of sperm-derived egg-activating factor and analysis of molecular mechanisms of intracellular Ca^<2+> increase at fertilization of mammalian eggs

这项研究是阐明精子诱导细胞内Ca^2+增加的分子机制的一个步骤，Ca ^2+是受精时卵子激活的关键信号。通过显微注射技术、Ca^2+图像分析和激光共聚焦扫描显微镜观察，获得了以下结果：1）作为现象学观察，我们建立了在配子融合抑制剂jaspisin存在下，精子不进入海胆卵，精子-卵融合仅瞬时形成的实验条件。在精子结合部位记录到局部Ca^2+升高，与正常受精时遍布整个卵子的Ca^2+波分开。sprem诱导的局部Ca^<2+>升高的时空方面被详细描述。2）将小鼠精子注射到卵子中（细胞质内精子注射，ICSI）引起重复的瞬时Ca^<2+升高（Ca^<2+离子），如受精时所见，星星 关于我们 精子注射后15至30分钟，并持续数小时。每一次Ca^2+的升高几乎都是在整个卵子中同步发生的。结果表明，即使ICSI绕过精卵结合，精子胞浆中的一种因子也会从注射的精子中泄漏到卵细胞质中，并引起Ca^2+振荡。3）我们发现，注射前体精子，即无鞭毛的圆形精子细胞，（圆形精子细胞注射，ROSI），不能在卵细胞中诱导任何Ca^<2+>反应，但联合注射肌醇1，4，5-三磷酸盐（IP_3）受体参与卵的激活（受精），并与雌、雄原核的形成有关。大约25%的两细胞胚胎移植到养母体内，发育成正常的后代。新生儿长成正常成年人后，再繁衍出正常的第二代。4）我们从仓鼠和海鞘精子中部分纯化了一种胞质蛋白，当注射到仓鼠、小鼠和海鞘卵中时，这种蛋白具有诱导Ca^2+振荡的活性。将精子因子显微注射到卵细胞的局部区域，结果表明，外周细胞质对精子因子更敏感，产生Ca^<2+>增加（可能是由于内质网通过IP_3受体释放Ca^<2+>所致），这些结果表明，在受精时，精子胞质因子可以通过精子和卵子之间形成的胞质连续性被引入卵子胞质的皮质区，并诱导卵子中Ca^2+的释放。本研究为今后的进一步研究提供了有用的信息。少

This study was performed as a step to elucidate the molecular mechanism by which the sperm induces an increase in intracellular Ca^<2+>, the pivotal signal for egg activation at fertilization. The following results were obtainde by microinjection techniques, Ca^<2+> image analysis, and confocal laser scanning microscopy.1)As a phenomenological observation, we established the experimental condition in which sperm-egg fusion is formed only transiently without sperm entry into the sea urchin egg in the presence of a gamete fusion inhibitor, jaspisin. A localized Ca^<2+> rise restricted at the sperm binding site was recorded, separated fron the Ca^<2+> wave which spreads throughout the egg at normal fertilization. Spatiotemporal aspects of the sprem-induced localized Ca^<2+> rise were characterized in detail.2) Injection of a mouse spermatozoon into an egg (intracytoplsmic sperm injection, ICSI) caused repetitive transient Ca^<2+> rises (Ca^<2+> oscillalions) as seen at fertilization, star … More ting 15 to 30 minutes after sperm injection and persisting for several hours. Each Ca^<2+> rise occurred almost synchronously in the whole egg. The results suggested that a sperm cytosolic factor leaks out from the injected spermatozoon into the egg cytoplasm and can induce Ca^<2+> oscillations, even if sperm-egg binding is bypassed by ICSI.3) We found that injection of a precursor sperm, round spermatids without flagellum (round spermatid injection, ROSI), can not induce any Ca^<2+> response in the egg but that combined injection of a potent agonist of the inositol 1,4,5-trisphosphate (IP_3) receptor resulted in egg activation (fertilization) associated with male and female pronucleus formation. About 25% of two-cell embryos transplanted into foster mothers developed to normal offspring. The newborn infants grew up to normal adults, which reproduced normal second generations.4)We partially purified a cytosolic protein from hamster and ascidian spermatozoa that possesses the activity of inducing Ca^<2+> oscillations when injected into hamster, mouse, and ascidian eggs. Microinjection of the sperm factor into a localized region of the egg showed that the peripheral cytoplasm is more sensitive to the sperm factor to generate Ca^<2+> increase (possibly due to Ca^<2+> release from the endoplasmic reticulum through IP_3 receptors), compared with the central region of the egg.These results suggested that, at fertilization, a sperm cytosolic factor could be introduced into the cortical region of the egg cytoplasm through cytoplasmic continuity formed between the sperm and egg, and induce Ca^<2+> release in the egg. This study provided useful information for advancing further studies in future. Less