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

这项研究是为了阐明精子诱导细胞内Ca^<2+>增加的分子机制，Ca^<2+>是卵子受精时激活的关键信号。通过显微注射技术、Ca^<2+>图像分析和共聚焦激光扫描显微镜获得了以下结果。1)从现象上观察，我们建立了在配子融合抑制剂茉莉素存在的情况下，精子不进入海胆卵的情况下，精子-卵子仅短暂融合的实验条件。记录到局限于精子结合位点的局部Ca^<2+>上升，与正常受精时遍布整个卵子的Ca^<2+>波分离。本文详细分析了spremam诱导的局域Ca^<2+>上升的时空特征。2)将小鼠精子注射到卵子中（卵胞浆内精子注射，ICSI）会引起反复的短暂性Ca^<2+>升高（Ca^<2+>振荡），在精子注射后15至30分钟，持续数小时。每个Ca^<2+>的上升几乎在整个鸡蛋中同步发生。结果表明，即使icsi绕过精卵结合，精子胞质因子也会从注射的精子中泄漏到卵细胞质中，并诱导Ca^<2+>振荡。3)我们发现，注射一个前体精子，没有鞭毛的圆形精子（圆形精子注射，ROSI），不能在卵中诱导任何Ca^<2+>反应，但联合注射肌醇1,4,5-三磷酸（IP_3）受体的强效激动剂可导致卵激活（受精），并与雄性和雌性原核形成相关。移植到养母体内的双细胞胚胎中，约有25%发育成正常的后代。新生的婴儿成长为正常的成年人，繁殖正常的第二代。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，该蛋白具有诱导Ca^<2+>振荡的活性。将精子因子微注射到卵细胞的局部区域表明，与卵细胞中心区域相比，周围细胞质对精子因子产生Ca^<2+>的增加更敏感（可能是由于Ca^<2+>通过IP_3受体从内质网释放）。这些结果表明，在受精时，一种精子胞质因子可能通过在精子和卵子之间形成的细胞质连续性被引入卵质皮质区，并诱导卵子释放Ca^<2+>。本研究为今后进一步的研究提供了有益的信息。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