The encounter of oocyte and sperm is the key event initiating embryonic development in mammals. Crucial functions of this existential interaction are determined by proteolytic enzymes, such as acrosin, carried in the sperm head acrosome, and ovastacin, stored in the oocyte cortical granules. Ovastacin is released upon fertilisation to cleave the zona pellucida, a glycoprotein matrix surrounding the oocyte. This limited proteolysis hardens the oocyte envelope, and thereby provides a definitive block against polyspermy and protects the developing embryo. On the other hand, acrosin, the renowned and most abundant acrosomal protease, has been thought to enable sperm to penetrate the oocyte envelope. Depending on the species, proteolytic cleavage of the zona pellucida by acrosin is either essential or conducive for fertilisation. However, the specific target cleavage sites and the resulting physiological consequences of this proteolysis remained obscure. Here, we treated native mouse zonae pellucidae with active acrosin and identified two cleavage sites in zona pellucida protein 1 (ZP1), five in ZP2 and one in ZP3 by mass spectrometry. Several of these sites are highly conserved in mammals. Remarkably, limited proteolysis by acrosin leads to zona pellucida remodelling rather than degradation. Thus, acrosin affects both sperm binding and mechanical resilience of the zona pellucida, as assessed by microscopy and nanoindentation measurements, respectively. Furthermore, we ascertained potential regulatory effects of acrosin, via activation of latent pro-ovastacin and inactivation of fetuin-B, a tight binding inhibitor of ovastacin. These results offer novel insights into the complex proteolytic network modifying the extracellular matrix of the mouse oocyte, which might apply also to other species.

中文翻译：

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顶体蛋白的有限蛋白水解影响小鼠透明带的精子结合和机械弹性

卵母细胞和精子的相遇是哺乳动物胚胎发育的关键事件。这种存在相互作用的关键功能是由蛋白水解酶决定的，例如精子头顶体中携带的顶糖素和储存在卵母细胞皮质颗粒中的卵母细胞素。Ovastacin 在受精时释放以切割透明带，这是一种围绕卵母细胞的糖蛋白基质。这种有限的蛋白水解使卵母细胞包膜变硬，从而提供了对多精症的最终阻断并保护了发育中的胚胎。另一方面，人们认为顶体蛋白酶是著名且最丰富的顶体蛋白酶，它可以使精子穿透卵母细胞的包膜。根据物种的不同，顶体素对透明带的蛋白水解切割对于受精是必不可少的或有利于受精的。然而，这种蛋白水解的特定靶点切割位点和由此产生的生理后果仍然不清楚。在这里，我们用活性顶体素处理天然小鼠透明带，并通过质谱鉴定了透明带蛋白 1 (ZP1) 中的两个切割位点、ZP2 中的五个和 ZP3 中的一个。其中一些位点在哺乳动物中是高度保守的。值得注意的是，顶糖蛋白的有限蛋白水解导致透明带重塑而不是降解。因此，顶糖苷影响精子结合和透明带的机械弹性，分别通过显微镜和纳米压痕测量进行评估。此外，我们通过激活潜在的前 ovastacin 和灭活 ovastacin 的紧密结合抑制剂 fetuin-B 确定了 crosin 的潜在调节作用。