Institute of Molecular Physiology, Cell and Matrix Biology, Johannes Gutenberg-University Mainz, Mainz, German.
Biointerface Laboratory, Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University Medical Faculty, Aachen, German.
Mol Hum Reprod. 2021 Mar 24;27(4). doi: 10.1093/molehr/gaab022.
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.
卵母细胞和精子的相遇是启动哺乳动物胚胎发育的关键事件。这种生存相互作用的关键功能是由蛋白酶决定的，如精子头部顶体中的顶体酶，以及卵母细胞皮质颗粒中储存的卵透明带酶。卵透明带酶在受精时被释放出来，以切割包围卵母细胞的糖蛋白基质——透明带。这种有限的蛋白水解作用使卵母细胞的包膜变硬，从而提供了一个明确的阻止多精入卵的障碍，并保护发育中的胚胎。另一方面，顶体酶，一种著名的、最丰富的顶体蛋白酶，被认为能使精子穿透卵母细胞的包膜。根据物种的不同，顶体酶对透明带的蛋白水解作用对于受精是必不可少的或有利的。然而，这种蛋白水解作用的特定靶标裂解位点和由此产生的生理后果仍然不清楚。在这里，我们用活性顶体酶处理天然的小鼠透明带，并通过质谱法鉴定出透明带蛋白 1（ZP1）中有两个裂解位点，ZP2 中有五个，ZP3 中有一个。这些位点中的几个在哺乳动物中高度保守。值得注意的是，顶体酶的有限蛋白水解作用导致透明带的重塑，而不是降解。因此，顶体酶影响精子的结合和透明带的机械弹性，这分别通过显微镜和纳米压痕测量来评估。此外，我们还通过激活潜伏的前卵透明带酶和失活卵透明带酶的紧密结合抑制剂胎球蛋白-B，确定了顶体酶的潜在调节作用。这些结果为我们提供了对复杂的蛋白水解网络的新认识，这些网络改变了小鼠卵母细胞的细胞外基质，这可能也适用于其他物种。
