Molecular Biology of Mammalian Fertilization

我们评估了Acr3-EGFP精子与正常卵子和huZP2卵子的结合情况，在这些卵子中，人ZP2取代了小鼠ZP2，但受精后仍未分裂。观察到的Acr3-EGFP精子与来自huZP2救援小鼠的胚胎的从头结合支持精子-卵子识别的“透明带支架”模型，在该模型中，完整的ZP2决定了一个支持精子结合的三维结构，与受精和皮质颗粒胞吐无关。令人惊讶的是，无论精子是在受精前还是受精后加入，在未切割的人类ZP2存在下，绑定的精子的顶体24小时都保持完好。顶体完整的存在表明精子与透明带的结合不足以诱导顶体胞吐。滤器穿透分析提出了另一种机制，即穿透到透明带基质中启动触发顶体反应所需的机械感觉信号转导。 在小鼠和人类中，唯一记录的与透明带块时间相关的透明带修饰是ZP2的裂解，其结果是N-端肽(30kD)通过二硫键与亲本片段保持共价连接(S)。幸运的是，在huZP2救援小鼠中，ZP2仍然没有被切割，在这种情况下，人ZP2取代了内源性小鼠ZP2，在单细胞胚胎中只存在两个原核，它们在体外进展到两细胞阶段，活体幼崽的出生证明了对多精受精的有效阻断。尽管精子在受精后继续结合，但卵黄周间隙中没有过多的精子表明，这种阻塞的部分原因是透明带。如果正确，受精后出现完整的huZP2表明，用人源化的透明带观察到的透明带块可能与huZP2的切割无关。因此，似乎不需要切割ZP2来阻止受精后精子穿透透明带。

We assessed Acr3-EGFP sperm binding to normal and huZP2 rescue eggs in which human ZP2 replaces mouse ZP2, but remains uncleaved after fertilization. The observed de novo binding of Acr3-EGFP sperm to embryos derived from huZP2 rescue mice supports a 'zona scaffold' model of sperm-egg recognition in which intact ZP2 dictates a three-dimensional structure supportive of sperm binding, independent of fertilization and cortical granule exocytosis. Surprisingly, the acrosomes of the bound sperm remain intact for ≥ 24 hours in the presence of uncleaved human ZP2 regardless of whether sperm are added before or after fertilization. The persistence of intact acrosomes indicates that sperm binding to the zona pellucida is not sufficient to induce acrosome exocytosis. A filter penetration assay suggests an alternative mechanism in which penetration into the zona matrix initiates a mechanosensory signal transduction necessary to trigger the acrosome reaction. The only documented modification of the zona pellucida that temporally correlates with the zona block in mice and humans is cleavage of ZP2 which results in an N-terminal peptide (30 kD) that remains covalently linked to the parental fragment via a disulfide bond(s). Fortuitously, ZP2 remains uncleaved in huZP2 rescue mice in which human ZP2 replaces endogenous mouse ZP2 and the presence of only two pronuclei in one-cell embryos, their progression to the two-cell stage in vitro, and the birth of live pups attests to an effective block to polyspermy. Even though sperm continue to bind after fertilization, the absence of excessive sperm in the perivitelline space suggests that part of this block is attributable to the zona pellucida. If correct, the presence of intact huZP2 following fertilization suggests that the zona block observed with the humanized zona pellucida may be independent of huZP2 cleavage. Thus, it appears that cleavage of ZP2 is not required to block post-fertilization sperm penetration of the zona pellucida.