Molecular Biology of Mammalian Fertilization

我们评估了Acr3-EGFP精子与正常卵子和huZP2拯救卵子的结合情况，其中人类ZP2取代了小鼠ZP2，但在受精后仍未分裂。观察到的Acr3-EGFP精子与来自huZP2救援小鼠的胚胎的重新结合支持了精子-卵子识别的“带状支架”模型，在该模型中，完整的ZP2决定了支持精子结合的三维结构，独立于受精和皮质颗粒胞分泌。令人惊讶的是，无论精子是在受精前还是受精后加入，在未分裂的人类ZP2存在的情况下，结合精子的顶体在24小时内保持完整。完整顶体的持续存在表明精子与透明带的结合不足以诱导顶体胞吐。过滤器渗透试验表明了另一种机制，即渗透到带基质中启动触发顶体反应所必需的机械感觉信号转导。

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.