Molecular Biology of Mammalian Fertilization

精子在受精前识别小鼠透明带（ZP1, ZP2, ZP3），而在受精后不识别。一些生化变化已被推断为解释这种二分法，但只有ZP2的蛋白水解裂解已被实验证明。我们提出了一个模型，其中ZP2的卵裂状态决定了透明带是允许的（完整的ZP2）还是不允许的（分裂的ZP2）来识别精子-卵子。为了证实ZP2在精卵识别中的核心作用，我们通过微尺度Edman降解确定了其裂解位点，并通过DNA重组在裂解位点突变了一个moZP2的基因组克隆。建立了具有突变ZP2的转基因小鼠系（mutZP2）， 6个创始系均表达正常和突变的ZP2蛋白。将两条线交叉到Zp2零背景中产生mutZP2拯救小鼠。3-4周龄mutZP2救援雌性卵巢组织学无显著差异，尽管救援小鼠的卵细胞带较正常卵细胞更薄。MutZP2救援小鼠在体内具有生殖能力，精子与卵子结合并使其受精。然而，正如该模型所预测的那样，尽管受精并有效地阻止了多精现象，精子仍继续粘附在2细胞胚胎周围的带基质表面。ZP2切割阻断精卵识别蛋白的分子基础仍在研究中。

Sperm recognize the mouse zona pellucida (ZP1, ZP2, ZP3) prior to, but not after, fertilization. Several biochemical changes have been inferred to account for this dichotomy, but only proteolytic cleavage of ZP2 has been experimentally demonstrated. We have proposed a model in which cleavage status of ZP2 determines whether the zona pellucida is permissive (intact ZP2) or non-permissive (cleaved ZP2) for sperm-egg recognition. To confirm the central role of ZP2 in sperm-egg recognition, the cleavage site was determined by microscale Edman degradation and a genomic clone of moZP2 was mutated at the cleavage site by DNA recombineering. Transgenic mouse lines with the mutant ZP2 (mutZP2) were established and each of six founder lines expressed both normal and mutated ZP2 proteins. Two lines were crossed into the Zp2 null background to generate mutZP2 rescue mice. Ovarian histology of 3-4 week old mutZP2 rescue females was unremarkable, although ovulated eggs from rescue mice had a thinner zona compared to normal eggs. MutZP2 rescue mice were fertile in vivo and sperm bound to and fertilized their eggs. However, as predicted by the model, sperm continued to adhere to the surface of the zona matrix surrounding 2-cell embryos despite fertilization and an effective block to polyspermy. The molecular basis by which ZP2 cleavage blocks sperm-egg recognitioin remains under investigation.