Molecular Biology of Mammalian Fertilization

精子在受精前识别小鼠透明带(ZP1、ZP2、ZP3)，但在受精后不识别。几种生化变化已被推断为解释这种二分性，但只有ZP2的蛋白水解性裂解已被实验证明。我们提出了一个模型，在这个模型中，ZP2的切割状态决定了透明带对于精卵识别是允许的(完整的ZP2)还是不允许的(切割的ZP2)。为了证实ZP2在精卵识别中的中心作用，用微量Edman降解法确定了切割位置，并通过DNA重组工程在切割位置突变了moZP2的基因组克隆。建立了含突变ZP2的转基因小鼠株系，6个方正株均表达正常和突变的ZP2蛋白。将两条线交叉到Zp2缺失背景中，产生了muZP2救援小鼠。3-4周龄的muZP2救援雌鼠的卵巢组织学未见明显变化，尽管从救援小鼠排出的卵子比正常卵子的卵带更薄。拯救MutZP2的小鼠在体内是有生育能力的，精子与其卵子结合并受精。然而，正如模型预测的那样，尽管受精并有效地阻止了多精受精，精子仍继续附着在2-细胞胚胎周围的透明带基质表面。ZP2裂解阻止精子-卵子识别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.