Immunohistochemical localization of zona pellucida proteins ZPA, ZPB and ZPC in human, cynomolgus monkey and mouse ovaries

The zona pellucida of mammalian oocytes plays an important role in binding and activation of sperm cells during the molecular events leading to fertil ization. The genes coding for the three zona pellucida glycoproteins ZPA, Z PB, and ZPC of various species including mouse, dog, and human have been cl oned and sequenced by several groups. However, it has remained a matter of debate as to whether the oocytes alone or in conjunction with the surroundi ng granulosa cells express and deposit these proteins to form the zona pell ucida matrix. Addressing this unresolved issue, we assessed the expression and localization of all three zona pellucida proteins in ovaries of human, cynomolgus monkey and mice using immunohistochemical methods. In addition, oocyte-specific expression of ZPC from the primordial stage onward was conf irmed by in situ hybridization. In sections of human ovaries, ZPA, ZPB, and ZPC proteins were immunohistochemically detected in the cytoplasms of prim ordial oocytes and during later stages of folliculogenesis in the zona pell ucida matrices of oocytes. In sections fixed with formalin, a clear homogen eous ring was visible around the oocyte and no staining of granulosa cells was observed. In contrast, staining of ZP proteins was also observed betwee n granulosa cells when Bouin's reagent had been used for tissue fixation. T hus, the original zona pellucida architecture was better preserved by forma lin fixation. We further demonstrated that dissolution of the zona pellucid a of isolated bovine oocytes occurred after they were exposed to Bouin's re agent. In summary, these results demonstrate that in mice, monkeys and huma ns, zona proteins are expressed and assembled exclusively by the oocyte and not by the granulosa cells. Previously observed results of ZP expression b y an involvement of granulosa cells might therefore be the result of an imp roper fixation of the tissues leading to the disruption of the zona pelluci da. Additionally this study highlights the importance of choosing the corre ct fixative for immunohistochemistry, not only for the usual reason of reta ining antigenicity, but rather to retain the entire architectural structure .