Molecular Biological Studies On The Sperm Receptor Of Zona Pellucida

PART I THE INFLUENCE OF FERTILIZATION AND ACTIVATION ON SPERM RECEPTOR OF MOUSE ZONA PELLUCIDA Fertilization is the core step in reproductive process. The key to fertilizaton regulation lies in the sperm-zona interaction. Zona pelLucida is a glycoprotein coat surrounding oocyte and early embryo, which is consisting of ZP1,ZP2 and ZP3 in most mammals. ZP3 is proved to be the primary receptor of sperm, and the O-linked oligosaccarides of the ZP3 glycoprotein form the important component of sperm receptor. But what is the structure of the sperm receptor exactly? What changes and modifications of zona pellucida will occur during fertilization to prevent from polyspermy? Whether these changes occur during oocyte activation? All these remain unclear. In this study, an effort will be concentrated on the analysis of modifications of zona pellucida caused by fertilization and artificial activation. Chaptor 1 Studies on the Optimum Age of Mouse Oocytes for Effective Activation by ethanol stimulation Objective To compare the effect of egg age on the in vitro fertilization rate and activation rate. Methods I. Superovulation 9 Female mice were superovulated with of Pregnant Mare's Serum Gonadotropin (PMSG) and Human Chorionic Gonadotropin (HCG). The Oocyte-Cumulus cell Complexes (OCCs) were recovered and cultured in M16 medium for artificial activation or in vitro fertilization. 2. Artificial Activation The OCC mass stimulated by 8% of ethanol were treated with 0. 3 mg/mL hyaluronidase to remove the cumulus cells. The pronucleus formation or immediate cleavage into 2-cell stage was regarded as the parameter of act i vat ion. 3. In Vitro Fertilization(IVF) OCC mass was co-cultured with the capacitated sperm in Whittingham's IVF medium, The percentage of eggs cleaved into 2-cell stage on the next day was regarded as a parameter of fertilization rate. Results The average activation rates and average percentages of immediate cleavage are shown in table 1. The highest value were obtained in 2Ohr egg. The rates of in vitro fertilization of oocytes varied with the ages of oocytes. The average fertilization rates are shown in table 2. Eggs with l5hr of age revealed a better fertilization rate. Conclusions: The comparisons of activation rates and fertilization rates were made between oocytes at different ages. Results showed that oocytes at different ages had different activation and fertilization rates when stimulated by sperm or ethanol. The activation rate of oocytes increased 10 with the ageing of oocyte, up to the highest average of 81.6%, but decreased after 20 hr post-hCG. Oocytes at 20 hr post-hCG exhibited the highest immediate cleavage rate(48.O%) after stimulated by ethanol. On the other hand, 13-15 hr oocytes exhibited higher fertilization rates, the older oocytes were more difficult to be fertilized by sperm in vitro. These suggest that the oocyte can be activated in different ways, the mechanism of fertilization might be different from that of...