In the present studies, we demonstrated that successful sperm penetration was achieved in mR1ECM with modifications in which PVA was replaced with BSA and the NaCl concentration was increased to 100-130 mM. In hamsters, mice, and cattle, sperm penetration of oocytes in vitro was achieved when cumulus-oocyte complexes were cocultured in media not containing BSA. In the rat, in contrast, previous findings showing that BSA is essential in order to result in sperm penetration have not yet been overturned. Miyamoto and Chang also reported that in the presence of glucose, BSA was the most important component for in vitro fertilization of rat oocytes and that addition of lactate and pyruvate facilitated the process. In the present studies, however, neither simply replacing PVA with BSA, or replacing PVA with BSA and increasing the concentration of sodium lactate, resulted in successful sperm penetration in mR1ECM. Since we confirmed the necessity of supplementing with BSA in mKRB, we basically used mR1ECM-BSA in the other experiments. buy birth control online
The fertilizability of spermatozoa also appears to be higher in hypertonic rather than hypotonic solution in the rabbit, mouse, and hamster. In rats, although penetration in hypotonic solution has not been examined, a high proportion of oocytes were penetrated in standard isotonic (309 mOsm) and slightly hypertonic (317-397 mOsm) solution regulated by adding NaCl. Therefore, epididymal rat spermatozoa appear to be more capacitated in media with elevated isotonic strength. In the present studies, we found that sperm penetration was observed only when the osmolarity of mR1ECM-BSA was increased to the level in mKRB (310 mOsm) or more (see Table 5) by adding NaCl but not by adding sorbitol. This means that not high os-molarity itself, but rather relatively high NaCl concentrations (100-130 mM), are beneficial for sperm penetration in the rat. In the present studies, we did not determine whether a high NaCl condition affected each step of sperm penetration such as sperm capacitation, penetration through zona pellucida, and fusion with oocytes. It has been reported that the fertilizing ability of rabbit spermatozoa was improved by treatment with hypertonic (380 mOsm) medium before insemination. It is possible that the effect of high NaCl condition could be mainly on sperm penetration.