Analysis of inhibitory mechanism of fertilization between different species

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First, we tried to establish the in vitro fertilization (IVF) technique of the Japanese field vole. It is improved by sperm concentration (1-2x10^7 cell/ml for preincubation and 1x10^6 cell/ml for insemination) and hypotaurine (0.1-1 mM). Preincubation of spermatozoa with 1 mM hypotaurine pripor to oocytes insemination did not have a significant effect on fertilization rate. At 96 h after insemination, most cultured embryos developed to the 2-cell stage (68-83%), but development to blastocyst was very low (0-3%) for all treatments. Althogh 43 living offspring were produced after transfer of IVF pronuclear embryos to recipients, the delivery rate of recipients was very low (21%). The litter size of pseudopregnat recipients that produced a litter was 4 to 11, and the rate of in vitro development of transferred embryos in the pregnant recipients was 41%. It would appear that hypotaurine affects IVF rather than spermatozoa preincubation and that IVF embryos can develop to normal offspring. Next, we investigated the mechanism of penetration by field vole spermatozoa of mouse and hamster zonae pellucidae. The field vole spermatozoa that can penetrate the zonae pellucidae of the mouse and hamster could not penetrate the zonae of the rat, mastomys and Mongolian gerbil. The vole spermatozoa that bound to the zona oaf field vole oocytes underwent the acrosome reaction before passing through it. In contrast, the vole spermatozoa that bound to the zonae of mouse and hamster oocytes penetrated the zonae without any sign of the acrosome reaction. The presence or absence of proteinase/hyaluronidase inhibitors in the medium did not make any difference to zona penetration by acrosome-intact vole spermatozoa. These observations suggest that field vole spermatozoa can cross mouse and hamster zonae mechanically without assistance from zona-hydrolysing enzymes.