Analysis of inhibitory mechanism of fertilization between different species

首先，我们尝试建立了日本田鼠的体外受精(IVF)技术。精子密度(1-2×10~(-7)细胞/毫升(预培养)和1×10~(-6)细胞/毫升(人工授精))和次牛磺酸(0.1-1毫米)可提高精子密度。用1 mM亚牛磺酸溶液预先孵育精子至卵母细胞受精，对受精率无显著影响。在受精后96h，大多数培养胚胎发育到2-细胞期(68-83%)，但所有处理的囊胚率都很低(0-3%)。尽管IVF原核胚胎移植给受体后产生了43个存活后代，但受体的受精率很低(21%)。每窝产仔的假孕受体产仔数为4~11窝，移植胚胎体外发育率为41%。看来，亚牛磺酸影响试管受精，而不是精子预培养，试管受精胚胎可以发育成正常的后代。接下来，我们研究了田鼠精子对小鼠和金黄地鼠透明带精子的穿透机制。田鼠精子能穿透小鼠和仓鼠的透明带，不能穿透大鼠、沙鼠和长爪沙鼠的透明带。与卵透明带田鼠卵母细胞结合的田鼠精子在通过前经历了顶体反应。相比之下，与小鼠和仓鼠卵母细胞的卵带结合的田鼠精子穿透了卵带，没有任何顶体反应的迹象。在培养液中是否存在蛋白酶/透明质酸酶抑制剂对顶体完整的田鼠精子穿透透明带没有任何影响。这些观察表明，田鼠的精子可以在没有透明带水解酶的帮助下机械地跨越小鼠和仓鼠的透明带。

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.