类固醇激素对牛输卵管上皮细胞分泌的调节及其分泌物对小鼠胚胎发育的影响

Effect of Steroid Hormones on Cultured in vitro Bovine Oviductal Epithelial Cell Secretion and Influence of Secretory Product on Development of Pronuclear Mouse Embryos Cultivated In vitro

模拟发情期（０～６ｄ）母牛外周血浆雌激素和孕酮变化水平，在添加相应水平１７β－雌二醇和孕酮的ＴＣＭ－１９９液中，培养间情期牛输卵管上皮细胞（ＢＯＥＣ）。５％ＳＤＳ－ＰＡＧＥ分析ＢＯＥＣ分泌物，发现上皮细胞受类固醇激素作用分泌的２类蛋白质分子量与自然发情期（０～６ｄ）分泌的特异蛋白相似。证明类固醇激素可以调节和启动间情期ＢＯＥＣ的分泌活动。当雌二醇浓度高达１ｍｇ／Ｌ时，即使不加孕酮，ＢＯＥＣ仍能分泌这２类蛋白质。在培养小鼠原核胚的ＣＺＢ培养液内添加牛输卵管上皮细胞分泌蛋白（ＢＯＥＰ），与添加间情期牛输卵管冲洗蛋白（ＢＯＰ）和小牛输卵管冲洗蛋白（ＣＯＰ）相比，能显著提高通过２－细胞阶段胚胎的百分率和桑囊形成率，表明ＢＯＥＰ能较好地促进胚胎的分裂和发育。但ＢＯＥＰ组的桑囊形成率显著低于对照组，表明ＢＯＥＰ中可能缺少某些低于Ｍｒ１．０×１０４的蛋白因子的协调作用，以及含有Ｍｒ３．０×１０４～５．６×１０４的分泌蛋白的抑制作用而阻碍胚胎分裂与发育。

Bovine oviductal epithelial cells(BOEC) during diestrus were cultured in vitro two days in TCM 199 media which were supplemented with estradiol 17 β and progesterone whose concentrations were identical with those ranged from day 0 (oestrus) up to day 6 in natural estrus bovine blood serum. After the secretory product of BOEC was separated by SDS polyacrylamide gel electrophoresis, it was divided into two kinds of macromolecular proteins of 3.0×10 4～5 6×10 4 and 8.6×10 4～9 4×10 4 respectively. During diestrus, the oviduct cells secreted the same pattern of proteins as their counterparps in vivo following the addition and action of exotic steroid hormones. The results indicated that the exogenous steroid hormones could regulate and promote the secretory activity of bovine oviduct cells during diestrus. When the estradiol 17 β concentration rose to 1 mg/L, though progesterone was not added the oviduct cells also secreted these two kinds of macromolecular proteins. The early mouse embryos(1 cell) were cultured in CZB medium supplemented with: a) fetal bovine serum (control), b) secreted proteins of BOEC(BOEP), c) dry diestrus bovine oviductal lumen flushings obtained with dialysis and lyophilization or d) dry calf oviductal lumen flushings obtained similarly to c. The percentage of embryos that passed two cell block stage(PPT) and attained morula and blastocyst stage(PMB) in Group b, c and d was 65.2%, 38.8%, 35.7% and 24.6%, 7.4%, 10.3% respectively, and the significant differences were shown between a and b or a and c (P<0.01). These findings suggested that the secretory product affected positively early mouse embryo development. Compared with the control group (79.4% PPT and 47.0% PMB), the BOEP group provided insignificant difference between a and b in PPT values (P>0.05), but significant difference in PMB values (P< 0.01 ) so that it can be concluded that BOEP dialyzed to remove all fractions less than 1.0×10 4 in molecular weight(MW) was devoid of some low MW protein factors and their associated action or 3 0×10 4～5 6×10 4 proteins secreted by oviduct cells might inhibit further development of cleaved mouse embryos to certain extent.