摘要
本研究系统探讨了卵泡细胞对牛卵母细胞体外成熟的影响。颗粒细胞及其单层细胞对卵母细胞成熟后的分裂率(分别为85.4%和89.4%,对照为88.5%)及囊胚发育率(分别为35.9%和41.2%,对照为37.1%)无明显影响(P>0.05),然颗粒细胞的条件液则使卵母细胞的分裂率(81.4%,对照为88.5%)明显降低(P<0.05)。加入5×10~6/mL 内膜细胞、内膜细胞的单层细胞或25%条件液到成熟培养系统中,卵母细胞的囊胚发育率均有所提高,分别为44.1%,42.6%及39.8%。进一步探讨应用内膜细胞条件液的几个问题发现:条件液在成熟液中的合适比例为50%,收集的适宜细胞培养阶段为3～4d;条件液制备前应加入10%的血清,不加促性腺激素。这些结果表明:内膜细胞可分泌一些有利于卵母细胞成熟的生长因子。这些生长因子分泌的质和量可能取决于内膜细胞的培养阶段、血清浓度及外源激素的种类。
The effects of follicular cells on the in vitromaturation of bovine oocytes were examined.The granulosa cells in either suspension or monolayers did not affect the frequency of oocytes undergoing cleavage(85.4% and 89.4% resp.,vs 88.5%,P>0.05)and development to blastocysts(35.9% and 41.2% resp.,vs 37.1%, P>0.05).However,the cleavage rate of oocytes was decreased by addition of the granulosa cell conditioned medium to the maturation medium(81.4%,vs 88.5%).The introduction of thecal cells either in suspension or in the form of the monolayer or conditioned medium to the maturation system resulted in an increase in the proportion of oocytes developing to blastocysts(44.1%,42.6% and 39.8% resp.).Several questions in the application of thecal cell conditioned medium were further determined.The optimum proportion of thecal cell conditioned medium in the maturation medium was proved to be 50%.The conditioned medium should be prepared by culturing 2～4 days of thecal cells in the medium supplemented with 10% serum but without hormones.These results indicate that thecal cells can secrete some growth factors that may favour the in vitro maturation of bovine oocytes.The quality and quantity of these factors secreted by thecal cells is dependent on the cell culture stages,presence or absence of hormones or serum.
关键词
牛
卵母细胞
体外受精
卵泡细胞
cattle
thecall cells
granulosa cells
oocytes
conditioned medium
monolayers
in vilro fertilization
