The Expression And Regulatory Mechanisms In Ovarian Granulosa Cells

Objectives: TRAP-ELISA method was used to analyze the activity of telomerase in ovarian granulosa cells and to study the effect of some important factors (such as HCG, FSH, dbcAMP, Verapamil and protooncogene ) that regulate ovarian function on telomerase activity. Furthermore, the relationship among the production of E2,P0 by ovarian granulosa cells, the proliferation of ovarian granulosa cells and the telomerase activity were also investigated. It may provide some important information about ovarian function and regulating mechanisms, provide new methods to cure and prevent premature senility of ovary.Methods: Preantral and preovulatory ovarian models were prepared by extrogenesis DES and PMSG, respectively, and then granulosa cells were collected. The activity of telomerase were analyzed by TRAP-ELISA and non-denaturing acrylamide gel silver electrophoresis. The level of E2, P0 in culture medium were determined by radioimmunoassay and the proliferation index of granulosa cells were determined by MTT.Results: (1) Telomerase activity were detected in normal ovary, and the telomerase activity in granulosa cells of preantral ovary was higher than of the preovulatory ovarian granulosa cells. OD450－OD690 value was 1.420±0.4237 in the preovulatory ovarian granulosa cells, and 2.123±0.009 in preantral ovarian granulosa cells, which indicated a significant difference between them( P<0.05).(2) The telomerase activity can be significantly enhanced by FSH, HCG,dbcAMP and Verapamil treatments(P<0.01), and be significantly reduced by antisense-c-myb ODN treatment(P<0.01).(3)The results of MTT assay showed that the proliferation rate of granulosa cells in preantral ovary was significantly higher than that of in preantrory ovarian granulosa cells(P<0.05). In both preantral ovarian granulosa cells and preovulatory ovarian granulosa cells, FSH may increase granulosa cells proliferation significantly(P<0.01), while antisense-hTERT ODN inhibit granulosa cells proliferation significantly(P<0.01). (4)Telomerase activity under different factors in preantral ovary are directly related with the serection of E2 r=0.953, P<0.01.Conclusions: (1)The telomerase activity can be detected in normal ovary, and the telomerase activity in granulosa cells of preantral ovary is higher than that of preovulation.(2) the activity of telomerase can be regulated by FSH, HCG, dbcAMP, Verapamil and antisense c-mybODN. (3)The antisense hTERT ODN can significantly inhibited the ovarian granulosa cells' proliferation of indicated by MTT assay. (4)In preantral ovarian granulosa cells, telomerase activity was directly related with the secretion of E2, and none concerned with the secretion of P0. In preovulatory ovarian granulosa cells, telomerase activity had none concerned with the secretion of P0 and E2.