Apoptosis Induced By Hyperandrogen In The Cultured Mouse Ovarian Granulosa Cells And Effects Of HSP10

Objective: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder affecting women of reproductive age, with the clinical manifestation of persistent anovulation and hyperandrogenism as the characterize, and the pathophysiology have yet to be determined. Recent studies have shown there was an imbalance between pro-apoptotic and anti-apoptotic in ovarian granulose cell (GC) of PCOS, and the GCs apoptosis was closely related with hyperandrogenic condition. By differential protein mass spectrum in our laboratory, we identify 69 protein differentially expressed between normal and PCOS ovaries, and among this group the most interesting protein is HSP10, which is down-regulated in PCOS ovary. Numerial studies indicate HSP10 are expressed in many tissues and involved in various biologic functions, such as cell apoptosis and proliferation, inflammatory and immune, reproductive and so on. In this study, we induce apoptosis in mouse ovarian GCs cultured in vitro by exogenous highandrogen, and observe expression of HSP10 under highandrogen; we achieve down-regulation and over-expression of HSP10 in mouse ovarian GCs cultured in vitro by use of mouse HSP10 recombinant adenovirus-delivered siRNA and over-expression vecter we have constructed succesfuliy, and then discuss the role of HSP10 on pathophysiology of mouse ovarian GCs apoptosis induced by highandrogen.Methods: (1) TUNEL analysis was used to detect and compare human GCs apoptosis in ovarian paraffin sections between normal and PCOS patients. (2) Mouse ovarian GCs cultured in vitro were treated with different concentrations of testosterone, MTT and Flow cytometry was used to detect GCs apoptosis, Western Blot was used to detect expression of active Caspase-3, Real time-PCR was used to detect expression of HSP10. (3) AdCMV-H1-siRNA/HSP10 and AdCMV-HSP10 has been successfully constructed by our group transfected AD293 cells for virus amplification, and extracted protein of mouse GCs infected AdCMV-H1- siRNA/HSP10 or AdCMV-HSP10, Western Blot was used to detect expression of HSP10, MTT was used to detect cell viability, and Flow cytometry was used to detect cell cycle and apoptosis. (4) Cultured mouse GCs were infected by AdCMV-H1-siRNA/HSP10, Western Blot and Real time-PCR was used to detect expression of proliferation and apoptosis-related factors, such as Ki67, ERK, p-ERK, Bcl-2, Bax, Caspase-9, Caspase-3. Mouse ovarian GCs were treated with PD98059, inhibitor of ERK, Western Blot and Real time-PCR was used to detect expression of apoptosis-related factors, such as p-ERK, Bcl-2, Caspase-9, Caspase-3. Mouse ovarian GCs were infected by AdCMV-HSP10, after forty-eight hours later, were treated with PD, the same methods were used to detect the expression of the apoptosis-related factors.Results: (1) Compared with control group, the ovarian GCs apoptosis index of PCOS patients was significantly increased (p<0.05). (2) Compared with control group, the cell viability and the expression of HSP10 in 10-6M and 10-5M testosterone-treated groups were significantly lower, however, the apoptosis and the expression of active Caspase-3 was higher (P<0.05). (3) HSP10 recombinant adenovirus vector was transfected into mouse granulosa cells by this platform. Compared with control group, HSP10 gene knocked down in mouse granulosa cells infected with AdCMV-siRNA/HSP10, the interfere efficiency was 76%, and overexpressed 2.5-fold in mouse granulosa cells infected with AdCMV-HSP10, (P<0.05). Compared with control group, knocking down of HSP10 in mouse GCs, the cell viability was lower; the cell cycle was arrested by G2, and significantly inhibited the expression of proliferation-related factors, such as p-ERK, Ki67, and antiapoptosis factor, such as Bcl-2; and significantly promoted the expression of proapoptosis factors, such as Caspase-9, Caspase-3 (P<0.05). (4) Compared with control group, the expression of antiapoptosis factors p-ERK, Bcl-2 was lower, and the expression of proapoptosis factors Caspase-9, Caspase-3 was higher in 50 uM PD98059 group (P<0.05). Compared with the 50 uM PD98059 group, up-regulating the expression of HSP10, the role of PD98059 on the apoptosis-related factors was inhibited (P<0.05).Conclusion: (1) The ovarian GCs apoptosis was increased in PCOS patients, which may be related to the pathophysiology of hyperandrogenism. (2) In vireo, the mouse GCs apoptosis was increased, and the expression of HSP10 was decreased in the high level of testosterone; the GCs apoptosis was increased when down-regulating the expression of HSP10, while the apoptosis was decreased when up-regulating the expression of HSP10. (3) Highandrogen inhibited the expression of HSP10, and then induced the GCs apoptosis through the mitochondrial apoptosis signal pathway. (4) These results enhanced our understanding to the pathophysiology of hyperandrogenism in PCOS, especially the regulation of HSP10 on molecular mechanism of GCs apoptosis.