Expression Of Mfn2 In Premature Ovarian Insufficiency And The Relationship With Premature Ovarian Insufficiency

Part I Expression of Mfn2 in premature ovarian insufficiencyObjectives: To examine the expression of Mfn2 in premature ovarian insufficiency and to determine the association between the Mfn2 expression and the occurrence of premature ovarian insufficiency.Methods: Follicular fluid was collected after ovary extraction through puncture in IVF-ET patients that were divided into two groups: normal ovarian function group(Normal group)and primary ovarian insufficiency group(POI group). Centrifugation with isometric Percoll was used to separate human granulosa cells; the protein expression of Mfn2 gene in the granular cell was detected by western blotting; To detect Mfn2 m RNA in the granular cell in the two groups by Quantitative Real-time PCR; the fluorescence microscope was used to observe the apoptosis of granulosa cell after Annexin V- FITC/PI staining.Results: Compared with the normal group, the expression of Mfn2 protein and m RNA in the POI group were significantly lower; the apoptosis of granulosa cell was increased.Conclusion: Low levels of Mfn2 may be related with the function of ovarian granulosa cells and involved in the occurrence of POI.Part II The screening of Mfn2 interfering sequence and the optimal transfection concentrationObjectives: To confirm the optimum concentration and to select effective sequences after transfection of human granulosa cells with Mfn2-si RNA.Methods: Three pairs of Mfn2-siRNA sequences and one pairs of Cy3-siRNA sequenc es were designed and synthesized. Cy3-si RNA sequences were transfected into human ovarian granulosa cells with different concentration observed by fluorescent microscope to determine an optimal concentration. The optimal concentration was used for transf ection and experiment is divided into Blank, Regent, Cy3-si RNA, Mfn2-si RNA1, Mfn2-si RNA2 and Mfn2-si RNA3 groups. q RT-PCR and Western blot were used to detect m RNA and protein expressions of Mfn2,respectively.The most effective si RNA sequenc e was evaluated based on the expression level of Mfn2.Results: Granulosa cell was observed by fluorescence microscope after transfected by Cy3-si RNA of different concentrations, results showed that 50 n M and 100 n M have the strongest fluorescence intensity. Expression of Mfn2 m RNA and protein was decreased after transfected by Mfn2-si RNA of different sequences, and especially Mfn2-si RNA3.Conclusion: 50 n M was the optimal transfection concentration, and Mfn2-si RNA3 was the best transfection sequence.Part III The mechanisms of Mfn2 on the process of premature ovarian insufficiencyObjectives: To explore the effect of Mfn2 gene silence on mitochondrial function, apoptosis related genes expressions and granulosa cell apoptosis and elucidate the role of Mfn2 in primary ovarian insufficiency(POI).Methods: Lip2000 as a medium, Mfn2-si RNA and Cy3-si RNA were respectively transfected to Granulosa cells in patients with normal ovarian function. The experiments consist of two series: Cy3-si RNA group and Mfn2-si RNA group. q RT-PCR and Western Blot were used to investigate the expression of Mfn2 m RNA and protein, in order to determine the effective silence; Cell apoptosis and mitochondrial membrane potential were observed and measured by inverted microscope and flow cytometry; Western Blot was used to detect expression levels of Cyt C,Caspases9, Caspases3, Bax and Bcl2 protein, in order to declare the signal pathway of granulosa cell apoptosis.Results: In the Mfn2-si RNA group, both Mfn2 m RNA and protein levels were decreased significantly; the mitochondrial membrane potential was decreased and granulosa cell apoptosis index increased; As well as the expression of Bcl2 was decreased, the rest four apoptosis-associated proteins expressions were all increased.Conclusion: The expression of Mfn2 was obviously lower in the Mfn2-siRNA group and the mitochondrial membrane potential decreased followed with cell apoptosis increased. We speculated that Mfn2 might involve in the pathogenesis of POI through regulating cell energy metabolism and cell apoptosis.