Expression Of Mitofusin2in Pcos Rat And Its Mechanism Of Effect On Rat Follicle Development

Polycystic ovary syndrome (PCOS) is the most prevalent femaleendocrinopathy and the largest single cause of anovulatory infertility.PCOS is classically characterized by chronic anovulation,hyperandrogenism, and a polycystic ovarian morphology revealed byultrasonography. PCOS has been defined as a metabolic syndromeassociated with obesity, insulin resistance, type2diabetes, dyslipidemia,hypertension, and other cardiovascular diseases. However, its etiologyremains unclear. At present, studies of the pathogenesis of PCOS focus onthe combination of genetic factors, environmental factors andmicroenvironment changes in ovary.Follicular dysplasia is one of the pathophysiological features of PCOS.Some studies have shown that abnormalities of follicular growth anddevelopment may be the pathological basis of the anovulation and clinicalendocrine changes in PCOS, and the follicular dysplasia may be associatedwith the apoptosis of ovarian granulosa cells, but the mechanism stillremains unknown. Mitofusin-2(Mfn2) is a transmembrane GTPase embedded in themitochondrial outer membrane that mediates mitochondrial fusion. Mfn2deficiency and mutations have been linked to human neurodegenerativediseases, such as Charcot-Marie-Tooth type2A and other Hereditary Motorand Sensory Neuropathies. Increasing researches have shown that Mfn2regulates both mitochondrial fusion and mitochondrial apoptotic signalingand is involved in the pathogenesis of disease conditions such as obesity,type2diabetes, insulin resistance, and the survival of different epithelialcancer cell lines. Recent data showed that mice that are deficient in eitherMfn1or Mfn2died early in the embryonic stages of development, probablybecause of underlying mitochondrial defects. However, there is no reportabout the involvement of Mfn2in the development of follicles of patientswith PCOS. Therefore, we designed the experiment to examine the role ofrMfn2in the pathogenesis of PCOS.Part one：The expression of rMfn2in the ovaries of rat modelswith polycystic ovary syndromeObjective: To examine the expression of mitofusin2in the ovaries of ratmodels with polycystic ovary syndrome and to determine the associationbetween rMfn2expression and the occurrence of polycystic ovarysyndrome.Methods: Female Sprague Dawley rats (age:2months) were administeredletrozole in order to develop the polycystic ovary model. The control group included rats that were not treated with letrozole. Histological changes inthe rat ovaries were observed by hematoxylin and eosin staining. Serumlevels of estradiol (E2), testosterone (T), progesterone (P), folliclestimulating hormone (FSH) and luteinizing hormone (LH) were detected byradioimmunoassay. Immunohistochemistry analysis were performed todetect the localization and expression of rMfn2、Bcl-2and Bax in theovarian tissues. Real-time polymerase chain reaction and western blottingwere performed to determine the expression rMfn2in the ovarian tissues.Results: Compared with the control group, the body weight of modelgroup were lower, whereas the ovarian weight and ovarian weight-bodyweight ratio were significantly higher (P <0.05). Testosterone and LHlevels were markedly higher whereas E2and P levels were significantlylower in the model group than in the control group (P <0.05), as nosignificance in FSH levels in two groups (P>0.05). The ovarian specimensof rats in the control group exhibited follicles at various stages ofdevelopment, including secondary follicles, Graafian follicles, and freshcorpora lutea, whereas ovarian specimens of rats in the model groupshowed typical PCOS-related changes such as follicle atresia and manylarge cystic follicles with an increased number of scant granulosa cells andwithout corpora lutea. Immunostaining showed that rMfn2was highlyexpressed in granulosa cells, follicular fluid, theca internal layer, corporalutea, and ovarian stroma in rats of two groups; however, rMfn2and Bcl-2 expressions in granulosa cells was lower in the model group than that of thecontrol group, while Bax expression was higher in the model group (P <0.05). Protein and mRNA expression of rMfn2in granulosa cells in modelgroup were lower than that in control rats (P <0.05).Conclusion: The PCOS rat model induced by letrozole was successful.Apoptosis in granulosa cell of PCOS rat may increase. Down-regulation ofrMfn2may be related with the apoptosis of rat granulosa cells and involvedin the occurrence of PCOS. Part two：The construction and identification of lentivirus vectorsmediated rMfn2gene overexpressionObjective: To construct lentivirus vectors targeting rMfn2geneoverexpression (lenti-GFP-rMfn2).Methods: pLenO-GTP plasmid system linearization was carried out.RMfn2gene was amplified by PCR and recombinated homologously intothe linearized plasmid system. The plasmid was constructed viatransformation and colony positive clone identification. Double restrictiondigestion and DNA sequencing were used to identify the recombinantplasmid. The correct recombinant plasmid were co-transfected into293Tcells to collect and purify lentivirus virus particles. Meanwhile, flowcytometry was used to detect the ratio of living cells to determin the titer of virus.Results: The double restriction digestion and DNA sequencing showed thatwe successfully constructed the correct recombinant plasmid, and the greenfluorescence was visible in which transfected293T cells. These datassuggest that lenti-GFP-rMfn2was successfully constructed, with a titer of2.2×108TU/ml.Conclusion: Lentivirus vectors mediated rMfn2gene overexpression wasconstructed successfully. Part three：Effect and Mechanism of rMfn2on ovarian granulosa cellsof ratsObjective: To investigate the effect and mechanism of rMfn2on theproliferation and apoptosis of ovarian granulosa cells in vitro.Methods: Primary granulose cells isolated and collected from SD rat ovarywere cultured and identifited in vitro. Immunofuorescence was used todetect the expression of rMfn2in the ovarian granulose cells. Lentivirusvectors mediated rMfn2gene overexpression (lenti-GFP-rMfn2) weretransfected into granulosa cells in vitro, and western blotting analysis wasconducted to examine tranfection effectiveness of rMfn2in ovary. MTTassay was used to draw the cell growth curve, and flow cytometrytechnology was used to detect the cell cycle and cell apoptosis rate. Theexpression of rMfn2、Akt、p-Akt、Bcl-2and Bax proteins was detected by western blotting.Results: RMfn2expression was detected in cytoplasm of granulosa cells.Western blotting analysis showed that lenti-GFP-rMfn2transfected theovarian granulosa cells successfully. MTT assay showed that theproliferation rate of granulosa cells in MFN group (rMfn2geneoverexpression group) was higher than that in CON group (blank controlgroup) and GFP group (lenti-GFP group)(P<0.05). Cell cycle detectionshowed that in MFN group the proportion of granulosa cells in G1phasedecreased obviously, as the proportion of granulosa cells in S and G2phases increased (P<0.05). Cell apoptosis rate detection showed that thecells apoptosis rate is very low in CON group、GFP and MFN group, as nostatistically difference in three groups (p>0.05). Western blotting resultsindicated that Bcl-2protein expression significantly increased in MFNgroup, as Bax protein expression decreased, the statistical difference issignificance (P<0.05); p-Akt protein expression in MFN groupsignificantly increased than that in other two groups (P<0.05), as Aktexpression showed no significant difference in three groups (p>0.05).Conclusion: rMfn2may promote granulosa cell proliferation throughactivation of PI3K/Akt mediated signaling pathway, leading to the increaseof Bcl-2and decrease of Bad and Bax. Part four：Effect of rMfn2on follicle development of normalrats and PCOS ratsObjective: To observe the effect of rMfn2on follicle development innormal rats and rats with polycystic ovary syndrome.Methods: Sixty Female Sprague Dawley rats were randomly assigned tofour groups: CON group (Control, only injected with saline), GFP group(injected with lenti-GFP), MFN group (injected with lenti-GFP-rMfn2),ML group (orally administered letrozole on30days after the injection oflenti-GFP-rMfn2for23consecutive days). LT group (orally administeredletrozole for23consecutive days) had induced in Part one. The expressionand location of rMfn2in ovaries and other tissues was observed by thefluorescence microscope. Real time RT-PCR and western blotting wereused to quantitative analysis the expression of rMfn2、ER、PR、FSHR andLHR. Histological changes in the rat ovaries were observed by hematoxylinand eosin staining. Serum levels of estradiol (E2), testosterone (T),progesterone (P), follicle stimulating hormone (FSH) and luteinizinghormone (LH) were determined by radioimmunoassay.Results: RMfn2was overexpressed in rat ovaries in MFN group on days30after transfection. Intraovary microinjection of lenti-GFP-rMfn2resulted in a significant time-dependent overexpression of rMfn2in ratovary, enhanced with time prolongation after infection and maintainedefficient and strong expression until60days after infection. HE stainingshowed that the ovarian specimens of rats in MFN group exhibited fresh corpora lutea、 primordial follicle and antral follicles; in ML group, largecystic follicles were still found, but the number obviously decreasedcompared with LT group, and exhibited a few corpora lutea, primordialfollicle and antral follicles, even Graafian follicles. Serum levels of E2、P inMFN group were obviously higher than that in CON group、ML group andLT group, but T level is far lower than the other three groups (P<0.05);compared with LT group, E2, P levels in ML group increased obviously,LH level decreased (P<0.05), FSH and T levels showed no significantdifference in two groups (p>0.05). The protein expression of ER, PR washigher in MFN group than that in GFP group (P<0.05), there was nosignificant difference in protein expression of FSHR and LHR among thetwo group (P>0.05).Conclusion: RMfn2overexpression in rat ovary in vivo may changereproductive endocrine function, and promote the follicular development;rMfn2overexpression may inhibit the polycystic ovary induced byletrozole.