Objective:To investigate the expression of placenta expressed transcript 1(Plet1)in ovarian tissue of the letrozole-induced model rats of polycystic ovary syndrome(PCOS)and its effect on the proliferation of rat ovarian granulosa cells,and to clarify the possible mechanism by which Plet1 may contribute to the pathogenesis of PCOS.Methods:The ovarian tissue samples from the rats collected in previous studies were used and divided into control and PCOS groups.Real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting methods were used to detect the expression levels of Plet1 mRNA and protein in ovarian tissue of the rat in two groups.Additionally,twenty-four rats underwent vaginal smear cytology were divided into four groups by estrous cycle phase:proestrus,estrus,metestrus and diestrus.RT-qPCR was used to detect the expression level of Plet1 mRNA in ovarian tissue of the rats in various groups,and immunohistochemistry(IHC)method was used to detect the location of Plet1 expression in the rat ovarian tissue in various groups.The rat ovarian granulosa cells were transfected and divided into control group,si-Plet1-rat-266 group,si-Plet1-rat-383 group,and si-Plet1-rat-554 group.Cell counting kit-8(CCK-8)method was used to assess the cell proliferation activities of rat ovarian granulosa cells in various groups,and RT-qPCR method was used to detect the expression levels of cyclin-d epend ent kinase 6(CDK6)and P53 mRNA in rat ovarian granulosa cells in various groups.Results:The RT-qPCR results revealed that Plet1 mRNA was expressed in the ovaries of normal rats,while no statistically significant difference was observed across estrous cycle phases(P＞0.05).The immunohistochemistry results showed that the expression of Plet1 protein was mainly localized in ovarian granulosa cells and luteal cells in the rat ovarian tissue.Compared with control group,the expression levels of Plet1 mRNA and protein in ovarian tissue of the rats in PCOS group were significantly decreased(P＜0.05).The RT-qPCR results showed that compared with control group,the expression level of Plet1 mRNA in ovarian granulosa cells in si-Plet1-rat-383 group was decreased(P＜0.01),exhibiting the most pronounced reduction.Compared with control group,the proliferation activity of rat ovarian granulosa cells in si-Plet1-rat-383 group was decreased(P＜0.05).Compared with control group,the expression levels of CDK6 and P53 mRNA in rat ovarian granulosa cells in si-Plet1-rat-383 group were significantly decreased(P＜0.05 or P＜0.01).Conclusion:Plet1 protein is predominantly expressed and localized in granulosa cells and luteal cells in normal rat ovarian tissue.Its expression is downregulated in the ovarian tissue of PCOS model rats,and interference with Plet1 gene expression may inhibit the proliferation of rat ovarian granulosa cells.

Objective:To discuss the effect of over-expression of solute carrier family 7 member 5(SLC7A5)gene on the apoptosis of ovarian granulosa cells of the rats,and to clarify its related mechanism.Methods:Four 3-week-old SPF grade SD female rats were used to extract the primary ovarian granulosa cells of the rats.These cells were divided into negative control group(NC group)and follicle-stimulating hormone receptor(FSHR)staining group(FSHR group).Immunofluorescence staining was used to detect the expressions of FSHR protein in the ovarian granulosa cells of the rats to identify the successful isolation of the primary ovarian granulosa cells of the rats.The ovarian granulosa cells were divided into control group(transfected with empty vector plasmid)and OE-SLC7A5 group(transfected with SLC7A5 over-expression plasmid).Real-time fluoresscence quantitative PCR(RT-qPCR)and Western blotting methods were used to verify the transfection efficiency of the cells;flow cytometry was used to detect the apoptotic rates and cell cycle percentages of the ovarian granulosa cells in two groups;RT-qPCR method was used to detect the expression levels of SLC7A5,cysteinyl aspartate specific proteinase(Caspase)-3,Caspase-8,and tumor necrosis factor-α(TNF-α)mRNA in the ovarian granulosa cells in two groups;Western blotting method was used to detect the expression levels of SLC7A5,Caspase-3,cleaved Caspase-3,Caspase-8,cleaved Caspase-8,and TNF-α proteins in the ovarian granulosa cells in two groups.Results:The fluorescence microscope observation results showed that the ovarian granulosa cells appeared spindle-shaped or irregular and specifically expressed FSHR.No FSHR green fluorescence was observed in NC group,while FSHR green fluorescence expression was observed in FSHR group,indicating successful isolation of primary ovarian granulosa cells of the rats.Compared with control group,the expression levels of SLC7A5 mRNA and protein in the ovarian granulosa cells in OE-SLC7A5 group were significantly increased(P<0.05),indicating successful transfection of SLC7A5 over-expression plasmid into the ovarian granulosa cells.The flow cytometry results showed that compared with control group,the apoptotic rate of the cells in OE-SLC7A5 group was significantly increased(P<0.05).Compared with control group,the percentage of the ovarian granulosa cells at S phase in OE-SLC7A5 group was significantly decreased(P<0.05).The RT-qPCR results showed that compared with control group,the expression levels of TNF-α,Caspase-3,and Caspase-8 mRNA in the ovarian granulosa cells in OE-SLC7A5 group were significantly increased(P<0.05).The Western blotting results showed that compared with control group,the expression levels of TNF-α,Caspase-8,cleaved Caspase-8,Caspase-3,cleaved Caspase-3,and SLC7A5 proteins in the ovarian granulosa cells in OE-SLC7A5 group were significantly increased(P<0.05).Conclusion:The increased expression of SLC7A5 protein promotes the apoptosis of the granulosa cells by upregulating the expressions of TNF-α,Caspase-8,and Caspase-3 apoptotic pathways.