Inhibitory effects of LATS2 overexpression on FSHR, STAR, CYP11A1, ESR1and ESR2 mRNA, and granulosa cell (GC) proliferation. (A) GCs of the prehierarchical follicles (6–8 mm in diameter) were cultured and transfected with reconstructed pYr-adshuttle-4-LATS2 vector (OE group), pYr-adshuttle-4 empty vector (negative control) and no plasmid (blank control), respectively. The expression of LATS2 with or without transfection of the pYr-adshuttle-4-LATS2 vector for 24 h was evaluated by RT-qPCR. (B) Expression of LATS2 protein with or without transfection of the pYr-adshuttle-4-LATS2 vector was examined by western blotting. The β-actin was utilized as the loading control. (C) Effects of LATS2 overexpression on the expression of FSHR, STAR, CYP11A1, ESR1, and ESR2 mRNA in the granulosa cells. (D) The influence of LATS2 overexpression on the expression of FSHR, STAR, CYP11A1, ESR1, and ESR2 proteins. (E) The results of overexpressing LATS2 on GC proliferation were analyzed using EdU cell proliferation assay. All cell nuclei show blue fluorescence indicating Hoechst 33342 staining; the EdU-labeled cells exhibited red fluorescence showing the proliferated cells with newly synthesized DNA (original magnification × 20). Scale bars, 25 μm. (F) Quantification of cell proliferation percentage after GCs being transfected with the pYr-adshuttle-4-LATS2 vector. Values were indicated as mean ± SEM, and bars with superscript symbol present the significant difference compared with control groups at **P < 0.01, * P < 0.05.