Abstract:

Objective： To discuss the differential expressions of calmodulin- dependent protein kinase Ⅱ （CaMKⅡ） in ovarian tissue of the normal mice and the mice with polycystic ovary syndrome （PCOS），and to clarify the effect of CaMKⅡ on the apoptosis of ovarian granulosa cells of the PCOS mice. Methods The dissolved dehydroepiandrosterone （DHEA） was used to induce the PCOS mouse models by subcutaneous injection， and the mice in control group were injected with the equal volume of castor oil. Enzyme-linked immunosorbent assay（ELISA） method was used to detect the serum testosterone level of mice in two groups； vaginal smear was used to monitor the changes of estrous cycle of the mice in two groups； HE staining was used to observe the pathomorphology of ovarian tissue of the mice in two groups； immunofluorescence staining was used to detect the localization and fluorescence intensity of CaMKⅡ protein in ovarian tissue of the mice in two groups； Western blotting method was used to detect the expression levels of CaMKⅡ and cysteine-containing aspartate protein hydrolase 3 （Caspase-3） proteins in ovarian tissue of the mice in two groups. The short hairpin RNA （sh-RNA）-CaMKⅡ lentivirus was used to transfect the human ovarian granulosa cells （KGN cells） to establish the stable transfection system. The experiment was divided into blank control， sh-CaMKⅡ-1，and sh-CaMKⅡ-2 groups.The expression levels of CaMKⅡ and Caspase-3 proteins in the KGN cells in various groups were detected by Western blotting method. Results Compared with control group， the serum total testosterone and free testosterone levels of the mice in PCOS group were significantly increased （P<0.01）， the estrous cycle was disordered and stagnated in the middle of estrus， and the ovarian tissue showed the vacuolated follicles with fewer layers of granulosa cells， indicating that the PCOS mouse model was successfully established.The CaMKⅡ protein was expressed in the oocytes， granulosa cells， and mesenchymal cells in ovarian tissue； compared with control group， the fluorescence intensity of CaMKⅡ and expression level of protein in ovarian tissue of the mice in PCOS group were significantly decreased （P<0.01）， and the expression level of Caspase-3 protein in ovarian tissue of the mice was significantly increased （P<0.01）.Compared with blank control group， the expression levels of CaMKⅡ protein in the KGN cells in sh-CaMKⅡ-1 and sh-CaMKⅡ-2 groups were significantly decreased （P<0.01）， and the expression levels of Caspase-3 protein were significantly increased （P<0.01）. Conclusion Decreasing the CaMKⅡ protein expression level in ovarian tissue of the PCOS mice induces the increasing of Caspase-3 protein expression level， thereby promoting the apoptosis of granulosa cells.

Key words: Calmodulin-dependent protein kinases Ⅱ, Polycystic ovary syndrome, Animal model, Cysteinyl aspartate specific proteinase-3