Study Of Effects Of PKC Inhibitor Staurosporine On The Development Of Preantral Follicles In Mouse

Mammalian follicular development is a complex process. It mainly includes oocyte growth, meiosis, granulosa cell proliferation and differentiation. Along with, it has a collaborative role in development and regulation of oocyte and granulosa cells. Protein kinase C (PKC) superfamily consists of multiple members, mainly divided into three categories, classical PKC, model PKC and atypical PKC. Members of PKC superfamily play an important role in the regulation, proliferation and differentiation of various cell types. The activation of PKC of cumulus-oocyte complexes promotes the oocytes to resume meiosis but the activation of PKC in oocyte inhibits oocyte maturation. Conclusion from previous studies shows that the role of the PKC signaling pathway in the overall development of follicles is still vague. In the present study we used in-vitro culture model of mice preantral follicles by adding staurosporine (PKC inhibitor) to find the effects of PKC inhibition on follicular growth, proliferation and apoptosis of granulosa cells during different stages of preantral follicles. The aim of the present study was to find the regulation mechanism of PKC involved in follicular development and granulosa cell function by detection of subcellular localization of cell gap junction and the expression of Smads family members especially smad4during preantral follicle development. The main findings are given below:1. Inhibition of PKC seriously affects the development of mouse preantral follicles in vitro cultured. We stripped preantral follicles from12days female Kunming mice for in-vitro culture and1μM of PKC inhibitor staurosporine was added to the culture medium. After48hours, changes in follicular cavity were observed under the microscope. With staurosporine granulosa cells in follicles became round, the edges of follicles became blurred, oocyte atrophied and died after48hours with staurosporine injection. In order to study the role of PKC in the mature follicle in vitro ovulation, we cultured preantral follicles to antral stage and after that culture medium was replaced with maturation medium. After16hours the ovulation of oocyte (whether GVBD or extruded the first polar body) was observed. Furthermore, staurosporine inhibited ovulation or exhausted oocyte death.2. Inhibition of PKC has a significant effect on the proliferation of granulosa cells. During the growth of granulosa cells (after72hours), different concentrations (0.2μM,0.5μM,1μM) of staurosporine was added to the culture. After14hours, we observed the changes in granulosa cells and detected apoptosis rate by flow cytometry. The morphology of granular cells changed with the increased inhibitor concentration and the cell membrane was having filamentous extension protruding outward. At the same time, the early apoptotic rate also increased significantly, e.g. control was5.31%, while with0.2μM concentration apoptosis rate was16.67%, with0.5μM was26.55%and with1μM it was32.15%. The SPSS statistical analysis result shows that the apoptosis rate increased depending on increasing concentrations with a significant difference.3. Location of cell gap junction protein connexin43in preantral follicles in vitro development with inhibition of PKC had changed. After48hours treated with staurosporine of preantral follicles, we detected subcellular localization of connexin43by immunofluorescence cytochemistry. It was observed that Cx43is located between granulosa cells. With the inhibition of PKC, the cell gap junction channels were destroyed. Although the location of Cx43was still between granulosa cells, the signal was irrigular and weak. Compared with the localation of Cx43in granulosa cells of the control group, the location of Cx43in inhibited group was still on the cell membrane but Cx43can’t form the gap junction channels. At the same time, the expression of phosphorylated Cx43in granulosa cells also decreased. These results suggested that, after inhibition of PKC, the cell gap junction channels were destroyed and communication as well as transportation between granulosa cells was blocked which seriously affected development of granulosa and follicular cells.4. Expression of member of Smad family i.e.Smad4in preantral follicles had changed after PKC inhibition. In the early development of mammalian follicles, TGFβ family members, GDF9and BMP15synthesized and secreted by oocyte itself while those binding with Smads complex synthesized by surrounding granulosa cells. It plays an important role in the proliferation and differentiation of granulosa cells. Through Western blot we found that the expression of Smad4in preantral follicles with PKC inhibition has increased. These results suggest that PKC inhibition might block the communication between oocyte and granulosa cells. Inhibition of PKC may inhibit the proliferation and differentiation of granulosa cells. Finally, more Smad4were secreted by the granulosa cells to interact with GDF9and BMP15from oocyte to promote proliferation and differentiation of granulosa cells. Time extension with staurosporine treatment increased granulosa cells apoptosis and other unusual abnormalities leading either to decrease synthesis or degradation of Smad4. So the expression level of Smad4was significantly decreased.In summary, during mammalian preantral follicular development, PKC signaling pathway plays an important role. The effect is possibly through its substrate regulated gap junction protein Cx43expression and localization or joined communication and exchange between the oocyte and granulosa cells of follicles. In conclusion, PKC plays a regulating role in granulosa cell proliferation and apoptosis, oocytes growth and so on.