The Role And Mechanism Of FSH On The ER Stress Mediated Ovarian Granulosa Cell Apoptosis

Objective Ovarian vitrification and cryopreservation is an important method for the preservation of fertility in young cancer patients.However,ischemia and hypoxia injury during transplantation is easy to cause loss and apoptosis of follicles after transplantation,which affects the survival and quality of eggs.The results of the previous study showed that 0.3 IU / mL FSH(Follicle-stimulating hormone)could reduce follicular apoptosis during ovarian cryopreservation,which may be achieved by regulating the ERS pathway.Granulocytes play an important role in follicular maturation,and the dialogue and interaction between granulosa cells and oocytes may be the cause of follicular initiation.Cx43 is an important gap junction protein in granulosa cells,which directly maintains the supply and signal expression between granulosa cells and granulocytes.The preliminary study suggests that FSH can promote follicular survival by increasing the expression of connexin 43,Play the role of anti-apoptotic,but the specific mechanism is unknown.In this study,we designed the rat ovarian granulosa cells induced by TM(tunicamycin)as a model to investigate whether FSH inhibited the apoptosis of ovarian granulosa cells through the regulation of endoplasmic reticulum stress and down-regulated by RNA interference And to explore the mechanism of endoplasmic reticulum stress-mediated apoptosis of ovarian granulosa cells,so as to further reveal the anti-apoptotic mechanism of FSH in ovarian vitrification during cryopreservation.Methods 21-day-old SD rats were ovariated and cultured in vitro.The ovarian granulosa cells were isolated and cultured by morphological observation and cell immunofluorescence of granulosa cell-specific marker molecule FSHR.The application of endoplasmic reticulum stress inducing agent tunicamycin,TM)induced the endoplasmic reticulum stress model of granulosa cells and screened the best TM concentration.The use of FSH and endoplasmic reticulum stress-specific inhibitor TUDCA inhibited TM-induced endoplasmic reticulum stress signaling-mediated granulocyte The expression of granulosa cell related genes and proteins was detected by real-time PCR and western blot.The results showed that FSH inhibited the apoptotic signaling pathway mediated by endoplasmic reticulum.The expression of CX43 gene was down-regulated by RNA interference technique.The CX43 gene and protein expression were detected by real-time quantitative PCR and western blot.The apoptotic rate of granulosa cells was detected by flow cytometry.The expression of caspase-12,CHOP and p-JNK,cleaved caspase-3,were detected by immunofluorescence.The apoptotic cells induced by down-regulation of CX43 were detected by activating endoplasmic reticulum Stress pathway of the apoptotic signal.Results(1)The optimal concentration of TM was 10μg / mL by detecting cell proliferation curve and endoplasmic reticulum stress-related molecules.The optimal intervention concentration of FSH was 0.3IU / mL.The optimal intervention concentration of TUDCA was: 0.5 mg / mL.(2)The results of flow cytometry showed that FSH could decrease the apoptosis rate of ovarian granulosa cells induced by TM.The results of GRP78 protein expression in FSH treatment group showed that the expression of GRP78 in granulosa cells was significantly lower than that in TM group(p <0.01).The results of CHOP protein analysis showed that the CHOP expression of granule cells in FSH treated group was significantly lower than that in TM group(P <0.001).The results of quantitative analysis of ATF6 protein showed that the expression of ATF6 in FSH-treated group was higher than that in TM group(p <0.05).These results suggest that FSH can protect the apoptosis of ovarian granulosa cells induced by TM by ERS,which may be achieved by inducing ERS to carry out UPR protection pathway.(3)The results of real-time PCR,western blot and flow cytometry revealed that the CX43 gene and protein level were significantly down-regulated and the apoptosis of ovarian granulosa cells was increased after lentivirus interfered with ovarian granulosa cells.(4)The expressions of caspase-12,CHOP and p-JNK were detected in the expression of caspase-12,CHOP and p-JNK,and the expression of caspase-12,CHOP and p-JNK were observed.Consistent with the apoptotic marker protein cleaved caspase-3 is also located in the apoptotic ovarian granulosa nucleus,indicating that endoplasmic reticulum stress pathway is involved in granulocyte apoptosis.Conclusion(1)FSH could inhibit the apoptosis of ovarian granulosa cells induced by endoplasmic reticulum stress.(2)FSH may inhibit the endoplasmic reticulum stress-mediated granulocyte cell apoptosis by modulating CX43.