Study On Ovarian Endoplasmic Reticulum Stress Induced By Vitrification

Objective To discuss whether endoplasmic reticulum stress of vitrified ovarian tissueis activated, and whether ovarian follicle loss existing in the the late period is associated withcell apoptosis induced by endoplasmic reticulum by the research of expression level ofGRP78, CHOP and caspase-12in the vitrified ovarian tissue.Method the ovaries peeled the outer membrane of ovaries from4weeks female ICRmice are cutted in half longitudinally and were randomly assigned into9groups(fresh group,TM group, TM+TUDCA group, vitrified group, vitrified+TUDCA group, vitrified+FSHgroup, protective agent penetrated group, protective agent+TUDCA group, protectiveagent+FSH group). The protein expression level of GRP78, CHOP and Caspase-12wasdetected by immunohistochemistry and the mRNA expression level of the three moleculeswas detected by Q-PCR. The cell apoptosis in cryopreserved ovarian tissue was detected byTUNEL.Results Immunohistochemistry detected the expression level of GRP78and CHOP inovarian tissue. The expression of ERS chaperone GRP78in vitrified group were significantlyincreased compared with fresh control group and appear statistical significance (P <0.05). Theexpression level of CHOP which is the protein related ERS apoptosis in the vitrified group was higher than fresh control group and showed statistically significance(P <0.05). Thevitrified group compared with1-1and10-1groups appeared no statistically difference andthere was also no significant difference between20-1and30-1(P>0.05). In the process tobuild the model of TUDCA alleviate endoplasmic reticulum stress, the fresh control groupacts as negative control group and the group induced by10μg/mL TM acts as positive control.Then Mix0.5mM,1mM,5mM and10mM TUDCA with10μg/mL TM respectively andcultured mouse ovaries in the mixtrue for12h, immunohistochemistry detected the expressionlevels of GRP78in ovaries.The results show that the expression levels of GRP78proteindecreased gradually with the concentrations of TUDCA increased, especially the expressionlevel of GRP78and the ovarian cell morphology in the group of10mM restored to freshgroup substantially. Real-time PCR Real-time PCR results showed that: the expression levelsof protective agent permeated group GRP78, CHOP, Caspase-12mRNA were significantlyup-regulated compared with fresh control group; GRP78, CHOP mRNA expression levels invitrified group were significantly increased compared with the fresh control group, theincrease rate of CHOP is more obvious, and the expression level of Caspase-12mRNA waslower than the fresh control group. Expression level of GRP78in the protective agentpenetrated adding FSH group increased compared with that without FSH and the expressionlevel of CHOP and Caspase-12were lower than those in protection agent penetrated group.Expression levels of GRP78and Caspase-12in the vitrified+FSH group were higher than thevitrified group and the expression level of CHOP was lower than that in the vitrified group.TUNEL assay results showed that green fluorescent located in the part of the apoptotic cells’nucleus, the red fluorescent stained by PI located in nucleus of all cells. Apoptosis level ofCells in protective agent permeated group was higher than those in vitrified group, and addingTUDCA and FSH in the protective agent penetrated group, the cryopreserved group and theTM group, the cell apoptosis level were significantly lower than the untreated group.Conclusion ovarian endoplasmic reticulum stress could induced by both protective agent penetrated and frozen processing and endoplasmic reticulum stress was significantdownward effect by TUDCA. Cells Apoptosi in protective agent penetrated group mainlycaused by ERS, endoplasmic reticulum stress level and apoptosis induced by endoplasmicreticulum stress mitigatied by adding FSH; ovarian vitrify after penetrated by protective agentacts protective effect in the vitrified group, vitrify could mitigate ERS caused by protectiveagent. Cells apoptosis in ovarian was not due to the acts of ERS and was mediated by otherapoptosis pathways.