The Role Of Endoplasmic Reticulum Stress And Autophagy In Goat Corpus Luteum Regression

In mammals,the corpus luteum?CL?,one of transient endocrine tissue that produces progesterone for maintaining pregnancy,develops from the follicular granulosa cells and theca cells after ovulation.The maintenance and regression of CL are closely related to estrus cycle,reproductive tract development,pregnancy maintenance and delivery.Previous study has proved that prostaglandin F2??PGF2??,a luteolysis hormone produced locally in the endometrial luminal epithelium and CL,causes CL regression.Cell apoptosis is involved in the maintenance and regression of CL.The endoplasmic reticulum?ER?is one of the essential organelles of cells,which is the site of synthesis and processing of protein and cholesterol.The homeostasis of ER is necessary for progesterone synthesis.Excess protein accumulation and Ca²⁺imbalance in ER cause ER stress,meanwhile,unfolded protein response is activated to resist the ER stress and restore ER homeostasis by inhibiting protein translation and promoting protein folding.However,when the UPR is insufficient to restore ER homeostasis in response to severe ER stress,the UPR will trigger cell apoptosis to kills abnormal cells and protect the body's health.In addition,it has been proved that UPR is also involved in the regulation of autophagy,which is also one of the factors affecting cell survival.Previous studies have shown that ER stress occurs in the regressive corpus luteum,but the UPR pathway were differently activated in rat and cattle.This suggests that UPR is involved in the regulation of CL regression,but the regulatory mechanisms of UPR are different in different species.Moreover,the underlying mechanism by which UPR regulates CL regression is still unclear.In this present study,the different stage of luteal phase of goat CLs were collected to detect the changes of the level of ER stress,apoptosis,and autophagy by western blot,immunohistochemistry and real-time PCR.Using immortalized goat luteal cells as a model,apoptosis of goat luteal cells was induced by PGF2?in vitro to simulate CL regression.The underlying regulatory role of ER stress and autophagy in PGF2?-induced goat CL regression was studied by flow cytometry,RNA interference,western blot,immunofluorescence staining.The main results are as follows:?1?ER stress was involved in goat CL maintenance and regression.The expression of GRP78 was present in the ovaries of both non-pregnant and pregnant goats and localization of GRP78 was in the CL and follicular cells.The protein of GRP78,phosphorylated-IRE1,ATF4,phosphorylated-EIF2S1,EIF2S1,cleaved ATF6,and LC3-II were highly expressed in the early and late luteal phases of the goat CL,but lower at the mid luteal phase of the goat CL.All of three UPR sensors were activated in late luteal phase of the goat CL.Increased expression of LC3-II and decreased expression of P62 indicated that autophagy occurred in late luteal phase of the goat CL.Moreover,increased cleavage of Caspase3 proved that cell apoptosis was involved in regression of late luteal phase of the goat CL.?2?1?M PGF2?induced goat luteal cells apoptosis and increased the expression of GRP78,EIF2S1,ATF4,phosphorylated-EIF2S1,phosphorylated-IRE1,cleaved ATF6,LC3-II and Bax.PGF2?treatment also decreased the expression of P62 and Bcl-2.Combined with these results,ER stress,autophagy and mitochondrial pathway were involved in PGF2?-induced goat luteal cells apoptosis.?3?we used ER stress inducer Tm to induce the same level of ER stress in goat luteal cells compared with the cell treated with PGF2?.The results showed that Tm treatment also promoted goat luteal cells apoptosis and increased the expression of Bax,cleaved Caspase9and cleaved Caspase3 and decreased the expression of Bcl-2.In addition,4-PBA decreased the apoptotic rate of goat luteal cell treated with Tm or PGF2?and inhibited Tm-or PGF2?-induced ER stress.The expression of Bcl-2 was partially recovered and the cleavage of Caspase3/9 were decreased when the ER stress was inhibited by 4-PBA.Meanwhile,4-PBA treatment decreased the expression of LC3-II,which indicated that inhibition of ER stress decreased the level of autophagy.When CQ inhibited PGF2?-induced autophagy,the luteal cell apoptotic rate was significantly increased.However,100?M CQ has no effect on PGF2?-induced ER stress of goat luteal cells.?4?the apoptotic rate of goat luteal cells treated with PGF2?was significantly decreased when IRE1 were knocked down by si IRE1.The expression of Bcl-2 was partially recovered in IRE1 knockdown luteal cells.However,although si ATF6 translation could significantly decreased the expression of ATF6,it had no effect on the apoptosis of goat luteal cells.In addition,the expression of EIF2S1 and p-EIF2S1 were decreased by transducing sh EIF2S1lentivirus.EIF2S1 knockdown observably increased apoptosis of goat luteal cells treated with PGF2?.The decreased expression of LC3-II indicated that autophagy was lowered when EIF2S1 were knocked down in goat luteal cells.In conclusion,our study revealed that ER stress-mediated UPR plays an important role in regulation of goat CL structural regression induced by PGF2?.The activated IRE1promoted mitochondrial pathway activation by inhibiting the expression of Bcl-2 in goat luteal cells.Furthermore,ER stress-mediated UPR promotes autophagy to inhibit goat luteal cell apoptosis by activating the PERK signaling pathway.These finding may provide new insight into the mechanistic pathways of regulating regression of goat CL.