Study On Mechanism Of Glycine Regulating Endoplasmic Reticulum Stress Induced By Imbalance Of Calcium Homeostasis In Porcine Oocytes

High-quality mature oocytes are essential for post-fertilization preimplantation embryonic developmental potential.However,changes in the microenvironment could imbalance cellular homeostasis that induced stress responses.Such as endoplasmic reticulum stress（ERS）which is imbalanced in calcium homeostasis caused a decreased quality of oocytes matured in vitro.In this study,we investigated the effect and mechanism of glycine on ERS induced by calcium homeostasis in in vitro porcine oocytes maturation.（1）ExperimentⅠThe role of glycine on endoplasmic reticulum（ER）function of porcine oocytes.The results showed that the addition of glycine could improve the average cumulus diameter and the first polar body excretion rate.Further experiments showed that ER level in the glycine group was significantly higher than that in the control group.The detection of ERS-related genes by real-time fluorescence quantification（q PCR）showed that the addition of glycine significantly down-regulated CHOP,Xbp1,and ATF4 genes.Calcium fluorescence staining showed that adding glycine could significantly decline the levels of cytosolic calcium([Ca²⁺]_i)and mitochondrial calcium([Ca²⁺]_m),but increase the levels of ER calcium([Ca²⁺]_ER)in porcine oocytes in vitro maturation.This experiment demonstrated that glycine regulates ER function and calcium homeostasis in porcine oocytes.（2）ExperimentⅡThe regulation effect of glycine on ERS induced by calcium homeostasis imbalance in porcine oocytes.We induced an ER stress model with thapsigargin（TG）to explore whether Glycine can reverse the ER stress induced by TG treatment and whether it is associated with calcium regulation.The results showed that glycine could significantly increase the maturation rate and ER level,and down-regulate the expression of ERS-related genes（CHOP,Xbp1,ATF4,ATF6,Grp78）in TG treated porcine oocytes.Ca²⁺levels were found to significantly reduce[Ca²⁺]_i and[Ca²⁺]_m levels,increase[Ca²⁺]_ER levels in the glycine group.And added glycine could also significantly down-regulate calreticulin CALR gene expression compared with TG treatment.In addition,the cellular immunofluorescence results demonstrated that the addition of glycine restored the intracellular distribution of calcium channel protein inositol triphosphate receptor 1（IP₃R1）,voltage-dependent anion channel 1（VDAC1）,but also promoted the IP₃R1 and VDAC1 to form cortical clusters.Adding glycine remarkably down-regulated the expression of IP₃R1,glucose regulatory protein 75（Grp75）,and VDAC1.Further studies showed that the addition of glycine could ameliorate the TG-induced decrease of mitochondrial membrane potential,significantly reduce the level of reactive oxygen species,also significantly down-regulate the expression of apoptosis-related genes（Bax,Cyto C,Caspase 3）to reduce apoptosis.The results of parthenogenetic activation（PA）showed that glycine could improve cleavage rate,blastocyst rate,and cell number after TG treatment.This experiment confirmed that glycine has a good effect on ERS induced by TG in porcine oocytes.（3）ExperimentⅢThe possible pathways of glycine to improve ERS were explored.Based on the above findings,we targeted the calcium channel protein IP₃R1.And we compared the effects of the IP₃R1 specific inhibitors Xestospondin C（Xe C）and glycine on ERS.The results showed that the addition of glycine or Xec could improve the decrease in the average cumulus diameter and the first polar body excretion rate caused by TG-induced ERS.And they also reversed the effects of TG on ER and Ca²⁺levels in oocytes.There was no significant difference between the glycine group and the Xec group.At last,the detection of early embryonic development ability after PA found that there was no significant difference in cleavage rate,blastocyst rate,and cell number among the control group,Xec group,and glycine group.This experiment confirmed that glycine and Xec had similar effects which regulated Ca²⁺homeostasis and ER function by affecting the distribution and expression of IP₃R1 during in vitro maturation of porcine oocytes.In conclusion,glycine can ameliorate ER stress in TG-exposed porcine oocytes by affecting the distribution and expression of IP₃R1 and regulating calcium homeostasis,further enhancing the developmental potential of porcine oocytes and early embryo in vitro.