Regulatory Mechanism Of Glycine On Porcine Oocyte Dysplasia Induced By MBP Exposure

The production of high quality gametes is a prerequisite for livestock reproduction and germplasm resource conservation.Exposure to plasticizers in the environment is one of the high risk factors that cause bottleneck and meiosis abnormalities in oocytes in vitro.At present,the metabolites of plasticizer with the highest content in serum and follicle fluid detected are Mibutyl phthalate（MBP）,which has a stable structure,may accumulate in organisms,and may potentially harm the reproductive system of humans and animals.Therefore,it is necessary to study the mechanism of MBP exposure on oocyte dysplasia and find ways to improve it.Previous studies have found that exogenous glycine supplementation can significantly improve the efficiency of oocyte maturation（IVM）and in vitro fertilization（IVF）.The purpose of this study was to investigate whether glycine can improve oocyte dysplasia induced by MBP exposure and to clarify the mechanism.The main results were as follows:（1）Detection of MBP residues in serum and follicular fluid of sows.Serum and follicle fluid samples of sows collected in three large-scale feedlots,with 5 samples per plant,were detected by ELISA and significantly higher than those in contral group,indicating that sows were at risk of continued exposure to plasticizers during growth.（2）To explore the effect of MBP exposure on porcine oocyte maturation.In vitro exposure models of MBP（50μM,100μM,200μM）were established.Compared with the control group,cumulus diffusion diameter and oocyte maturation rate were significantly decreased after exposure to MBP,and ROS fluorescence intensity was significantly increased,showing dose-induced damage.The model concentration of MBP exposure in vitro was determined to be 50μM by the fatality rate of half（LD50）,and the effect of MBP exposure on the molecular regulatory mechanism of oocytes was investigated by single-cell transcriptome sequencing（SMART-seq）.GO functional enrichment analysis showed that MBP exposure mainly affected cytoskeletal tissue and cytoplasmic microtubule structure,which resulted in impaired oocyte development.（3）Regulation of glycine on porcine oocyte developmental abnormalities induced by MBP exposure.It was found that exogenous supplementation of 6 m M glycine could significantly improve the abnormal cumulus diffusion diameter and maturation rate of oocytes induced by MBP exposure,and significantly improve the abnormal spindle and chromosome morphology caused by MBP exposure.Mitochondria-endoplasmic reticulum colocalization test results showed that,compared with the control group,MBP exposure significantly enhanced the mitochondria-endoplasmic reticulum interaction and down-regulated the expression of interacting proteins MFN1 and MFN2.The detection of Ca²⁺level showed that glycine could significantly improve the abnormal levels of[Ca_]i²⁺and[Ca²⁺]_minduced by MBP exposure,and down-regulate the expressions of apoptosis-related genes Bax,Caspase 3 and AIFM1,thus reducing cell apoptosis.The results indicated that glycine could improve the meiosis and apoptosis of porcine oocytes induced by MBP exposure by regulating the interaction between mitochondria and endoplasmic reticulum.（4）Regulation of glycine on early embryonic developmental abnormalities induced by MBP exposure.It was found that glycine could significantly improve the abnormal expression levels of H3K36me3 and SETD2 protein in MBP exposed MⅡoocytes,and significantly reduce the expression level of DNA break markerγH2AX,and further significantly improve the MBP exposed IVF cleavage rate,blastocyst rate and cell number reduction.The early embryo development reached the control level.In conclusion,glycine can significantly improve the MBP exposure induced developmental abnormalities of porcine oocytes and early embryos.This study systematically explores the effects of MBP exposure on the quality of porcine oocytes and embryonic development ability,and establishes effective technical methods to alleviate the harm,which will provide a theoretical basis for improving the effects of plasticizer exposure on reproductive performance of female animals.