Effect Of Insulin On Luteinization Of Porcine Granulosa Cells

As a more common complication endemic to late pregnancy and early lactation,mammalian insulin resistance was one of the key obstacles affecting the fertility of perinatal sows and restricting the development of the pig industry.Studies have shown that insulin can affect mammalian fertility by influencing luteal formation,and granulosa cell luteinization was essential for lutein formation and functional maintenance,but the effect of insulin on granulosa cell luteinization was not yet clear.In this study,the effects of insulin on the proliferation,cell viability,hormone secretion and luteinization of porcine granulosa cells were detected by Ed U,CCK-8and ELISA,and autophagy was studied in combination with transcriptome sequencing,MDC staining,electron microscopy analysis and Western Blot(WB),especially the effect of changes in autophagosomes number on insulin-induced luteinization of porcine granulosa cells.This study provided theoretical basis and data support for further elucidating the pathogenesis of perinatal insulin resistance,the formulation of corresponding treatment regimens,and the mechanism of insulin regulation of follicle development.The results were as follows:1.Insulin regulated the function of porcine granulosa cellsThe effect of insulin on porcine granulosa cells function was measured by ELISA,EDU,and CCK-8.The results showed that insulin could promote the proliferation of porcine granulosa cells and promote the secretion of steroid hormones by porcine granulosa cells;insulin(2 μg/m L)treatment of porcine granulosa cells for3 d could induce luteinization of porcine granulosa cells: the concentration of progesterone was significantly increased(P<0.01),the expression of the luteinization-related genes STAR and 3β-HSD was significantly increased(P< 0.01),and the collection of porcine granulosa cells and insulin-induced porcine granulosa cells after luteinization was performed for transcriptome sequencing,and the results showed that in the process of insulin-induced porcine granulosa cells luteinization,the differential genes were enriched in MAPK,STAT and ERK.In addition to signaling pathways such as m TOR,Lysosome,Phagosomes and mitophagy,which were Autophagy-related signaling pathways.This suggested that autophagy played a role in insulin-induced luteinization of porcine granulosa cells.2.Autophagy played roles in insulin-induced luteinization of porcine granulosa cellsThe detection of autophagy levels during insulin-induced porcine granulosa cells luteinization by QRT-PCR,MDC staining,transmission electron microscopy and WB showed that in the process of insulin-induced porcine granulosa cells luteinization,although the number of autophagosomes increased,the decrease in p62 and LC3 expression levels indicated a decrease in autophagy levels.Effects of autophagosome degradation inhibitor chloroquine(CQ)and autophagosome production inhibitor 3-methyladenine(3-MA)on the level of luteinization of porcine granulosa cells after 2days of insulin induction were detected by ELISA and QRT-PCR.The results showed that the inhibition of autophagy increased the level of luteinization of porcine granulosa luteinizated cells,and inhibition of autophagosome degradation significantly increased the level of luteinization of porcine granulosa luteinizated cells.CCK-8,Ed U,and flow cytometry detected the effects of CQ on porcine granulosa luteinizated cells proliferation and apoptosis after 2 days of insulin induction.The results showed that inhibiting autophagosome degradation would promote the proliferation of porcine granulosa luteinizated cells and inhibit the apoptosis of porcine granulosa luteinizated cells.The above results showed that insulin promoted the proliferation of porcine granulocytes and the secretion of steroid hormones,and could induce luteinization of porcine granulosa cells luteinization.In the process of insulin-induced luteinization of porcine granulosa cells,the number of autophagosomes increased and the level of autophagy decreased;inhibiting autophagosome degradation(autophagosome increase)promoted the proliferation of porcine granulosa luteinizated cells and inhibited the apoptosis of porcine granulosa luteinizated cells,and thus promoted the process of luteinization.