The Role And Mechanism Of Luman In The Testosterone Secretion Of Mouse Leydig Cell

Testosterone,secreted by testis Leydig cells,was the most important steroid hormone in male animals.Testosterone plays an important role in maintaining secondary sexual characteristics and promoting sperm maturation.There are multiple factors and pathways involved in the regulation of testosterone synthesis.Luman/CREB3,basic leucine-zipper(b-ZIP)family transcription factor,was widely expressed in animals.The expression of Luman in mouse uterus and ovary was correlated with reproductive cycle.In mouse ovarian granulosa cells,Luman knock-down caused a decrease of estrogen and progesterone secretion.In adrenal gland,Luman knockout increased the secretion of glucocorticoid and enhance the activity of glucocorticoid receptors.These results indicated that Luman plays an important role in the process of hormone secretion.Luman also act as a bridge between the three endoplasmic reticulum stress(ERS)pathways.However,the expression of Luman in male mouse reproductive system was not clear.In this study,Western-blotting,RT-qPCR,ELISA,Immunohistochemistry(IHC),Immunofluorescence(IF),RNAi,testicular micro-injection,Flow cytometry(FCM)and dual-luciferin reporting system were used to explore(1)the expression pattern of Luman in male mice reproductive system;(2)the location and expression of Luman in mouse testicular Leydig cells and its effect on testosterone secretion;(3)the mechanism of Luman on regulating testosterone secretion;(4)the role of Luman played in hCG-induced ERS in MLTC-1 cell.1.IHC,IF,RT-qPCR and Western blotting results indicated that Luman was widely expressed in mouse testes,epididymis,vas deferens,seminal vesicles,coagulated glands and preputial glands,etc.,and the expression relatively high in epididymis,testis and vas deferens.In the epididymis,Luman both expressed in the wall of the epididymis and sperm.In sperm,Luman was mainly expressed in the tail of mature sperm and the expression level increased with the increase of sperm maturation.In testis,Luman was specifically expressed in testicular Leydig cells and the expression dispalyed an age dependent increase manner.2.To explore the function of Luman in MLTC-1,primary Leydig cells(PLC)and in vivo testicular Leydig cells,ELISA RT-qPCR,Western-blotting,IF and RNAi were used.RT-qPCR,Western-blotting and IF results indicated that shLuman significantly knock-down80% Lman protein in MLTC-1.Luman knock-down increased testosterone secretion and StAR,Cyp11a1,3β-HSD and Cyp17a1 expression in MLTC-1.ELISA,RT-qPCR andWestern-blotting results showed taht in vivo shLuman significantly decreased Luman expression and increased the serum testosterone concentration.RT-qPCR,Western-blotting and IF results indicated that si-Luman significantly knock-down 75% Luman protein in PLC,with an interference rate of.Luman knock-down increased testosterone secretion and StAR,Cyp11a1,3β-HSD and Cyp17a1 expression in PLC.3.RT-qPCR,ELISA and dual-luciferin reporting system were used to investigate the mechanism of Luman regulating testosterone secretion in MLTC-1.Luman knock-down up-regulated the expression of steroidogensis genes by increasing the activity of steroid hormone synthesis related nuclear receptors Nur-77 and SF-1 promoters;Luman knock-down increased the activity of LH/CG receptor promoter and active the adenylate cyclase,which increased the intracellular cAMP concentration.Then,the increased cAMP activated the PKA pathway,which up-regulated the expression of steroidogenesis genes expression.4.RT-qPCR and FCM results showed that Luman knock-down significantly reduced the proportion of MLTC-1 cells in S phase by up-regulating the expression of Cyclin A1,Cyclin B1 and Cyclin D2..Western-blotting results indicated that 10 IU/mL and 20 IU/mL hCG casuaed an significant increase of GRP78,ATF6,p-EIF2S1 and pIRE1 in MLTC-1 and the expression of these proteins displayed an hCG treat time related increase manner.Western blotting,RT-qPCR,ELISA results indicated that,with 10 IU/mL hCG treatment,Luman knock-down significantly increased the testosterone secretion of MLTC-1.Also,Luman knock-down up-regulated the expression of Cyp11a1,p-IRE1,p-EIF2S1 and BCL-2,and down-regulated the expression of GRP78,CHOP and Caspase-3.These results indicated that Luman knock-down increased the expression of UPR related proteins and apoptotic inhibitors,reduced the expression of apoptotic proteins and factors,promoted cell survival and function recovery,and increased the secretion of testosterone.This experiment revealed the expression pattern of Luman in male mice reproductive system,explored the mechanism of Luman on regulating testosterone secretion,and supplemented the role of Luman played in ERS.It provided a starting point for further study of the role Luman played in mammalian reproductive process and a new direction for understanding the relationship between ERS and endocrine.