| Mammalian RF amide-related peptide 3（RFRP-3）can affect the histological changes of the epididymis,the number of epididymal epithelial cells and germ cells,the production of sperm and the levels of testosterone,thus affecting the reproduction of male mammals.Leydig cells（LC）,located in the loose connective tissue between the seminiferous tubules,are mainly involved in the synthesis and secretion of testosterone.The changes in the number and morphology of LC,and the decreased ability to synthesize and secrete testosterone can lead to serious male reproductive diseases.The main site of synthesis of testosterone in LC is endoplasmic reticulum（ER）,and the changes in endoplasmic reticulum homeostasis will affect the synthesis of testosterone.Steroid synthase catalyzed the synthesis of testosterone,and its expression is regulated by nuclear receptors SF-1 and Nur77.However,there are few studies on the effect of RFRP-3 on LC and the synthesis of testosterone in LC.There is no relevant report on the role of nuclear receptors and endoplasmic reticulum stress（ERS）in the effects of RFRP-3 on Leydig cell testosterone synthesis.（1）To detect the effect of RFRP-3 on mouse testicular mesenchymal cell（TM3）,different concentrations of RFRP-3(0,10-12,10-10,10-8,10-6 M)were used to treat TM3for 24 h or 48 h.The concentration and response time of RFRP-3 was screened by MTS,and then the effect of RFRP-3 on the proliferation and apoptosis of TM3 was detected by flow cytometry.Finally,the expression level of the proliferation gene Ki-67 was detected by q RT-PCR.MTS results showed that compared with other groups,the survival rate of TM3 was significantly reduced by 10-8 M RFRP-3 for 48 h（P<0.01）.FCM results showed that the percentage of cells in the G1 phase in 10-8 M RFRP-3group was higher than that in the control group（P<0.05）,while the percentage of cells in the G2+S phase was decreased（P<0.05）.The apoptosis rate of 10-8 M RFRP-3 group was also significantly increased compared with the control group（P<0.01）.The results of q RT-PCR showed that,compared with the control group,the expression level of proliferative gene Ki-67 mRNA in 10-8 M RFRP-3 group was significantly decreased（P<0.01）.These results suggested that RFRP-3 could induce apoptosis in TM3.（2）To investigate the role of nuclear receptors SF-1 and Nur77 on the effect of RFRP-3 on testosterone synthesis in LC,TM3 were treated with 10-8 M RFRP-3 for 48h in vitro,and the levels of testosterone,the synthetase genes of testosterone and the levels of protein were detected by ELISA,q RT-PCR and Western blot.Then,the mRNA expression levels of nuclear receptor gene SF-1 and Nur77 were detected by q RT-PCR.Finally,the recombinant plasmid of SF-1 and Nur77 promoter was transfected based on RFRP-3,and the activities of SF-1 and Nur77 promoter were detected by double luciferase reporter gene assay.Compared with the control group,the results showed that the testosterone level in the experimental group was decreased（P<0.05）,and the mRNA expression levels of testosterone biosynthesis-related enzymes genes（St AR,P450scc,3β-HSD and CYP17a1）in the experimental group were significantly declined（P<0.01）.The expression level of CYP17a1 protein in the experimental group was also decreased（P<0.05）.Compared with the control group,the mRNA expression levels of nuclear receptor genes SF-1 and Nur77 were significantly decreased（P<0.01）.The relative luciferase activity was significantly reduced in the 10-8 M RFRP-3 group transfected with p SF-1-Luc or p Nur77-Luc promoter recombinant plasmid compared with the control phase of the recombinant plasmid（P<0.05）.These results indicated that RFRP-3 reduced the transcriptional expression of SF-1 and Nur77 by inhibiting the transcriptional activity of SF-1 and Nur77 promotors,and thereby reduced the expression of testosterone biosynthesis related enzyme genes and proteins,and ultimately reduced testosterone synthesis in TM3.（3）In order to investigate the role of ER stress in the effect of RFRP-3 on LC apoptosis and testosterone synthesis,we added 10-8 M RFRP-3 to TM3 for 48 h in vitro.The expression levels of genes and proteins,related to ER stress pathway and apoptosis markers（Caspase 7,Caspase 9,Caspase 12）,were induced by ER stress,which were detected by q RT-PCR and Western blot.The ER stress pathway inhibitor 4-PBA was added,and q RT-PCR was used to detect the expression levels of ER stress pathway related genes and testosterone biosynthesis related enzymes.Compared with the control group,the mRNA expression levels of ER stress pathway-related genes GRP94,ATF4,CHOP,IRE1α,ATF6 and e IF2αin the experimental group were significantly increased（P<0.01）,and the expression levels of ER stress pathway-related proteins GRP78 and CHOP in the experimental group were also significantly increased（P<0.01）.Compared with the control group,the mRNA expression levels of ER stress marker apoptosis genes Caspase 7,Caspase 9 and Caspase 12 in 10-8 M RFRP-3 group were significantly increased（P<0.01）.mRNA levels of ER stress pathway-related genes GRP94,ATF4,CHOP,IRE1αand e IF2αin RFRP-3+4-PBA group were lower than those in RFRP-3group（P<0.05）.Compared with RFRP-3 group,the expression levels of testosterone biosynthesis related enzyme St AR and CYP17a1 genes in RFRP-3+4-PBA group were up-regulated（P<0.05）,and the expression levels of testosterone biosynthesis related enzyme P450scc and 3β-HSD gene were increased,but there was no significant difference.These results suggested that RFRP-3 could cause TM3 ER stress and ER stress-induced apoptosis,but PBA pretreatment could alleviate ER stress of TM3 cells,weaken the inhibitory effect of RFRP-3 on key enzymes of testosterone synthesis to a certain extent,and improve testosterone secretion.In this study,we confirmed that endoplasmic reticulum stress may play a role in RFRP-3 inhibited testosterone synthesis in TM3.In summary,RFRP-3 could inhibit the transcription of nuclear receptors in mouse LC,thereby inhibiting the expression of testosterone synthesis-related enzyme genes,suggesting that nuclear receptors may be involved in the inhibition of testosterone synthesis by RFRP-3.The endoplasmic reticulum stress pathway is involved in the process of RFRP-3 inducing the apoptosis of LC and inhibiting the synthesis of testosterone. |
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