Molecular Mechanism On Circadian Clock Gene BMAL1 Regulation Of Testosterone Synthesis In Goat Leydig Cells

The circadian clock,as an important regulatory system,exists in nearly all cells of living organisms,which orchestrates various physiological functions with a cycle of appropriate 24h by regulating rhythmic expression of clock-controlled genes.The circadian clock protein Brain and Muscle ARNT-like 1（BMAL1）is a member of the base-helix-loop-helix（b HLH）-PAS transcription factor family,acting as a core transcriptional factor that constitutes the positive and negative feedback mechanism of circadian clock transcription and translation.BMAL1 binds to the other circadian clock protein CLOCK to form BMAL1/CLOCK heterodimer,which plays a positive role in the feedback loop.Previous studies have found that Bmal1 was expressed in the testis of wild-type mouse.Moreover,the protein BMAL1displayed a rhythmic expression pattern with a period of 24 h in mouse Leydig cells（LCs）,and a significant decline of serum testosterone levels was detected in male Bmal1^-/-mice,indicating that BMAL1 plays an important role in regulating testosterone synthesis and male reproduction.In addition,existing evidence proved that serum testosterone concentrations underwent diurnal changes in rat,mouse,and humans.However,as a ruminant,it is unclear whether there is circadian rhythmicity of goat serum testosterone levels,and whether BMAL1is involved in regulation of testosterone synthesis in the goat LCs.In this study,we proposed a hypothesis that circadian clock in goat LCs regulates testosterone synthesis mediated by BMAL1.The goat testes and LCs were taken as research models.Several cell biological and molecular biological methods,such as Kronos AB-2550real time bioluminescence monitoring system,RT-q PCR,Western blotting,ELISA,Immunohistochemistry,flow cytometry,and Dual luciferase report assay were used to detect the molecular mechanism on circadian clock gene BMAL1 regulation of testosterone synthesis in goat LCs in vivo and in vitro.The results are as following:1.ELISA results revealed that the goat serum testosterone levels exhibited robust diurnal rhythmic changes,with peak time at ZT11.2.Immunohistochemical staining showed that BMAL1 is highly expressed in goat LCs.RT-q PCR and Western blotting methods were further used to detect the m RNA and protein levels of circadian clock genes and steroidogenic genes at ZT1 and ZT13 of goat testes.The results showed that there was no significant difference at the m RNA expression levels of BMAL1,PER2,DBP and CYP11A1 in goat testes between ZT1 and ZT13,while the levels of St AR,HSD3B2 and HSD17B3 increased significantly at ZT13.Moreover,in line with the m RNA expression level,the expression level of BMAL1 protein remained constant between ZT1 and ZT13,while the levels of St AR,HSD3B2 and HSD17B3 significantly increased at ZT13.Therefore,it is reasonable to speculate that the high serum testosterone levels of goat in ZT13 may be related to the increased m RNA expression of steroidogenic genes（St AR,HSD3B2,HSD17B3）compared to ZT1.2.Further experiments demonstrated that both 22R-OH-cholesterol and h CG addition stimulated testosterone synthesis in primary goat LCs with a dose-dependent manner,indicating that the purified goat LCs possess the biological function of testosterone synthesis.Then,RT-q PCR was used to detect the circadian rhythmicity of the circadian clock genes and steroidogenesis-related genes in dexamethasone-synchronized LCs.The results showed that both circadian clock genes（BMAL1,PER1,PER2,DBP,NR1D1）and steroidogenesis-related genes（SF1,NUR77,St AR,HSD3B2,CYP17A1,CYP11A1,HSD17B3）exhibited rhythmic expression patterns in goat LCs.In addition,BMAL1-Luc oscillations with a cycle of 24.1±0.5 h were also observed in goat LCs with p LV6-BMAL1-Luc plasmid transfection.Thus,all these results suggest that there is a circadian clock system in cultured goat LCs in vitro.3.In order to detect whether circadian clock gene BMAL1 regulates the production of testosterone in goat LCs,goat LCs were treated by BMAL1 si RNA and BMAL1 overexpresion plasmids,and RT-q PCR,Western blotting and ELISA methods were adopted to investigate the effect of BMAL1 expression change on steroidogenesis-related genes expression and testosterone synthesis.The results showed that BMAL1 si RNA not only downregulated the m RNA levels of PER2,NR1D1,DBP,SF1,NUR77,GATA4,St AR and HSD3B2,but also reduced the protein levels of St AR,HSD3B2 and cell supernatant testosterone production.Conversely,BMAL1 overexpression significantly increased the m RNA and protein levels of St AR and HSD17B3,and stimulated the production of testosterone in the goat LCs.Bioinformatics analysis revealed that the promoter regions of steroidogenesis-related genes（SF1,NUR77,GATA4,DAX1,St AR,CYP17A1,CYP11A1,HSD3B2 and HSD17B3）contained circadian clock-related E-box,RORE,and D-box elements.Dual luciferase report assay further proved that BMAL1 directly activated HSD17B3 gene promoter activity in goat LCs at the transcriptional level.To sum up,the current study revealed that not only the serum testosterone levels of ruminant goat displayed diurnal rhythmic changes,but also the expressions of circadian clock genes and steroidogenesis-related genes in goat LCs exhibited robust circadian rhythmicity.More importantly,this study demonstrated that circadian clock gene BMAL1 is involved in testosterone synthesis in goat LCs by regulating the transcription of steroidogenic genes（HSD17B3）.This study provides the early theoretical basis for exploring the mechanism of BMAL1 in ruminant reproductive endocrine function,and opens up new ideas for exploring new techniques of male reproductive regulation based on the biological clock theory.