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Testosterone is produced by Leydig cells (LCs) and undergoes diurnal changes in serum levels in rats, mice, and humans, but little is known in goats. The present study revealed that goat serum testosterone levels displayed diurnal rhythmic changes (peak time at ZT11.2). Immunohistochemical staining showed that BMAL1, a circadian clock protein, is highly expressed in goat LCs. ELISA revealed that both hCG (0–5 IU/ml) and 22R‐OH‐cholesterol (0–30 μM) addition stimulated testosterone synthesis in primary goat LCs in a dose‐dependent manner. Treating goat LCs with hCG (5 IU/ml) significantly increased intracellular cAMP levels. Additionally, real‐time quantitative polymerase chain reaction (PCR) analysis revealed that the circadian clock (BMAL1, PER1, PER2, DBP, and NR1D1) and steroidogenesis‐related genes (SF1, NUR77, StAR, HSD3B2, CYP17A1, CYP11A1, and HSD17B3) showed rhythmic expression patterns in goat LCs following dexamethasone synchronization. Several Bmal1‐Luc circadian oscillations were clearly observed in dexamethasone‐treated goat LCs transfected with the pLV6‐Bmal1‐Luc plasmid. BMAL1 knockdown significantly downregulated mRNA levels of PER2, NR1D1, DBP, StAR, HSD3B2, SF1, NUR77, and GATA4, and dramatically decreased StAR and HSD3B2 protein levels and testosterone production. In contrast, BMAL1 overexpression significantly increased the mRNA and protein expression levels of StAR and HSD17B3 and enhanced testosterone production. Reporter assays revealed that goat BMAL1, or in combination with mouse CLOCK, activated goat HSD17B3 transcription in vitro. These data indicate that BMAL1 contributes to testosterone production by regulating transcription of steroidogenesis‐related genes in goat LCs, providing a basis for further exploring the underlying mechanism by which the circadian clock regulates ruminant reproductive capability. [ABSTRACT FROM AUTHOR]

Zearalenone (ZEA), a mycotoxin, is known to impair reproductive capability by disrupting the synthesis and secretion of testosterone by Leydig cells (LCs), although the mechanism is unknown. Robust rhythmicity of circadian clock and steroidogenic genes were identified in LCs. The aim of this study was to examine whether ZEA significantly attenuated the transcription of core clock genes (Bmal1, Dbp, Per2, and Nr1d1) as well as steroidogenic genes (StAR, Hsd3b2, and Cyp11a1) in mouse testis Leydig cell line (TM3). Western blotting confirmed declines in BMAL1, NR1D1, and StAR protein levels. ZEA also suppressed secreted testosterone levels. In primary LCs, isolated from PER2::LUCIFERASE reporter gene knock in mice, ZEA diminished the amplitude of PER2::LUC expression, and induced a phase shift and period extension. In primary LCs, ZEA also suppressed the expression levels of core clock and steroidogenic genes, reduced protein levels of BMAL1, and decreased testosterone secretion. In vivo expression of core clock and steroidogenic genes were reduced in testes of mice exposed to ZEA for 1 week leading to decreased serum testosterone levels. In summary, data suggest that ZEA may impair testosterone synthesis through attenuation of the circadian clock in LCs culminating in reproductive dysfunction in male mammals. [ABSTRACT FROM AUTHOR]

The circadian clock system plays an important role in regulating testosterone synthesis in mammals. Male Bmal1 −/− mice are infertile with low serum testosterone levels and decreased expression of testicular steroidogenic genes, suggesting that circadian clock genes regulate testosterone biosynthesis by activating steroidogenic gene transcription. However, whether the circadian clock regulates testosterone production via other genes remains unknown. Using Bmal1 −/− mice and their wild-type (WT) siblings, we aimed to identify additional genes by which the circadian clock regulates testosterone synthesis. WT and Bmal1 −/− mouse testes sections had similar normal morphologies, although there was a decrease in testicular spermatozoa in the Bmal1 −/− mice. Low serum testosterone levels were detected in the Bmal1 −/− mice. RNA sequencing identified 37 and 48 genes that were differentially expressed between WT and Bmal1 −/− mouse testes at circadian time (CT2 and CT14), respectively. The cholesterol metabolism pathway was significantly enriched in the KEGG pathway analysis, and there was lower expression of three apolipoprotein genes (Apoa1 , Apoa2 , and Apoc3) at CT2 in the testes of Bmal1 −/− mice than in those of WT mice. These decreases in Apoa1 , Apoa2 , and Apoc3 expression were verified by quantitative polymerase chain reaction analysis, which also revealed downregulation of the expression of the circadian clock (Per2 , Dbp , and Nr1d1) and steroidogenic (StAR , Cyp11a1 , and Hsd17b3) genes. The expression of circadian clock genes was relatively stable in WT mice over a 20-h period, whereas there was clear circadian rhythmic expression of Apoa1 , Apoa2 , Apoc3 , StAR , Cyp11a1 , Hsd3b2 , and Hsd17b3. Bmal1 −/− mice showed severely reduced expression of testicular circadian clock genes at three time points (CT4, CT12, and CT20), and a reduction in mRNA expression levels of Apo (Apoa1 , Apoa2 , and Apoc3) and steroidogenic (StAR , Cyp11a1 , Hsd3b2 , and Hsd17b3) genes. Oil Red O staining showed decreased lipid aggregation in the Leydig cells of Bmal1 −/− mouse testes. Considering the vital role of Apo genes in high-density lipoprotein formation and cholesterol transport, the present data suggest that the circadian clock system regulates testosterone production by orchestrating the rhythmic expression of Apo genes. These data extend our understanding of the role of the circadian clock in regulating testosterone production in mammals. • Robust rhythmic expression of apolipoprotein genes was detected in WT mice testes. • The rhythmic expression of apolipoprotein genes was lost in Bmal1 −/− mice testes. • Reduced expression of apolipoprotein genes was observed in Bmal1 −/− mice testes. • The cholesterol metabolism pathway is severely impaired in Bmal1 −/− mice testes. • Lipid accumulation in Bmal1 −/− mice Leydig cells was clearly attenuated. [ABSTRACT FROM AUTHOR]

The circadian system performs an important role in mammalian reproduction with significant effects on hormone secretion. Nuclear receptor subfamily 1 group D member 1 (NR1D1) functions as a transcriptional repressor in the circadian system and affects granulosa cells (GCs), but how it regulates estrogen synthesis has not been clarified. We investigated the effect of NR1D1 on estrogen synthesis and found that NR1D1 was highly expressed in GCs, mainly in cell nuclei. Additionally, the expression of NR1D1 and estrogen synthesis key genes CYP19A1 , CYP11A1 and StAR showed rhythmic changes in porcine ovarian GCs. Activation of NR1D1 enhances its ability to inhibit the transcriptional activity of CYP19A1 by binding to the RORE on the CYP19A1 promoter, resulting in a decrease in estradiol content. Interference with NR1D1 can eliminate the transcriptional inhibition of CYP19A1 and promote the synthesis of estradiol. The results suggest that the hormone secretion of the ovary itself is also regulated by the biological clock, and any factors that affect the circadian rhythm can affect the endocrine and reproductive performance of sows, so the natural rhythm of sows should be maintained in production. • NR1D1 was highly expressed in granulosa cells, mainly in cell nuclei. • NR1D1 inhibits the synthesis of estradiol in granulosa cells. • NR1D1 regulates estrogen synthesis through affecting the transcription of CYP19A1. [ABSTRACT FROM AUTHOR]

The circadian clock and autophagy are essential biological mechanisms involved in regulating many physiological processes. Accumulating evidence has revealed that autophagic activity is regulated by the circadian clock system. However, whether autophagy regulates the circadian clock system remains unclear. In this study, rapamycin and AICAR, two classical activators of autophagy, were used to create autophagy activation models in BMAL1-dLuc U2OS cell line. The results showed that the mRNA expression of MAP1LC3B and ATG5 were significantly upregulated after autophagy activation, whereas the mRNA expression of circadian clock genes (BMAL1 , PER2 , REV-ERBα , and DBP) were significantly decreased. Consistent with these data, the relative ratio of LC3-II/LC3-I and the protein level of ATG5 were increased after rapamycin or AICAR treatment. In contrast, BMAL1 and REV-ERBα levels were decreased. Notably, the mRNA expression of circadian clock genes (BMAL1 , PER2 , REV-ERBα , and DBP) and autophagy-related genes (MAP1LC3B and ATG5) showed rhythmic expression patterns in both untreated and rapamycin/AICAR-treated U2OS cells. Moreover, the autophagy inhibitor 3-methyladenine partially reversed the inhibitory effects of autophagy on circadian clock genes expression and BMAL1-Luc oscillations. Another critical finding was that ATG5 knockout alleviates the inhibitory effect of rapamycin-mediated autophagy activation on the circadian clock oscillators in U2OS cells. Collectively, our data indicate that autophagy activation attenuates the circadian clock oscillators in U2OS cells via the ATG5 pathway. • Autophagy activation results in damped circadian clock genes expression in U2OS cells. • 3-Methyladenine alleviates the inhibition of autophagy on BMAL1-Luc oscillations. • ATG5 knockout attenuates the inhibitory effect of autophagy activation on cell clock. [ABSTRACT FROM AUTHOR]

Bisphenol A (BPA) is an endocrine-disrupting compound that impairs testosterone synthesis in male mammals. A circadian clock gene deficiency leads to diminished fertility and even infertility in male mice. However, whether circadian clock signaling pathways mediate the suppressive effect of BPA on testosterone synthesis in Leydig cells (LCs) remains unknown. The present study aims to detect the effect of BPA on cellular circadian clock and testosterone synthesis in mouse LCs, and examine the mechanisms underlying NR1D1 signaling. BPA treatment significantly attenuated the transcription levels of Nr1d1 and steroidogenic genes (Hsd3b2 and Hsd17b3) in TM3 cells, but increased other circadian clock gene levels (Per2 and Dbp). BPA treatment also significantly downregulated NR1D1 and StAR protein expression, but upregulated BMAL1 protein expression in TM3 cells. Furthermore, there was a marked decline in testosterone production in BPA-treated TM3 cells. Intraperitoneal injection of BPA profoundly reduced NR1D1 and StAR protein levels and steroidogenic gene transcription levels (Cyp11a1 , Hsd3b2 , and Hsd17b3), while enhancing BMAL1 protein and other circadian clock gene (Per2 and Dbp) levels in mouse testes. Notably, serum testosterone levels were also drastically reduced in BPA-treated mice. Moreover, SR9009, an NR1D1 agonist, augmented testosterone production in TM3 cells via elevated expression of steroidogenic genes (StAR, Cyp11a1 and Hsd17b3). Conversely, Nr1d1 knockdown inhibited testosterone accumulation and attenuated steroidogenic gene expression. Moreover, treatment with SR9009 partially reversed the BPA effect on the circadian clock and testosterone production. Taken together, our study demonstrates that BPA perturbs testosterone production, at least partially, via inhibiting NR1D1 signaling in LCs. • BPA attenuates testosterone synthesis in Leydig cells in vivo and in vitro. • BPA administration alters PER2::LUC oscillations in primary Leydig cells. • SR9009, an agonist of NR1D1, augments testosterone production in TM3 cells. • Knock-down of Nr1d1 represses testosterone production in TM3 cells. • BPA perturbs testosterone synthesis via inhibiting NR1D1 signaling in Leydig cells. [ABSTRACT FROM AUTHOR]