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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., (© 2021 Wiley Periodicals LLC.)

Glyphosate is a broad-spectrum herbicide that impairs testosterone synthesis in mammals. Leydig cells (LCs), the primary producers of testosterone, demonstrate rhythmic expression of circadian clock genes both in vivo and in vitro. The nuclear receptor NR1D1 is an important clock component that constitutes the subsidiary transcriptional/translational loop in the circadian clock system. Nr1d1 deficiency resulted in diminished fertility in both male and female mice. However, whether NR1D1 is involved in the glyphosate-mediated inhibition of testosterone synthesis in LCs remains unclear. Here, the involvement of NR1D1 in glyphosate-mediated inhibition of testosterone synthesis was investigated both in vitro and in vivo. Glyphosate exposure of TM3 cells significantly increased Nr1d1 mRNA levels, but decreased Bmal1, Per2, StAR, Cyp11a1, and Cyp17a1 mRNA levels. Western blotting confirmed elevated NR1D1 and reduced StAR protein levels following glyphosate exposure. Glyphosate exposure also reduced testosterone production in TM3 cells. In primary LCs, glyphosate exposure also upregulated Nr1d1 mRNA levels and downregulated the mRNA levels of other clock genes (Bmal1 and Per2) and steroidogenic genes (StAR, Cyp17a1, Cyp11a1, and Hsd3b2), and inhibited testosterone synthesis. Moreover, glyphosate exposure significantly reduced the amplitude and shortened the period of PER2::LUCIFERASE oscillations in primary LCs isolated from mPer2 Luciferase knock-in mice. Four weeks of oral glyphosate upregulated NR1D1 at both the mRNA and protein levels in mouse testes, and this was accompanied by a reduction in StAR expression. Notably, serum testosterone levels were also drastically reduced in mice treated with glyphosate. Moreover, dual-luciferase reporter and EMSA assays revealed that in TM3 cells NR1D1 inhibits the expression of StAR by binding to a canonical RORE element present within its promoter. Together, these data demonstrate that glyphosate perturbs testosterone synthesis via NR1D1 mediated inhibition of StAR expression in mouse LCs. These findings extend our understanding of how glyphosate impairs male fertility., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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 .

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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]