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 mPer2knock-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. Luciferase
