Northwest A&F University, Yangling 712100, Shaanxi, China; Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China; Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China.
Department of Physiology, Key Laboratory of Cellular Physiology, Ministry of Education, Shanxi Medical University, Taiyuan, China.
Sci Total Environ. 2021 Sep 1;785:147323. doi: 10.1016/j.scitotenv.2021.147323. Epub 2021 Apr 25.
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 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.
草甘膦是一种广谱除草剂，可损害哺乳动物的睾丸酮合成。Leydig 细胞（LCs）是睾丸酮的主要生产者，在体内和体外均表现出昼夜节律基因的节律性表达。核受体 NR1D1 是昼夜节律钟系统中重要的时钟组成部分，构成了辅助转录/翻译循环。Nr1d1 缺陷导致雌雄小鼠的生育能力下降。但是，NR1D1 是否参与草甘膦介导的 LCs 中睾丸酮合成的抑制作用尚不清楚。在这里，研究了 NR1D1 在草甘膦介导的睾丸酮合成抑制中的作用，包括在体外和体内。TM3 细胞暴露于草甘膦可显著增加 Nr1d1 mRNA 水平，但降低 Bmal1、Per2、StAR、Cyp11a1 和 Cyp17a1 mRNA 水平。Western blot 证实草甘膦暴露后 NR1D1 升高和 StAR 蛋白水平降低。草甘膦暴露还降低了 TM3 细胞中的睾丸酮产生。在原代 LCs 中，草甘膦暴露也上调了 Nr1d1 mRNA 水平，并下调了其他时钟基因（Bmal1 和 Per2）和类固醇生成基因（StAR、Cyp17a1、Cyp11a1 和 Hsd3b2）的 mRNA 水平，并抑制了睾丸酮合成。此外，草甘膦暴露显著降低了从 mPer2 敲入小鼠分离的原代 LCs 中 PER2::LUCIFERASE 振荡的振幅和缩短周期。口服草甘膦 4 周可上调小鼠睾丸中的 Nr1d1 mRNA 和蛋白质水平，同时 StAR 表达减少。值得注意的是，用草甘膦处理的小鼠血清睾丸酮水平也明显降低。此外，双荧光素酶报告和 EMSA 测定表明，在 TM3 细胞中，NR1D1 通过与启动子内的典型 RORE 元件结合抑制 StAR 的表达。综上所述，这些数据表明，草甘膦通过 NR1D1 介导的抑制 StAR 表达来破坏小鼠 LCs 中的睾丸酮合成。这些发现扩展了我们对草甘膦如何损害男性生育力的理解。
