Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, People's Republic of China.
Key Laboratory of Animal Biotechnology of the Ministry of Agriculture and Rural affairs, Northwest A&F University, Yangling, People's Republic of China.
Am J Physiol Cell Physiol. 2022 Feb 1;322(2):C231-C245. doi: 10.1152/ajpcell.00267.2021. Epub 2021 Dec 22.
Autophagy of granulosa cells (GCs) is involved in follicular atresia, which occurs repeatedly during the ovarian development cycle. Several circadian clock genes are rhythmically expressed in both rodent ovarian tissues and GCs. Nuclear receptor subfamily 1 group D member 1 (NR1D1), an important component of the circadian clock system, is involved in the autophagy process through the regulation of autophagy-related genes. However, there are no reports illustrating the role of the circadian clock system in mouse GC autophagy. In the present study, we found that core circadian clock genes (, , , and ) and an autophagy-related gene () exhibited rhythmic expression patterns across 24 h in mouse ovaries and primary GCs. Treatment with SR9009, an agonist of NR1D1, significantly reduced the expression of , , and in mouse GCs. ATG5 expression was significantly attenuated by SR9009 treatment in mouse GCs. Conversely, knockdown increased ATG5 expression in mouse GCs. Decreased NR1D1 expression at both the mRNA and protein levels was detected in the ovaries of mice, along with elevated expression of ATG5. Dual-luciferase reporter assay and electrophoretic mobility shift assay showed that NR1D1 inhibited transcription by binding to two putative retinoic acid-related orphan receptor response elements within the promoter. In addition, rapamycin-induced autophagy and ATG5 expression were partially reversed by SR9009 treatment in mouse GCs. Taken together, our current data demonstrated that the circadian clock regulates GC autophagy through NR1D1-mediated inhibition of ATG5 expression, and thus, plays a role in maintaining autophagy homeostasis in GCs.
颗粒细胞（GCs）的自噬参与卵泡闭锁，这在卵巢发育周期中反复发生。几种生物钟基因在啮齿动物卵巢组织和 GCs 中呈节律性表达。核受体亚家族 1 组 D 成员 1（NR1D1）是生物钟系统的重要组成部分，通过调节自噬相关基因参与自噬过程。然而，目前尚无报道表明生物钟系统在小鼠 GC 自噬中的作用。在本研究中，我们发现核心生物钟基因（、、、和）和一个自噬相关基因（）在小鼠卵巢和原代 GCs 中呈现 24 小时的节律性表达模式。NR1D1 的激动剂 SR9009 显著降低了小鼠 GCs 中、和的表达。SR9009 处理显著减弱了小鼠 GCs 中 ATG5 的表达。相反，敲低增加了小鼠 GCs 中 ATG5 的表达。在 小鼠的卵巢中，NR1D1 的表达在 mRNA 和蛋白水平均降低，同时 ATG5 的表达升高。双荧光素酶报告基因检测和电泳迁移率变动分析显示，NR1D1 通过与启动子内两个假定的视黄酸相关孤儿受体反应元件结合来抑制的转录。此外，雷帕霉素诱导的自噬和 ATG5 表达在小鼠 GCs 中被 SR9009 部分逆转。综上所述，我们目前的数据表明，生物钟通过 NR1D1 介导的抑制 ATG5 表达来调节 GC 自噬，从而在维持 GCs 自噬平衡中发挥作用。
