Circadian clock regulates granulosa cell autophagy through NR1D1-mediated inhibition of ATG5

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 (Bmal1, Per2, Nr1d1, and Dbp) and an autophagy-related gene (Atg5) 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 Bmal1, Per2, and Dbp in mouse GCs. ATG5 expression was significantly attenuated by SR9009 treatment in mouse GCs. Conversely, Nr1d1 knockdown increased ATG5 expression in mouse GCs. Decreased NR1D1 expression at both the mRNA and protein levels was detected in the ovaries of Bmal1-/- mice, along with elevated expression of ATG5. Dual-luciferase reporter assay and electrophoretic mobility shift assay showed that NR1D1 inhibited Atg5 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.