REVERBA couples the circadian clock to Leydig cell steroidogenesis.

ABSTRACT

The involvement of the molecular clock in regulating cell physiological processes on a specific time scale is a recognized concept, yet its specific impact on optimizing androgen production in Leydig cells has been unclear. This study aimed to confirm the role of the REVERBA (NR1D1) gene in controlling the transcription of key genes related to Leydig cell steroid production. We investigated daily variations by collecting Leydig cells from rats at various times within a 24-h period. Chromatin immunoprecipitation study showed a time-dependent pattern for genes linked to steroid production (Nur77, Star, Cyp11a1, and Cyp17a1), which closely matched the 24-h REVERBA levels in Leydig cells, peaking between zeitgeber time (ZT) 7-11. To understand the physiological significance of REVERBA's interaction with promoters of steroidogenesis-related genes, Leydig cells from rats at two different times (ZT7 and ZT16; chosen based on REVERBA expression levels), were treated with either an agonist (GSK4112) or an antagonist (SR8278). The results revealed that the REVERBA agonist stimulated gene transcription, while the antagonist inhibited it, but only when REVERBA was sufficiently present, indicating a reliance on REVERBA's circadian fluctuation. Moreover, this REVERBA-dependent stimulation had a clear impact on testosterone production in the culture medium, underscoring REVERBA's involvement in the circadian regulation of testosterone. This study indicates that REVERBA, in addition to being a core component of the cellular clock, plays a key role in regulating androgen production in Leydig cells by influencing the transcription of critical steroidogenesis-related genes.