Rev-erb alpha, in conjunction with its ligands, regulates the expression of circadian clock genes in rat mature granulosa cells

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The nuclear receptor Rev-erbα has been identified as a link between circadian rhythms and metabolic processes. In addition, heme has been proved as a ligand of Rev-erbα. However, little is known about the function of Rev-erbα in ovarian circadian oscillators. In the present study, we characterized the function of Rev-erbα in mature granulosa cells (GCs) using its ligands. Immature Per2-dLuc transgenic rats were injected with eCG to prepare GCs. Per2-dLuc oscillations were measured by real-time monitoring system. Gene expression levels were examined by RT-qPCR. Heme (10 μM) significantly decreased the amplitude of Per2 oscillations in GCs with LH syncronzation compared with the Cont group. In addition, heme treatment signigicantly reduced the mRNA levels of core clock genes across 24 h. GSK4112 (GSK; agonist) and SR8278 (SR; antagonist) were used to further dissect the function of Rev-erbα. Both of them (10 μM, 2 h shock) elicited the precense of Per2 oscillations, although the period and amplitude did not shown a significant defference. Interestingly, GSK significantly induced a phase advance shift compared with SR treatment. Moreover, either GSK or SR were found to significant lengthen and shorten, respectively, the period of Per2 oscillations of GCs after LH synchronization compared with DMSO treament. LH plus DMSO treatment (Cont) entrained all examined genes (Bmal1, Per2, Rev-erbα, Dbp, and Cox2) with robust rhythmic expression, whereas both GSK and SR were found to impair the rhythmic expression of examined genes. These finding indicate that Rev-erbα plays an important role in ovarin circadian oscillators.