Inhibition Of Luteinizing Hormone Signaling Pathways In Preovulatory Avian Follicles Regulates Circadian Clock Expression In Granulosa Cell

Ovulation in birds is triggered by a surge of luteinizing hormone (LH), and the ovulatory cycle is affected by the circadian rhythms of clock genes transcription levels in follicles. The influence of LH signaling cascades action on circadian clock genes was investigated using granulosa cells of preovulatory follicles with inhibitors and LH treatments from domestic hens cultured in a serum-free system. The main results are as follows:The expression of core oscillators (Bmal1, Clock, Cry1, Per2, and Rev-erbβ), clock-controlled gene (Star), Egr-1 and LHr were measured by quantitative real-time PCR. Peaks for Clock, Cry1, Per2, and Rev-erbβ genes were observed at CT12 in control groups. Significant changes in clock genes transcription levels were observed over 24 h, and the expression of Egr-1 gene was upregualted by LH treatment and peaked at CT12, All of this indicating that cell-autonomous rhythms always exist in granulosa cells. Intriguingly, the transcript levels of clock genes increased with LH treatment during 24 h of culture; especially the expression of Bmall and Per2 genes were upregulated rapidly and LH changed the cycle time of clock genes. they peaked 4 h in advance of controls and second but weaker oscillations were also observed. It appeared that LH changed the amplitude of cell-autonomous rhythm and cycle time of clock genes.To further investigate the interrelation of LH signaling cascades pathways and the influence of LH signaling action on circadian rhythm, inhibitors of cyclic adenosine monophosphate (cAMP), p38 mitogen-activated protein kinases (p38MAPK) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathways were used. With LH treatment the expression of Areg, EGFr and Egr-1 were upregulated comparing with control groups. But the transcript level of Areg decreased by blocking ERK1/2 pathway. The expression of Egr-1 were affected by among the three pathways. The transcript levels of clock genes were suppressed by blocking cAMP, but increased with similar expression patterns by blocking the p38MPAK and ERK1/2 pathways over 24 h. Thus, the influence of LH signaling cascades in chicken ovulation is mediated by the cAMP pathway and also involves the p38MAPK and ERK1/2 pathways.