Circadian rhythm patterns of NO-cGMP signaling are moderately synchronized by melatonin in testosterone-producing Leydig cells

Abstract

Summary. It is established that pineal melatonin is involved in circadian regulation of testosterone secretion from Leydig cells. However, the precise routes of this regulatory involvement are still unknown. As cGMP is also regarded as modulator of steroidogenesis, we sought to study the effects of melatonin on cGMP variations and the expression of genes that encode elements of NO-cGMP signaling pathways in Leydig cells from adult rats. The melatonin effect was tested in vivo on pinealectomized and melatonin-replaced rats and ex vivo on primary culture of Leydig cells. Pinealectomy increased amplitude of serum testosterone circadian oscillation which was restored by melatonin treatment. Real-time quantitative PCR analysis revealed a circadian transcriptional pattern of Nos2 (gene encoding NO producer) and Pde5a (gene encoding cGMP remover) in Leydig cells. Pinealectomy increased transcription of Nos3 and Pde5ain certain time points (ZT11 and ZT5, respectively). Altered patterns of gene transcription were restored by melatonin-replacement. The transcription of Gucy1a3, Gucy1b3 (genes encoding subunits of cGMP producer) and Prkg1 (gene encoding the main effector of NO-cGMP signaling pathway) was not affected by pineal abolition. Although, pinealectomy affected expression of some genes of NO-cGMP signaling, the circadian variation of cGMP in Leydig cells was not significantly changed. Ex vivo studies revealed that the effect of melatonin on nitrite and testosterone production in Leydig cells is not direct but most probably mediated through the reproductive axis. Altogether, obtained results revealed circadian rhythm of NO-cGMP signaling in rat Leydig cells which is slightly synchronized by melatonin.