CAMP/PKA/CREB signaling pathway-mediated effects of melatonin receptor genes on clock gene expression in Bactrian camel ovarian granulosa cells

Abstract

The cAMP/PKA/CREB pathway is involved in the regulation of melatonin during important physiological activities in mammals. However, the regulation of circadian clock genes in ovarian granulosa cells remains unclear. Herein, we determined the relationship between melatonin and biological clock genes using cultured Bactrian camel ovarian granulosa cells. The enzyme-linked immunosorbent assays showed that the cAMP content was reduced when melatonin receptor (MT) genes or cryptochrome (Cry) genes were overexpressed; the quantitative polymerase chain reaction and western blot analyses revealed that the expression levels of all circadian clock genes (GNB2, PKA, CREB, Per1/2/3, and Clock) except Cry1/2 decreased significantly at 24 h. Cellular immunolocalization analysis showed that melatonin receptors were localized in the cell membrane and cytoplasm; the CRY protein was mainly localized in the nucleus. Overall, our findings indicated that the rhythmic regulation of ovarian granulosa cells was consistent with the regulatory action of the central circadian clock.