Zhao S-Q, Gao Y, Zhang Y, Yang X-P, Yang Z
College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China; Basic Experimental Teaching Center, Gansu Agricultural University, Lanzhou 730070, China; Gansu Key Laboratory of Animal Generational Physiology and Reproductive Regulation, Gansu Agricultural University, Lanzhou 730070, China.
Gansu Key Laboratory of Animal Generational Physiology and Reproductive Regulation, Gansu Agricultural University, Lanzhou 730070, China.
Domest Anim Endocrinol. 2021 Jul;76:106609. doi: 10.1016/j.domaniend.2021.106609. Epub 2021 Jan 29.
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.
环磷酸腺苷/蛋白激酶A/环磷腺苷反应元件结合蛋白（cAMP/PKA/CREB）信号通路参与哺乳动物重要生理活动期间褪黑素的调节。然而，卵巢颗粒细胞中昼夜节律基因的调控仍不清楚。在此，我们使用培养的双峰驼卵巢颗粒细胞确定了褪黑素与生物钟基因之间的关系。酶联免疫吸附测定表明，当褪黑素受体（MT）基因或隐花色素（Cry）基因过表达时，环磷酸腺苷含量降低；定量聚合酶链反应和蛋白质免疫印迹分析显示，除Cry1/2外，所有生物钟基因（GNB2、PKA、CREB、Per1/2/3和Clock）的表达水平在24小时时均显著下降。细胞免疫定位分析表明，褪黑素受体定位于细胞膜和细胞质中；CRY蛋白主要定位于细胞核中。总体而言，我们的研究结果表明，卵巢颗粒细胞的节律性调节与中枢生物钟的调节作用一致。
