Melatonin modulated GPX5 and PTGDS expression in Bactrian camel epididymis mainly via receptor MT1†.

Melatonin (Mel), an important mediator of photoperiodic annual rhythm regulation and seasonal reproduction in animals, directly modulates the expression of specific genes in the epididymis and protects sperm from oxidative damage. Bactrian camel is a dominant species in desert and semi-desert areas, exhibiting the unique reproductive regulation patterns. However, the underlying regulation mechanism of Mel on Bactrian camel is still unclear. This study isolated the epididymal caput epithelial cells of Bactrian camels and investigated the expression of specific genes involving sperm protection after Mel treatment and overexpression/knockdown of Mel receptor MT1/MT2 using real-time quantitative PCR assay (qPCR), ELISA, and western blotting assay. The results showed that MT1, MT2, clock genes cryptochrome 1/2 (Cry1/Cry2) were all positively expressed in the epididymal lumen epithelial cells, peritubular myoid cells, and luminal spermatozoa. Intriguingly, Mel treatment activated receptor MT1 in epididymal caput epithelial cells, indicating that Mel treatment regulated genes expression mainly via MT1-dependent manner. Mel treatment or overexpression of MT1 both increased secretion of glutathione peroxidase 5 (GPX5) and prostaglandin D2 synthase (PTGDS), and MT1 silencing induced downregulation of GPX5 and PTGDS expression, indicating that the expression of GPX5 and PTGDS were regulated by Mel-MT1. Overexpression of MT1 or MT2 promoted Cry2 expression, and overexpression of Cry2 also activated the MT1/MT2 expression by feedback regulation. Finally, the double luciferase reports assay showed that the activation of MT1 by Cry2 occurred during transcription. These results help to understand the regulatory effect of Mel on the epididymis in Bactrian camels.