Melatonin promotes progesterone secretion in sheep luteal cells by regulating autophagy via the AMPK/mTOR pathway

The corpus luteum is primarily responsible for the production and secretion of progesterone. Melatonin has been established to regulate autophagy and induce progesterone secretion in luteal cell. However, whether melatonin affects progesterone secretion by interfering with autophagy is yet to be reported. In the present study, the expression levels of melatonin receptors (MT1 and MT2), autophagy-related protein Beclin1 (Bec1), microtubuleassociated protein light chain 3 B (LC3B), progesterone and steroidogenic acute regulatory protein (StAR), and cytochrome P450scc (CYP11A1) were analyzed in the corpus luteum of sheep at different stages (early, middle, and late); specifically, enzyme-linked immunosorbent assays, immunohistochemical staining, and western blotting were utilized for this expression analysis. In addition, to determine whether melatonin regulated progesterone secretion via the regulation of autophagy, luteal cells were cultured before being exposed to different concentrations of melatonin (0.01-100 nM) and the autophagy inhibitor chloroquine (50 mu M). Next, luteal cells were treated with the melatonin receptor inhibitors 4-phenyl-2-propionamidotetralin (1 mu M) and luzindole (1 mu M) before detecting Bec1, LC3B2, AMPK/mTOR, and progesterone secretion levels to ascertain whether the effect of melatonin on autophagy and progesterone secretion is mediated by its corresponding receptors in luteal cells. Finally, to determine the significance of the AMPK/mTOR pathway in this process, an AMPK inhibitor, Compound C (10 mu M), was added to luteal cells. Overall, the highest expression of melatonin receptors, autophagy and progesterone secretion was observed in the middle-phase corpus luteum; additionally, melatonin promoted autophagy, at least partially, through its receptor-mediated AMPK/mTOR pathway, which thereby promoting progesterone secretion in luteal cells in vitro. Ultimately, this study is the first to clarify the important role of autophagy in the melatonin-mediated regulation of progesterone secretion in the corpus luteum of sheep; it also lays a foundation for further exploration into the role of melatonin in regulating sheep's ovarian function.