Melatonin Promotes Ubiquitination of Phosphorylated Pro-Apoptotic Protein Bcl-2-Interacting Mediator of Cell Death-Extra Long (Bim(EL)) in Porcine Granulosa Cells

Melatonin (N-acetyl-5-methoxytryptamine) is found in ovarian follicular fluid, and its concentration is closely related to follicular health status. Nevertheless, the molecular mechanisms underlying melatonin function in follicles are uncertain. In this study, melatonin concentration was measured in porcine follicular fluid at different stages of health. The melatonin concentration decreased as the follicles underwent atresia, suggesting that melatonin may participate in the maintenance of follicular health. The molecular pathway through which melatonin may regulate follicular development was further investigated. The pro-apoptotic protein Bim(EL) (Bcl-2-interacting mediator of cell death-Extra Long), a key protein controlling granulosa cell apoptosis during follicular atresia, was selected as the target molecule. Bim(EL) was downregulated when porcine granulosa cells were cultured in medium containing 10(-9) M melatonin and isolated cumulus oocyte complexes (COCs) or follicle stimulating hormone (FSH). Interestingly, ERK-mediated phosphorylation was a prerequisite for the melatonin-induced decline in Bim(EL), and melatonin only promoted the ubiquitination of phosphorylated Bim(EL), and did not affect the activities of the lysosome or the proteasome. Moreover, the melatonin-induced downregulation of Bim(EL) was independent of its receptor and its antioxidant properties. In conclusion, melatonin may maintain follicular health by inducing Bim(EL) ubiquitination to inhibit the apoptosis of granulosa cells.