Abstract
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 BimEL (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. BimEL 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 BimEL, and melatonin only promoted the ubiquitination of phosphorylated BimEL, and did not affect the activities of the lysosome or the proteasome. Moreover, the melatonin-induced downregulation of BimEL was independent of its receptor and its antioxidant properties. In conclusion, melatonin may maintain follicular health by inducing BimEL ubiquitination to inhibit the apoptosis of granulosa cells.
Highlights
During mammalian follicular development, only a limited number of follicles are selected to ovulate, with the remainder undergoing atresia at different stages
The BimEL protein in the surrounding cumulus granulosa cells decreased significantly after porcine cumulus oocyte complexes (COCs) were treated with 10−9 M melatonin for 42–44 h (Figure 1A)
Melatonin significantly decreased the BimEL level when primary granulosa cells were cocultured with COCs (Figure 1B)
Summary
Only a limited number of follicles are selected to ovulate, with the remainder undergoing atresia at different stages. The Bcl-2 family, which includes both anti-apoptotic (Bcl-2, B cell lymphoma/leukemia X (Bcl-X)) and pro-apoptotic (Bcl-2 interacting domain (Bid), Bim, Bax, Bak) proteins, are key regulators of apoptosis, and members of the Bcl-2 protein family play pivotal roles in follicular growth and atresia [2,5,6]. Previous studies indicated that melatonin could induce apoptosis under the regulation of Bim in many different cancer cell lines, including human hepatocellular carcinoma cells, breast carcinoma MDA-MB231 cells, and renal cancer Caki cells [11,25,26]. These findings imply that BimEL has diverse functions in different cell types. We explored the relationship between melatonin and BimEL in porcine follicle granulosa cells
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