Abstract
BackgroundMelatonin is an amine hormone that plays an important role in regulating mammalian reproduction. This study aimed to investigate the expression pattern of melatonin synthesis enzymes AANAT and HIOMT and melatonin receptors MT1 and MT2 in sheep cumulus–oocyte complexes (COCs) as well as the change of melatonin level in follicular fluid (FF) during antral follicle development. In this research, we also study the effect of β-estradiol (E2) on MT1 and MT2 expression as well as melatonin synthesis in COCs so as to lay the foundation for further exploration of the regulation mechanism of melatonin synthesis in the ovary.MethodsCOCs and FF were collected from different size (large follicles (diameter ≥ 5 mm), medium follicles (diameter 2–5 mm), and small follicles (diameter ≤ 2 mm)) of antral follicles in sheep ovaries. To assess whether E2 regulates melatonin synthase and its receptors expression in sheep COCs and whether it is mediated through estrogen receptor (ER) pathway. The collected COCs were cultured in vitro for 24 h and then treat with 1 μM E2 and/or 1 μM ICI182780 (non-selective ER antagonist). The expression of AANAT, HIOMT, MT1 and MT2 mRNA and protein were determined by qRT-PCR and western blot. The melatonin level was determined by ELISA.ResultsThe expression of AANAT, HIOMT, MT1 and MT2 were significantly higher expression in the COCs of small follicles than in those of large follicles (P < 0.05). However, the melatonin level was significantly higher in large follicle FF than in small follicle FF (P < 0.05). Further, the expression of AANAT, HIOMT, MT1, and MT2 and melatonin production were decreased by E2 treatment (P < 0.05), but when ICI182780 was added, the expression of AANAT, HIOMT, MT1, and MT2 and melatonin production recovered (P < 0.05).ConclusionsWe suggest that sheep COCs can synthesize melatonin, but this ability is decreased with increasing follicle diameter. Furthermore, E2 play an important role in regulated the expression of MT1 and MT2 as well as melatonin synthesis in sheep COCs through the ER pathway.
Highlights
Melatonin is an amine hormone that plays an important role in regulating mammalian reproduction
The lowest melatonin level was seen in the small follicles (19.54 ± 1.64 pg/ml, P < 0.05), but there was no significant difference between medium follicles and large follicles
The analysis showed that the melatonin level in the small follicular fluid (FF) was significantly lower than that in the medium FF and the large FF (P < 0.05)
Summary
Melatonin is an amine hormone that plays an important role in regulating mammalian reproduction. The granulosa cells, including those forming the cumulus oophorus [13, 14], and the oocytes [15] have been reported to detect the melatoninsynthesizing enzymes, and studies have found that human [16] and bovine [13] cumulus–oocyte complexes (COCs) can synthesize melatonin. Rather, these cells use the melatonin they produce for their own benefit or for that of their neighboring cells as an antioxidant and as an autocrine or paracrine agent [17]
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