Wang SJ, Liu WJ, Wu CJ, Ma FH, Ahmad S, Liu BR, Han L, Jiang XP, Zhang SJ, Yang LG. rules of StAR manifestation and P4 production in hGL cells, our results may serve to improve current strategies used to treat medical Mc-MMAD infertility. fertilization (IVF), premature luteinization is definitely defined as an increase in serum P4 levels before or on the day of human being chorionic gonadotropin (hCG) administration. Several studies possess shown that premature luteinization is definitely associated with decreased implantation and pregnancy rates [2, 3]. In contrast, insufficient ovarian P4 production (i.e. luteal phase deficiency) is definitely associated with dysfunction of the secretory endometrium, which compromises successful embryo implantation and growth . Therefore, a precise rules of P4 secretion in hGL cells is required to maintain normal reproductive functions. Although pituitary luteinizing hormone (LH) takes on a central part in the induction of P4 secretion in the ovary, accumulating evidence suggests that P4 biosynthesis can also be controlled by locally-produced factors that exert their effects in an autocrine and/or paracrine fashion [5, 6]. Melatonin, a pineal hormone, Mc-MMAD regulates major physiological functions including the sleep-wake cycle, pubertal development, and seasonal adaptation . While most endogenous melatonin is definitely synthesized and released at night from the pineal gland, this hormone is also produced by extra-pineal organs such as the ovary, where it was shown to regulate reproductive functions through both receptor-mediated signaling influencing cellular metabolism, and receptor-independent Mc-MMAD actions like a scavenger for reactive oxygen and nitrogen varieties [8C10]. Research has shown that melatonin levels in serum are reduced with ageing [9, 11], potentially impacting reproductive potential in ladies. Melatonin functions on target cells by binding to and activating two membrane-bound G-protein-coupled receptors, MT1 (< 0.05). Melatonin-induced Celebrity expression is definitely mediated by MT1 and MT2 receptors To identify the cellular receptor(s) involved in melatonin-induced StAR manifestation in hGL cells, two melatonin receptor antagonists, 4-P-PDOT (MT2-selective) and luzindole (MT1/MT2-nonselective), were tested . As demonstrated in Number 2A, none of these inhibitors affected basal Celebrity mRNA levels. However, in the presence of melatonin, Celebrity mRNA upregulation was partially inhibited by pre-treatment with 4-P-PDOT, and abolished by pre-treatment with luzindole. Furthermore, western blot analyses showed that these antagonists also reduced StAR protein manifestation (Number 2B). These results indicate that both MT1 and MT2 mediate melatonin-induced upregulation of Celebrity manifestation in hGL cells. Open in a separate window Number 2 MT1 andMT2 melatonin receptors mediate melatonin-induced Celebrity expression in main hGL cells. Cells were pre-treated with vehicle control (DMSO), 10 M 4-P-PDOT, or 10 M luzindole for 30 min and then exposed to 500 M melatonin for 24 h. Celebrity mRNA (A) and protein (B) levels were examined by RT-qPCR and western blot, respectively. Results are indicated as the mean SEM of 4 self-employed experiments. Values without a common letter are significantly different (< 0.05). PI3K/AKT signaling mediates melatonin-induced Celebrity manifestation Upon binding to MT1/MT2 receptors, melatonin can activate the MEK/ERK1/2 and PI3K/AKT signaling pathways inside a cell type-dependent manner . Therefore, we examined the effect of melatonin on the activity of these two signaling pathways in hGL cells. As demonstrated in Number 3A, melatonin treatment improved phospho-AKT levels, indicating PI3K/AKT activation, but did not elicit ERK1/2 activation. We used amphiregulin like a positive control, since we have shown that it can activate ERK1/2 signaling in hGL cells . Next, we tested a specific PI3K inhibitor, LY294002, to further determine whether PI3K is required for melatonin-induced upregulation of Celebrity expression. As demonstrated in Number 3B and ?and3C,3C, pre-treatment with LY294002 partially attenuated melatonin-induced upregulation of Celebrity mRNA and protein levels. These results indicate that activation of the PI3K/AKT signaling pathway is definitely involved in melatonin-induced StAR manifestation in hGL cells. Open in a separate window Number 3 Melatonin-induced Celebrity expression is definitely partly mediated by PI3K/AKT Mc-MMAD activation. (A) hGL cells were treated with 500 M melatonin for 10 or 30 min, and both total and phosphorylated ERK1/2 and AKT manifestation was determined by western blot. Rabbit Polyclonal to AurB/C (phospho-Thr236/202) Cells treated with 100 ng/mL amphiregulin (AREG) were used as positive control for ERK1/2 phosphorylation. (B, C) hGL cells were pre-treated with vehicle control (DMSO) or 10 M.