After treatment, the cells were treated with 0

After treatment, the cells were treated with 0.5?mg/mL MTT and then incubated for an additional 4?h. granulosa cells and explored the effects of melatonin on PA-induced apoptosis. Methods Granulosa cells from immature female mice were cultured for 24?h in medium containing PA and/or melatonin. Then, the effects of PA alone or combined with melatonin on viability, apoptosis and endoplasmic reticulum (ER) stress in granulosa cells were detected by methyl thiazolyl tetrazolium (MTT) assay, flow cytometry assay and western blot. After 48?h of PA and/or melatonin treatment, the concentrations of estradiol (E2) and progesterone (P4) in the culture supernatants were measured with ELISA kits. Results In this study, we explored the effects of melatonin on cell viability and apoptosis in PA-treated mouse granulosa cells and uncovered the signaling pathways involved in these processes. Our results showed that 200-800?M PA treatment reduces cell viability, induces cell apoptosis, enhances the expression of apoptosis-related genes (Caspase 3 and B-cell lymphoma-2 (BCL-2) associated X protein (BAX)), and activates the expression of ER stress marker genes (glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP)). Melatonin treatment (1-10?M) suppresses 400?M PA-induced cell viability decrease, AGN 205327 cell apoptosis, Caspase 3 activation, and BAX, CHOP, and GRP78 expression. In addition, we found that 10?M melatonin successfully attenuated the 400?M PA-induced estrogen (E2) and progesterone (P4) decreases. Conclusions This study suggests that PA triggers cell apoptosis via ER stress and that melatonin protects AGN 205327 cells against apoptosis by inhibiting ER stress in mouse granulosa cells. Electronic supplementary material The online version of this article (10.1186/s13048-019-0519-z) contains supplementary material, which is available to authorized users. Keywords: Palmitic acid, Melatonin, Endoplasmic reticulum stress, Mouse granulosa cell, Apoptosis Background Palmitic acid (PA) is one of the most common fatty acids in animal and human follicular fluid (FF) and blood serum [1C3]. The PA level in mammalian FF is reported to be approximately 10??4?M [3C5]. Recently, increasing evidence has shown that elevated PA levels may be associated with infertility in humans [6, 7]. Animal model studies have reported relations between higher PA levels and decreased rates of fertilization, cleavage, and blastocyst formation [3, 8, 9]. Granulosa cells play essential roles in follicular development, oocyte maturation and sex hormone secretion [10C12]. The exposure of granulosa cells to PA inhibits cell proliferation and decreases steroidogenesis. PA impairs fertility by suppressing human granulosa cell survival and inducing apoptosis [13, 14]. Therefore, ameliorating the toxic effects of PA on granulosa cells may be an effective method to treat human infertility. To date, the exact molecular mechanism of PA-induced granulosa cell apoptosis, however, has not been fully understood. Our previous studies have suggested that ER stress is involved in granulosa cell apoptosis [15, 16]. However, it remains elusive whether ER stress is involved in PA-induced granulosa cell apoptosis. The ER plays an important role in the folding, transport, and processing of newly synthesized proteins and the biosynthesis of cholesterol, steroids, and other lipids, which is essential for the maintenance of homeostasis in organisms. The Rabbit Polyclonal to ACTBL2 accumulation of unfolded or misfolded proteins in the ER lumen can affect ER homeostasis and trigger a protective mechanism known as the unfolded protein response (UPR). Three ER transmembrane proteins, protein kinase RNA (PKR)-like ER kinase (PERK), inositol-requiring enzyme-1 (IRE-1), and activating transcription factor-6 (ATF6), are involved in ER stress and are associated with glucose-regulated protein 78 (GRP78, an ER chaperone) [17]. The primary objective of the UPR is to re-establish homeostasis and alleviate ER stress by increasing the protein folding capacity and decreasing the unfolded protein load. However, when ER stress fails to manage AGN 205327 misfolded and unfolded proteins, cell apoptosis is induced [18]. Previous studies have reported that melatonin inhibits cell apoptosis by attenuating ER stress [19C21]. Melatonin is an important endogenous hormone involved in the biological clock, the circadian rhythm and reproductive physiology. Its actions are mediated via two types of receptors, MT1 and MT2, which are expressed in not only the pineal gland but also other parts of the organism, including granulosa cells [22C24]. Increasing evidence from in vitro cultured cell and animal studies has shown the beneficial effects of melatonin on female reproductive processes, such as follicle growth [25, 26], embryonic development [27] and oocyte maturation [25]. Dynamic changes in the porcine intrafollicular melatonin concentration correlate with the progress of follicular atresia. Normally, melatonin levels might positively correlate with follicular growth [28]. High levels of melatonin were found in human preovulatory FF [29]. A recent study revealed that the intrafollicular melatonin concentration decreases as follicular atresia progresses, whereas the percentage of apoptotic granulosa cells increases [26]. The initiation of granulosa cell apoptosis during porcine follicular atresia may be related to an ER stress response, and melatonin can inhibit apoptosis and stimulate progesterone production by granulosa cells [26, 30]. PA has been demonstrated to induce apoptosis in human granulosa.