Ovarian granulosa cells (GCs) are a critical approach to investigate the mechanism of gene regulation during folliculogenesis. The objective of this study was to investigate the role of MT2 in bovine GCs, and assess whether MT2 silencing affected GCs response to melatonin. We found that MT2 silencing significantly decreased the secretion of progesterone and estradiol, and increased the concentration of inhibin B and activin B. To further reveal the regulatory mechanism of MT2 silencing on steroids synthesis, it was found that the expression of CYP19A1 and CYP11A1 enzymes (steroid hormone synthesis) were down-regulated, while genes related to hormonal synthesis (StAR, RUNX2, INHA and INHBB) were up-regulated without affecting the expression of INHBA, suggesting that MT2 silencing may regulate hormone abundance. Furthermore, MT2 silencing significantly increased the expression of TGFBR3 and BMP6, and decreased the expression of LHR and DNMT1A without significant difference in the expression of FSHR and EGFR. In addition, MT2 silencing didn’t affect the effect of melatonin on increasing the expression of DNMT1A, EGFR, INHBA and LHR, and progesterone level, or decreasing INHA, TGFBR3 and StAR expression, and production of inhibin B. Moreover, MT2 silencing could disrupt the role of melatonin in decreasing the FSHR, INHBB and BMP6 expression, and activin B secretion. In conclusion, these results reveal that melatonin and MT2 are essential regulator of bovine GCs function by modulating reproduction-related genes expression, hormones secretion and other regulators of folliculogenesis.
Granulosa cells (GCs) play an important role in ovarian follicle growth, development, and follicular atresia. In the present study, we investigated the effects of Melatonin on bovine GCs, and asked if MTNR1A was involved in their response to this indole hormone. Our results indicated that Melatonin inhibited GC apoptosis by up-regulating the expression of BCL2, BCL-XL, GPX4, and SOD1, and down-regulating the expression of BAX, CASP3, and TP53. Moreover, Melatonin modulated bovine GC function by decreasing the expression of INHA, INHBB, FSHR, and TGFBR3, and the abundance of Inhibin β and Activin B, while increasing the expression of LHR, INHBA, and secretion of progesterone by GCs. In contrast, knockdown of MTNR1A significantly increased the expression of BAX, CASP3, TP53, INHA, FSHR, and TGFBR3, as well as Inhibin β abundance, while decreasing the expression of BCL2, GPX4, SOD1, and LHR, and production of progesterone and estradiol; no effect was observed on the expression of BCL-XL, INHBA, or INHBB. These results suggest that Melatonin and MTNR1A play an important role in modulating bovine GC function by regulating cellular progression, apoptosis, hormones secretion, and reproduction-related genes. Furthermore, altered expression of MTNR1A could affect how bovine GCs respond to Melatonin.
Melatonin is well known as a powerful free radical scavenger and exhibits the ability to prevent cell apoptosis. In the present study, we investigated the role of melatonin and its receptor MTNR1B in regulating the function of bovine granulosa cells (GCs) and hypothesized the involvement of MTNR1B in mediating the effect of melatonin on GCs. Our results showed that MTNR1B knockdown significantly promoted GCs apoptosis but did not affect the cell cycle. These results were further verified by increasing the expression of pro-apoptosis genes (BAX and CASP3), decreasing expression of the anti-apoptosis genes (BCL2 and BCL-XL) and anti-oxidant genes (SOD1 and GPX4) without affecting cell cycle factors (CCND1, CCNE1 and CDKN1A) and TP53. In addition, MTNR1B knockdown did not disrupt the effects of melatonin in suppressing the GCs apoptosis or blocking the cell cycle. Moreover, MTNR1B knockdown did not affect the role of melatonin in increasing BCL2, BCL-XL, and CDKN1A expression, or decreasing BAX, CASP3, TP53, CCND1 and CCNE1 expression. The expression of MTNR1A was upregulated after MTNR1B knockdown, and melatonin promoted MTNR1A expression with or without MTNR1B knockdown. However, despite melatonin supplementation, the expression of SOD1 and GPX4 was still suppressed after MTNR1B knockdown. In conclusion, these findings indicate that melatonin and MTNR1B are involved in BCL2 family and CASP3-dependent apoptotic pathways in bovine GCs. MTNR1A and MTNR1B may coordinate the work of medicating the appropriate melatonin responses to GCs.
Granulosa cells play an essential physiological role in mediating the follicle development and survival or apoptosis of granulosa cells dictate the follicle development or atresia. The aim of this study was to investigate the role of high dose (10−5 M) and low dose (10−9 M) melatonin in bovine granulosa cells, and assess whether MT1 and MT2 inhibiter affect granulosa cells response to melatonin. We found that the high dose (10−5 M) and low dose (10−9 M) both could act as an essential role in modulating granulosa cells apoptosis, cell cycle and antioxidant. The beneficial effect could be related to that melatonin promoted the expression of Bcl2, Bcl-xl, SOD1 and GPX4, and inhibited Bax, caspase-3 and p53 expression. Moreover P21 expression was decreased in granulosa cells treated with the high dose (10−5 M) melatonin and increased in that treated with the low dose (10−9 M) melatonin. To further reveal the role of MT1 and MT2 in mediating the effect of melatonin on granulosa cells apoptosis, cell cycle and antioxidant, we found that the luzindole and 4P-PDOT did not affect the effect of high dose (10−5 M) melatonin on regulating Bcl2, Bax, caspase-3, SOD1, GPX4 and p53 expression, while blocked its effect on modulating Bcl-xl and P21expression. However, luzindole and 4P-PDOT disturbed the effect of low dose (10−9 M) melatonin on regulating Bcl2, Bax, caspase-3, Bcl-xl, SOD1, GPX4, and p53 expression. In conclusion, these results reveal that the effect of low dose (10−9 M) melatonin on granulosa cells apoptosis are mediated by MT1 and MT2, and the high dose (10−5 M) melatonin affect the granulosa cells apoptosis by other pathway, besides MT1 and MT2. Moreover MT1 and MT2 may work in concert to modulate bovine granulosa cells function by regulating cellular progression and apoptosis.
Vascular endothelial growth factor (VEGF)-dependent angiogenesis has a crucial role in the corpus luteum formation and their functional maintenances in mammalian ovaries. A previous study by our group reported that activation of hypoxia‑inducible factor (HIF)‑1α signaling contributes to the regulation of VEGF expression in the luteal cells (LCs) in response to hypoxia and human chorionic gonadotropin. The present study was designed to test the hypothesis that HIF prolyl‑hydroxylases (PHDs) are expressed in LCs and overexpression of PHD2 attenuates the expression of VEGF induced by hypoxia in LCs. PHD2-overexpressing plasmid was transfected into LC2 cells, and successful plasmid transfection and expression was confirmed by reverse transcription quantitative polymerase chain reaction and western blot analysis. In addition, the present study investigated changes of HIF‑1α and VEGF expression after incubation under hypoxic conditions and PHD2 transfection. PHD2 expression was significantly higher expressed than the other two PHD isoforms, indicating its major role in LCs. Moreover, a significant increase of VEGF mRNA expression was identified after incubation under hypoxic conditions, which was, however, attenuated by PHD2 overexpression in LCs. Further analysis also indicated that this hypoxia‑induced increase in the mRNA expression of VEGF was consistent with increases in the protein levels of HIF‑1α, which is regulated by PHD-mediated degradation. In conclusion, the results of the present study indicated that PHD2 is the main PHD expressed in LCs and hypoxia‑induced VEGF expression can be attenuated by PHD2 overexpression through HIF‑1α‑mediated mechanisms in LCs. This PHD2-mediated transcriptional activation may be one of the mechanisms regulating VEGF expression in LCs during mammalian corpus luteum development.
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