Luzindole and 4P-PDOT block the effect of melatonin on bovine granulosa cell apoptosis and cell cycle depending on its concentration

Abstract

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.

Keywords: Bovine; Gene regulation; Granulosa cell; Melatonin; Melatonin receptor antagonist.

Figure 1. Effects of high dose (10⁻⁵ M) and low dose (10⁻⁹ M) melatonin and melatonin receptor antagonist supplementation on the cell cycle related genes (CyclinD1, CyclinE1 and p21).

The mRNA levels of CyclinD1 (A), CyclinE1 (B) and P21 (C) were examined by real-time PCR in granulosa cells at 48 h after melatonin supplementation in the absence/presence of luzindole or 4P-PDOT. The quantity of mRNA was normalized to that of β-actin. The statistical differences were performed using one-way ANOVA. Statistical differences among the samples were labeled with different letters (P < 0.05). About 60 bovine ovaries were used in this study. The experiment was repeated three times independently. Abbreviations: C, control; M, melatonin; ML, melatonin and luzindole; MP, melatonin and 4P-PDOT.

Figure 2. Effects of high dose (10⁻⁵ M) and low dose (10⁻⁹ M) melatonin and melatonin receptor antagonist supplementation on granulosa cells apoptosis related genes (Bcl2, caspase-3, Bcl-xl, Bax and p53).

The mRNA abundance of Bcl2 (A), Bcl-xl (B), p53 (C), Bax (D) and caspase-3 (E) were examined by real-time PCR at 48 h and corresponding protein abundance (F) were detected by Western blot after melatonin supplementation in the absence/presence of luzindole or 4P-PDOT. mRNA abundance was normalized to that of β-actin. The statistical differences were performed using one-way ANOVA. Statistical differences among the samples were labeled with different letters (P < 0.05). About 60 bovine ovaries were used in this study. The experiment was repeated three times independently. Abbreviations: C, control; M, melatonin; ML, melatonin and luzindole; MP, melatonin and 4P-PDOT.

Figure 3. Effects of high dose (10⁻⁵ M) and low dose (10⁻⁹ M) melatonin and melatonin receptor antagonist supplementation on granulosa cells antioxidant related genes expression (SOD1 and GPX4).

The mRNA abundance of GPX4 (A) and SOD1 (B) were examined by real-time PCR at 48 h after melatonin supplementation in the absence/presence of luzindole or 4P-PDOT. mRNA abundance was normalized to that of β-actin. The statistical differences were performed using one-way ANOVA. Statistical differences among the samples were labeled with different letters (P < 0.05). About 60 bovine ovaries were used in this study. The experiment was repeated three times independently. Abbreviations: C, control; M, melatonin; ML, melatonin and luzindole; MP, melatonin and 4P-PDOT.