Melatonin regulates chicken granulosa cell proliferation and apoptosis by activating the mTOR signaling pathway via its receptors

Abstract

Melatonin is a key regulator of follicle granular cell maturation and ovulation. The mammalian target of rapamycin ( mTOR ) pathway plays an important role in cell growth regulation. Therefore, our aim was to investigate whether the mTOR signaling pathway is involved in the regulation of melatonin-mediated proliferation and apoptotic mechanisms in granulosa cells. Chicken follicle granular cells were cultured with melatonin (0, 2, 20, or 200 μmol/L) for 48 h. The results showed that melatonin treatment enhanced proliferation and suppressed apoptosis in granular cells at 20 μmol/L and 200 μmol/L ( P < 0.05) by upregulation of cyclin D1 ( P < 0.01) and Bcl-2 ( P < 0.01) and downregulation of P21, caspase-3, Beclin1, and LC3-II ( P < 0.01). The effects resulted in the activation of the mTOR signaling pathway by increasing the expression of avTOR, PKC, 4E-BP1, S6K ( P < 0.05), p-mTOR, and p-S6K. We added an mTOR activator and inhibitor to the cells and identified the optimal dose (10 μmol/L MHY1485 and 100 nmol/L rapamycin) for subsequent experiments. The combination of 20 μmol/L melatonin and 10 μmol/L MHY1485 significantly enhanced granulosa cell proliferation ( P < 0.05), while 100 nmol/L rapamycin significantly inhibited proliferation and enhanced apoptosis ( P < 0.05), but this action was reversed in the 20-μmol/L melatonin and 100-nmol/L rapamycin cotreatment groups ( P < 0.05). This was confirmed by mRNA and protein expression that was associated with proliferation, apoptosis, and autophagy ( P < 0.05). The combination of 20 μmol/L melatonin and 10 μmol/L MHY1485 also activated the mTOR pathway upstream genes PI3K, AKT1, and AKT2 and downstream genes PKC, 4E-BP1, and S6K ( P < 0.05), as well as protein expression of p-mTOR and p-S6K. Rapamycin significantly inhibited the mTOR pathway–related genes mRNA levels ( P < 0.05). In addition, activation of the mTOR pathway increased melatonin receptor mRNA levels ( P < 0.05). In conclusion, these findings demonstrate that melatonin regulates chicken granulosa cell proliferation and apoptosis by activating the mTOR signaling pathway via its receptor.

The Effect of Oxidative Stress on the Chicken Ovary: Involvement of Microbiota and Melatonin Interventions

“…Our results also indicated that the SIRT1 was increased by melatonin addition in vitro and decreased FoxO1 and P53 expression. As reported previously, melatonin was able to protect mouse granulosa cells against oxidative damage by inhibiting FoxO1-mediated autophagy [ 72 , 73 ].…”

Resveratrol Alleviating the Ovarian Function Under Oxidative Stress by Alternating Microbiota Related Tryptophan-Kynurenine Pathway

“…As observed in the current study, IDO1 in the jejunum was enhanced by the OS challenge and reversed by the dietary supplementation of RV, which may suggest that the tryptophan-kynurenine pathway was also involved in this process. Tryptophan is also the precursor of melatonin, which can be synthesized in the intestine ( 66 , 67 ). Therefore, the decreasing levels of tryptophan in serum may be the result of the higher Mel in serum.…”

Grade follicles transcriptional profiling analysis in different laying stages in chicken

“…Follicular development is a complex physiological process, regulated by diverse genes and endocrine hormones [ 3 ]. Previous studies revealed the effect of GPR12 [ 20 ], GREM1 [ 31 ], BMP4 [ 32 ], RAC1 [ 33 ], FOXO3 [ 34 ], bFGF [ 35 , 36 ], Melatonin [ 37 , 38 ], CSAL1 and CSAL3 [ 39 ] on follicular development. RNA-seq has empowered many research areas and has led to new discoveries throughout the mRNA field [ 40 ].…”