Melatonin alleviates ovarian function damage and oxidative stress induced by dexamethasone in the laying hens through FOXO1 signaling pathway.

Oxidative stress can trigger follicular atresia, and decrease follicles quantity in each development stage, thereby alleviating reproductive activity. The induction of oxidative stress in chickens through intraperitoneal injection of dexamethasone is a reliable and stable method. Melatonin has been shown to mitigate oxidative stress in this model, but the underlying mechanism remains unclear. Therefore, this study aimed to investigate whether melatonin can recover aberrant antioxidant status induced by dexamethasone and the specific mechanism behind melatonin-dependent protection. A total of 150 healthy 40-wk-old Dawu Jinfeng laying hens with similar body weights and laying rates were randomly divided into three groups, with five replicates per group and 10 hens per replicate. The hens in the control group (NS) received intraperitoneal injections of normal saline for 30 d, the dexamethasone group (Dex+NS) received 20 mg/kg dose of dexamethasone for the first 15 d, followed by the 15 d of normal saline treatment. While in the melatonin group (Dex+Mel), dexamethasone (20 mg/kg dose) was injected intraperitoneally in the first 15 d, and melatonin (20 mg/kg/d) was injected in the last 15 d. The results showed that dexamethasone treatment significantly enhanced oxidative stress (P < 0.05), while melatonin not only inhibited the oxidative stress but also notably enhanced the antioxidant enzymes superoxide dismutase (SOD), catalase activity (CAT), glutathione peroxidase (GSH-Px), and antioxidant genes CAT, superoxide dismutase 1 (SOD1), glutathione peroxidase 3 (GPX3), and recombinant peroxiredoxin 3 (PRDX3) expression (P < 0.05). Melatonin treatment also markedly reduced 8-hydroxy deoxyguanosine (8-OHdG), malondialdehyde (MDA), and reactive oxygen species (ROS) levels (P < 0.05) and apoptotic genes Caspase-3, Bim, and Bax in the follicle. In the Dex+Mel group, the Bcl-2 and SOD1 protein levels were also increased (P < 0.05). Melatonin inhibited the forkhead Box Protein O1 (FOXO1) gene and its protein expression (P < 0.05). In general, this investigation revealed that melatonin might decrease oxidative stress and ROS by enhancing antioxidant enzymes and genes, activating the antiapoptotic genes, and inhibiting the FOXO1 pathway in laying hens.

The relationship between the mTOR signaling pathway and ovarian aging in peak-phase and late-phase laying hens.

The molecular mechanisms underlying reproductive aging in avian species are poorly understood. Previous studies have shown the importance of mechanistic target of rapamycin (mTOR) signaling pathway in aging. In this study, we investigated the relationship between the mTOR signaling pathway and ovarian aging in the peak phase and late phase of egg production in laying hens. The egg production rate and egg quality were tracked for 5 consecutive weeks in 30-week-old and 70-week-old Dawu Jinfeng hens (N = 30/group). During the peak phase (week 35) and late phase (week 75), antioxidant and immune indicators were detected by enzyme-linked immunosorbent assay, and mTOR signaling-related genes (CLIP-170, GRB10, LIPIN-1, ATG1, 4E-BP1, S6K, PKC, RHO, and SGK1) were detected in the follicles by quantitative reverse transcription-PCR technology. The protein expression of key genes (mTOR, PKC, 4EBP1) was evaluated by Western blot analysis. The egg production rate and the antioxidant indexes superoxide dismutase and glutathione peroxidase and the levels of total antioxidant capacity and immunoglobulins (IgM and IgG) were significantly higher at week 35 than those at week 75 (P < 0.01), while malondialdehyde levels were significantly lower (P < 0.01). At week 75, there were fewer follicles in the different stages of development than were detected at week 35. The number of white follicles (large and small) and primary follicles were significantly higher at week 75 than those detected at week 35 (P < 0.01). The mRNA expression of avTOR, CLIP-170, GRB10, LIPIN-1, 4E-BP1, S6K, RHO, and SGK genes in small white follicles (SWF), large white follicles (LWF), F3, F1, and ovary at week 75 was lower than that in the hens at week 35 (P < 0.05). The mRNA expression in small yellow follicle (SYF) was significantly higher than that at week 35 (P < 0.05), while the mRNA expression of ULK1 in SWF, LWF, F3, F1, and ovary at week 75 was higher than that of hens at week 35 (P＜0.01), and SYF was lower (P < 0.05). Treatment of chicken granulosa cells with the mTOR agonist MHY1485 significantly enhanced granulocyte proliferation (P < 0.01) and inhibited apoptosis (P < 0.01) and significantly increased avTOR, S6K, 4E-BP1, and PKC gene expression (P < 0.01). The protein expression levels of mTOR, S6K, p-mTOR, and p-S6K were consistent with mRNA expression levels. The mTOR activity is age-specific, and a compensatory enhancement of the mTOR signaling cascade can regulate ovarian follicular development in aged laying hens.

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

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.

Melatonin regulates the ovarian function and enhances follicle growth in aging laying hens via activating the mammalian target of rapamycin pathway.

The signal pathway of target of rapamycin (TOR) plays an important role in regulating cell growth and proliferation, follicular development, and ovulation. Melatonin (N-acetyl-5-methoxytryptamine) (MT) is involved in the regulation of many physiological functions in animals. Recent studies have shown that MT affects the number and the degree of maturation of follicles in the ovary, but there are few studies concerning its mechanism. Therefore, the aim of this study was to investigate the mechanism of TOR signal pathway in the regulation of ovarian function by MT in aging laying hens. In the present study, a total of 60 hens (70-week-old) were randomly divided into 2 groups: control group and melatonin group (M). Melatonin was administered intraperitoneally at a dose of 20 mg/kg/D for 28 D in the M group. The results showed that MT significantly increased the levels of the antioxidant enzymes superoxide dismutase and total antioxidant capacity (P < 0.01) as well as levels of immunoglobulin (IgA, IgG, and IgM) (P < 0.05) and the reproductive hormones estradiol and luteinizing hormone (P < 0.01) in the plasma and also increased the numbers of middle white follicles and small white follicles (P < 0.05) and decreased the level of reactive oxygen species in plasma (P < 0.01) in laying hens. There were higher expression levels in MT receptor A (P < 0.05), melatonin receptor B (P < 0.01), and tuberous sclerosis complex 2 (P < 0.01). Activation of TOR, 4E binding protein-l (4E-BP1), and ribosomal protein 6 kinase (P < 0.01) was found in the M. The levels of mTOR and p-mTOR protein were increased in the M (P < 0.05). The mTORC1-dependent 4E-BP1 and p-4E-BP1 were increased in the M (P < 0.05). This study indicated that MT may enhance follicle growth by increasing levels of antioxidant enzymes and reproductive hormones and by activating the mTOR and downstream components in aging laying hens.