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

Abstract

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.

Keywords: aging; follicle; hen; mTOR; melatonin.

Figure 1

Effects of melatonin treatment on MT，ROS levels, and antioxidant enzymes in the plasma. Diagram displaying MT，ROS levels, and antioxidant enzymes of animal groups. Data are shown as means ± SD; n = 12 chicken/group. *P < 0.05 indicates significance from respective control values, **P < 0.01 indicates extremely significance from respective control values. MT, melatonin; ROS, reactive oxygen species; SOD, superoxide dismutase; T-AOC, total antioxidant capacity; CON, control group; M, melatonin group.

Figure 2

Effects of melatonin treatment on reproduction index in the plasma. Diagram displaying FSH, LH, E2, and P4 levels of animal groups. Data are shown as means ± SD; n = 12 chicken/group. **P < 0.01 indicates extremely significance from respective control values. E2, estradiol; P4, progesterone; FSH, follicle-stimulating hormone; LH, luteinizing hormone; CON, control group; M, melatonin group.

Figure 3

Effects of melatonin treatment on immune index in the plasma. Diagram displaying IgA, IgG, and IgM of animal groups. Data are shown as means ± SD; n = 12 chicken/group. *P < 0.05 indicates significance from respective control values. IgA, immunoglobulin A; IgG, immunoglobulin G; IgM, immunoglobulin M.

Figure 4

Effect of melatonin treatment on laying rate and ovarian follicular numbers. Comparison of the laying rate (A) and number of follicles (B) between the control groups (CON) and melatonin groups (M). Data are shown as means ± SD; Laying rate: n = 30 chicken/group, numbers of follicle: n = 6 chicken/group. *P < 0.05 indicates significance from respective control values.

Figure 5

Effects of melatonin treatment on MELRA, MELRB, TSC1, TSC2, avTOR, and 4E-BP1, S6K and PKC mRNA expression in the ovary. Expressions melatonin receptors (A and B)and activations of TOR upstream components TSC1, TSC2(C and D) and its downstream components PKC, 4E-BP1, and S6K (E-H) mRNA expression between the control groups (CON) and melatonin groups (M). N = 6 chicken/group. *P < 0.05 indicates significance from respective control values, **P < 0.01 indicates extremely significance from respective control values.

Figure 6

Effects of melatonin treatment on TOR and downstream components protein expression in the ovary. Expressions and activations of TOR (A and B) and its downstream components 4E-BP1 (C and D) and PKC (E and F) expression between the control groups (CON) and melatonin groups (M). N = 6 chicken/group. **P < 0.01 indicates extremely significance from respective control values.