Abstract
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.