College of Animal Science and Technology, Hebei Agricultural University, Baoding, Hebei 071001, China.
College of Animal Science and Technology, Hebei Agricultural University, Baoding, Hebei 071001, China.
Poult Sci. 2021 Jan;100(1):334-347. doi: 10.1016/j.psj.2020.10.005. Epub 2020 Oct 12.
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.
禽类生殖衰老的分子机制尚不清楚。先前的研究表明，雷帕霉素靶蛋白（mTOR）信号通路在衰老中具有重要作用。本研究探讨了 mTOR 信号通路与产蛋高峰期和产蛋后期大武金凤母鸡（N=30/组）卵巢衰老之间的关系。在 30 周龄和 70 周龄大武金凤母鸡中，连续 5 周跟踪产蛋率和蛋品质。在产蛋高峰期（第 35 周）和后期（第 75 周），通过酶联免疫吸附试验检测抗氧化和免疫指标，并通过定量逆转录聚合酶链反应技术检测卵泡中 mTOR 信号相关基因（CLIP-170、GRB10、LIPIN-1、ATG1、4E-BP1、S6K、PKC、RHO 和 SGK1）。通过 Western blot 分析评估关键基因（mTOR、PKC、4EBP1）的蛋白表达水平。产蛋率和抗氧化指标超氧化物歧化酶和谷胱甘肽过氧化物酶以及总抗氧化能力和免疫球蛋白（IgM 和 IgG）水平在第 35 周时显著高于第 75 周（P<0.01），而丙二醛水平显著降低（P<0.01）。第 75 周时，不同发育阶段的卵泡数量少于第 35 周。第 75 周时，白色卵泡（大和小）和初级卵泡的数量明显高于第 35 周（P<0.01）。第 75 周时，小白色卵泡（SWF）、大白色卵泡（LWF）、F3、F1 和卵巢中的 avTOR、CLIP-170、GRB10、LIPIN-1、4E-BP1、S6K、RHO 和 SGK 基因的 mRNA 表达均低于第 35 周（P<0.05）。小黄色卵泡（SYF）中的 mRNA 表达显著高于第 35 周（P<0.05），而第 75 周时 SWF、LWF、F3、F1 和卵巢中的 ULK1 mRNA 表达高于第 35 周（P<0.01），SYF 较低（P<0.05）。用 mTOR 激动剂 MHY1485 处理鸡颗粒细胞显著增强了粒细胞增殖（P<0.01）并抑制了凋亡（P<0.01），并显著增加了 avTOR、S6K、4E-BP1 和 PKC 基因的表达（P<0.01）。mTOR、S6K、p-mTOR 和 p-S6K 的蛋白表达水平与 mRNA 表达水平一致。mTOR 活性具有年龄特异性，对 mTOR 信号级联的代偿性增强可以调节老龄产蛋母鸡的卵巢卵泡发育。
