College of Animal Science and Technology, Hebei Agricultural University, Baoding Hebei 071001, China.
Baoding Livestock Husbandry Workstation, Baoding Hebei 071001, China.
Poult Sci. 2024 Jun;103(6):103656. doi: 10.1016/j.psj.2024.103656. Epub 2024 Mar 15.
Follicular atresia in chickens reduces the number of follicles that can further develop, leading to decrease egg laying. Endoplasmic reticulum stress (ERS) can initiate a unique pathway inducing the apoptosis of follicular granulosa cells, thus reducing egg laying. Melatonin (MEL) is involved in the regulation of follicle development, ovulation, and oocyte maturation, and is closely related to follicle fate. Mammalian target of Rapamycin (mTOR) signaling pathway plays an important role in cell growth regulation, and that there is a possible crosstalk between melatonin and mTOR activity in granular cells maturation and ovulation. This study aimed to investigate whether MEL inhibits ERS and follicular granulosa cell apoptosis by regulating ATF4 to activate mTOR signaling pathway in chickens. Frist, we established an in vitro ERS cell model using tunicamycin (TM). The results showed that different concentrations of TM exhibited dose-dependent inhibition of cell activity and induction of granulosa cells (P<0.01). Therefore, we chose 5 µg/mL of TM and a treatment time for 6 h as the optimal concentration for the following experiments. Then we investigate whether melatonin can inhibit ERS. TM treatment decreased the cell viability and Bcl-2 expression, increasing ROS levels and the mRNA expression of Grp78, ATF4, CHOP, PERK, eIF-2α, and BAX (P<0.01), whereas TM+MEL treatment significantly inhibited these changes (P<0.01). Then we explored whether melatonin protects follicular granulosa cells from ERS-induced apoptosis through the mammalian target of rapamycin (mTOR) signaling pathway by regulating ATF4, we found that ATF4 knockdown inhibited ERS by decreasing the expression of ERS-related genes and proteins and activating mTOR signaling pathway by increasing the protein expression of p4E-BP1 and pT389-S6K (P<0.001), while these changes were promoted by TM+si-ATF4+MEL treatment (P<0.01). These results indicate that MEL could alleviate TM-induced ERS by regulating ATF4 to activate mTOR signaling pathway in follicular granulosa cells, thus providing a new perspective for prolonging the laying cycle in chickens.
鸡的卵泡闭锁会减少能够进一步发育的卵泡数量，导致产蛋量下降。内质网应激（ERS）可以启动诱导卵泡颗粒细胞凋亡的独特途径，从而减少产蛋量。褪黑素（MEL）参与调节卵泡发育、排卵和卵母细胞成熟，与卵泡命运密切相关。哺乳动物雷帕霉素靶蛋白（mTOR）信号通路在细胞生长调节中发挥重要作用，并且在颗粒细胞成熟和排卵中，褪黑素和 mTOR 活性之间可能存在相互作用。本研究旨在探讨褪黑素是否通过调节 ATF4 激活 mTOR 信号通路来抑制 ERS 和卵泡颗粒细胞凋亡。首先，我们使用衣霉素（TM）建立了体外 ERS 细胞模型。结果表明，不同浓度的 TM 表现出剂量依赖性的细胞活性抑制和颗粒细胞诱导（P<0.01）。因此，我们选择 5μg/mL 的 TM 和 6 h 的处理时间作为后续实验的最佳浓度。然后，我们研究了褪黑素是否可以抑制 ERS。TM 处理降低了细胞活力和 Bcl-2 表达，增加了 ROS 水平和 Grp78、ATF4、CHOP、PERK、eIF-2α 和 BAX 的 mRNA 表达（P<0.01），而 TM+MEL 处理显著抑制了这些变化（P<0.01）。然后，我们通过调节 ATF4，探讨了褪黑素是否通过哺乳动物雷帕霉素（mTOR）信号通路保护卵泡颗粒细胞免受 ERS 诱导的凋亡，我们发现 ATF4 敲低通过降低 ERS 相关基因和蛋白的表达来抑制 ERS，并通过增加 p4E-BP1 和 pT389-S6K 的蛋白表达来激活 mTOR 信号通路（P<0.001），而 TM+si-ATF4+MEL 处理则促进了这些变化（P<0.01）。这些结果表明，MEL 可以通过调节 ATF4 激活 mTOR 信号通路来减轻 TM 诱导的 ERS，从而为延长鸡的产蛋周期提供了新的视角。
