Melatonin alleviates endoplasmic reticulum stress and follicular granulosa cell apoptosis by regulating ATF4 to activate mTOR signaling pathway in chickens.

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.

Melatonin alleviates endoplasmic reticulum stress to improve ovarian function by regulating the mTOR pathway in aged laying hens.

Granular cell apoptosis is a key factor leading to follicular atresia and decreased laying rate in aged laying hens. Endoplasmic reticulum stress (ERS) induced cell apoptosis is a new type of apoptosis pathway. Previous studies have shown that the ERS pathway is involved in the regulation of follicular development and atresia, and can be regulated by mTOR. Melatonin (MEL) can protect the normal development of follicles, but the precise mechanism by which MEL regulates follicular development is not yet clear. So, we investigated the potential relationship between MEL and ERS and mTOR signaling pathway in vivo through intraperitoneal injection of MEL in aged laying hens. The results show that the laying rate, ovarian follicle number, plasma MEL, E2, LH, FSH concentrations, as well as the mRNA expression of mTOR signaling-associated genes TSC1, TSC2, mTOR, 4E-BP1, and S6K in old later-period chicken control (Old-CN) group was significantly decreased (P < 0.01). In contrast, the ERS-related of plasma and granular cell layer mRNA expression of Grp78, CHOP, and Caspase-3 was significantly increased (P < 0.01). While both of the effects were reversed by MEL. Then, aging granulosa cells were treated with MEL in vitro, followed by RNA seq analysis, and it was found that 259 and 322 genes were upregulated and downregulated. After performing GO enrichment analysis, it was found that DEGs significantly contribute to the biological processes including cell growth and apoptosis. Using pathway enrichment analysis, we found significant overrepresentation of cellular processes related to mTOR signaling and endoplasmic reticulum (ER) stress, involving genes such as GRB10, SGK1, PRKCA, RPS6KA2, RAF1, PIK3R3, FOXO1, DERL3, HMOX1, TLR7, VAMP7 and INSIG2. The obtained results of RT-PCR showed consistency with the RNA-Seq data. In summary, the underlined results revealed that MEL has significantly contributed to follicular development via activating the mTOR signaling pathway-related genes and alleviating ERS-related genes in laying hens. The current study provides a theoretical background for enhancing the egg-laying capability of hens and also providing a basis for elucidating the molecular mechanism of follicular selection.