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

Abstract

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.

Keywords: chicken; granulosa cell; mTOR signaling pathway; melatonin; transcriptome analysis.

Figure 1

Effects of MEL on plasma MEL(A), FSH(B), LH(C), E₂(D), and plasma mRNA expression of Grp78(E), Caspase-3(F), CHOP(G) level between the young peak period chicken control group (Young-CN), later period chicken control group (Old-CN), and melatonin (MEL) group. The results are presented as the means ± SEM. *P < 0.05, ^⁎⁎P < 0.01 represents significant differences compared with the Young-CN group. ^#P < 0.05, ^##P < 0.01 represent significant differences compared with the Old-CN group.

Figure 2

Comparison of the mRNA expression of Grp78(A), CHOP(B), Caspase-3(C), eIF-2α(D), TSC1(E), TSC2(F), mTOR(G), 4E-BP1(H) and S6K(I) in follicles between the young peak period chicken control group (Young-CN), later period chicken control group (Old-CN), and melatonin (MEL) group. The results are presented as the means ± SEM. *P < 0.05, ^⁎⁎P < 0.01 represents significant differences compared with the Young-CN group. ^#P < 0.05, ^##P < 0.01 represent significant differences compared with the Old-CN group.

Figure 3

Summary of sequencing results. (A) The volcano diagram of differentially expressed genes. Upregulated genes are shown in red, downregulated genes are shown in green, and genes with no significant difference in expression are indicated in blue; significance was indicated by P-value < 0.05. (B) GO enrichment analysis of DEGs in the yellow follicle granular layer tissues. The 3 main categories used for GO analysis are shown. The y-axis and x-axis indicate subcategory name and gene number, respectively. (C) The GO analysis of up-regulate differentially expressed gene—molecular function. Different colors represent different significance, the deeper the color, the more significant. (D) The GO analysis of up-regulate differentially expressed gene—biological process. KEGG enrichment analysis of DEGs in the granulosa cells. (E) The significantly enriched 30 KEGG pathways are presented. The y-axis and x-axis indicate pathway name and enrichment factor, respectively. The size of the circle indicates gene number.

Figure 4

qRT-PCR validation of upregulated DEGs identified by transcriptome analysis.

Figure 5

qRT-PCR validation of downregulated DEGs identified by transcriptome analysis.