Research On The Regulatory Mechanism Of MTOR Signaling Pathway Mediated Melatonin To Delay Ovary Aging In Late-phase Laying Hen

The performance of laying hens is affected by many factors.Among them,ovarian aging in the late-phase and the decrease in the number of follicles are the main reasons for the decrease in egg production rate.Melatonin is of great significance to protect the normal development of follicles.The mTOR signaling pathway plays an important role in regulating cell growth and proliferation.Therefore,this research combined multi-omics,fluorescence quantitative PCR and Western Blot and other technologies to systematically study the melatonin-mTOR signaling pathway-ovarian function regulatory pathway,and explore the related genes of melatonin and mTOR pathway in chicken ovary aging,analysis the mechanism of the mTOR signaling pathway in the melatonin-mediated ovarian aging process,which provide a theoretical basis for further research on follicular hierarchy to establish and extend the cycle of late-phase laying hens.Thirty 30-day-old and 70-week-old Dawu Jinfeng hens were selected,and the egg production rate and egg quality were tracked for 5 consecutive weeks.Antioxidant indicators and immune indicators were detected by ELISA method,HE staining to detect changes in the tissue structure of follicles at different developmental stages,Detection of mTOR signaling pathway-related genes(CLIP-170,GRB10,LIPIN-1,ULK1,4E-BP1,S6K,PKC,RHO,and SGK1)using real-time quantitative PCR technology during peak egg production(35 week-old)and late egg production(75 week-old)relative expression in follicles at different developmental stages.The relative expression in the protein was measured using Western Blot to detect the protein expression of key genes(mTOR,PKC,4E-BP1).The results showed that compared with 35-week-old laying hens,the egg production rate of 75-week-old laying hens,the levels of antioxidant indexes SOD,GSH-Px,T-AOC and immunoglobulin IgM,IgG were extremely significantly reduced(P<0.01),the level of MDA was extremely significantly increased(P<0.01).The number of follicles of different grades was less than 35-week-old at 75-week-old,and the number of large white follicles,small white follicles,and primary follicles reached a extremely significant level compared with 35-week-old(P<0.05).The granular cell layer,membrane layer and connective layer of SWF,LWF,SYF and POF in laying hens at 75-week-old were all extremely significantly reduced compared with the 35-week-old(P<0.01).Compared with 35-week-old laying hens,75-week-old laying hens mTOR signaling pathway downstream genes CLIP-170,GRB10,LIPIN-1,4E-BP1,S6K,RHO,SGK1 mRNA expression levels in SWF,LWF,F1,F3 and ovarian expression levels significantly decreased(P<0.05),mRNA expression levels in SYF follicles significantly increased(P<0.05).The expression of ULK1 gene in SWF,LWF,F3,F1 and ovary of 75-week-old laying hens was extremely significantly higher than that of 35-week-old laying hens(P<0.01),and the SYF of small yellow follicles was significantly decreased(P<0.05).The results suggest that the gene expression pattern of mTOR signaling pathway is age-specific.The experiment selected 60 laying hens(70 weeks old)and were randomly divided into 2 groups:control group and melatonin group.Group melatonin was administered intraperitoneally with melatonin at a dose of 20 mg/kg/d for 28 d.The results showed that MT significantly increased the levels of the antioxidant enzymes superoxide dismutase and total antioxidant capacity(P<0.01)as well as levels of immunoglobulin(IgA,IgG,and IgM)(P<0.05)and the reproductive hormones estradiol and luteinizing hormone(P<0.01)in the plasma and also increased the numbers of middle white follicles and small white follicles(P<0.05)and decreased the level of reactive oxygen species in plasma(P<0.01)in laying hens.The mRNA expression of mTOR,4E-binding protein-1(4E-BP1)and ribosomal protein 6 kinase(S6K)in the melatonin group was significantly up-regulated(P<0.01),and the levels of mTOR,p-mTOR,4E-BP1 and p-4E-BP1 protein were increased in the M(P<0.05).This study indicated that MT may promote follicle growth by increasing levels of antioxidant enzymes and reproductive hormones and by activating the mTOR signaling pathway in late-phase laying hens.The experiment selected melatonin-treated(200umol/L)and untreated layer follicle granulosa cells as the research object for transcriptome sequencing analysis.A total of 582 differentially expressed genes were screened by sequencing.Compared with the control group,259 genes were up-regulated and 322 genes were down-regulated in melatonin-treated chicken follicle granulosa cells.After performing GO enrichment analysis on differential genes,it was found that differentially expressed genes mainly involved biological processes such as cell proliferation and apoptosis.KEGG analysis showed that it mainly gathered in mTOR signaling pathway,PI3K-Akt signaling pathway,Foxo signaling pathway,MAPK signaling pathway.etc.Real-time quantitative PCR detection of differentially expressed genes was consistent with the transcriptome results.The screened differentially expressed genes GRB10.SGK1.PRKCA,RPS6KA2,RAF1,PIK3R3,WNT16 regulate the physiological function of cells in the mTOR signaling pathway,and screened the differentially expressed genes CDKA1A,STAR,IL1B,BOK,FAT,BMP5,AIFM3,PAWR and FOXO1 are involved in regulating the proliferation and apoptosis of follicles.This indicates that the mTOR signaling pathway is involved in the regulation of melatonin-mediated proliferation of chicken follicle granulosa cells,and melatonin may play a regulatory role through the PI3K-Akt-mTOR cascade signaling pathway.In the experiment,small yellow follicles(8-10mm in diameter)were selected as the research object,granulosa cells were isolated,and granular cells were cultured with melatonin(0,2,20 or 200 umol/L)for 48 hours.The results showed that melatonin treatment enhanced proliferation and suppressed apoptosis of granular cell at 20umol/L and 200umol/L(P<0.05)by up-regulation of Cyclin D1(P<0.01),Bcl-2(P<0.01)and down-regulation of P21,Caspase-3,Beclinl,LC3-Ⅱ(P<0.01).Melatonin treatment increased the expression of mTOR,PKC,4E-BP1,S6K mRNA and p-TOR,p-S6K protein(P<0.05).Add mTOR activator and inhibitor to cells and screened the optimal dose(10 umol/L MHY1485 and 100nM rapamycin),and co-culture with 20umol/L melatonin for subsequent experiments.The results showed that compared with the control group,20umol/L melatonin+10 umol/L MHY1485 co-culture,granulosa cell proliferation was significantly enhanced(P<0.05);100nM rapamycin significantly inhibited granulosa cells proliferation and enhanced granulosa cells apoptosis(P<0.05),However,in the 20umol/L melatonin+100nM rapamycin co-culture groups,the effect was reversed(P<0.05).Cyclin D1,Bcl-2 protein up-regulation and P21,Caspase-3,Beclinl,LC3-Ⅱ protein down-regulation also verified this result(P<0.05).The 20umol/L melatonin+10 umol/L MHY1485 activated the mTOR signaling pathway upstream gene PI3K,AKT1,AKT2 and downstream genes PKC,4E-BP1,S6K(P<0.05)and protein expression of p-mTOR,p-S6K.Rapamycin significantly inhabited the mTOR signaling pathway-related genes mRNA levels(P<0.05).In addition,activation of the mTOR signaling pathway increased the mRNA level of melatonin receptors(P<0.05).These findings finally revealed that melatonin regulates chicken granulosa cells proliferation and anti-apoptosis by activating the mTOR signaling pathway.In summary,the results of the study indicate that melatonin inhibits the apoptosis of follicle granulosa cells through the mTOR signaling pathway,enhances the body’s antioxidant properties,and delays the ovarian aging in the late-phase laying hen.This experiment provides a scientific basis for further understanding of the mechanism of declining ovarian function and prolonging the laying cycle of the late-phase laying hens.