Effects Of Exogenous Valine On Mouse Testis Structure And Molecular Pathways

Branched chain amino acids(BCAA),including Valine(Val),Leucine(Leu)and Isoleucine(Ile),are essential nutrients for animal growth and metabolic health,which could affect fat deposition and muscle development.Val can function to promote fat deposition,increase the content of triglycerides in serum,affect liver lipid metabolism,inhibit the expression of lipolytic genes,and influence animal obesity.Evidences have shown that obesity leads to male infertility through affecting the hypothalamic-pituitary-gonadal(HPG)axis.Excess adipose tissue can reduce testosterone production,change the relative ratio of testosterone and estrogen,and therefore damage spermatogenesis.The effect of val on male reproduction and underlying molecular signal pathways are unclear yet.Therefore,the present study used ICR(Institute of Cancer Research)male rats at the age of 4-5 week as research model,to explored the effect of val on testicular tissue structure and molecular pathways via adding val at different concentrations into drinking water,providing new understandings regarding for regulation of animal male reproduction by valine.The main results are as follows:(1)Effects of Val supplement on the growth and development of mice and testis tissueICR male mouse were supplemented with different concentrations of Val through drinking water for 21 days.It was showed that Val treatment unchanged the body weight of mice and the weight of left and right testis(P>0.05),but the morphology of sertoli cells and germ cells in testis tissue were significantly changed,and the space between the vas deferens within the testis was expanded by Val supplement in a dose dependent manner.TUNEL assay andγ-H2AX immunofluorescence staining showed that the proportion of TUNEL positive cells within testis was significantly higher in0.45%Val group than that in Control group(P<0.05),and the percentage ofγ-H2AX positive cells was also significantly higher in 0.45%Val group than that in Control and0.30%Val groups(P<0.05).The cells with positive apoptotic signals were mainly spermatogenic and sperm cells.RT-qPCR showed the expression levels of Caspase3(P<0.05)and Caspase9(P<0.01)m RNAs to be significantly higher in 0.45%Val group than that in Control group;Immunofluorescence staining confirmed the fluorescence level of CASPASE9 protein in testicular tissue to be significantly higher in 0.45%Val group than that in Control and 0.30%Val groups,mainly distributed in spermatogenic cells.Therefore,0.45%Val supplement can induce apoptosis of spermatogenic cells within testis by activating Caspase pathway,thus damage testis tissue.(2)Effects of Val on autophagy and nucleic acid methylation pathways of mouse testisRT-qPCR results showed that 0.45%Val significantly reduced the m RNA levels of autophagy related genes Atg5(P<0.01)and Lamp2(P<0.01),nucleic acid methylation(DNA 5m C and RNA m~6A)related genes Dnmt1(P<0.01),Dnmt3a(P<0.01),Tet2(P<0.001),Tet3(P<0.05),Mettl14(P<0.01),Alkbh5(P<0.001),Fto(P<0.001),Ythdf1(P<0.01)Ythdf2(P<0.05),Ythdf3(P<0.001),Igfbp1(P<0.01)and Igfbp2(P<0.05),as compared to Control group.Western blotting showed that 0.45%Val significantly reduced protein abundance of ALKBH5(P<0.001)and YTHDF3(P<0.05).Immunofluorescence showed that 0.45%Val significantly decreased the abundance of FTO protein located in leydig cells and at the edge of tubules.These data indicate that0.45%Val could affect testicular development via reducing the expression of genes related to autophagy and nucleic acid methylation pathways.(3)Transcriptome altered by 0.45%Val treatment of mouse testisRNA-seq identified 537(26 up-regulated and 511 down-regulated)differently expressed genes(DEGs)(|log2Fold Change|≥1,Padj.≤0.05)between 0.45%Val group and Control group,which were enriched in GO terms of steroid and lipid metabolism,hormone metabolism,reproductive development,apoptosis,antioxidant activity,transcriptional regulation activity etc.in KEGG pathways of in cholesterol metabolism,protein digestion and absorption,steroid biosynthesis signal pathway etc.Nine DEGs(Cd36,Scd1,Insl3,Anxa5,Lcn2,Hsd17b3,Cyp11a1,Cyp17a1 and Agt)involved in important pathways of lipid and hormone metabolism,reproductive development and apoptosis function were selected for RT-qPCR validation.Except for Insl3 without significant changes,all other eight genes showed consistent expression pattern with RNA-seq results.The transcriptome results showed that 0.45%Val supplement could inhibit the expression of many genes,and mediate the damage effect on male mice testicular tissue through multiple vital signal pathways.Taken together,the results from the present study showed that exogenous supplement of 0.45%Val for three weeks could inhibit the expression of many genes in genome-wide,affect multiple signal pathways and biological processes,and finally induce testicular tissue damage in mice by activating apoptosis pathways.These results provide the basis and references for deep investigation of Val and animal male reproduction.