l-valine supplementation disturbs vital molecular pathways and induces apoptosis in mouse testes

The branched-chain amino acids (BCAAs: leucine, isoleucine and valine) are essential for animal growth and metabolic health. However, the effect of valine on male reproduction and its underlying molecular mechanism remain largely unknown. Here, we showed that l-valine supplementation (0.30% or 0.45%, water drinking for 3 weeks) did not change body and testis weights, but significantly altered morphology of sertoli cells and germ cells within seminiferous tubule, and enlarged the space between seminiferous tubules within mouse testis. l-valine treatment (0.45%) increased significantly the Caspase3/9 mRNA levels and CASPASE9 protein levels, therefore induced apoptosis of mouse testis. Moreover, gene expression levels related to autophagy (Atg5 and Lamb3), DNA 5 mC methylation (Dnmt1, Dnmt3a, Tet2 and Tet3), RNA m6A methylation (Mettl14, Alkbh5 and Fto), and m6A methylation binding proteins (Ythdf1/2/3 and Igf2bp1/2) were significantly reduced. Protein abundances of ALKBH5, FTO and YTHDF3 were also significantly reduced, but not for ATG5 and TET2. Testis transcriptome sequencing detected 537 differentially expressed genes (DEGs, 26 up-regulated and 511 down-regulated), involved in multiple important signaling pathways. RT-qPCR validated 8 of 9 DEGs (Cd36, Scd1, Insl3, Anxa5, Lcn2, Hsd17b3, Cyp11a1, Cyp17a1 and Agt) to be decreased significantly, consistent with RNA-seq results. Taken together, l-valine treatment could disturb multiple signaling pathways (autophagy and RNA methylation etc.), and induce apoptosis to destroy the tissue structure of mouse testis.