Valine induces testis apoptosis through altering autophagy and nucleic acid methylation in mouse

The branched-chain amino acids (BCAA) are required for animal growth and metabolic health. However, as one kind of BCAA, the effects and molecular signals of valine on male reproduction were unknown. Valine supplementation by drinking water (0.45%) for 21 days unchanged body and testis weight as compared to Control group, but significantly disturbed morphology of sertoli cells and germ cells within seminiferous tubule and enlarged the space inter seminiferous tubules within testis. TUNEL assay and γH2AX immunostaining showed that 0.45% valine supplement induced apoptosis of mouse testis through upregulating the expression of Caspase 3 and Caspase 9. The mRNA levels of autophagy related genes of Atg5 and Lamb3 were significantly reduced in 0.45% valine group as compared to Control group, but protein level of ATG5 was unchanged. The mRNA levels of DNA 5mC methylation related genes of Dnmt1, Dnmt3a, Tet2 and Tet3 were significantly reduced by 0.45% valine supplement, but protein level and localization of Tet2 was without significant difference. 0.45% valine supplement significantly reduced the mRNA levels of RNA m6A methylation related genes of Mettl14, Alkbh5 and Fto, as well as decreased protein level and changed localization of Alkbh5 and FTO. For m6A binding proteins, 0.45% valine treatment significantly decreased the mRNA levels of YTHDF1, YTHDF2, YTHDF3, IGF2BP1 and IGF2BP2, suppressed protein level of YTHDF3. Taken together, valine could disturb the autophagy and the nucleic acid methylation (DNA 5mC and RNA m6A) pathways to induce apoptosis, and therefore change the tissue structure of mouse testis.