Ablation of Slc22a14 causes male infertility in mice, but the underlying mechanisms remain unknown. Here, we discovered that SLC22A14 is a riboflavin transporter localized at the mitochondrial inner membranes of spermatozoa midpiece, and showed via genetic, biochemical, multi-omic and nutritional evidence that riboflavin transport deficiency suppresses the oxidative phosphorylation and reprograms spermatozoa energy metabolism by disrupting flavoenzyme functions. Specifically, we found that fatty acid β-oxidation (FAO) is defective with significantly reduced levels of acyl-carnitines and metabolites from TCA cycle, but accumulated triglycerides and free fatty acids in Slc22a14 knockout spermatozoa. We demonstrated that Slc22a14-mediated FAO is essential for spermatozoa energy generation and motility. Furthermore, sperm from wildtype mice treated with riboflavin deficient diet mimics those in Slc22a14 knockout mice, confirming that altered riboflavin level causes spermatozoa morphological and bioenergetic defects. Beyond substantially advancing our understanding of spermatozoa energy metabolism, our study provides an attractive target for development of male contraceptives.
