SLC22A14 is a mitochondrial riboflavin transporter required for sperm oxidative phosphorylation and male fertility

Ablation of Slc22a14 causes male infertility in mice, but the underlying mechanisms remain unknown. Here, we show that SLC22A14 is a riboflavin transporter localized at the inner mitochondrial membrane of the spermatozoa mid-piece and show by 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 find that fatty acid β-oxidation (FAO) is defective with significantly reduced levels of acyl-carnitines and metabolites from the TCA cycle (the citric acid cycle) but accumulated triglycerides and free fatty acids in Slc22a14 knockout spermatozoa. We demonstrate that Slc22a14-mediated FAO is essential for spermatozoa energy generation and motility. Furthermore, sperm from wild-type mice treated with a riboflavin-deficient diet mimics those in Slc22a14 knockout mice, confirming that an 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 the development of male contraceptives. [Display omitted] •Slc22a14 deficiency results in decreased sperm motility and male infertility•Slc22a14 ablation disrupts fatty acid β-oxidation and flavoenzyme activity•Slc22a14 is a riboflavin transporter located at inner mitochondrial membrane in sperm Long-chain fatty acid β-oxidation is an important energy source during epididymal maturation of spermatozoa. Kuang et al. show that Slc22a14 is a riboflavin transporter located at the inner mitochondrial membrane. Slc22a14 deficiency in the spermatozoa mid-piece disrupts the riboflavin transport and subsequently alters flavoenzyme-mediated bioenergetic metabolism, resulting in male infertility.