Functional analysis of genes enriched in male reproductive organs: validation via fertility assessment of 14 knockout mouse lines

Tissue-restricted gene expression serves as a key indicator of functional importance within specific organs. Despite advances in high-throughput sequencing and genome editing over the past decade, this principle remains the gold standard for identifying candidate genes for functional studies. In reproductive biology, testis-restricted expression is anticipated to indicate genes crucial for gametogenesis, given the conserved features shared between spermatogenesis and oogenesis. Concurrently, rapid evolution of spermatogenesis-related genes is well documented and thought to arise through duplication and subsequent mutation of ubiquitously expressed genes. In this study, we established 14 knockout (KO) mouse lines for male reproductive organ-enriched genes, all of which exhibited normal growth and fertility in natural mating assays. These genes share sequence similarity with previously characterized gene families or functionally defined paralogs. These observations indicate that while these genes acquired enriched expressions in male reproductive organ following their evolutionary origin, they may represent: (1) currently non-functional loci, (2) genes with redundant functions compensated by paralogs, or (3) genes undergoing loss of function.