DNAH3 interacts with DNALI1 is required for sperm flagellum function and male fertility

The inner dynein arm (IDA) plays a crucial role in regulating ciliary and flagellar beating; however, the molecular mechanisms underlying IDA regulation remain largely unclear. Whole-exome sequencing (WES) was performed to identify candidate pathogenic variants in an infertile man with asthenoteratozoospermia. A knockout mouse model was generated using CRISPR/Cas9 to investigate the pathophysiological effects of the identified variant. Phenotypic characterization included semen parameter analysis (e.g., sperm concentration, motility), morphological assessments (electron microscopy, immunostaining, histology), and mechanistic studies (e.g., scRNA-seq, proteomics, co-immunoprecipitation, mass spectrometry, computational prediction). A homozygous missense variant in DNAH3 was identified in the patient. DNAH3, an IDA component, is predominantly expressed in post-meiotic cells, specifically within the sperm flagella. Notably, Dnah3 knockout (Dnah3−/−) mice exhibited abnormal flagellar morphology and male infertility. Immunofluorescence and transmission electron microscopy revealed that the absence of IDAs in Dnah3−/− male mice. Additionally, scRNA-seq analysis indicated that Dnah3 deficiency in elongating spermatids significantly alters the expression of genes related to sperm motility. Combined comparative proteomics and co-immunoprecipitation analyses demonstrated that DNAH3 forms a complex with DNALI1. Furthermore, DNAH3-associated male infertility in human and mice was resolved by intracytoplasmic sperm injection (ICSI). Our findings suggest that the interaction between DNAH3 and DNALI1 is critical for flagellum assembly. DNAH3 is a candidate gene for the genetic diagnostic of severe asthenoteratozoospermia and primary male infertility.