Prenatal Low Testosterone Levels Induced by DNAH8 Dysfunction Leads to Urethral Fusion and Male Differentiation Abnormalities

Background: Hypospadias development is influenced by prenatal androgen levels, with genetic factors typically playing a significant role. Through whole-exome sequencing, we found that rare damaging variants in DNAH8 (dynein axonemal heavy chain 8) were significantly enriched in hypospadias cases. However, the role of DNAH8 deficiency in hypospadias pathogenesis remains unclear. Objectives: This study aimed to clarify the function of DNAH8 in urethral development and fusion. Materials and Methods: Using CRISPR/Cas9, we generated DNAH8 knockout mice and employed a multi-disciplinary approach to evaluate urogenital development, male differentiation, testosterone levels, steroid biosynthesis gene expression, and cellular changes in fetal testes and external genitalia. Results:DNAH8 knockout mice presented abnormal masculinization phenotype, and fetal mice exhibited urethral fusion defects and hypoplastic glans during early urethral development. DNAH8 knockout was found to reduce prenatal testosterone levels and steroid biosynthesis in the testes. Based on single-cell sequencing and multicolor immunofluorescence, we demonstrated that in the early stage of fetal testis development, the loss of DNAH8 function affected the differentiation of Sertoli and steroidogenic cell lineages, thereby impairing testosterone synthesis ability during the masculinization programming window. Meanwhile, we identified two key distal glans cell populations that cause abnormal urethral fusion and hypoplastic glans. Furthermore, DNAH8 knockout could synergistically interact with low-dose endocrine-disrupting chemicals, increasing the incidence of urethral fusion defects at E16.5, and led to clear hypospadias phenotypes at E18.5. Conclusions: Loss of DNAH8 delays differentiation of Sertoli and steroidogenic lineages, reduces prenatal testosterone, and, with environmental exposure, increases hypospadias risk.