Deletion of an evolutionarily conserved TAD boundary impacts spermatogenesis in mice

Abstract Spermatogenesis is a complex process that can be disrupted by genetic and epigenetic changes, potentially leading to male infertility. Recent research has rapidly increased the number of protein coding mutations causally linked to impaired spermatogenesis in humans and mice. However, the role of non-coding mutations remains largely unexplored. As a case study to evaluate the effects of non-coding mutations on spermatogenesis, we first identified an evolutionarily conserved topologically associated domain (TAD) boundary near two genes with important roles in mammalian testis function: Dmrtb1 and Lrp8. We then used CRISPR-Cas9 to generate a mouse line where 26 kb of the boundary was removed including a strong and evolutionarily conserved CTCF binding site. ChIP-seq and Hi-C experiments confirmed the removal of the CTCF site and a resulting mild increase in the DNA–DNA interactions across the domain boundary. Mutant mice displayed significant changes in testis gene expression, higher frequency of histological abnormalities, a drop of 47–52% in efficiency of meiosis, a 15–18% reduction in efficiency of spermatogenesis, and consistently, a 12–28% decrease in daily sperm production compared to littermate controls. Despite these quantitative changes in testis function, mutant mice show no significant changes in fertility. This suggests that non-coding deletions affecting testis gene regulation may have smaller effects on fertility compared to coding mutations of the same genes. Our results demonstrate that disruption of a TAD boundary can have a negative impact on sperm production and highlight the importance of considering non-coding mutations in the analysis of patients with male infertility.