N-6 adenine-specific DNA methyltransferase 1 is essential for meiosis prophase I progression during spermatogenesis

Mammalian sexual reproduction critically relies on the generation of haploid gametes through a specialized cell division process known as meiosis. Here, we demonstrate that N-6 Adenine-Specific DNA methyltransferase 1 (N6AMT1) plays a crucial role in the progression of meiosis during spermatogenesis, as follows. Gene expression profiling revealed that N6AMT1 was expressed in germ cells throughout the entire process of spermatogenesis, with a peak in expression levels in spermatocytes at the prophase I stage of meiosis. Germ cell-specific deletion of N6amt1 in mice resulted in male subfertility as well as a significant reduction in sperm count. Notably, N6amt1-null spermatocytes exhibited meiotic arrest at prophase I and extensive apoptosis. Chromosome spreading assays revealed that N6amt1 loss impaired meiotic sex chromosome inactivation (MSCI) and delayed DNA double-strand break (DSB) repair, both crucial for proper meiotic progression. Correspondingly, transcriptomic analysis identified a substantial upregulation of genes mapping to sex chromosomes in N6amt1-null mutants, consistent with failures in MSCI. Moreover, N6AMT1 deficiency significantly augmented the expression of genes involved in the p53 pathway. Incorporating single-cell analysis, we found that the differentially expressed genes following the knockout of N6amt1 were primarily high-variability genes in pachytene stage spermatocytes, indicating that the absence of N6AMT1 leads to gene expression disorders during the pachytene stage. Taken together, our findings demonstrate that N6AMT1 is crucial to male fertility in mice, through molecular mechanisms for meiotic progression during spermatogenesis, including MSCI and DSB repair.