Loss of N-6 adenine-specific DNA methyltransferase 1 leads to meiotic prophase abnormalities and male sub-fertility in mice

Mammalian sexual reproduction critically relies on the generation of haploid gametes following 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. N6AMT1 was expressed in germ cells throughout the entire process of spermatogenesis, with a peak in mRNA 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. Correspondingly, transcriptomic analysis identified a substantial increase in transcript levels for genes mapping to sex chromosomes in N6amt1-null mutants, consistent with disruptions in MSCI. Moreover, N6AMT1 deficiency led to a significant upregulation in the steady-state mRNA levels of genes involved in the p53 pathway and functionally activated p53 signaling. Through integrated analysis of data from single-cell RNA sequencing (RNA-seq) and bulk RNA-seq experiments, we found that knockout of N6amt1 primarily affected the transcriptomic profiles of normal pachytene spermatocytes. Taken together, our findings demonstrate that N6AMT1 is required for quantitatively normal male fertility in mice and involved in the molecular mechanisms for meiotic progression during spermatogenesis, including MSCI and DSB repair.