The Roles of N⁶-Methyladenosine Modification and Its Regulators in Male Reproduction

Infertility affects an estimated 10 to 15 percent of couples worldwide, with approximately half of the cases attributed to male-related issues. Most men diagnosed with infertility exhibit symptoms such as oligospermia, asthenospermia, azoospermia, and compromised sperm quality. Spermatogenesis is a complex and tightly coordinated process of germ cell differentiation, precisely regulated at transcriptional, posttranscriptional, and translational levels to ensure stage-specific gene expression during the development of spermatogenic cells and normal spermiogenesis. N⁶-methyladenosine (m⁶A) stands out as the most prevalent modification on eukaryotic mRNA, playing pivotal roles in various biological processes, including mRNA splicing, transportation, and translation. RNA methylation modification is a dynamic and reversible process primarily mediated by “writers”, removed by “erasers”, and recognized by “readers”. In mammals, the aberrant methylation modification of m⁶A on mRNA is associated with a variety of diseases, including male infertility. However, the precise involvement of disrupted m⁶A modification in the pathogenesis of human male infertility remains unresolved. Intriguingly, a significant correlation has been found between the expression levels of m⁶A regulators in the testis and the severity of sperm concentration, motility, and morphology. Aberrant expression patterns of m⁶A regulatory proteins have been detected in anomalous human semen samples, including those of oligospermia, asthenozoospermia, and azoospermia. Furthermore, the examination of both sperm samples and testicular tissues revealed abnormal mRNA m⁶A modification, leading to reduced sperm motility and concentration in infertile men. Consequently, it is hypothesized that dysregulation of m⁶A modification might serve as an integral link in the mechanism of male infertility. This paper presents a comprehensive review of the recent discoveries regarding the spatial and temporal expression dynamics of m⁶A regulators in testicular tissues and the correlation between deregulated m⁶A regulators and human male infertility. Previous studies predominantly utilized constitutive or conditional knockout animal models for testicular phenotypic investigations. However, gene suppression in additional tissues could potentially influence the testis in constitutive knockout models. Furthermore, considering the compromised spermatogenesis observed in constitutive animals, distinguishing between the indirect effects of gene depletion on testicular development and its direct impact on the spermatogenic process is challenging, due to their intricate relationship. Such confounding factors might compromise the validity of the findings. To address this challenge, an inducible and conditional gene knockout model may serve as a superior approach. To date, nearly all reported studies have concentrated solely on the level changes of m⁶A and its regulators in germs cells, while the understanding of the function of m⁶A modification in testicular somatic cells remains limited. Testicular somatic cells, including peritubular myoid cells, Sertoli cells, and Leydig cells, play indispensable roles during spermatogenesis. Hence, comprehensive exploration of m⁶A modification within these cells as an additional crucial regulatory mechanism is warranted. In addition, exploration into the presence of unique methylation mechanisms or m⁶A regulatory factors within the testes is warranted. To elucidate the role of m⁶A modification in germ cells and testicular somatic cells, detailed experimental strategies need to be implemented. Among them, manipulation of the levels of key enzymes involved in m⁶A methylation and demethylation might be the most effective approach. Moreover, comprehensive analysis of the gene expression profiles involved in various signaling pathways, such as Wnt/β-catenin, Ras/MAPK, and Hippo, in m⁶A-modified germ cells and testicular somatic cells can provide more insight into its regulatory role in the spermatogenesis process. Further research in this area could provide valuable insights for developing innovative strategies to treat male infertility. Finally, considering the mitigation impact of m⁶A imbalance regulation on disease, investigation concerning whether restoring the equilibrium of m⁶A modification regulation can restore normal spermatogenesis function is essential, potentially elucidating the pivotal clinical significance of m⁶A modulation in male infertility.