Spermatogenesis is a continual process that occurs in the testes, in which diploid spermatogonial stem cells (SSCs) differentiate and generate haploid spermatozoa. This highly efficient and intricate process is orchestrated at multiple levels. N⁶-methyladenosine (m⁶A), an epigenetic modification prevalent in mRNAs, is implicated in the transcriptional regulation during spermatogenesis. However, the dynamics of m⁶A modification in non-rodent mammalian species remains unclear. Here, we systematically investigated the profile and role of m⁶A during spermatogenesis in pigs. By analyzing the transcriptomic distribution of m⁶A in spermatogonia, spermatocytes, and round spermatids, we identified a globally conserved m⁶A pattern between porcine and murine genes with spermatogenic function. We found that m⁶A was enriched in a group of genes that specifically encode the metabolic enzymes and regulators. In addition, transcriptomes in porcine male germ cells could be subjected to the m⁶A modification. Our data show that m⁶A plays the regulatory roles during spermatogenesis in pigs, which is similar to that in mice. Illustrations of this point are three genes (SETDB1, FOXO1, and FOXO3) that are crucial to the determination of the fate of SSCs. To the best of our knowledge, this study for the first time uncovers the expression profile and role of m⁶A during spermatogenesis in large animals and provides insights into the intricate transcriptional regulation underlying the lifelong male fertility in non-rodent mammalian species.

精子发生是睾丸中发生的一个持续过程，其中二倍体精原干细胞（SSC）分化并产生单倍体精子。这个高效且复杂的过程是在多个层面上精心策划的。 N ⁶ -甲基腺苷(m ⁶ A) 是 mRNA 中普遍存在的一种表观遗传修饰，与精子发生过程中的转录调控有关。然而，非啮齿类哺乳动物中 m ⁶ A 修饰的动态仍不清楚。在这里，我们系统地研究了 m ⁶ A 在猪精子发生过程中的概况和作用。通过分析精原细胞、精母细胞和圆形精子细胞中 m ⁶ A 的转录组分布，我们确定了具有生精功能的猪和鼠基因之间全局保守的 m ⁶ A 模式。我们发现 m ⁶ A 富含一组专门编码代谢酶和调节因子的基因。此外，猪雄性生殖细胞中的转录组可以进行 m ⁶ A 修饰。我们的数据表明，m ⁶ A 在猪的精子发生过程中发挥着调节作用，这与小鼠的精子发生过程类似。三个基因（ SETDB1 、 FOXO1和FOXO3 ）可以说明这一点，它们对于决定 SSC 的命运至关重要。 据我们所知，这项研究首次揭示了大型动物精子发生过程中 m ⁶ A 的表达谱和作用，并为非啮齿类哺乳动物终生雄性生育力背后复杂的转录调控提供了见解。