As a ubiquitous reversible epigenetic RNA modification, N6-methyladenosine (m6A) plays crucial regulatory roles in multiple biological pathways. However, its functional mechanisms in sex determination and differentiation during gonadal development of chicken embryos are not clear. Therefore, we established a transcriptome-wide m6A map in the female and male chicken left gonads of embryonic day 7 (E7) by methylated RNA immunoprecipitation sequencing (MeRIP-seq) to offer insight into the landscape of m6A methylation and investigate the post-transcriptional modification underlying gonadal differentiation. The chicken embryonic gonadal transcriptome was extensively methylated. We found 15,191 and 16,111 m6A peaks in the female and male left gonads, respectively, which were mainly enriched in the coding sequence (CDS) and stop codon. Among these m6A peaks, we identified that 1013 and 751 were hypermethylated in females and males, respectively. These differential peaks covered 281 and 327 genes, such as BMP2, SMAD2, SOX9 and CYP19A1, which were primarily associated with development, morphogenesis and sex differentiation by functional enrichment. Further analysis revealed that the m6A methylation level was positively correlated with gene expression abundance. Furthermore, we found that YTHDC2 could regulate the expression of sex-related genes, especially HEMGN and SOX9, in male mesonephros/gonad mingle cells, which was verified by in vitro experiments, suggesting a regulatory role of m6A methylation in chicken gonad differentiation. This work provided a comprehensive m6A methylation profile of chicken embryonic gonads and revealed YTHDC2 as a key regulator responsible for sex differentiation. Our results contribute to a better understanding of epigenetic factors involved in chicken sex determination and differentiation and to promoting the future development of sex manipulation in poultry industry.

中文翻译：

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m6A甲基化调控鸡胚性腺性别分化

作为一种普遍存在的可逆表观遗传 RNA 修饰，N6-甲基腺苷 (m6A) 在多种生物学途径中起着至关重要的调节作用。然而，其在鸡胚性腺发育过程中的性别决定和分化的功能机制尚不清楚。因此，我们通过甲基化 RNA 免疫沉淀测序 (MeRIP-seq) 在胚胎第 7 天 (E7) 的雌性和雄性鸡左侧性腺中建立了一个转录组范围的 m6A 图，以深入了解 m6A 甲基化的前景并研究转录后修改潜在的性腺分化。鸡胚性腺转录组被广泛甲基化。我们分别在雌性和雄性左侧性腺中发现了 15,191 和 16,111 个 m6A 峰，主要富集在编码序列 (CDS) 和终止密码子中。在这些 m6A 峰中，我们发现 1013 和 751 分别在女性和男性中高甲基化。这些差异峰涵盖了 281 和 327 个基因，如 BMP2、SMAD2、SOX9 和 CYP19A1，它们主要与发育、形态发生和功能富集的性别分化相关。进一步分析表明，m6A甲基化水平与基因表达丰度呈正相关。此外，我们发现YTHDC2可以调节雄性中肾/性腺混合细胞中性相关基因，尤其是HEMGN和SOX9的表达，这已通过体外实验得到证实，表明m6A甲基化在鸡性腺分化中具有调节作用。这项工作提供了鸡胚性腺的全面 m6A 甲基化谱，并揭示了 YTHDC2 是负责性别分化的关键调节因子。我们的研究结果有助于更好地了解参与鸡性别决定和分化的表观遗传因素，并促进家禽业性别操纵的未来发展。