College of Animal Science and Technology, Southwest University, Rongchang, Chongqing, 402460, China; Beef Cattle Engineering and Technology Research Center of Chongqing, Southwest University, Rongchang, Chongqing, 402460, China.
College of Animal Science and Technology, Southwest University, Rongchang, Chongqing, 402460, China; Beef Cattle Engineering and Technology Research Center of Chongqing, Southwest University, Rongchang, Chongqing, 402460, China; Chongqing Key Laboratory of Herbivore Science, Rongchang, Chongqing, 402460, China.
Theriogenology. 2022 Jul 15;187:51-57. doi: 10.1016/j.theriogenology.2022.04.022. Epub 2022 Apr 27.
N6-methyladenosine (mA) is the most common RNA methylation modification in mammals, which is controlled in the male germline to ensure coordinated gene expression in the entire process of spermatogenesis. Dzo is the male offspring of a cross between the domestic cattle (Bos taurus) and yak (Bos grunniens), and is sterile. This study aimed to investigate whether mA-associated genes are linked with dzo male sterility. The mRNA expression pattern of mA-associated genes and spermatogenesis-related genes modified by mA was characterized in cattle, yak, and dzo. Compared with fertile cattle and yak, mA erasing (ALKBH5 and FTO), writing (METTL14, WTAP, and ZC3H13), and reading (YTHDC2, YTHDF1, and YTHDF2) were testis-specifically downregulated in infertile dzo. The expression of mA target genes in spermatogonial self-renewal and proliferation (BCL6B, FOXO1, TAF4B, and FGFR1) and differentiation genes (DNMT3B and SOHLH2) were dereguleted in dzo testis. Immunofluorescent staining showed that intense ALKBH5 immunoreactivity was present in spermatogonia, primary spermatocytes, and round spermatids of cattle and yak testis. However, the number of ALKBH5 immunoreactive-positive cells were significantly reduced in dzo testis, especially in primary spermatocytes and round spermatids. Whole genome bisulfite-seq data showed that the promoter regions of FTO and YTHDC2 genes were hypermethylated in dzo testis. Moreover, bta-miR-200a was significantly downregulated in dzo testis, and it targeted the mA-associated genes such as ALKBH5, FTO, WTAP, and YTHDF2. In conclusion, mRNA of ALKBH5 was testis-specifically downregulated in dzo, which may be because fewer specific spermatogenic cells express this gene. The role of mA-associated genes in dzo male sterility and the interaction of DNA methylation and miRNA with mA-associated protein expression need to be further explored.
N6-甲基腺苷（m6A）是哺乳动物中最常见的 RNA 甲基化修饰，它在雄性生殖细胞中受到调控，以确保精子发生过程中整个过程中的基因表达协调。犏牛是黄牛（Bos taurus）和牦牛（Bos grunniens）杂交的雄性后代，是不育的。本研究旨在探讨 m6A 相关基因是否与犏牛雄性不育有关。在牛、牦牛和犏牛中，对 m6A 相关基因的 mRNA 表达模式以及 mA 修饰的精子发生相关基因进行了特征描述。与可育的牛和牦牛相比，不育的犏牛中，m6A 清除（ALKBH5 和 FTO）、写入（METTL14、WTAP 和 ZC3H13）和读取（YTHDC2、YTHDF1 和 YTHDF2）在睾丸中特异性地下调。在犏牛睾丸中，精子发生自我更新和增殖（BCL6B、FOXO1、TAF4B 和 FGFR1）和分化基因（DNMT3B 和 SOHLH2）的 mA 靶基因的表达失调。免疫荧光染色显示，牛和牦牛睾丸的精原细胞、初级精母细胞和圆形精子中存在强烈的 ALKBH5 免疫反应性。然而，在犏牛睾丸中，ALKBH5 免疫反应性阳性细胞的数量明显减少，尤其是在初级精母细胞和圆形精子中。全基因组亚硫酸氢盐测序数据显示，犏牛睾丸中 FTO 和 YTHDC2 基因的启动子区域超甲基化。此外，bta-miR-200a 在犏牛睾丸中显著下调，它靶向 m6A 相关基因，如 ALKBH5、FTO、WTAP 和 YTHDF2。总之，在犏牛中，ALKBH5 的 mRNA 特异性地下调，这可能是因为较少的特定生精细胞表达这种基因。m6A 相关基因在犏牛雄性不育中的作用以及 DNA 甲基化和 miRNA 与 m6A 相关蛋白表达的相互作用需要进一步探索。
