Landscape of m6A Modification in Testicular Tissues of Normal and Cryptorchid Tianhua Mutton Sheep

Cryptorchidism is a common male reproductive disorder generally characterized by spermatogenic failure. Nevertheless, the molecular mechanisms underlying infertility induced by cryptorchidism in sexually mature rams remain insufficiently characterized. N6-methyladenosine (m6A), the most prevalent internal RNA modification, plays a dynamic role in testicular development and spermatogenesis. In the present study, testicular tissues collected from normal and cryptorchid Tianhua mutton sheep were used to construct a transcriptome-wide m6A methylation atlas of the testis. Histological observation showed significantly reduced numbers of Sertoli and Leydig cells and diminished seminiferous tubule diameters in cryptorchid testes. RNA sequencing (RNA-seq) results demonstrated the dysregulated tissue-level mRNA expression of m6A regulators in cryptorchid testes. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) revealed 2080 differentially methylated m6A peaks, with 1449 hypermethylated and 631 hypomethylated m6A peaks in cryptorchid testes. The majority of these peaks were concentrated in the CDS region, followed by the stop codons and 3′ UTR. The integrative analysis of MeRIP-seq and RNA-seq data revealed that differentially expressed and m6A methylated genes were enriched in the apoptosis, AMPK, FoxO, TNF, and MAPK signaling pathways. Seven candidate genes, including RRM2, GTSE1, ATM, GORAB, INSR, HSPA1L, and MAPK9 were screened. In conclusion, this study identified differences in the mRNA abundance of m6A regulators and regional m6A enrichment between normal and cryptorchid sheep testes. These tissue-level differences may partly reflect changes in cellular composition. The findings provide a basis for further investigation of the potential involvement of m6A regulation in cryptorchidism-associated spermatogenic impairment.