As the hybrid offspring of cattle and yak, cattle–yaks suffer from male sterility, manifesting as cascading spermatogenic failure. Despite the Y chromosome’s pivotal role in spermatogenesis, the absence of a high-quality yak Y assembly has long impeded mechanistic understandings from this perspective. Here, a near-complete 42.4 Mb yak Y chromosome is constructed through a multi-stage assembly strategy that integrates de novo assembly with pangenome graph construction and Hi-C guided refinement. By developing a rigorously standardized gene annotation pipeline for precise cross-species comparison, we find that yaks have undergone a greater expansion of Y-linked ampliconic genes than cattle. Integrating this ampliconic landscape with short-read and full-length transcriptomics further demonstrates that yaks exhibit a drastic 2-to-4-fold increase in transcriptionally active copies of spermatogenesis-related ampliconic genes (including TSPY1, ZNF280BY, HSFY and PRAMEY) relative to cattle. Given negligible homology outside the pseudoautosomal region and conservation of key meiotic proteins, we propose a ‘cis-trans regulatory mismatch’ model driven by divergent Y-linked amplification as a working hypothesis to explain the primary genetic mechanism of cattle–yak male sterility. Together, these findings offer critical insights for addressing cattle–yak male sterility and establish the Y chromosome as an active driver of reproductive isolation beyond its traditional degenerate characterization.

The yak (Bos grunniens) thrives under chronic hypoxia and cold on the Qinghai–Tibet Plateau, yet a cross-tissue view of post-transcriptional regulation in this species remains limited. Here, we integrated multi-tissue RNA-seq and miRNA-seq data (tissues pooled from three Maiwa yaks) to construct and compare tissue-specific competing endogenous RNA (ceRNA) networks, while explicitly addressing a major source of false positives in ceRNA inference—misclassified lncRNA candidates with translational signatures. We cataloged 10,037 high-confidence lncRNAs (9360 non-redundant), 234 circRNAs, and 1030 miRNAs across six tissues. We then used Ribo-seq as an orthogonal quality-control layer to remove lncRNA candidates showing clear ribosome-association signals prior to network construction. Using a shared-target strategy (7mer-m8 seed matches; a ceRNA edge required ≥5 shared miRNAs), we assembled ceRNA networks for liver, lung, spleen, testis, and small intestine; skeletal muscle was excluded owing to insufficient Ribo-seq support for consistent filtering. Network topology varied substantially across tissues, with the testis network exhibiting the highest connectivity. ceRNA edges showed minimal overlap between tissues, indicating strong tissue dependence, whereas miRNA load/use profiles were moderately concordant, supporting a hierarchical conserved core—variable periphery organization. Importantly, the Ribo-seq–filtered lncRNA set provides a separate pool of ribosome-associated candidates for targeted follow-up, although ribosome association alone does not establish stable micropeptide production. Together, our results deliver a multi-tissue ceRNA resource and a reproducible, evidence-aware workflow for prioritizing candidate regulators while reducing annotation-driven false positives in yak.