Y-Linked Expression Signatures Distinguish Dysfunctional Testicular States in Sheep

The sheep Y chromosome encodes genes essential for testicular development and spermatogenesis, yet their transcriptional dynamics across developmental and pathological states remain largely uncharacterized. Here, we profiled Y-linked gene expression using RNA-seq datasets from Hu sheep testes across postnatal development (0, 3, 6, 12 months) and from divergent testicular (large, normal, small, cryptorchid) and epididymal (large, small) weights. Developmental trajectory analysis of 134 expressed Y-linked genes revealed three distinct patterns: progressive activation from birth to sexual maturity (dominated by multicopy families ZNF280BY, HSFY, PRAMEY, TSPY3), progressive decrease, and stage-specific expression. Differential expression identified 41 Y-linked genes consistently downregulated in small, cryptorchid, and pre-pubertal testes compared with normal or large mature testes. Hierarchical clustering showed that 6-month-old small testes transcriptionally clustered with cryptorchid testes, not with immature or normal testes. In the epididymis, only one Y-linked gene was differentially expressed between large and small groups. Among X-Y gametolog pairs, most showed positive expression correlations in testes, whereas DDX3X/DDX3Y exhibited a significant negative correlation (ρ = -0.450, p = 0.0185); strikingly, this pair reversed to a positive correlation in the epididymis (ρ = 0.607, p = 3.29 × 10^-5). In conclusion, Y-linked transcriptional profiles distinguish testicular pathology from normal maturation, and DDX3X/DDX3Y regulation is strictly tissue-specific.