Transient Pulses and Transcriptomic Rigidity: Dynamic miRNA Expression Patterns in Ovine Ovarian Development Reveal Molecular Strategies Underlying Seasonal Reproduction

Ovine fecundity is a key economic trait constraining livestock production efficiency, and the marked differences in reproductive strategies among breeds provide an ideal model for dissecting the molecular mechanisms underlying seasonal reproduction and prolificacy. In this study, we investigated Ujumqin sheep, a typical seasonally breeding, single-litter breed, by collecting ovarian tissues at three critical developmental stages—3 months (pre-pubertal), 6 months (puberty initiation), and 12 months (sexual maturity)—and performed whole-transcriptome sequencing to systematically characterize the dynamic expression trajectories of miRNAs, lncRNAs, circRNAs and mRNA during ovarian development. Unexpectedly, we found that miRNA expression changes did not follow a monotonic linear trajectory, but rather exhibited a highly nonlinear “transient pulse” pattern: pairwise comparisons revealed 42 and 44 differentially expressed miRNAs (DEMs) between 3 vs. 6 months and 6 vs. 12 months, respectively, yet only 21 DEMs between 3 vs. 12 months—an “inverted-V” distribution. Cross-validation by Mfuzz temporal clustering and Venn diagram intersection analyses pinpointed 10 core miRNAs that synchronously elevated during the puberty initiation window (3 → 6 months) and returned to baseline by sexual maturity (6 → 12 months). Target gene enrichment of these 10 miRNAs converged significantly in Wnt, cAMP, FoxO, and oxytocin signaling pathways—suggesting that these miRNAs are associated with molecular programs related to follicular development and hormone signaling at the 6-month puberty initiation window. We further constructed a competing endogenous RNA (ceRNA) regulatory network centered on these 10 core miRNAs and resolved two potential functional axes, lncRNA MSTRG.20588-miR-211-CTNNB1 and circRNA3387-miR-320d-ATG7, directly linking the transient pulse signals to follicle dominance selection and granulosa cell quality control. An exploratory Cross-breed comparison revealed that the prolific breed Small-Tailed Han sheep exhibited 227 DEMs at the puberty initiation window (approximately 5.4-fold more than Ujumqin sheep), with the early changes largely sustained into adulthood. In contrast, the majority of differential changes observed in Ujumqin sheep at 6 months were reversed and essentially “reset” by 12 months. Based on these findings, we propose a conceptual framework of “transcriptomic rigidity” versus “transcriptomic plasticity” to distinguish the two breeds: Ujumqin sheep ovaries tend to reset the transcriptome to a quiescent state after completing puberty initiation—a “rigidity” strategy that may represent the molecular hallmark of seasonal breeding breeds. Collectively, this study provides new conceptual tools for understanding the molecular regulation of seasonal reproduction in sheep and offers potential molecular targets for genetic improvement of reproductive performance in single-litter breeds.