The objective of this study is to investigate the molecular regulatory mechanisms of non-coding RNA (ncRNA) during the developmental process of multi-litter sheep ovaries and identify key regulatory genes that enhance the reproductive capacity of sheep. This study selected Small-Tail Han sheep (multi-litter sheep) and Ujumuqin sheep (single-litter sheep) as comparative models, constructed the expression profiles of ncRNAs and mRNAs in ovarian tissues, identified differentially expressed (DE) lncRNAs, circRNAs, miRNAs, and mRNAs, and performed target gene prediction along with functional and signaling pathway enrichment analyses. Reproduction-related pathways were further screened to construct competing endogenous RNA (ceRNA) regulatory networks (lncRNA–miRNA–mRNA and circRNA–miRNA–mRNA). Finally, the dual-luciferase reporter gene assay system was employed to perform the functional validation of the relevant targeted regulatory effects. A comprehensive screening identified 411 DE lncRNAs, 322 DE circRNAs, 26 DE miRNAs, and 29 DEGs from the ovarian tissues of Ujumqin and Small-Tail Han sheep. The results of the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses demonstrated that the DE target genes were significantly enriched in pathways associated with cell dedifferentiation, the positive regulation of embryonic development, glycosaminoglycan biosynthesis, Hippo signaling, and other signaling pathways. To identify genes associated with reproductive processes, we performed differential expression screening followed by pathway enrichment analysis, which revealed significant enrichment in reproductive regulatory pathways. Based on these findings, we constructed a ceRNA regulatory network incorporating 22 DEGs, 17 DE lncRNAs, three DE circRNAs, and one DE miRNA. Our analysis revealed that oar-let-7a is involved in signaling pathways such as oocyte meiosis and Hippo, suggesting it may serve as a key miRNA regulating the trait of multiple offspring. The dual-luciferase reporter assay was employed to confirm that oar-let-7a directly targets and regulates the expression of CPEB1. Additionally, it was demonstrated that circRNA803 and lncRNA MSTRG.19726 function as molecular sponges to competitively bind and regulate oar-let-7a. These findings suggest that oar-let-7a mediates the expression of CPEB1 via circRNA803 and lncRNA MSTRG.19726 sponge adsorption, thereby regulating the process of follicular dominance in sheep. The qRT-PCR method was employed to validate the expression patterns of nine randomly selected DEGs, and the results corroborated the reliability of the RNA-seq sequencing data. This study investigated the coordinated regulatory mechanism of DE ncRNAs and their corresponding target genes, identifying a ceRNA network, circRNA803/lncRNA MSTRG.19726-oar-let-7a-CPEB1, which plays a critical role in regulating the process of follicular dominance in sheep. These findings provide fundamental data for uncovering the reproductive potential of sheep and facilitate a comprehensive understanding of their reproductive characteristics, which hold significant guiding implications for enhancing reproductive efficiency.

(1) Background: This study aims to systematically identify key candidate genes and the regulatory networks governing ovarian development in sheep breeds with divergent fecundity. Focusing on elucidating the central regulatory roles of these factors during distinct ovarian developmental stages in highly prolific breeds, the research seeks to reveal the mechanism by which multilevel regulatory networks synergistically determine ewe reproductive capacity. (2) Methods: This study utilized the ovaries from the low-fertility sheep breed Ujumqin sheep, the high-fertility sheep breed Small-tailed Han sheep, and various developmental stages of Small-tailed Han sheep as research subjects. Through whole-transcriptome sequencing analysis, differentially expressed mRNAs(DEGs) and non-coding RNAs (ncRNAs) were screened, and a ceRNA regulatory network was constructed and subjected to bioinformatic analysis. The dual-luciferase reporter gene detection system was employed to validate the targeting relationships within the obtained key circRNA-miRNA-mRNA networks. Finally, qRT-PCR was used to verify the accuracy of the sequencing results. (3) Results: The results revealed that the different fecundity groups constructed a ceRNA network comprising 116 differentially expressed circRNAs (DECs), 46 differentially expressed miRNAs (DEMs), and 82 DEGs. Similarly, the groups representing different ovarian developmental stages constructed a ceRNA network consisting of 186 DECs, 143 DEMs, and 338 DEGs. Functional enrichment analysis identified several reproduction-related signaling pathways, such as the MAPK, JAK-STAT, and WNT signaling pathways (in the different fecundity groups), and the MAPK, Ras, WNT, and Hippo signaling pathways (in the different ovarian developmental stage groups). Through comprehensive analysis of the reproduction-related pathways and screening of genes co-regulating the ceRNA networks in both comparison groups, a circRNA-miRNA-mRNA regulatory network was constructed. This analysis ultimately identified circRNA2058-miR-9226-5p-MET as playing a core regulatory role. The targeting relationship was validated using the dual-luciferase reporter assay system. The results demonstrated that miR-9226-5p mediates the expression of MET through sponging by circRNA2058. The accuracy of the sequencing results was further confirmed by qRT-PCR analysis of 8 randomly selected circRNAs and 8 miRNAs. (4) Conclusions: This study innovatively deciphered the synergistic regulatory network involving DE ncRNAs and their target genes. We identified, for the first time, a pivotal ceRNA regulatory axis: circRNA2058-miR-9226-5p-MET, which functions as a critical molecular switch driving the follicular dominance process in sheep. This discovery establishes a molecular foundation for precisely targeting core regulators of ovine reproductive efficiency, deepens our understanding of the core regulatory mechanisms governing sheep reproductive biology, and offers significant guidance for innovating strategies to enhance sheep reproductive efficiency.

(1) Background: This study aims to systematically identify key candidate genes and the regulatory networks governing ovarian development in sheep breeds with divergent fecundity. Focusing on elucidating the central regulatory roles of these factors during distinct ovarian developmental stages in highly prolific breeds, the research seeks to reveal the mechanism by which multilevel regulatory networks synergistically determine ewe reproductive capacity. (2) Methods: This study utilized the ovaries from the low-fecundity sheep breed Ujumqin sheep, the high-fecundity breed small-tailed Han sheep, and various developmental stages of small-tailed Han sheep as research subjects. Through whole-transcriptome sequencing analysis, differentially expressed mRNAs(DEGs) and non-coding RNAs (ncRNAs) were screened, and a ceRNA regulatory network was constructed and subjected to bioinformatic analysis. The dual-luciferase reporter gene detection system was employed to validate the targeting relationships within the obtained key circRNA-miRNA-mRNA networks. Finally, qRT-PCR was used to verify the accuracy of the sequencing results. (3) Results: Our analysis constructed two distinct ceRNA networks: one from different fecundity groups (116 DECs, 46 DEMs, 82 DEGs) and another from different ovarian stages (186 DECs, 143 DEMs, 338 DEGs). Functional enrichment revealed key reproduction-related pathways, including Mitogen-Activated Protein Kinase(MAPK), Janus Kinase-Signal Transducer and Activator of Transcription(JAK-STAT), and WNT signaling in the fecundity comparison, and MAPK, Ras, WNT, Hippo signaling in the developmental stage comparison. Integrated analysis identified a core circRNA-miRNA-mRNA network, pinpointing circRNA2058-miR-9226-5p-MET as a central regulatory axis. The dual-luciferase assay confirmed that circRNA2058 acts as a sponge for miR-9226-5p, thereby mediating MET expression. qRT-PCR validation of randomly selected RNAs confirmed the sequencing reliability. (4) Conclusions: this study deciphers a synergistic regulatory network and identifies, for the first time, the pivotal circRNA2058-miR-9226-5p-MET ceRNA axis as an potential critical molecular switch driving follicular dominance in sheep. This discovery provides a molecular foundation for targeting core regulators of ovine reproductive efficiency and offers significant insights for innovative strategies in enhancing sheep reproduction.

Celastrol (CSL), a natural triterpenoid extracted from Tripterygium wilfordii, demonstrates a wide range of biological activities. In this study, we explored whether CSL alleviates kidney damage in spontaneously hypertensive rats (SHRs) through the modulation of the Nrf2/Ho-1 pathway, a crucial target in renal injury models. A total of 40 male SHRs, aged 6–8 weeks, were randomly allocated to four groups: the control group (CON, serving as the healthy control), the spontaneously hypertensive rat group (SHR), the SHR group treated with low-dose CSL (L-CSL + SHR, 0.5 mg/kg/d), and the SHR group treated with high-dose CSL (H-CSL + SHR, 1 mg/kg/d). All drugs were formulated using physiological saline as the solvent and administered via intraperitoneal injection. The control group received an equivalent volume of physiological saline via intraperitoneal injection, and all groups underwent continuous daily administration for 6 weeks. The results indicated that, in comparison with the control group, the serum levels of angiotensin, angiotensin-converting enzyme, and aldosterone in the SHR group were relatively high, and CSL treatment further downregulated these indices. Simultaneously, CSL downregulated pro-inflammatory factors (tumor necrosis factor-α and interleukin-1β) and upregulated interleukin-6. Regarding renal function-related indicators, CSL reduced malondialdehyde levels and enhanced the activities of antioxidant enzymes, such as superoxide dismutase, glutathione peroxidase, and catalase. Moreover, CSL inhibited the overexpression of Keap1. Significantly, the mRNA levels of Nrf2, Nqo1, and Ho-1 in the CSL-treated groups were notably higher than those in the SHR group. These findings suggest that CSL mitigates renal pathological damage in SHR by activating the Nrf2/Ho-1 pathway, offering a potential therapeutic approach for hypertension-induced renal injury.

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.