Long non-coding RNA (LncRNA) have been identified as important regulators in the hypothalamic-pituitary-ovarian axis associated with sheep prolificacy. However, their expression pattern and potential roles in the pituitary are yet unclear. To explore the potential mRNAs and lncRNAs that regulate the expression of the genes involved in sheep prolificacy, we used stranded specific RNA-seq to profile the pituitary transcriptome (lncRNA and mRNA) in high prolificacy (genotype FecB BB, litter size = 3; H) and low prolificacy sheep (genotype FecB B+; litter size = 1; L). Our results showed that 57 differentially expressed (DE) lncRNAs and 298 DE mRNAs were found in the pituitary between the two groups. The qRT-PCR results correlated well with the RNA-seq results. Moreover, functional annotation analysis showed that the target genes of the DE lncRNAs were significantly enriched in pituitary function, hormone-related pathways as well as response to stimulus and some other terms related to reproduction. Furthermore, a co-expression network of lncRNAs and target genes was constructed and reproduction related genes such as SMAD2, NMB and EFNB3 were included. Lastly, the interaction of candidate lncRNA MSTRG.259847.2 and its target gene SMAD2 were validated in vitro of sheep pituitary cells. These differential mRNA and lncRNA expression profiles provide a valuable resource for understanding the molecular mechanisms underlying Hu sheep prolificacy.

The pituitary gland directly regulates the reproduction of domestic animals. Research has increasingly focused on the potential regulatory mechanism of non-coding RNA in pituitary development. Little is known about the differential expression pattern of lncRNAs in Hu sheep, a famous sheep breed with high fecundity, and its role in the pituitary gland between the follicular phase and luteal phase. Herein, to identify the transcriptomic differences of the sheep pituitary gland during the estrus cycle, RNA sequencing (RNA-Seq) was performed. The results showed that 3529 lncRNAs and 16,651 mRNAs were identified in the pituitary gland. Among of them, 144 differentially expressed (DE) lncRNA transcripts and 557 DE mRNA transcripts were screened in the follicular and luteal phases. Moreover, GO and KEGG analyses demonstrated that 39 downregulated and 22 upregulated genes interacted with pituitary functions and reproduction. Lastly, the interaction of the candidate lncRNA XR_001039544.4 and its targeted gene LHB were validated in sheep pituitary cells in vitro. LncRNA XR_001039544.4 and LHB showed high expression levels in the luteal phase in Hu sheep. LncRNA XR_001039544.4 is mainly located in the cytoplasm, as determined by FISH analysis, indicating that XR_001039544.4 might act as competing endogenous RNAs for miRNAs to regulate LHB. LncRNA XR_001039544.4 knockdown significantly inhibited LH secretion and cell proliferation. LncRNA XR_001039544.4 may regulate the secretion of LH in the luteal-phase pituitary gland via affecting cell proliferation. Taken together, these findings provided genome-wide lncRNA- and mRNA-expression profiles for the sheep pituitary gland between the follicular and luteal phases, thereby contributing to the elucidation of the molecular mechanisms of pituitary function.

Pituitary gonadotropins play a pivotal role in reproduction. Long noncoding RNAs (lncRNAs) have been identified as important regulators in the hypothalamic–pituitary–ovarian (HPO) axis associated with reproduction. However, the contributions of lncRNAs to pituitary gonadotropin secretion remain largely unknown. Therefore, this work was performed to uncover the functional mechanisms of the novel lncRNA TCONS_00083279 (lncRNA SM2) and its potential targeting pathway oar-miR-16b/TGF-beta/SMAD2, which is associated with gonadotropin secretion in sheep pituitary cells. In the present study, the lncRNA SM2 showed high expression levels in the sheep pituitary gland, and it was located in both the nucleus and the cytoplasm of pituitary cells. lncRNA SM2 knockdown inhibited pituitary cell proliferation and FSH and LH secretion. The function of the lncRNA SM2 was sponged by oar-miR-16b, and this regulated the growth and gonadotropin secretion of pituitary cells by modulating SMAD2, as shown by the dual-luciferase reporter assay. FSH and LH levels were both upregulated by SMAD2 overexpression. Moreover, the levels of the lncRNA SM2, SMAD2 and TGFR1, as well as FSH and LH, in sheep pituitary cells increased significantly under gonadotropin-releasing hormone (GnRH) stimulation (p < 0.05). This work illustrates that the lncRNA SM2 regulates gonadotropin secretion in the Hu sheep anterior pituitary by targeting the oar-miR-16b/TGF-β/SMAD2 signaling pathway, providing a valuable resource for understanding the molecular mechanisms underlying sheep reproduction.

The complement 1q binding protein C (C1QBP), also known as p32, is highly expressed in rapidly growing tissues and plays a crucial role in cell proliferation and apoptosis. However, there are no data interpreting its mechanisms in muscle development. To investigate the role of p32 in sheep muscle development, an 856 bp cDNA fragment of p32 containing an 837 bp coding sequence that encodes 278 amino acids was analyzed. We then revealed that the expression of p32 in the longissimus and quadricep muscles of fetal sheep was more significantly up-regulated than expression at other developmental stages. Furthermore, we found that the expression of p32 was increased during myoblasts differentiation in vitro. Additionally, the knockdown of p32 in sheep myoblasts effectively inhibited myoblast differentiation, proliferation, and promoted cell apoptosis in vitro. The interference of p32 also changed the energy metabolism from Oxidative Phosphorylation (OXPHOS) to glycolysis and activated AMP-activated protein kinase (AMPK) phosphorylation in sheep myoblasts in vitro. Taken together, our data suggest that p32 plays a vital role in the development of sheep muscle and provides a potential direction for future research on muscle development and some muscle diseases.

Oxidative damage to skin fibroblast cells is a causative factor in many skin diseases. Previous studies have reported that lysine-specific demethylase 4D (Kdm4d) is involved in DNA replication, but its role on antioxidant capacity remains unclear. In the present study, we used goat fibroblast cells (GFCs) as the research model and identified 504 up-regulated and 1013 down-regulated genes following the knockdown of Kdm4d, respectively. The down-regulated genes of this enzyme were found to be enriched in the cell cycle, DNA replication, mitotic processes, and the oxidative phosphorylation pathway, as previously revealed from gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and gene set enrichment analysis (GSEA), suggesting vital roles of the Kdm4d enzyme in the cell cycle and in antioxidant regulation. To this end, we found the cell proliferation rate was significantly decreased after the knockdown of Kdm4d. Moreover, both the mRNA and protein expression levels of superoxide dismutase 2 (SOD2), one of the major antioxidant enzymes, was decreased, while the reactive oxygen species (ROS) level was significantly increased in Kdm4d knocked-down cells. In addition, the expression of γH2A histone family member X (γH2AX) increased significantly, indicating the presence of DNA double-strand breaks after the knockdown of the Kdm4d enzyme. In conclusion, the knockdown of Kdm4d inhibited DNA replication and the cell cycle, repressed the expression of SOD2, and increased the generation of ROS, which led to the production of DNA damage in GFCs. Our data will be helpful for understanding the mechanism underlying antioxidant capacity regulation in fibroblast cells.

Bivalent chromatin, marked by H3K4me3 and H3K27me3, contributes to maintaining developmental genes in a poised state. However, it remains unclear whether sheep embryonic stem cells (ESCs) establish bivalent domains. In this study, we identified bivalent chromatin regions and characterized the dynamics of H3K4me3, chromatin accessibility, and expression of bivalent chromatin-associated genes in sheep early embryos and ESCs. Using an overlap threshold of >200 bp between H3K4me3 and H3K27me3 peaks, we identified 40,543 bivalent regions in sheep ESCs. These regions preferentially occurred at promoters and were enriched in RNA processing, translation, and ribonucleoprotein complex biogenesis. By integrating Cleavage Under Targets and Tagmentation (CUT&Tag), Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), and RNA-seq data across preimplantation development from MII oocytes to hatching blastocysts, we found that ESC-defined bivalent regions underwent dynamic changes in H3K4me3 enrichment and chromatin accessibility, with distinct patterns across developmental stages. Bivalent-associated genes also exhibited stage-specific expression patterns, and key pluripotency- and lineage-associated transcription factors, including SOX2, SOX17, POU5F1, and ZFP42, showed distinct developmental expression profiles. Moreover, formative ESCs displayed higher transcriptional activity and chromatin accessibility at bivalent regions than that of primed ESCs. Together, these results characterize the genomic distribution of bivalent chromatin in sheep ESCs and its stage-specific remodeling during preimplantation development, providing a resource for further investigation of epigenetic regulation during early embryonic development in sheep.