GmNLP1 and GmNLP4 activate nitrate-induced CLE peptides NIC1a/b to mediate nitrate-regulated root nodulation.

Symbiotic nitrogen fixation is an energy-intensive process, to maintain the balance between growth and nitrogen fixation, high concentrations of nitrate inhibit root nodulation. However, the precise mechanism underlying the nitrate inhibition of nodulation in soybean remains elusive. In this study, CRISPR-Cas9-mediated knockout of GmNLP1 and GmNLP4 unveiled a notable nitrate-tolerant nodulation phenotype. GmNLP1b and GmNLP4a play a significant role in the nitrate-triggered inhibition of nodulation, as the expression of nitrate-responsive genes was largely suppressed in Gmnlp1b and Gmnlp4a mutants. Furthermore, we demonstrated that GmNLP1b and GmNLP4a can bind to the promoters of GmNIC1a and GmNIC1b and activate their expression. Manipulations targeting GmNIC1a and GmNIC1b through knockdown or overexpression strategies resulted in either increased or decreased nodule number in response to nitrate. Additionally, transgenic roots that constitutively express GmNIC1a or GmNIC1b rely on both NARK and hydroxyproline O-arabinosyltransferase RDN1 to prevent the inhibitory effects imposed by nitrate on nodulation. In conclusion, this study highlights the crucial role of the GmNLP1/4-GmNIC1a/b module in mediating high nitrate-induced inhibition of nodulation.

Bioinformatic validation and machine learning-based exploration of purine metabolism-related gene signatures in the context of immunotherapeutic strategies for nonspecific orbital inflammation.

Background: Nonspecific orbital inflammation (NSOI) represents a perplexing and persistent proliferative inflammatory disorder of idiopathic nature, characterized by a heterogeneous lymphoid infiltration within the orbital region. This condition, marked by the aberrant metabolic activities of its cellular constituents, starkly contrasts with the metabolic equilibrium found in healthy cells. Among the myriad pathways integral to cellular metabolism, purine metabolism emerges as a critical player, providing the building blocks for nucleic acid synthesis, such as DNA and RNA. Despite its significance, the contribution of Purine Metabolism Genes (PMGs) to the pathophysiological landscape of NSOI remains a mystery, highlighting a critical gap in our understanding of the disease's molecular underpinnings. Methods: To bridge this knowledge gap, our study embarked on an exploratory journey to identify and validate PMGs implicated in NSOI, employing a comprehensive bioinformatics strategy. By intersecting differential gene expression analyses with a curated list of 92 known PMGs, we aimed to pinpoint those with potential roles in NSOI. Advanced methodologies, including Gene Set Enrichment Analysis (GSEA) and Gene Set Variation Analysis (GSVA), facilitated a deep dive into the biological functions and pathways associated with these PMGs. Further refinement through Lasso regression and Support Vector Machine-Recursive Feature Elimination (SVM-RFE) enabled the identification of key hub genes and the evaluation of their diagnostic prowess for NSOI. Additionally, the relationship between these hub PMGs and relevant clinical parameters was thoroughly investigated. To corroborate our findings, we analyzed expression data from datasets GSE58331 and GSE105149, focusing on the seven PMGs identified as potentially crucial to NSOI pathology. Results: Our investigation unveiled seven PMGs (ENTPD1, POLR2K, NPR2, PDE6D, PDE6H, PDE4B, and ALLC) as intimately connected to NSOI. Functional analyses shed light on their involvement in processes such as peroxisome targeting sequence binding, seminiferous tubule development, and ciliary transition zone organization. Importantly, the diagnostic capabilities of these PMGs demonstrated promising efficacy in distinguishing NSOI from non-affected states. Conclusions: Through rigorous bioinformatics analyses, this study unveils seven PMGs as novel biomarker candidates for NSOI, elucidating their potential roles in the disease's pathogenesis. These discoveries not only enhance our understanding of NSOI at the molecular level but also pave the way for innovative approaches to monitor and study its progression, offering a beacon of hope for individuals afflicted by this enigmatic condition.

Study on the mechanism of S100A4-mediated cancer oncogenesis in uveal melanoma cells through the integration of bioinformatics and in vitro experiments.

BACKGROUND: The elevated metastasis rate of uveal melanoma (UM) is intricately correlated with patient prognosis, significantly affecting the quality of life. S100 calcium-binding protein A4 (S100A4) has tumorigenic properties; therefore, the present study investigated the impact of S100A4 on UM cell proliferation, apoptosis, migration, and invasion using bioinformatics and in vitro experiments. METHODS: Bioinformatic analysis was used to screen S100A4 as a hub gene and predict its possible mechanism in UM cells, and the S100A4 silencing cell line was constructed. The impact of S100A4 silencing on the proliferative ability of UM cells was detected using the Cell Counting Kit-8 and colony formation assays. Annexin V-FITC/PI double fluorescence and Hoechst 33342 staining were used to observe the effects of apoptosis on UM cells. The effect of S100A4 silencing on the migratory and invasive capabilities of UM cells was assessed using wound healing and Transwell assays. Western blotting was used to detect the expression of related proteins. RESULTS: The present study found that S100A4 is a biomarker of UM, and its high expression is related to poor prognosis. After constructing the S100A4 silencing cell line, cell viability, clone number, proliferating cell nuclear antigen, X-linked inhibitor of apoptosis protein, and survivin expression were decreased in UM cells. The cell apoptosis rate and relative fluorescence intensity increased, accompanied by increased levels of Bax and caspase-3 and decreased levels of Bcl-2. Additionally, a decrease in the cell migration index and relative invasion rate was observed with increased E-cadherin expression and decreased N-cadherin and vimentin protein expression. CONCLUSION: S100A4 silencing can inhibit the proliferation, migration, and invasion and synchronously induces apoptosis in UM cells.

Glutamine metabolism-related genes and immunotherapy in nonspecific orbital inflammation were validated using bioinformatics and machine learning.

BACKGROUND: Nonspecific orbital inflammation (NSOI) is an idiopathic, persistent, and proliferative inflammatory condition affecting the orbit, characterized by polymorphous lymphoid infiltration. Its pathogenesis and progression have been linked to imbalances in tumor metabolic pathways, with glutamine (Gln) metabolism emerging as a critical aspect in cancer. Metabolic reprogramming is known to influence clinical outcomes in various malignancies. However, comprehensive research on glutamine metabolism's significance in NSOI is lacking. METHODS: This study conducted a bioinformatics analysis to identify and validate potential glutamine-related molecules (GlnMgs) associated with NSOI. The discovery of GlnMgs involved the intersection of differential expression analysis with a set of 42 candidate GlnMgs. The biological functions and pathways of the identified GlnMgs were analyzed using GSEA and GSVA. Lasso regression and SVM-RFE methods identified hub genes and assessed the diagnostic efficacy of fourteen GlnMgs in NSOI. The correlation between hub GlnMgs and clinical characteristics was also examined. The expression levels of the fourteen GlnMgs were validated using datasets GSE58331 and GSE105149. RESULTS: Fourteen GlnMgs related to NSOI were identified, including FTCD, CPS1, CTPS1, NAGS, DDAH2, PHGDH, GGT1, GCLM, GLUD1, ART4, AADAT, ASNSD1, SLC38A1, and GFPT2. Biological function analysis indicated their involvement in responses to extracellular stimulus, mitochondrial matrix, and lipid transport. The diagnostic performance of these GlnMgs in distinguishing NSOI showed promising results. CONCLUSIONS: This study successfully identified fourteen GlnMgs associated with NSOI, providing insights into potential novel biomarkers for NSOI and avenues for monitoring disease progression.

Antioxidant potential of chlorogenic acid in Age-Related eye diseases.

Oxidative stress is an important mechanism of aging, and in turn, aging can also aggravate oxidative stress, which leads to a vicious cycle. In the process of the brain converting light into visual signals, the eye is stimulated by harmful blue-light radiation directly. Thus, the eye is especially vulnerable to oxidative stress and becomes one of the organs most seriously involved during the aging process. Cataracts, age-related macular degeneration (AMD), glaucoma, diabetic retinopathy (DR), and dry eye are inextricably linked to the aging process and oxidative stress. Chlorogenic acid (CGA) has been demonstrated to have antioxidant and anti-inflammatory activities, and its validity has been established experimentally in numerous fields, including cardiovascular disease, metabolic disorders, cancers, and other chronic diseases. There has previously been evidence of CGA's therapeutic effect in the field of ophthalmopathy. Considering that many ophthalmic drugs lead to systemic side effects, CGA may act as a natural exogenous antioxidant for patients to take regularly, controlling their condition while minimizing side effects. In this paper, in vitro and in vivo studies of CGA in the treatment of age-related eye diseases are reviewed, and the prospects of CGA's antioxidant application for the eye are discussed. The aim of this review is to summarize the relevant knowledge and provide theoretical support for future research.

What drives the changing characteristics of phytoplankton in urban lakes: Climate, hydrology, or human disturbance?

Phytoplankton in shallow urban lakes are influenced by various environmental factors. However, the long-term coupling effects and impact pathways of these environmental variables on phytoplankton remain unclear. This is an emerging issue due to high urbanization and the resultant complex climate, lake hydrology and morphology, human interference, and water quality parameter changes. This study used Tangxun Lake, the largest urban lake in the Yangtze River Economic Belt, as an example to assess for the first time the individual contributions and coupled effects of four environmental variables and fourteen indicators on chlorophyll-a (Chla) concentrations under two scenarios from 2000 to 2019. Additionally, the influence pathways between the environmental variables and Chla concentration were quantified. The results indicated that the Chla concentration was most affected by lake hydrology and morphology, as were the total nitrogen, total phosphorus, and transparency. Especially after urbanization (2015-2019), the coupling effect of human interference, lake hydrology and morphology, and water quality parameters was strongest (18%). This is mainly due to fluctuations in the lake water level and an increase in the shape index of lake morphology, large amounts of nutrients were input, which reduced lake transparency and indirectly changed the Chla content. In addition, due to the rapid development of Wuhan city, the expansion of construction land has led to an increase in impervious surface area and a decrease in lake area. During periods of intense summer rainfall, a substantial amount of pollutants entered the lakes through surface runoff, resulting in decreased lake transparency, and elevated concentrations of nitrogen and phosphorus, indirectly increasing the Chla content. This study provides a scientific basis for aquatic ecological assessment and pollution control in urban shallow lakes.

CREB1 Is Involved in miR-134-5p-Mediated Endometrial Stromal Cell Proliferation, Apoptosis, and Autophagy.

The successful establishment of endometrial receptivity is a key factor in ensuring the fertility of ewes and their economic benefits. Hu sheep have attracted attention due to their high fecundity and year-round estrus. In this study, we found that in the luteal phase, the uterine gland density, uterine coefficient, and number of uterine caruncles of high-fertility Hu sheep were higher than those of low-fertility Hu sheep. Thousands of differentially expressed genes were identified in the endometrium of Hu sheep with different fertility potential using RNA sequencing (RNA-Seq). Several genes involved in endometrial receptivity were screened using bioinformatics analysis. The qRT-PCR analysis further revealed the differential expression of cAMP reactive element binding protein-1 (CREB1) in the Hu sheep endometrium during the estrous cycle. Functionally, our results suggested that CREB1 significantly affected the expression level of endometrial receptivity marker genes, promoted cell proliferation by facilitating the transition from the G1 phase to the S phase, and inhibited cell apoptosis and autophagy. Moreover, we observed a negative linear correlation between miR-134-5p and CREB1 in the endometrium. In addition, CREB1 overexpression prevented the negative effect of miR-134-5p on endometrial stromal cell (ESC) growth. Taken together, these data indicated that CREB1 was regulated by miR-134-5p and may promote the establishment of uterine receptivity by regulating the function of ESCs. Moreover, this study provides new theoretical references for identifying candidate genes associated with fertility.

Purine metabolism-related genes and immunization in thyroid eye disease were validated using bioinformatics and machine learning.

Thyroid eye disease (TED), an autoimmune inflammatory disorder affecting the orbit, exhibits a range of clinical manifestations. While the disease presentation can vary, cases that adhere to a prototypical pattern typically commence with mild symptoms that subsequently escalate in severity before entering a phase of stabilization. Notably, the metabolic activity of cells implicated in the disease substantially deviates from that of healthy cells, with purine metabolism representing a critical facet of cellular material metabolism by supplying components essential for DNA and RNA synthesis. Nevertheless, the precise involvement of Purine Metabolism Genes (PMGs) in the defensive mechanism against TED remains largely unexplored. The present study employed a bioinformatics approach to identify and validate potential PMGs associated with TED. A curated set of 65 candidate PMGs was utilized to uncover novel PMGs through a combination of differential expression analysis and a PMG dataset. Furthermore, GSEA and GSVA were employed to explore the biological functions and pathways associated with the newly identified PMGs. Subsequently, the Lasso regression and SVM-RFE algorithms were applied to identify hub genes and assess the diagnostic efficacy of the top 10 PMGs in distinguishing TED. Additionally, the relationship between hub PMGs and clinical characteristics was investigated. Finally, the expression levels of the identified ten PMGs were validated using the GSE58331 and GSE105149 datasets. This study revealed ten PMGs related with TED. PRPS2, PFAS, ATIC, NT5C1A, POLR2E, POLR2F, POLR3B, PDE3A, ADSS, and NTPCR are among the PMGs. The biological function investigation revealed their participation in processes such as RNA splicing, purine-containing chemical metabolism, and purine nucleotide metabolism. Furthermore, the diagnostic performance of the 10 PMGs in differentiating TED was encouraging. This study was effective in identifying ten PMGs linked to TED. These findings provide light on potential new biomarkers for TED and open up possibilities for tracking disease development.

Non-targeted Metabolomics Analysis Reveals Distinct Metabolic Profiles Between Positive and Negative Emotional Tears of Humans: A Preliminary Study.

Background Basal, reflex, and emotional tears differ in chemical components. It is not yet known whether chemical differences exist in tears of different emotions. We investigated the biochemical basis of emotional tears by performing non-targeted metabolomics analyses of positive and negative emotional tears of humans. Methods Samples of reflex, negative, and positive emotional tears were obtained from 12 healthy college participants (11 females and one male). Untargeted metabolomics was performed to identify metabolites in different types of tears. The differentially altered metabolites were screened and assessed using univariate and multivariate analyses. Results The orthogonal partial least squares discriminant analysis model showed that reflex, negative, and positive emotional tears were clearly separated. A total of 133 significantly differentially expressed metabolites of electrospray ionization source (ESI-) mode were identified between negative and positive emotional tears. The top 50 differentially expressed metabolites between negative and positive emotional tears were highly correlated. Pathway analysis revealed that secretion of negative emotional tears was associated with some synapses in the brain, regulation of a series of endocrine hormones, including the estrogen signaling pathway, and inflammation activities, while secretion of positive emotional tears was correlated with biotin and caffeine metabolism. Conclusions It is indicated that metabolic profiles of reflex, positive, and negative emotional tears of humans are distinct, and secretion of the tears involves distinct biological activities. Therefore, we present a chemical method for detecting human emotions, which may become a powerful tool for the diagnosis of mental diseases and the identification of fake tears.

Genome-wide association study for biomass accumulation traits in soybean.

Soybean is one of the most versatile crops for oil production, human diets, and feedstocks. The vegetative biomass of soybean is an important determinant of seed yield and is crucial for the forage usages. However, the genetic control of soybean biomass is not well explained. In this work, we used a soybean germplasm population, including 231 improved cultivars, 207 landraces, and 121 wild soybeans, to investigate the genetic basis of biomass accumulation of soybean plants at the V6 stage. We found that biomass-related traits, including NDW (nodule dry weight), RDW (root dry weight), SDW (shoot dry weight), and TDW (total dry weight), were domesticated during soybean evolution. In total, 10 loci, encompassing 47 putative candidate genes, were detected for all biomass-related traits by a genome-wide association study. Among these loci, seven domestication sweeps and six improvement sweeps were identified. Glyma.05G047900, a purple acid phosphatase, was a strong candidate gene to improve biomass for future soybean breeding. This study provided new insights into the genetic basis of biomass accumulation during soybean evolution. Supplementary information: The online version contains supplementary material available at 10.1007/s11032-023-01380-6.

PPP2R2A promotes testosterone secretion in Hu sheep Leydig cells via activation of the AKT/mTOR signaling pathway.

The serine-threonine protein phosphatase 2A (PP2A) is a heterotrimeric enzyme complex that plays a vital role in regulating male reproductive activities. However, as an essential member of the PP2A family, the physiological functions of PP2A regulatory subunit B55α (PPP2R2A) in testis remain inconclusive. Hu sheep are noted for their reproductive precocity and fertility, and are ideal models for the study of male reproductive physiology. Here, we analyzed the expression patterns of PPP2R2A in the male Hu sheep reproductive tract at different developmental stages and further investigated its role in testosterone secretion and its underlying mechanisms. In this study, we found that there were temporal and spatial differences in PPP2R2A protein expression in the testis and epididymis, especially the expression abundance in the testis at 8 months old (8M) was higher than that at 3 months old (3M). Interestingly, we observed that PPP2R2A interference reduced the testosterone levels in the cell culture medium, which is accompanied by a reduction in Leydig cell proliferation and an elevation in Leydig cell apoptosis. The level of reactive oxygen species in cells increased significantly, while the mitochondrial membrane potential (ΔΨm) decreased significantly after PPP2R2A deletion. Meanwhile, the mitochondrial mitotic protein DNM1L was significantly upregulated, while the mitochondrial fusion proteins MFN1/2 and OPA1 were significantly downregulated after PPP2R2A interference. Furthermore, PPP2R2A interference suppressed the AKT/mTOR signaling pathway. Taken together, our data indicated that PPP2R2A enhanced testosterone secretion, promoted cell proliferation, and inhibited cell apoptosis in vitro, all of which were associated with the AKT/mTOR signaling pathway.

Dietary spirulina supplementation modifies rumen development, fermentation and bacteria composition in Hu sheep when consuming high-fat dietary.

Introduction: This study aims to investigate the long-term effects of spirulina supplementation in a high-fat diet (HFD) on rumen morphology, rumen fermentation, and the composition of rumen microbiota in lambs. Spirulina is a blue-green microalgae that has been shown to have high nutritional value for livestock. Methods: Fifty-four lambs were randomly divided into three groups: a normal chow diet (NCD) group, a high-fat diet (HFD) group, and a high-fat diet supplemented with 3% spirulina (HFD+S) group. Rumen morphology, rumen fermentation, and rumen microbiota were analyzed at the end of the study. Results: Spirulina supplementation improved the concentration of volatile fatty acids and rumen papilla length. Additionally, there was a tendency for an increase in rumen weight and an upregulation of the genes Claudin-1, Claudin-4, and Occludin in the HFD+S group. Pyrosequencing of the 16S ribosomal RNA gene also showed that spirulina supplementation significantly changed the rumen microbiota composition in the HFD group, with a decrease in richness and diversity. Specifically, the relative abundance of Prevotella 9 and Megasphaera was significantly increased in the HFD group compared to the NCD group, while spirulina supplementation reversed these changes. Discussion: This study suggests that 3% spirulina supplementation can improve rumen development and fermentation, and effectively relieve rumen microbe disorders in lambs caused by a high-fat diet. However, further research is needed to confirm the findings and to examine the long-term effects of spirulina supplementation in different types of livestock and under different dietary conditions.

Exploration the role of INHBA in Hu sheep granulosa cells using RNA-Seq.

Activin/inhibin is an important factor for the fecundity of Hu sheep, and it is involved in follicular development in ovaries. Inhibin subunit beta A (INHBA) participates in the synthesis of activin A and inhibin A. In this study, we also noted a positive correlation between INHBA level and the secretion of both activin A and inhibin A in culture medium. Nevertheless, both knockdown and overexpression of INHBA downregulated the expression of Inhibin Subunit Alpha (INHA). Based on RNA-Sequencing, we further examined the effect and molecular mechanism of INHBA knockdown in GCs on mRNA expression. A total of 1,687 differentially expressed genes (DEGs) were identified (Fold change ≥ 2; False-discovory-rates (FDR) ≤ 0.01), of which 602 genes were upregulated and 1,087 genes were downregulated in the INHBA interference group compared with the control groups. Gene Ontology (GO) enrichment indicated that these DEGs were mainly involved in the regulation of cell cycle, protein serine/threonine kinase activity, and actin cytoskeleton reorganization. Moreover, DEGs were significantly enriched in 40 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including P53, progesterone-mediated oocyte maturation, and PI3K-AKT signaling pathways. We also noted a positive correlation between INHBA level and many PI3K/Akt/mTOR pathway-related genes at the gene or/and protein expression. Overall, this study may contribute to a better understanding of the roles of INHBA on GCs of prolific sheep, as well as the molecular effect of low INHBA expression on GCs, clarifying some reproductive failures.

Identification and characterization of unique and common lncRNAs and mRNAs in the pituitary, ovary, and uterus of Hu sheep with different prolificacy.

LncRNAs are regarded as regulators in various animal reproductive physiological processes. However, the regulation of lncRNAs in the reproductive organ development of Hu sheep with different prolificacy remains unknown. Herein, numerous tissue-unique and -common differentially expressed lncRNAs (DELs) and differentially expressed genes (DEGs), and fecundity-unique DELs and DEGs were identified among different comparison groups at horizontal and vertical levels. Moreover, the tissue-unique and -common, and fecundity-unique female reproduction-associated DEGs and DELs were screened, and the interaction networks were constructed. Furthermore, MSTRG.43442.1 was mainly present in the cytoplasm of tested cells. The key genes ADAMTS1 and DCN were mainly localized in the granulosa cells, pituitary cells and/or endometrial epithelial cells of ovary, pituitary and/or uterus. Overall, this study identified large numbers of unique and common DELs and DEGs in the female reproductive organs of Hu sheep with different prolificacy and provided new insights into understanding the regulation of Hu sheep fecundity.

Elimination of an unfavorable allele conferring pod shattering in an elite soybean cultivar by CRISPR/Cas9.

Pod shattering can lead to devastating yield loss of soybean and has been a negatively selected trait in soybean domestication and breeding. Nevertheless, a significant portion of soybean cultivars are still pod shattering-susceptible, limiting their regional and climatic adaptabilities. Here we performed genetic diagnosis on the shattering-susceptible trait of a national registered cultivar, Huachun6 (HC6), and found that HC6 carries the susceptible genotype of a candidate Pod dehiscence 1 (PDH1) gene, which exists in a significant portion of soybean cultivars. We next performed genome editing on PDH1 gene by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). In T2 progenies, several transgene-free lines with pdh1 mutations were characterized without affecting major agronomic traits. The pdh1 mutation significantly improved the pod shattering resistance which is associated with aberrant lignin distribution in inner sclerenchyma. Our work demonstrated that precision breeding by genome editing on PDH1 holds great potential for precisely improving pod shattering resistance and adaptability of soybean cultivars.

A study on the safety and therapeutic effect of Xiaoyaosan on depressive disorder related dry eye disease in a murine animal model.

This study was done to observe the safety and effect of Xiaoyaosan (XYS) on the stimulated depressive disorder (DD) related dry eye disease (DED) in mice. Normal control (NC) group, Vehicle group, and drug treatment groups, including Sertraline Hydrochloride (SH), XYS low-dose (XYS-LD), medium-dose (XYS-MD), and high-dose (XYS-HD), were established. The drug quality of XYS was assessed by high-performance liquid chromatography (HPLC). XYS toxicity in kidney and liver was assessed with Hematoxylin & Eosin (H&E) staining. Serum enzyme-linked immunosorbent assay (ELISA) and body weights were used to evaluate the depression status of mice. Tear production, corneal sensitivity, Oregon Green Dye (OGD) staining, and corneal confocal microscopy were used to assess ocular surface changes. H&E staining was also used to assess pathological cornea and lacrimal gland changes. HPLC results showed that XYS complied with Chinese drug quality standards. The drug treatment groups observed no drug toxicity reactions in the liver and kidney. SH and XYS groups improved DD-related serological indices as compared with Vehicle. Body weight was enhanced in mice with XYS groups compared to Vehicle and SH. Mice with XYS treatments showed increased tear production and corneal sensitivity, decreased corneal OGD staining scores, improved morphology, and decreased inflammatory cell infiltration in the cornea and lacrimal gland. In conclusion, XYS had no drug toxicity, improved serological indices, and ocular surface pathological changes in DD-related DED mice. XYS is safe and may have a therapeutic effect on DD-related DED.

LncRNA-412.25 activates the LIF/STAT3 signaling pathway in ovarian granulosa cells of Hu sheep by sponging miR-346.

Although long non-coding RNAs (lncRNAs) are reported to regulate follicular development and reproductive disease pathogenesis, the underlying mechanisms have not been elucidated. In this study, lncRNA expression profiling of different-sized healthy follicles from Hu sheep with different prolificacy revealed 50 613 lncRNAs. Numerous lncRNAs were differentially expressed among different comparison groups. This study characterized one novel transcript, lncRNA-412.25 (from healthy follicles with a diameter of >5 mm), which was predominantly expressed in the high prolificacy group and localized to the cytoplasm of granulosa cells (GCs). LncRNA-412.25 knockdown promoted and inhibited Hu sheep GC apoptosis and proliferation, respectively. Interestingly, lncRNA-412.25 could directly bind to miR-346, which can target the gene of leukemia inhibitory factor (LIF). Knockdown of lncRNA-412.25 promoted GC apoptosis by downregulating LIF expression, where this effect was attenuated by miR-346. Moreover, the miR-346 inhibitor mitigated the lncRNA-412.25 knockdown-induced downregulation of phosphorylated protein of signal transducer and activator of transcription 3 (STAT3), which was validated using immunofluorescence analysis. Our results demonstrated that lncRNA-412.25 regulates GC proliferation and apoptosis in Hu sheep by binding to miR-346 and then activating the LIF/STAT3 pathway. These findings provide novel insights into the mechanisms underlying prolificacy in sheep.

The Novel Competing Endogenous Long Noncoding RNA SM2 Regulates Gonadotropin Secretion in the Hu Sheep Anterior Pituitary by Targeting the Oar-miR-16b/TGF-ß/SMAD2 Signaling Pathway.

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-ß/SMAD2 signaling pathway, providing a valuable resource for understanding the molecular mechanisms underlying sheep reproduction.

Integrative Genome-Wide DNA Methylome and Transcriptome Analysis of Ovaries from Hu Sheep with High and Low Prolific.

DNA methylation plays an important role in biological processes by affecting gene expression. However, how DNA methylation regulates phenotypic variation in Hu sheep remains unclear. Therefore, we generated genome-wide DNA methylation and transcriptomic profiles in the ovaries of Hu sheep with different prolificacies and genotypes (FecBB and FecB+). Results showed that ovary DNA methylome and transcriptome were significantly different between high prolificacy and low prolificacy Hu sheep. Comparative methylome analyses identified 10,644, 9,594, and 12,214 differentially methylated regions and 87, 1,121, and 2,375 genes, respectively, showing differential expression levels in three different comparison groups. Female reproduction-associated differentially methylated regions-related genes and differentially expressed genes were enriched, thereby the respective interaction networks were constructed. Furthermore, systematical integrative analyses revealed a negative correlation between DNA methylation around the transcriptional start site and gene expression levels, which was confirmed by testing the expression of integrin ß2 subunit (ITGB2) and lysosome-associated protein transmembrane-4 beta (LAPTM4B) in vivo and in vitro. These findings demonstrated that DNA methylation influences the propensity for prolificacy by affecting gene expression in the ovaries, which may contribute to a greater understanding of the epigenome and transcriptome that will be useful for animal breeding.