Deficiency of SLC26A3 promotes jejunal barrier damage in metabolic disease-susceptible transgenic pigs.

Intestinal disorders are common in metabolic syndrome. However, their pathogenesis is still not fully understood. Pig and human intestines are highly similar in terms of associated metabolic processes. Here, we successfully constructed a metabolic disease-susceptible transgenic (TG) Bama pig model by knocking in three humanized disease risk genes with the CRISPR/Cas9 technique to assess its potential as a model for human intestinal diseases and explore the possible pathological mechanisms involved. We found that jejunal barrier integrity was disrupted and that the infiltration of inflammatory cells increased in TG pigs after high-fat and high-sucrose diet (HFHSD) treatment. We revealed significant differences in the transcriptome, associated microbiome profiles and microbial metabolite short-chain fatty acid (SCFA) content of the jejunum of TG pigs. Notably, we found that SLC26A3 was significantly downregulated in TG pigs. Knockdown or overexpression of the SLC26A3 gene in IPEC-J2 cells significantly affected the expression of MUC2, MUC13 and occludin. Furthermore, in vitro experiments further verified that CDX2 directly regulated the expression of SLC26A3. Mechanistically, CDX2 mediated intestinal barrier function by enhancing the expression of SLC26A3 by binding to its promoter region between -1120 and - 1070 bp. TG pigs represent a promising model that provides new insights into preclinical research on human intestinal metabolic diseases associated with metabolic disorders and revealed that SLC26A3 may be a potential therapeutic target for intestinal metabolic diseases.

Single-nucleus transcriptomics reveal cardiac cell type-specific diversification in metabolic disease transgenic pigs.

Cardiac damage is widely present in patients with metabolic diseases, but the exact pathophysiological mechanisms involved remain unclear. The porcine heart is an ideal material for cardiovascular research due to its similarities to the human heart. This study evaluated pathological features and performed single-nucleus RNA sequencing (snRNA-seq) on myocardial samples from both wild-type and metabolic disease-susceptible transgenic pigs (previously established). We found that transgenic pigs exhibited lipid metabolism disturbances and myocardial injury after a high-fat high-sucrose diet intervention. snRNA-seq reveals the cellular landscape of healthy and metabolically disturbed pig hearts and identifies the major cardiac cell populations affected by metabolic diseases. Within metabolic disorder hearts, metabolically active cardiomyocytes exhibited impaired function and reduced abundance. Moreover, massive numbers of reparative LYVE1+ macrophages were lost. Additionally, proinflammatory endothelial cells were activated with high expression of multiple proinflammatory cytokines. Our findings provide insights into the cellular mechanisms of metabolic disease-induced myocardial injury.

Glucose restriction enhances oxidative fiber formation: A multi-omic signal network involving AMPK and CaMK2.

Skeletal muscle is a highly plastic organ that adapts to different metabolic states or functional demands. This study explored the impact of permanent glucose restriction (GR) on skeletal muscle composition and metabolism. Using Glut4m mice with defective glucose transporter 4, we conducted multi-omics analyses at different ages and after low-intensity treadmill training. The oxidative fibers were significantly increased in Glut4m muscles. Mechanistically, GR activated AMPK pathway, promoting mitochondrial function and beneficial myokine expression, and facilitated slow fiber formation via CaMK2 pathway. Phosphorylation-activated Perm1 may synergize AMPK and CaMK2 signaling. Besides, MAPK and CDK kinases were also implicated in skeletal muscle protein phosphorylation during GR response. This study provides a comprehensive signaling network demonstrating how GR influences muscle fiber types and metabolic patterns. These insights offer valuable data for understanding oxidative fiber formation mechanisms and identifying clinical targets for metabolic diseases.

SnRNA-Seq of Pancreas Revealed the Dysfunction of Endocrine and Exocrine Cells in Transgenic Pigs with Prediabetes.

Diabetes poses a significant threat to human health. Exocrine pancreatic dysfunction is related to diabetes, but the exact mechanism is not fully understood. This study aimed to describe the pathological phenotype and pathological mechanisms of the pancreas of transgenic pigs (PIGinH11) that was constructed in our laboratory and to compare it with humans. We established diabetes-susceptible transgenic pigs and subjected them to high-fat and high-sucrose dietary interventions. The damage to the pancreatic endocrine and exocrine was evaluated using histopathology and the involved molecular mechanisms were analyzed using single-nucleus RNA-sequencing (SnRNA-seq). Compared to wild-type (WT) pigs, PIGinH11 pigs showed similar pathological manifestations to type 2 diabetes patients, such as insulin deficiency, fatty deposition, inflammatory infiltration, fibrosis tissue necrosis, double positive cells, endoplasmic reticulum (ER) and mitochondria damage. SnRNA-seq analysis revealed 16 clusters and cell-type-specific gene expression characterization in the pig pancreas. Notably, clusters of Ainar-M and Endocrine-U were observed at the intermediate state between the exocrine and endocrine pancreas. Beta cells of the PIGinH11 group demonstrated the dysfunction with insulin produced and secret decreased and ER stress. Moreover, like clinic patients, acinar cells expressed fewer digestive enzymes and showed organelle damage. We hypothesize that TXNIP that is upregulated by high glucose might play an important role in the dysfunction of endocrine to exocrine cells in PIGinH11 pigs.

CoQ10 improves meiotic maturation of pig oocytes through enhancing mitochondrial function and suppressing oxidative stress.

Coenzyme Q10 (CoQ10) is essential to many fundamental biological processes. However, the effect of CoQ10 on meiotic maturation of pig oocytes still remains elusive. In the present study we aimed to understand the effects of CoQ10 on porcine oocyte maturation, by supplementing different concentrations of CoQ10 (25, 50 and 100 µM) into the maturation medium. We showed that CoQ10 at 50 µM had better capacity to promote the nuclear maturation of pig oocytes derived from both small and large antral follicles. Though the cleavage and blastocyst rates of parthenotes stayed stable, 50 µM CoQ10 treatment could accelerate the development of parthenotes to blastocyst stage, and increase the average cell number of blastocyst. For cumulus-oocyte complexes from large antral follicles categorized by the brilliant cresyl blue (BCB) test, 50 µM CoQ10 treatment could specifically promote the nuclear maturation of poor-quality oocytes in the BCB-negative group. Mitochondrial function of oocytes treated by 50 µM CoQ10 could be boosted, through increasing the levels of mitochondrial membrane potential, ATP production and CoQ6, and changing the pattern of mitochondrial distribution as well. Moreover, 50 µM CoQ10 treatment suppressed the level of reactive oxygen species and reduced the percentage of oocytes with early apoptosis signal. Taken together, CoQ10 could improve the meiotic maturation of pig oocytes, especially for poor-quality oocytes, mainly through enhancing mitochondrial function and suppressing oxidative stress to reduce apoptosis.

Single cell RNA-seq reveals molecular pathways altered by 7, 12-dimethylbenz[a]anthracene treatment on pig oocytes.

Oocytes of better quality and developmental competence are highly demanded, which is affected by many intrinsic and external factors, including environmental pollutants. We have previously demonstrated that 7, 12-dimethylbenz [a]anthracene (DMBA) reduces the developmental competence of porcine oocytes, by desynchronizing nuclear and ooplasmic maturation. However, the underlying molecular mechanism remains obscure. Here we performed single cell RNA-seq to study the transcriptome changes in DMBA-treated porcine MII oocytes, and identified 19 protein-coding genes and 156 novel long non-coding RNAs (lncRNAs) with abundance to be significantly different (P < 0.05), which enriched in signaling pathways such as glycosphingolipid biosynthesis, nicotine addiction, basal transcription factors and nucleotide excision repair. RT-qPCR on oocyte pools confirmed ornithine aminotransferase (Oat) and serine/arginine-rich splicing factor 4 (Srsf4) to be significantly up- and down-regulated, respectively (P < 0.05). Treating porcine COCs with MAPK and PLC pathway inhibitors suppressed DMBA's effects on increasing PB1 extrusion rate. In addition, DMBA co-incubation with 250 µM vitamin C derivative (l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, AA2P) and 100 µM co-enzyme Q10 (CoQ10) could significantly reduce the DMBA-induced high ROS level, and partially alleviate the DMBA-induced high PB1 rate, whereas the cleavage and blastocyst rates of parthenotes derived from treated mature oocytes remained to be low. Collectively, our findings indicate that single cell RNA-seq can help reveal the dynamics of molecular signaling pathways for porcine oocytes treated by DMBA, and supplement of anti-oxidative reagents could not sufficiently rescue DMBA-induced defects of porcine oocytes.

Long noncoding RNA 2193 regulates meiosis through global epigenetic modification and cytoskeleton organization in pig oocytes.

Long noncoding RNAs (lncRNAs) regulate a variety of physiological and pathological processes. However, the biological function of lncRNAs in mammalian germ cells remains largely unexplored. Here we identified one novel lncRNA (lncRNA2193) from single-cell RNA sequencing performed on porcine oocytes and investigated its function in oocyte meiosis. During in vitro maturation (IVM), from germinal vesicle (GV, 0 hr), GV breakdown (GVBD, 24 hr), to metaphase II stage (MII, 44 hr), the transcriptional abundance of lncRNA2193 remained stable and high. LncRNA2193 interference by small interfering RNA microinjection into porcine GV oocytes could significantly inhibit rates of GVBD and the first polar body extrusion, but enhance the rates of oocytes with a nuclear abnormality. Moreover, lncRNA2193 knockdown disturbed cytoskeletal organization (F-actin and spindle), and decreased DNA 5-methylcytosine (5mC) and histone trimethylation (H3K4me3, H3K9me3, H3K27me3, and H3K36me3) levels. The lncRNA2193 downregulation induced a decrease of 5mC level could be partially due to the reduction of DNA methyltransferase 3A and 3B, and the elevation of 5mC-hydroxylase ten-11 translocation 2 (TET2). After parthenogenetic activation of MII oocytes, parthenotes exhibited higher fragmentation but lower cleavage rates in the lncRNA2193 downregulated group. However, lncRNA2193 interference performed on mature MII oocytes and parthenotes at 1-cell stage did not affect the cleavage and blasctocyst rates of pathenotes. Taken together, lncRNA2193 plays an important role in porcine oocyte maturation, providing more insights for relevant investigations on mammalian germ cells.

Lysosomal dysfunction disturbs porcine oocyte maturation and developmental capacity by disorganizing chromosome/cytoskeleton and activating autophagy/apoptosis.

Lysosome, an important organelle in eukaryotes, can sequester macromolecules submitted by the endocytosis and autophagy pathways for degradation and recycling. Massive macromolecular turnover is also vital to the growth and development of mammalian oocytes. However, the functional role of lysosomes in the meiotic maturation of mammalian oocytes remains largely unexplored. Here, by treating in vitro matured porcine cumulus-oocyte complexes (COCs) with chloroquine (CQ), a lysosome inhibitor, we showed that regardless of CQ concentration, lysosomal inhibition affected neither the extrusion of the first polar body (PB1), nor the ROS levels. However, CQ treatment dramatically decreased the rates of oocytes with normal chromosome alignment and cytoskeleton organization (P < 0.05), but boosted the rates of oocytes with apoptosis (P < 0.05). Subsequently, after pathenogenetic activation or in vitro fertilization, the death or fragmentation rates of oocytes treated by CQ (both 35 µM and 45 µM) were significantly higher (P < 0.05), whereas the rates of embryo cleavage, embryos developed to blastocysts, and average blastomere number per blastocyst, were all significantly lower (P < 0.05), respectively. Furthermore, CQ (35 µM) treatment activated the autophagy pathway by elevating the LC3 II/I ratio. Taken together, lysosomes could affect porcine oocyte maturation and subsequent developmental capacity partially through the chromosome organization/cytoskeleton assembly and autophagy/apoptosis pathways.

Reduced nucleic acid methylation impairs meiotic maturation and developmental potency of pig oocytes.

Oocyte meiosis is a complex process coordinated by multiple endocrinal and molecular circuits. Recently, N6-methyladenosine (m6A) epigenetic modification on RNA is revealed to be important for meiotic maturation. However, the molecular mechanism of how m6A modification exerts its effect on oocyte maturation is largely unknown. Here, we showed that endogenous m6A writers (Mettl3 and Wtap) and eraser (Fto) elevated their transcript levels during meiotic maturation of pig oocytes. From germinal vesicle (GV) to metaphase II (MII) stages, global m6A level significantly increased, and existed mostly in ooplasm. Methyl donor (betaine, 16 mM) treatment of porcine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM) significantly boosted nucleic acid m6A level within oocytes, but unchanged meiotic process and oocyte subsequent development. By contrast, methylation inhibitor (cycloleucine, 20 mM) reduced nucleic acid m6A level, and significantly decreased the germinal vesicle breakdown (GVBD) rate, the extrusion rate of the first polar body, and the cleavage and blastocyst rates of parthenotes. In addition, in cycloleucine-treated oocytes Wtap increased but Lin28 decreased their abundances significantly, along with the higher incidence of spindle defects and chromosome misalignment. Furthermore, pT161-CDK1 protein level in pig oocytes was confirmed to be decreased after cycloleucine treatment for 24 h. Taken together, chemical induced reduction of nucleic acid m6A methylation during pig oocyte meiosis could impair meiotic maturation and subsequent development potency, possibly through down-regulating pluripotency marker Lin28 mRNA abundance and disturbing MPF-regulated chromosome/spindle organization.

Heat shock protein 90α couples with the MAPK-signaling pathway to determine meiotic maturation of porcine oocytes.

Heat shock protein 90 (Hsp90) functions as a molecular chaperone in its interaction with clients to influence multiple cellular and physiological processes. However, our current understanding on Hsp90's relationship with mammalian oocyte maturation is still very limited. Here, we aimed to investigate Hsp90's effect on pig oocyte meiotic maturation. Endogenous Hsp90α was constantly expressed at both mRNA and protein levels in porcine maturing oocytes. Addition of 2 µM 17-allylamino-17-demethoxygeldanamycin (17-AAG), the Hsp90 inhibitor, to in vitro mature cumulus-oocyte complexes (COC) significantly decreased Hsp90α protein level (P < 0.05), delayed germinal vesicle breakdown (GVBD) (P < 0.05), and impeded the first polar body (PB1) extrusion (P < 0.01) of porcine oocytes. 2 µM 17-AAG treatment during in vitro maturation also decreased the subsequent development competence as indicated by the lower cleavage (P < 0.001) and higher fragmentation (P < 0.001) rates of parthenotes, whereas no effects on the percentage and average cell number of blastocysts were found. Immunodepletion of Hsp90α by antibody microinjection into porcine oocytes at germinal vesicle and metaphase II stages induced similar defects of meiotic maturation and parthenote development, to that resulted from 2 µM inhibitor 17-AAG. For oocytes treated by 2 µM 17-AAG, the cytoplasm and membrane actin levels were weakened (P < 0.01), and the spindle assembly was disturbed (P < 0.05), due to decreased p-ERK1/2 level (P < 0.05). However, the mitochondrial function and early apoptosis were not affected, as demonstrated by rhodamine 123 staining and Annexin V assays. Our findings indicate that Hsp90α can couple with mitogen-activated protein kinase to regulate cytoskeletal structure and orchestrate meiotic maturation of porcine oocytes.

Ascorbic acid induces global epigenetic reprogramming to promote meiotic maturation and developmental competence of porcine oocytes.

L-ascorbic acid (Vitamin C) can enhance the meiotic maturation and developmental competence of porcine oocytes, but the underlying molecular mechanism remains obscure. Here we show the role of ascorbic acid in regulating epigenetic status of both nucleic acids and chromatin to promote oocyte maturation and development in pigs. Supplementation of 250 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (AA2P) during in vitro maturation significantly enhanced the nuclear maturation (as indicated by higher rate of first polar body extrusion and increased Bmp15 mRNA level), reduced level of reactive oxygen species, and promoted developmental potency (higher cleavage and blastocyst rates of parthenotes, and decreased Bax and Caspase3 mRNA levels in blastocysts) of pig oocytes. AA2P treatment caused methylation erasure in mature oocytes on nucleic acids (5-methylcytosine (5 mC) and N 6 -methyladenosine (m6A)) and histones (Histone H3 trimethylations at lysines 27, H3K27me3), but establishment of histone H3 trimethylations at lysines 4 (H3K4me3) and 36 (H3K36me3). During the global methylation reprogramming process, levels of TET2 (mRNA and protein) and Dnmt3b (mRNA) were significantly elevated, but simultaneously DNMT3A (mRNA and protein), and also Hif-1α, Hif-2α, Tet3, Mettl14, Kdm5b and Eed (mRNA) were significantly inhibited. Our findings support that ascorbic acid can reprogram the methylation status of not only DNA and histone, but also RNA, to improve pig oocyte maturation and developmental competence.