Effects of hydroxychloroquine on the mucosal barrier and gut microbiota during healing of mice colitis.

OBJECTIVES: The present study aimed to evaluate the impact of hydroxychloroquine (HCQ) on the mucosal barrier and gut microbiota during the healing of mice colitis. METHODS: The body weight, colon length, colon Hematoxylin-Eosin (H&E) staining, occult blood in feces and serum inflammatory factor levels were measured to evaluate the function of HCQ on inflammatory process in colitis mice. The Alcian blue staining, immunohistochemistry, immunofluorescence and serum FITC-Dextran assay were performed to assess the intestinal mucosal permeability. And the composition and expression differences of intestinal microorganisms in feces were analyzed with 16S rDNA sequencing for exploration of HCQ impact on gut microbiota in colitis. RESULTS: The results showed that the administration of HCQ did not significantly alter the body weight, colon length, or fecal occult blood of the mice. However, HCQ treatment did lead to recovery of the structure and morphology of the intestinal mucosa, increased expression of tight junction proteins (E-cadherin and Occludin), decreased permeability of the intestinal mucosal barrier, increased serum IL-10, and decreased level of tumor necrosis factor-alpha (TNF-α). Additionally, HCQ was found to increase the abundance of Euryarchaeota, Lactobacillus_murinus and Clostridium_fusiformis, while decreasing the abundance of Oscillibacter, uncultured_Odoribacter, Bacterioidetes and Muribaculum. CONCLUSIONS: These findings support that HCQ plays a role in the treatment of mice colitis possibly by altering the gut microbiota.

Exploring nicotinamide adenine dinucleotide precursors across biosynthesis pathways: Unraveling their role in the ovary.

Natural Nicotinamide Adenine Dinucleotide (NAD+) precursors have attracted much attention due to their positive effects in promoting ovarian health. However, their target tissue, synthesis efficiency, advantages, and disadvantages are still unclear. This review summarizes the distribution of NAD+ at the tissue, cellular and subcellular levels, discusses its biosynthetic pathways and the latest findings in ovary, include: (1) NAD+ plays distinct roles both intracellularly and extracellularly, adapting its distribution in response to requirements. (2) Different precursors differs in target tissues, synthetic efficiency, biological utilization, and adverse effects. Importantly: tryptophan is primarily utilized in the liver and kidneys, posing metabolic risks in excess; nicotinamide (NAM) is indispensable for maintaining NAD+ levels; nicotinic acid (NA) constructs a crucial bridge between intestinal microbiota and the host with diverse functions; nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN) increase NAD+ systemically and can be influenced by delivery route, tissue specificity, and transport efficiency. (3) The biosynthetic pathways of NAD+ are intricately intertwined. They provide multiple sources and techniques for NAD+ synthesis, thereby reducing the dependence on a single molecule to maintain cellular NAD+ levels. However, an excess of a specific precursor potentially influencing other pathways. In addition, Protein expression analysis suggest that ovarian tissues may preferentially utilize NAM and NMN. These findings summarize the specific roles and potential of NAD+ precursors in enhancing ovarian health. Future research should delve into the molecular mechanisms and intervention strategies of different precursors, aiming to achieve personalized prevention or treatment of ovarian diseases, and reveal their clinical application value.

Mitophagy in mammalian follicle development and health.

Mitophagy, the cellular process that removes damaged mitochondria, plays a crucial role in maintaining normal cell functions. It is deeply involved in the entire process of follicle development and is associated with various ovarian diseases. This review aims to provide a comprehensive overview of mitophagy regulation, emphasizing its role at different stages of follicular development. Additionally, the study illuminates the relationship between mitophagy and ovarian diseases, including ovary aging (OA), primary ovarian insufficiency (POI), and polycystic ovary syndrome (PCOS). A detailed understanding of mitophagy could reveal valuable insights and novel strategies for managing female ovarian reproductive health.

Integrated gut microbiome and metabolome analysis reveals the inhibition effect of Lactobacillus plantarum CBT against colorectal cancer.

The microecological stability of the gut microbiota plays a pivotal role in both preventing and treating colorectal cancer (CRC). This study investigated whether Lactobacillus plantarum CBT (LP-CBT) prevents CRC by inducing alterations in the gut microbiota composition and associated metabolites. The results showed that LP-CBT inhibited colorectal tumorigenesis in azoxymethane/dextran sulfate sodium (AOM/DSS)-treated mice by repairing the intestinal barrier function. Furthermore, LP-CBT decreased pro-inflammatory cytokines and anti-inflammatory cytokines. Importantly, LP-CBT remodeled intestinal homeostasis by increasing probiotics (Coprococcus, Mucispirillum, and Lactobacillus) and reducing harmful bacteria (Dorea, Shigella, Alistipes, Paraprevotella, Bacteroides, Sutterella, Turicibacter, Bifidobacterium, Clostridium, Allobaculum), significantly influencing arginine biosynthesis. Therefore, LP-CBT treatment regulated invertases and metabolites associated with the arginine pathway (carbamoyl phosphate, carboxymethyl proline, L-lysine, 10,11-epoxy-3-geranylgeranylindole, n-(6)-[(indol-3-yl)acetyl]-L-lysine, citrulline, N2-succinyl-L-ornithine, and (5-L-glutamyl)-L-glutamate). Furthermore, the inhibitory effect of LP-CBT on colorectal cancer was further confirmed using the MC38 subcutaneous tumor model. Collectively, these findings offer compelling evidence supporting the potential of LP-CBT as a viable preventive strategy against CRC.

Ythdf2-mediated STK11 mRNA decay supports myogenesis by inhibiting the AMPK/mTOR pathway.

An emerging research focus is the role of m6A modifications in mediating the post-transcriptional regulation of mRNA during mammalian development. Recent evidence suggests that m6A methyltransferases and demethylases play critical roles in skeletal muscle development. Ythdf2 is a m6A "reader" protein that mediates mRNA degradation in an m6A-dependent manner. However, the specific function of Ythdf2 in skeletal muscle development and the underlying mechanisms remain unclear. Here, we observed that Ythdf2 expression was significantly upregulated during myogenic differentiation, whereas Ythdf2 knockdown markedly inhibited myoblast proliferation and differentiation. Combined analysis of high-throughput sequencing, Co-IP, and RIP assay revealed that Ythdf2 could bind to m6A sites in STK11 mRNA and form an Ago2 silencing complex to promote its degradation, thereby regulating its expression and consequently, the AMPK/mTOR pathway. Furthermore, STK11 downregulation partially rescued Ythdf2 knockdown-induced impairment of proliferation and myogenic differentiation by inhibiting the AMPK/mTOR pathway. Collectively, our results indicate that Ythdf2 mediates the decay of STK11 mRNA, an AMPK activator, in an Ago2 system-dependent manner, thereby driving skeletal myogenesis by suppressing the AMPK/mTOR pathway. These findings further enhance our understanding of the molecular mechanisms underlying RNA methylation in the regulation of myogenesis and provide valuable insights for conducting in-depth studies on myogenesis.

ALKBH5-PYCR2 Positive Feedback Loop Promotes Proneural-Mesenchymal Transition Via Proline Synthesis In GBM.

AlkB homolog 5, RNA demethylase (ALKBH5) is abnormally highly expressed in glioblastoma multiforme (GBM) and is negatively correlated with overall survival in GBM patients. In this study, we found a new mechanism that ALKBH5 and pyrroline-5-carboxylate reductase 2 (PYCR2) formed a positive feedback loop involved in proline synthesis in GBM. ALKBH5 promoted PYCR2 expression and PYCR2-mediated proline synthesis; while PYCR2 promoted ALKBH5 expression through the AMPK/mTOR pathway in GBM cells. In addition, ALKBH5 and PYCR2 promoted GBM cell proliferation, migration, and invasion, as well as proneural-mesenchymal transition (PMT). Furthermore, proline rescued AMPK/mTOR activation and PMT after silencing PYCR2 expression. Our findings reveal an ALKBH5-PYCR2 axis linked to proline metabolism, which plays an important role in promoting PMT in GBM cells and may be a promising therapeutic pathway for GBM.

Treatment of inflammatory bowel disease: Potential effect of NMN on intestinal barrier and gut microbiota.

Nicotinamide mononucleotide (NMN) exerts physiological effects in mammals through its conversion to nicotinamide adenine dinucleotide (NAD+). In this study, we established experimental models of colitis by mixing drinking water of C57BL/6J mice with dextran sodium sulphate (DSS), and then fed them with the same concentration of NMN or at the same time. After NMN treatment, we observed improved morphology of inflamed intestines, slightly restored length of colon, improved barrier function and reduced proinflammatory factors expression in serum. Also, significant alterations in the composition and abundance of intestinal flora in IBD mice were found. The abundance of Firmicutes, Verrucomicrobia, Akkermansia and Lactobacillus, considered as beneficial bacteria, increased, while Bacteroidetes and Muribaculaceae unclassifiably decreased. Taken together, these results suggest that NMN may improve intestinal inflammation, reduce intestinal mucosal permeability and repair gut flora dysbiosis in IBD.

Correlations of ALD, Keap-1, and FoxO4 expression with traditional tumor markers and clinicopathological characteristics in colorectal carcinoma.

Aldolase A (A-2) (ALD), Kelch-like-ECH associated protein-1 (Keap-1), and Forkhead box O4 (FoxO4) are key regulatory proteins, which have been proven to be involved in tumor development. However, the clinicopathological significance of ALD, Keap-1, and FoxO4 expressions in colorectal (colon) carcinoma (CRC) is not clearly known. We sought to explore the clinicopathological significance of ALD, Keap-1, and FoxO4 in CRC to provide evidences for potential monitoring index of CRC. Cases of 199 CRC patients were analyzed retrospectively. Evaluation of ALD, cAMP response element-binding protein-2, cyclo-oxygenase 2, FoxO4, Keap-1, and p53 expressions in CRC patients was accomplished with immunohistochemical technique. The patients were divided into negative and positive groups in accordance with immunohistochemical result. We compared the clinicopathological characteristics of the patients in the 2 groups, coupled with analysis of the relationship between 6 aforesaid proteins and clinicopathological characteristics. Herein, we confirmed the association of tumor location with the expression of ALD, Keap-1, and FoxO4. Also, tumor differentiation was observed to associate significantly with the expression of Keap-1, FoxO4, and Cox-2. The data also revealed that there was a correlation between smoking and expression of ALD, Keap-1, FoxO4, p53, and Cox-2. Nevertheless, insignificant difference was observed when clinicopathological characteristics were compared with cAMP response element-binding protein-2 expression. These findings suggest that ALD, Keap-1, and FoxO4 reinvolved in CRC development, and thus may be considered as potential monitoring protein for CRC.

The regulation of LncRNA GTL2 expression by DNA methylation during sheep skeletal muscle development.

DNA methylation has crucial roles in regulating the expression of genes involved in skeletal muscle development. However, the DNA methylation pattern of lncRNA during sheep skeletal muscle development remains unclear. This study investigated previous WGBS and LncRNA data in skeletal muscle of sheep (fetus and adult). We then focused on LncRNA GTL2, which is differentially expressed in skeletal muscle and has multiple DMRs. We found that the expression level of GTL2 decreased with age. GTL2 DMRs methylation levels were significantly higher in adult muscle than in fetal muscle. After 5AZA treatment, GTL2 expression was significantly increased in a dose-dependent manner.The dCas9-DNMT3A-sgRNA significantly reduced the expression level of GTL2 in cells, but increased GTL2 DMR methylation levels. The above studies indicate that dCas9-DNMT3A can effectively increase the methylation level in the DMR region of GTL2, the expression level of GTL2 is regulated by DNA methylation during muscle development.

Long-term treatment of Nicotinamide mononucleotide improved age-related diminished ovary reserve through enhancing the mitophagy level of granulosa cells in mice.

Ovarian aging affects the reproductive health of elderly women due to decline in oocyte quality, which is closely related to mitochondrial dysfunction. Nicotinamide mononucleotide (NMN), as a precursor of NAD+, effectively regulate mitochondria metabolism in mice. However, roles of NMN in improving age-related diminished ovary reserve remain to be determined. In present study, 4, 8, 12, 24, 40-week old female ICR mice were collected and a 20-week-long administration of NMN was conducted to 40-week-old mice (60WN), meanwhile the control group is given water (60WC). First, we found that 20-week-long administration of NMN to 40-week-old mice exhibited anti-aging and anti-inflammatory effects on organ structures, along with the improvement of estrus cycle condition and endocrine function. The number of primordial, primary, secondary, antral follicles and corpora luteum of ovaries in 60WN group was significantly increased compared with those in 60WC group. Additionally, the protein and gene expressions of P16 of ovaries were significantly reduced in 60WN group than in 60WC group. the mitochondria biogenesis, autophagy level, and proteases activity enhanced in granulosa cells after 20-week-administration of NMN. Present results indicate that NMN has the potential to save diminished ovary reserve by long-term treatment, providing a basis for exploring the role of NMN in anti-ovarian aging by enhancing the mitophagy level of granulosa cells.

NMN Maintains Intestinal Homeostasis by Regulating the Gut Microbiota.

The aim of this study was to determine the effects of long-term Nicotinamide mononucleotide (NMN) treatment on modulating gut microbiota diversity and composition, as well as its association with intestinal barrier function. In this study, C57BL/6J mice were fed different concentrations of NMN, and their feces were collected for detection of 16S rDNA and non-targeted metabolites to explore the effects of NMN on intestinal microbiota and metabolites. The results revealed that NMN increased the abundance of butyric acid-producing bacteria (Ruminococcae_UCG-014 and Prevotellaceae_NK3B31_group) and other probiotics (Akkermansia muciniphila), while the abundance of several harmful bacteria (Bilophila and Oscillibacter) were decreased after NMN treatment. Meanwhile, the level of bile acid-related metabolites in feces from the G1 group (0.1 mg/ml) was significantly increased compared to the control group, including cholic acid, taurodeoxycholic acid, taurocholic acid, glycocholic acid, and tauro-ß-muricholic acid. In addition, long-term NMN treatment affected the permeability of the intestinal mucosa. The number of goblet cells and mucus thickness increased, as well as expression of tight junction protein. These results demonstrate that NMN reduced intestinal mucosal permeability and exerts a protective effect on the intestinal tract. This study lays the foundation for exploring NMN's utility in clinical research.

FTO regulates myoblast proliferation by controlling CCND1 expression in an m6A-YTHDF2-dependent manner.

N6-Methyladenosine (m6A) modification is the most abundant chemical modification in mRNA, and it participates in various biological processes, such as cell differentiation and proliferation. However, little is known about the function of m6A demethylase fat mass and obesity-associated (FTO) in myoblast proliferation. Here, we demonstrated that knockdown of FTO can significantly inhibit myoblast proliferation and promote apoptosis. RNA sequencing analysis revealed that a lot of downregulated genes in FTO knockdown cells are associated with cell cycle and apoptosis. Furthermore, silencing FTO drastically decreased cyclin D1 (CCND1) expression through YTHDF2-mediated mRNA degradation, thereby delaying the progression of G1 phase, and leading to impaired myoblast proliferation. These findings unraveled that FTO regulates myoblast proliferation by controlling CCND1 expression in an m6A-YTHDF2-dependent manner, which highlights the critical roles of m6A modification in myoblast proliferation.

Reinterpreting sheep muscle strand-specific RNA sequencing data showing extensive 3'UTR extensions.

The more complex 3' UTR in higher organisms may have the function of increasing post-transcriptional gene regulation. Recent RNA sequencing technologies have provided us with the possibility to capture the complete 3' UTR landscape of different species and cells. However, no systematic analysis of sheep-related 3' UTR has been performed. Here, we conducted a detailed analysis of the 3' UTR with the primary goal of identifying intact 3' UTR landscapes in the sheep muscles of the three developmental stages. Based on strand-specific RNA sequencing (ssRNA-seq) data, we found that thousands of genes in sheep muscle are continuously transcribed after the UTR of the reference genome (Oar_v4.0). More than 66% of the 3' UTR extensions exhibit similar expression trends to their upstream gene exons. These 3' UTR extensions strongly enrich thousands of conserved microRNA binding sites. The 3' UTR extension-associated RNA of PFKM (PuaRNA) was predicted to be derived from the 3' UTR of PFKM. In sheep myocytes, myotubes and various tissues, the expression pattern of PuaRNA is positively correlated with that of PFKM. Taken together, these new 3' UTR annotations greatly extend the range of mammalian post-transcriptional regulatory networks, which have a particular impact on the regulation of sheep muscle development.

Analysis of candidate biomarkers and related transcription factors involved in the development and restoration of stress-induced gastric ulcer by transcriptomics.

Stress-induced gastric ulcer is one of the common complications affecting patients after trauma, mainly leading to gastrointestinal bleeding and perforation, and severe cases may be life-threatening. However, the molecular mechanism of stress-induced gastric ulcer remains unclear. In the present study, RNA-sequencing was performed on gastric tissues of normal rats (C), stress-induced gastric ulcer rats (T0), and rats recovered from gastric ulcer for 3 days (T3), and bioinformatics analysis was performed to determine changes in gene expression and biological pathways. The protein-protein interaction (PPI) networks of differentially expressed genes (DEGs) were constructed by STRING and visualized by the Cytoscape software. The associated transcriptional factor (TFs)-gene regulatory network of the hub DEGs was also constructed. Pairwise comparisons obtained 103 (T0_C), 127 (T3_T0), and 13 (T3_C) DEGs, respectively. Gene ontology (GO) enrichment analysis indicated DEGs in T0_C and T3_T0 were significantly enriched in response to oxygen-containing compound, response to organic substance, and response to external stimulus. Pathway analysis suggested that DEGs were enriched in TNF signaling pathway, PPAR signaling pathway, apoptosis, and IL-17 signaling pathway. Seven hub genes (Fos, Jun, Nfkbia, Dusp1, Pim3, Junb, and Fosb) were obtained from the PPI networks of T0_C and T3_T0. Key TFs with close interactions, such as Fos, Jun, Nfkbia, Junb, Egr1, and Fosb, were screened This study used RNA-sequencing and bioinformatics analysis to screen out genes associated with gastric ulcer, which can help reveal the molecular mechanism of gastric ulcer development and restoration, and provide reference for the treatment of human gastric ulcers.

Roles of vitamin D and its receptor in the proliferation and apoptosis of luteinised granulosa cells in the goat.

The objective of this study was to investigate the dose-dependent effect of 1α,25-(OH)2VD3 (Vit D3) on invitro proliferation of goat luteinised granulosa cells (LGCs) and to determine the underlying mechanisms of its action by overexpressing and silencing vitamin D receptor (VDR) in LGCs. Results showed that VDR was prominently localised in GCs and theca cells (TCs) and its expression increased with follicle diameter, but was lower in atretic follicles than in healthy follicles. The proliferation rate of LGCs was significantly higher in the Vit D3-treated groups than in the control group, with the highest proliferation rate observed in the 10nM group; this was accompanied by changes in the expression of cell cycle-related genes. These data indicate that Vit D3 affects LGC proliferation in a dose-dependent manner. Contrary to the VDR knockdown effects, its overexpression upregulated and downregulated cell cycle- and apoptosis-related genes respectively; moreover, supplementation with 10nM of Vit D3 significantly enhanced these effects. These results suggest that changes in VDR expression patterns in LGCs may be associated with follicular development by regulation of cell proliferation and apoptosis. These findings will enhance the understanding of the roles of Vit D3 and VDR in goat ovarian follicular development.

The Expression Pattern of p32 in Sheep Muscle and Its Role in Differentiation, Cell Proliferation, and Apoptosis of Myoblasts.

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.

YAP1 regulates PPARG and RXR alpha expression to affect the proliferation and differentiation of ovine preadipocyte.

Adipose tissue development is regulated by a serial of developmental signaling pathways. The Hippo pathway is a novel signaling cascade closely associated with adipogenesis. While most of Hippo pathway components had been verified that have a vital role in preadipocytes proliferation and differentiation, little is known about the function of Yes-associated protein 1 (YAP1) in mammalian adipose tissue development. Therefore, we investigated the role of YAP1 in ovine adipose tissue development by in vitro and in vivo experiments. We observed that the adipocyte size in subcutaneous adipose tissue increased with development. YAP1 expression increased during adipose tissue development, while decreased during the differentiation of ovine preadipocytes in vitro. YAP1 knockdown notably promoted lipid accumulation and suppressed ovine preadipocyte proliferation. In addition, we observed that YAP1 deficiency significantly upregulated peroxisome proliferator-activated receptor gamma (PPARG) and retinoid X receptor alpha (RXR alpha) expression. By contrast, overexpression of YAP1 led to the suppression of preadipocyte differentiation, lipid droplets formation, and PPARG expression. In brief, our findings demonstrated that YAP1 regulates the proliferation and differentiation of ovine preadipocyte via altering PPARG and RXR alpha expression.

Effects of algae supplementation in high-energy dietary on fatty acid composition and the expression of genes involved in lipid metabolism in Hu sheep managed under intensive finishing system.

This study aims to investigate the effect of algae supplementation in high-energy diet (HE diet) on lipid metabolism of intensive feeding sheep. The lambs were assigned to two groups and received a standard diet (ST diet, 8.40 MJ/kg) or a HE diet (9.70 MJ/kg) based on corn, wheat bran, soybean meal. Each group was divided into two subgroups: control and algae supplement (3%, DM basis). The body fat, serum cholesterol, and oleic acid in the liver and muscle increased in lambs receiving the HE diet. However, after supplementing the microalgae (Schizochytrium sp.) in the HE diet, these parameters were all reduced. In addition, EPA, DHA and CLA cis-9, trans-11 in muscle and liver increased. Algae supplementation also altered the expression of lipid metabolism-related genes, including up-regulated FADS2, ELOVL2, SCD, CPT1α and SREBF-1, and down-regulated PPARα and PPARγ. In conclusion, algae supplementation in ST and HE diets increased n-3PUFA and improved metabolic disorder caused by the HE diet.

Pituitary Transcriptomic Study Reveals the Differential Regulation of lncRNAs and mRNAs Related to Prolificacy in Different FecB Genotyping Sheep.

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.

Highly methylated Xist in SCNT embryos was retained in deceased cloned female goats.

X (inactive)-specific transcript (Xist) is crucial in murine cloned embryo development, but its role in cloned goats remains unknown. Therefore, in this study we examined the expression and methylation status of Xist in somatic cell nuclear transfer (SCNT) embryos, as well as in ear, lung, and brain tissue of deceased cloned goats. First, the Xist sequence was amplified and a differentially methylated region was identified in oocytes and spermatozoa. Xist methylation levels were greater in SCNT- than intracytoplasmic sperm injection-generated female 8-cell embryos. In addition, compared with naturally bred controls, Xist methylation levels were significantly increased in the ear, lung, and brain tissue of 3-day-old female deceased cloned goats, but were unchanged in the ear tissue of female live cloned goats and in the lung and brain of male deceased cloned goats. Xist expression was significantly increased in the ear tissue of female live cloned goats, but decreased in the lung and brain of female deceased cloned goats. In conclusion, hypermethylation of Xist may have resulted from incomplete reprogramming and may be retained in 3-day-old female deceased cloned goats, subsequently leading to dysregulation of Xist.