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.

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.

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.

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.

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.

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.

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.

Long noncoding RNAs exchange during zygotic genome activation in goat.

In mammals, their proper development during the early cleavage stages strongly relies on the gene products newly transcribed by zygotic genome activation (ZGA). Long noncoding RNAs (lncRNAs) have been characterized as key regulators of the ZGA process in mice and human. However, the ZGA stage has not yet been identified and epigenetic regulations of the ZGA process remain largely unknown in goats. Here, we show that ZGA occurred at the 8-cell stage in goats. During ZGA, trimethylation of H3K9 was dynamically changed but maintained strong staining in development arrested embryos. Using single-cell RNA sequencing, we identified 800 mRNAs and 250 lncRNAs as candidates of key molecules in goat preimplantation embryos. These mRNAs and lncRNAs were differentially expressed from 4- to 8-cell stage embryos and were strongly enriched in terms of retinoic acid receptor signaling pathway as well as signaling pathway regulating pluripotency of stem cells. In particular, we found that microinjection of siRNA against lnc_137 caused development arrest. Our results are consistent with the notion that lncRNAs play vital roles during ZGA, and the data presented here provide an excellent source for further ZGA lncRNA studies.

Comprehensive analysis of long noncoding RNA and mRNA expression patterns in sheep testicular maturation.

Long noncoding RNAs (LncRNAs) have been identified as important regulators of testis development; however, their expression patterns and roles in sheep are not yet clear. Thus, we used stranded specific RNA-seq to profile the testis transcriptome (lncRNAs and mRNAs) in premature and mature sheep. Hormone levels and the testis index were examined, and histological analyses were performed at five stages of testis development, 5-day-old (D5), 3-month-old (3M), 6-month-old (6M), 9-month-old (9M), and 2-year-old (2Y), the results of which indicate a significant difference in hormone levels and testis morphometries between the 3M and 9M stages (P < 0.05). Based on the comparison between 3M and 9M samples, we found 1,118 differentially expressed (DE) lncRNAs and 7,253 DE mRNAs in the testes, and qRT-PCR analysis showed that the results correlated well with the transcriptome data. Furthermore, we constructed lncRNA-protein-coding gene interaction networks. Forty-seven DE lncRNA-targeted genes enriched for male reproduction were obtained by cis- and trans-acting; 51 DE lncRNAs and 45 cis-targets, 2 DE lncRNAs and 2 trans-targets were involved in this network. Of these, 5 lncRNAs and their targets, PRKCD, NANOS3, SERPINA5, and CYP19A1, were enriched for spermatogenesis and male gonad development signaling pathways. We further examined the expression levels of 5 candidate lncRNAs and their target genes during testis development. Lastly, the interaction of lncRNA TCONS__00863147 and its target gene PRKCD was validated in vitro in sheep Leydig cells. This study provides a valuable resource for further study of lncRNA function in sheep testis development and spermatogenesis.

Characterization of RUNX1T1, an Adipogenesis Regulator in Ovine Preadipocyte Differentiation.

Runt-related transcription factor 1 translocation partner 1 (RUNX1T1), a potential novel regulator of adipogenesis, exists in two splice variants: a long (RUNX1T1-L) and a short (RUNX1T1-S) isoform. However, there is no data showing the existence of RUNX1T1 in ovine subcutaneous fat at different stages of developmental and its role on ovine adipogenesis. Therefore, the objectives of this study were to evaluate the presence of RUNX1T1 in subcutaneous fat of five-day-old to 24-month-old sheep and to investigate the role of RUNX1T1 in ovine adipogenesis. In this study, we detected a 1829 bp cDNA fragment of RUNX1T1 which contains a 1815 bp coding sequence that encodes 602-amino acid and 14 bp of 5' untranslated region, respectively. The amino acid sequence of RUNX1T1 has 31.18⁻94.21% homology with other species' protein sequences. During fat development, the RUNX1T1 protein expression was higher in subcutaneous fat of 24-month-old Hu sheep. In addition, the expression of RUNX1T1-L mRNA decreased first, then subsequently increased during ovine preadipocyte differentiation. Knockdown of RUNX1T1-L in ovine preadipocytes promoted preadipocyte differentiation and lipid accumulation. Taken together, our data suggests that RUNX1T1 is an important functional molecule in adipogenesis. Moreover, it showed for the first time that RUNX1T1-L was negatively correlated with the ovine preadipocyte differentiation.

Abnormal expression of DNA methyltransferases and genomic imprinting in cloned goat fibroblasts.

Somatic cell nuclear transfer (SCNT) is a useful way to produce cloned animals. However, SCNT animals exhibit DNA methylation and genomic imprinting abnormalities. These abnormalities may be due to the faulty epigenetic reprogramming of donor cells. To investigate the consequence of SCNT on the genomic imprinting and global methylation in the donor cells, growth patterns and apoptosis of cloned goat fibroblast cells (CGFCs) at passage 7 were determined. Growth patterns in CGFCs were similar to the controls; however, the growth rate in log phase was lower and apoptosis in CGFCs were significantly higher (P < 0.01). In addition, quantitative expression analysis of three DNA methyltransferases (Dnmt) and two imprinted genes (H19, IGF2R) was conducted in CGFCs: Dnmt1 and Dnmt3b expression was significantly reduced (P < 0.01), and H19 expression was decreased sixfold (P < 0.01); however, the expression of Dnmt3a was unaltered and IGF2R expression was significantly increased (P < 0.05). Finally, we used bisulfite sequencing PCR to compare the DNA methylation patterns in differentially methylated regions (DMRs) of H19 and IGF2R. The DMRs of H19 (P < 0.01) and IGF2R (P < 0.01) were both highly methylated in CGFCs. These results indicate that the global genome might be hypomethylated. Moreover, there is an aberrant expression of imprinted genes and DMR methylation in CGFCs.

A novel fluorescence reporter system for the characterization of dairy goat mammary epithelial cells.

Goat mammary epithelial cells (GMECs) are a useful model to understand the physiological function of mammary glands and to assess the efficiency of mammary-specific vectors. The aim of this study was to develop an effective and convenient way to evaluate the secretory capacity of GMECs in primary culture. In this study, we developed a reporter system using fluorescent proteins driven by the CSN2 (Capra hircus beta-casein) gene promoter to detect the secretory capacity of GMECs. Additionally, we evaluated the efficiency of the reporter system by determining the expression of cytoskeletal proteins and beta-casein protein. The results suggest that this reporter system provides an easy, convenient and effective method to detect the function of milk synthesis in GMECs. Primary cultures of GMECs were homogeneous and retained the function of milk synthesis, prompting their usefulness as a model for further studies.

Scd1 mammary-specific vector constructed and overexpressed in goat fibroblast cells resulting in an increase of palmitoleic acid and oleic acid.

Stearoyl-CoA desaturase-1 (Scd1) is a rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids. Overexpression of Scd1 in transgenic animals would modify the nutritional value of ruminant-derived foods by increasing the monounsaturated fatty acid (MUFA) and decreasing the saturated fatty acid (SFA) content. The aim of this study was to develop an effective Scd1 vector that is specifically expressed in dairy goat mammary glands. We successfully amplified the goat full length Scd1 cDNA and evaluated its activity in goat ear skin-derived fibroblast cells (GEFCs) by lipid analysis. In addition, we constructed a mammary gland-specific expression vector and confirmed efficient expression of Scd1 in goat mammary epithelial cells (GMECs) by qRT-PCR and Western blot analysis. Fatty acid analysis showed that Scd1-overexpression resulted in an increase in levels of palmitoleic acid (16:1n-7) and oleic acid (18:1n-9), from 1.73 ± 0.02% to 2.54 ± 0.02% and from 27.25 ± 0.13% to 30.37 ± 0.04%, respectively (both p<0.01) and the ratio of MUFA to SFA was increased. This work lays a foundation for the generation of Scd1 transgenic goats.

[Reprogramming mechanism and genetic stability of induced pluripotent stem cells (iPSCs)].

Induced pluripotent stem cells (iPSCs) were reprogrammed from somatic cells using specific transcription factors. Bypassing the ethical issue caused by embryonic stem cells (ESCs), iPSCs can be successfully induced from a variety of cells, which makes iPSCs a powerful research tool for developmental biology. iPSCs have also become indispensable to the research of life science due to their broad potential applications. However, it's a big challenge to obtain iPSCs with high quality and genetic stability. Here, we review the research progress of increasing the reprogramming mechanism and genetic stability of iPSCs in order to provide references of reprogramming efficiency of iPSCs, reducing the cost, and addressing key points of iPSCs quality control, further promoting clinical application of the iPSCs.

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.

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.

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.