SIRT6 Knockdown in Buffalo Fetal Fibroblasts Exacerbates Premature Senescence Caused by DNA and Telomere Damage.

As a gene with antiaging functions, sirtuin6 (SIRT6) belonging to the sirtuin family plays a vital role in DNA repair, telomerase function, and cellular senescence, as well as maintains epigenomic stability and promotes longevity. However, its role in cell senescence in large animals, such as buffaloes, remains unknown. Fibroblasts are commonly used for somatic reprogramming, and their physiological characteristics affect the efficiency of this process. We aimed to elucidate the role of SIRT6 in cellular senescence and proliferation and analyze its effect on the biological function of buffalo fibroblasts to help improve the efficiency of buffalo somatic cell reprogramming. The expression of SIRT6 and related DNA damage was measured in buffalo fibroblasts obtained at different developmental stages (in the fetus and at 3 and 10 years of age), and the effect of SIRT6 knockdown on the senescence of buffalo fetal fibroblast was investigated. An inverse relationship was observed between SIRT6 expression and senescence in buffalo fibroblasts obtained from animals of various ages. This was accompanied by decreased cell growth, viability, and increased DNA damage. Short hairpin RNA-mediated SIRT6 knockdown accelerated the senescence of buffalo fetal fibroblasts. It blocked the cell cycle during in vitro cell culture, which further enhanced DNA damage, particularly with respect to the telomeres. Collectively, our findings suggest that SIRT6 expression was closely associated with buffalo senescence in fibroblasts. These findings serve as a foundation to better understand the cellular functions of SIRT6 and also aid in selecting donor cells for buffalo somatic cell reprogramming.

CircRRAS2 promotes myogenic differentiation of bovine MuSCs and is a novel regulatory molecule of muscle development.

The proliferation and myogenic differentiation of muscle stem cells (MuSCs) are important factors affecting muscle development and beef quality. There is increasing evidence that circRNAs can regulate myogenesis. We found a novel circRNA, named circRRAS2 that is significantly upregulated in the differentiation phase of bovine MuSCs. Here, we aimed to determine its roles in the proliferation and myogenic differentiation of these cells. The results showed that circRRAS2 was expressed in several bovine tissues. CircRRAS2 inhibited MuSCs proliferation and promoted myoblast differentiation. In addition, chromatin isolation by using RNA purification and mass spectrometry in differentiated muscle cells identified 52 RNA-binding proteins that could potentially bind to circRRAS2, in order to regulate their differentiation. The results suggest that circRRAS2 could be a specific regulator of myogenesis in bovine muscle.HighlightsCircRRAS2 expression is higher in DM cells than in GM cells.CircRRAS2 could significantly inhibit the proliferation and apoptosis of bovine MuSCs.CircRRAS2 promotes the differentiation of bovine MuSCs into myotubes.CircRRAS2 may exert regulatory effects through multiple RNA binding proteins.

HDAC11 Regulates the Proliferation of Bovine Muscle Stem Cells through the Notch Signaling Pathway and Inhibits Muscle Regeneration.

Myogenesis is an essential process that can affect the yield and quality of beef. Transcriptional studies have shown that histone deacetylase 11 (HDAC11) was differentially expressed in muscle tissues of 6 and 18 month old Longlin cattle, but its role in the regulation of myogenesis remains unclear. This study aimed to determine the role of HDAC11 in the proliferation and differentiation of bovine muscle stem cells (MuSCs). HDAC11 promoted MuSC proliferation by activating Notch signaling and inhibited myoblast differentiation by reducing MyoD1 transcription. In addition, overexpression of HDAC11 inhibited the repair regeneration process of muscle in mice. HDAC11 was found to be a novel key target for the control of myogenesis, and this is a theoretical basis for the development of HDAC11-specific modulators as a new strategy to regulate myogenesis.

CircUBE2Q2 promotes differentiation of cattle muscle stem cells and is a potential regulatory molecule of skeletal muscle development.

BACKGROUND: The growth and development of muscle stem cells (MuSCs) are significant events known to affect muscle plasticity, disease, meat production, and meat quality, which involves the types and functions of mRNA and non-coding RNA. Here, MuSCs were cultured from Guangxi fetal cattle. RNA sequencing was used to analyze the RNA expression of mRNA and non-coding RNAs during the cell proliferation and differentiation phases. RESULTS: Two thousand one hundred forty-eight mRNAs and 888 non-coding RNAs were differentially expressed between cell proliferation and differentiation phases, including 113 miRNAs, 662 lncRNAs, and 113 circRNAs. RT-qPCR verified the differential expression levels of mRNAs and non-coding RNAs, and the differentially expressed circUBE2Q2 was subsequently characterized. Expression profile analysis revealed that circUBE2Q2 was abundant in muscle tissues and intramuscular fat. The expression of cricUBE2Q2 was also significantly upregulated during MuSCs myogenic differentiation and SVFs adipogenic differentiation and decreased with age in cattle muscle tissue. Finally, the molecular mechanism of circUBE2Q2 regulating MuSCs function that affects skeletal muscle development was investigated. The results showed that circUBE2Q2 could serve as a sponge for miR-133a, significantly promoting differentiation and apoptosis of cultured MuSCs, and inhibiting proliferation of MuSCs. CONCLUSIONS: CircUBE2Q2 is associated with muscle growth and development and induces MuSCs myogenic differentiation through sponging miR-133a. This study will provide new clues for the mechanisms by which mRNAs and non-coding RNAs regulate skeletal muscle growth and development, affecting muscle quality and diseases.

Follicle-stimulating hormone regulates glycolysis of water buffalo follicular granulosa cells through AMPK/SIRT1 signalling pathway.

Glycolysis in follicular granulosa cells (GCs) is the primary source of energy metabolism substrate of oocytes and is closely related to follicular development in mammals. Many physiological functions of GCs are regulated by follicle-stimulating hormone (FSH). In contrast, whether FSH regulates the glycolysis of GCs and its mechanism remains unclear. This study explored the correlation between FSH concentration and glycolysis level of GCs from different diameters of water buffalo follicles, and further explored the mechanism of FSH regulation in glycolysis in vitro cultured GCs. Results showed the variation trend of lactic acid concentration in follicular fluid and the expression level of glycolysis-related genes in GCs were consistent with the variation trend of FSH concentration in follicular fluid from follicles with different diameters. When GCs were treated with FSH in vitro, the expression level of glycolysis-related genes, lactate production and glucose uptake increased correspondingly (p < .05). Furthermore, we found that expression trend of AMPK/Sirtuin1 (SIRT1) pathway-related genes in GCs was consistent with the expression trend of glycolysis-related genes and was positively correlated with FSH concentrations in vivo or cultured in vitro. Activation of SIRT1 increased the expression level of glycolytic key proteins and lactic acid production in GCs, while inhibition of SIRT1 showed the opposite effect. In general, glycolysis in water buffalo GCs in vivo or cultured in vitro was positively correlated with FSH concentration. AMPK/SIRT1 pathway plays an important role in the regulation of FSH on glycolysis in GCs. Our findings will enrich the understanding of FSH regulating the development of water buffalo follicles.

Whole-Transcriptome Analysis of LncRNAs Mediated ceRNA Regulation in Granulosa Cells Isolated From Healthy and Atresia Follicles of Chinese Buffalo.

Granulosa cells (GCs) are the main supporting cells in follicles and play an important role in the regulation of oocyte maturation and follicular atresia. Accumulating evidence indicates that non-coding RNAs participate in regulation of the physiological function of GCs. However, whole-transcriptome analysis for GCs of buffalo has yet to be reported. In this study, healthy follicles (HFs) and atretic follicles (AFs) were defined according to the apoptosis rate of GCs and the hormone level in follicular fluid. GCs were collected from HFs and AFs (n = 15, 5 < n < 8 mm) for whole-transcriptome analysis using second-generation high-throughput sequencing. A total of 1,861 and 1,075 mRNAs, 159 and 24 miRNAs, and 123 and 100 lncRNAs, were upregulated and downregulated between HFs and AFs, respectively. Enrichment of functions and signaling pathways of these differentially expressed (DE) genes showed that most of DEmRNAs and targets of DEmiRNAs were annotated to the categories of ECM-receptor interaction and focal adhesion, as well as PI3K-AKT, mTOR, TGF-beta, Rap1, and estrogen signaling pathways. The competing endogenous RNA (CeRNA) network was also constructed based on the ceRNA theory which further revealed regulatory roles of these DERNAs in GCs of buffalo follicles. Finally, we validated that lnc4040 regulated the expression of Hif1a as miR-709 sponge in a ceRNA mechanism, suggesting their critical functions in GCs of buffalo follicles. These results show that lncRNAs are dynamically expressed in GCs of HFs and AFs, and interacting with target genes in a ceRNA manner, suggesting their critical functions in buffalo follicular development and atresia.

The Expression Profiles of mRNAs and lncRNAs in Buffalo Muscle Stem Cells Driving Myogenic Differentiation.

Buffalo breeding has become an important branch of the beef cattle industry. Hence, it is of great significance to study buffalo meat production and meat quality. However, the expression profiles of mRNA and long non-coding RNAs (lncRNA) molecules in muscle stem cells (MuSCs) development in buffalo have not been explored fully. We, therefore, performed mRNA and lncRNA expression profiling analysis during the proliferation and differentiation phases of MuSCs in buffalo. The results showed that there were 4,820 differentially expressed genes as well as 12,227 mRNAs and 1,352 lncRNAs. These genes were shown to be enriched in essential biological processes such as cell cycle, p53 signaling pathway, RNA transport and calcium signaling pathway. We also identified a number of functionally important genes, such as MCMC4, SERDINE1, ISLR, LOC102394806, and LOC102403551, and found that interference with MYLPF expression significantly inhibited the differentiation of MuSCs. In conclusion, our research revealed the characteristics of mRNA and lncRNA expression during the differentiation of buffalo MuSCs. This study can be used as an important reference for the study of RNA regulation during muscle development in buffalo.

Integration of transcriptomics and non-targeted metabolomics reveals the underlying mechanism of follicular atresia in Chinese buffalo.

Follicular atresia is a complex physiological process, which results in the waste of follicles and oocytes from the ovary. Elucidating the physiological mechanism of follicular atresia will hopefully reverse the fate of follicles, thereby improve the reproductive efficiency of female animals. However, there are still many gaps to be filled during the follicular atresia process. In this study, we first comprehensively summarized and compared a variety of methods to classify Chinese buffalo follicles with different extent of atresia. Then follicular fluid and granulosa cells from the corresponding follicles with different extent of atresia were collected for non-targeted metabolomics and transcriptomics analysis, respectively. After the detection and analysis of 129 follicles, a reasonable classification standard was formed: on the basis of morphological classification, the relative concentrations of estradiol (E2) and progesterone (PROG) in the follicular fluid were determined, follicles with an estradiol-to-progesterone (E2/PROG) ratio >5 were classified as healthy follicles (HF), 1≤ E2/PROG ≤5 as early atretic follicles (EF) and E2/PROG <1 as late atretic follicles (LF). Correspondingly, follicles with granulosa cells apoptosis rate less than 15 % were divided into HF, 15%-25% were classified as EF and more than 25 % were classified as LF. The integration analysis of non-targeted metabolomics and transcriptomics highlights the following three aspects: (1) Atresia seriously damaged the lipid metabolism homeostasis of follicle, in which PPARγ play important roles. (2) Energy metabolism and nucleotide metabolism of atretic follicles were inhibited. (3) Bilirubin is involved in follicular atresia, and it may be the main force to prevent lipid peroxidation in follicular cells. In summary, results of this study provide new understanding of the molecular mechanisms of Chinese buffalo follicular atresia.

Two Small Molecule Inhibitors Promote Reprogramming of Guangxi Bama Mini-Pig Mesenchymal Stem Cells Into Naive-Like State Induced Pluripotent Stem Cells.

Past researches have shown that pluripotency maintenance of naive and primed-state pluripotent stem cells (PSCs) depends on different signaling pathways, and naive-state PSCs possess the ability to produce chimeras when they are introduced into a blastocyst. Considering porcine is an attractive model for preclinical studies, many researches about pig induced pluripotent stem cells (piPSCs) have been reported. Some cytokines and small molecule compounds could transform primed piPSCs into naive state. However, there are no suitable culture conditions for generation of naive-state piPSCs with high efficiency; other small molecule compounds need further exploration. In this study, we investigated whether p38 MAPK and JNK signal pathway inhibitor SB203580 and SP600125 could be of benefit for acquiring naive-state piPSCs. By comparing reprogramming efficiencies under conditions of different donor cells and culture environment, we found that porcine bone marrow mesenchymal stem cells (PBMSCs) have higher efficiency on piPSC induction, and the culture condition of CHIR99021+PD0325901(2i)+Lif+bFGF is more suitable for subculturing of piPSCs. Our results also indicate that SB203580 and SP600125 could promote reprogramming of PBMSCs into naive-like state piPSCs. These results provide guidance for choosing donor cells, culture conditions, and research of different state iPSCs during the process of reprogramming pig somatic cells.

SQLE Promotes Differentiation and Apoptosis of Bovine Skeletal Muscle-Derived Mesenchymal Stem Cells.

In this study, Squalene epoxidase (SQLE) overexpression vector was transfected into bovine skeletal muscle-derived mesenchymal stem/stromal cells (MSCs) to study the molecular mechanism of SQLE regulating meat quality through myogenesis. We initially profiled the expression of SQLE in cattle embryos and adults, in the muscle tissue of four different cattle varieties, and in 11 different tissues/organs of Guangxi cattle variety. Subsequently, we isolated and cultured bovine skeletal muscle-derived MSCs and detected the expression of SQLE during cell proliferation and differentiation. Then, we constructed a bovine SQLE overexpression vector and transfected it into bovine skeletal muscle-derived MSCs by liposome transfection. Cell Counting Kit-8 (CCK-8), 5-ethynyl-20-deoxyuridine (EdU), flow cytometry, immunofluorescence, and quantitative polymerase chain reaction assays were used to characterize cell proliferation and differentiation in detail. The results showed that the relative expression level of bovine SQLE gene in brain tissue was the highest, and in adult muscle tissue was significantly higher than that in embryonic stage. Especially, the expression of SQLE was significantly upregulated in cell differentiation stage. Furthermore, the proliferation, cell cycle, apoptosis, and myoblast differentiation assays indicated that SQLE significantly promoted the differentiation and apoptosis of bovine skeletal muscle-derived MSCs, but inhibited their proliferation. In conclusion, our study reveals the role of SQLE in myoblast differentiation. These results will provide new clues for the regulation network of bovine muscle development.

Molecular Cloning, Identification, and Expression Patterns of Myostatin Gene in Water Buffalo (Bubalus Bubalis).

Myostatin (MSTN), also named growth differentiation factor 8 (GDF8), is a transforming growth factor-ß (TGF-ß) family member with a key role in the negative regulation of skeletal muscle growth. However, its role in ovarian folliculogenesis remains unclear. To provide us with a basis for understanding this role, we cloned MSTN and examined its expression patterns in water buffalo (Bubalus bubalis). The complete ORF of the water buffalo MSTN gene is 1,128 nucleotides, which encode a 375 amino acid protein and sharing 99% identity at the deducted amino acid level with that of Bos taurus. Protein sequence analysis showed that MSTN is a weakly acerbic extracellular protein, consisting of signal peptides at 18-19 sites, a TGF-ß propeptide, and a TGF-ß domain. RT-PCR analyses demonstrated that water buffalo MSTN was expressed in multiple tissues but not limited to muscle. Immunohistochemistry staining confirmed the presence of MSTN in oocytes and granulosal cells. To our knowledge, this is the first study to confirm the expression of MSTN in the water buffalo ovary, suggesting an additional role of MSTN in water buffalo folliculogenesis, along with its role in skeletal muscle growth regulation. Further study of the regulatory mechanism of MSTN in water buffalo reproduction is warranted. ABBREVIATIONS: MSTN, myostatin; ORF, open reading frame.