RESUMO
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.
Assuntos
MicroRNAs , Desenvolvimento Muscular , Animais , Bovinos , Diferenciação Celular/genética , China , MicroRNAs/genética , MicroRNAs/metabolismo , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Músculos/metabolismo , Mioblastos/metabolismo , RNA Mensageiro/genéticaRESUMO
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.
Assuntos
Búfalos , Hormônio Foliculoestimulante , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Búfalos/metabolismo , Células Cultivadas , Estradiol/metabolismo , Feminino , Hormônio Foliculoestimulante/metabolismo , Glicólise , Células da Granulosa/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismoRESUMO
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.
Assuntos
Búfalos , Sirtuínas , Animais , Búfalos/genética , Senescência Celular , Fibroblastos/metabolismo , Feto , DNA/metabolismo , Telômero/metabolismo , Sirtuínas/genética , Sirtuínas/metabolismoRESUMO
Lipopolysaccharide (LPS), also known as endotoxin, is a component of the outer membrane of gram-negative bacteria. LPS is released into the surrounding environment during bacterial death and lysis. Due to its chemical and thermal stability, LPS can be detected anywhere and easily exposed to humans and animals. Previous studies have shown that LPS causes hormonal imbalances, ovarian failure, and infertility in mammals. However, the potential mechanisms remain unclear. In this study, we investigated the effects and mechanisms of LPS on tryptophan degradation, both in vivo and in vitro. The effects of kynurenine, a tryptophan derivative, on granulosa cell function and reproductive performance were explored. Results showed that p38, NF-κB, and JNK signaling pathways were involved in LPS-induced Ido1 expressions and kynurenine accumulation. Furthermore, the kynurenine decreased estradiol production, but increased granulosa cell proliferation. In vivo, experiments showed that kynurenine decreased estradiol and FSH production and inhibited ovulation and corpus luteum formation. Additionally, pregnancy and offspring survival rates decreased considerably after kynurenine treatment. Our findings suggest that kynurenine accumulation disrupts hormone secretion, ovulation, corpus luteal formation, and reproductive performance in mammals.
Assuntos
Cinurenina , Ovário , Gravidez , Feminino , Humanos , Animais , Cinurenina/metabolismo , Ovário/metabolismo , Triptofano/metabolismo , Lipopolissacarídeos/farmacologia , Estradiol/metabolismo , Mamíferos/metabolismoRESUMO
MicroRNAs (miRNAs) are involved in many physiological processes such as signal transduction, cell proliferation and apoptosis. Many studies have shown that miRNAs can regulate the process of follicular development. Our previous studies found that the expression of miR-29c-5p in buffalo atretic follicles was much higher than that in healthy follicles, suggesting that this miRNA may participate in the process of buffalo follicular atresia. In this study, we aim to explore to the role and molecular mechanisms of miR-29c-5p on the functions of buffalo granulosa cells (GCs). GCs cultured in vitro were transfected with miR-29c-5p mimics and its inhibitor, respectively, and it was found that the mimics significantly increased the apoptotic rate of GCs. They also inhibited the proliferation of GCs and the secretion of steroid hormones. The effect of the inhibitor was opposite to that of the mimics. MiR-29c-5p was subsequently shown to target the inhibin subunit beta A, (INHBA). Overexpression of INHBA could promote the production of activin A and inhibin A, and then reverse the effect of miR-29c-5p on buffalo GCs. In conclusion, these results suggest that miR-29c-5p promotes apoptosis and inhibits proliferation and steroidogenesis by targeting INHBA in buffalo GCs. This may ultimately promote atresia in buffalo follicles.
Assuntos
Búfalos , MicroRNAs , Animais , Feminino , Apoptose/genética , Búfalos/genética , Proliferação de Células , Atresia Folicular/genética , Células da Granulosa/metabolismo , MicroRNAs/metabolismo , Folículo OvarianoRESUMO
The follicular fluid of mammals has a high abundance of bile acids and these have proven to be closely related to the follicular atresia. However, the origin and content of bile acids in follicular fluid and its mechanisms on follicular atresia remain largely unknown. In this work, we analyzed the origin of bile acids in buffalo follicles by using cell biology studies, and quantified the subspecies of bile acids in follicular fluid from healthy follicles (HF) and atretic follicles (AF) by targeted metabolomics. The function of differential bile acids on follicular granulosa cells was also studied. The results showed that the bile acids transporters were abundantly expressed in ovarian tissues, but the rate-limiting enzymes were not, which was consistent with the inability of cultured follicular cells to convert cholesterol into bile acids. Targeted metabolomics analysis revealed thirteen differential subspecies of bile acids between HF and AF. The free bile acids were significant down-regulated and their conjugated forms were significantly up-regulated in AF as compared to HF. Finally, cell biological validation found a specific differentially conjugated bile acid, glycodeoxycholic acid (GDCA), which could promote follicular granulosa cell apoptosis and reduce steroid hormone secretion. In summary, our studies suggest that bile acids in buffalo follicles are transported from the blood rather than being synthesized within the follicles. The conjugated bile acids such as GDCA, accumulate in buffalo follicles, and may accelerate atresia by promoting apoptosis of granulosa cells and inhibiting steroid hormone production. These results will provide new clues for studying the physiological role and mechanism of bile acids involved in buffalo follicular atresia.
Assuntos
Búfalos , Atresia Folicular , Animais , Apoptose/fisiologia , Ácidos e Sais Biliares , Estradiol/análise , Feminino , Ácido Glicodesoxicólico , Células da Granulosa , Metabolômica , EsteroidesRESUMO
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.
Assuntos
Histona Desacetilases , Mioblastos , Animais , Bovinos , Diferenciação Celular , Proliferação de Células , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Camundongos , Músculo Esquelético/metabolismo , Músculos/metabolismo , Regeneração/genética , Transdução de SinaisRESUMO
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.
RESUMO
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.
Assuntos
Búfalos/genética , Búfalos/metabolismo , Atresia Folicular/genética , Atresia Folicular/metabolismo , Animais , Apoptose , Metabolismo dos Carboidratos , Feminino , Perfilação da Expressão Gênica , Metabolismo dos Lipídeos , Metaboloma , Metabolômica , Nucleotídeos/metabolismo , Folículo Ovariano/metabolismo , TranscriptomaRESUMO
Follicular atresia is one of the main factors limiting the reproductive power of domestic animals. At present, the molecular mechanisms involved in porcine follicular atresia at the metabolic level remain unclear. In this study, we divided the follicles of Bama Xiang pigs into healthy follicles (HFs) and atretic follicles (AFs) based on the follicle morphology. The expression of genes related to atresia in granulosa cells (GCs) and the concentration of hormones in the follicular fluid (FF) from HFs and AFs were detected. We then used liquid chromatography-mass spectrometry-based non-targeted metabolomic approach to analyze the metabolites in the FF from HFs and AFs. The results showed that the content of estradiol was significantly lower in AFs than in HFs, whereas that of progesterone was significantly higher in AFs than that in HFs. The expression of BCL2, VEGFA, and CYP19A1 was significantly higher in HFs than in AFs. In contrast, the expression of BAX and CASPASE3 was significantly lower in HFs. A total of 18 differential metabolites (DMs) were identified, including phospholipids, bioactive substances, and amino acids. The DMs were involved in 12 metabolic pathways, including arginine biosynthesis and primary bile acid biosynthesis. The levels of eight DMs were higher in the HF group than those in the AF group (p < 0.01), and those of 10 DMs were higher in the AF group than those in the HF group (p < 0.01). These findings indicate that the metabolic characteristics of porcine AFs are lower levels of lipids such as phospholipids and higher levels of amino acids and bile acids than those in HFs. Disorders of amino acid metabolism and cholic acid metabolism may contribute to porcine follicular atresia.