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Follicular fluid (FF) plays an important role during follicular development and it contains several bioactive molecules including extracellular microRNAs (ECmiRNAs) that may mediate cell-cell communication during follicular development. Yet, the distribution patterns of ECmiRNAs in FF is not well characterized. This study aims to investigate the distribution of ECmiRNAs in two major fractions, namely exosomal and non-exosomal, of bovine follicular fluid (bFF). Exosomal and non-exosomal fractions from bFF were separated using Exoquick™ exosomes precipitation kit. miRNA expression was evaluated using the human miRCURY LNA™ Universal RT miRNA PCR array system. Transmission electron microscopy and immunoblotting revealed that the isolated vesicles were exosomes. The real-time PCR-based expression analysis revealed that 516 miRNAs were detected in the exosomal fraction of bFF, while 393 miRNAs were detected in the non-exosomal fraction. Among the detected miRNAs, a total of 370 miRNAs were detected in both fractions, while 145 miRNAs and 23 miRNAs were solely detected in exosomal and non-exosomal fractions, respectively. Exploratory pathway analysis showed that the genes targeted by exosomal and non-exosomal miRNAs to be involved in MAPK, Wnt, FoxO, TGF-beta, Oxytocin, ErbB, PI3K-Akt, Neurotrophin signalling pathways which are believed to be involved in follicular development, cell proliferation, and meiotic resumption. The results of our study demonstrated that besides the exosomal fraction, non-exosomal fractions can carry a significant amount of miRNAs in bFF where the exosomal fraction carries a significantly higher number of detectable miRNAs.
Assuntos
Líquido Folicular , MicroRNAs , Animais , Bovinos , Feminino , Líquido Folicular/metabolismo , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Fatores de Crescimento Neural/metabolismo , Ocitocina , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Crescimento Transformador betaRESUMO
Acylglycerophosphate acyltransferases (AGPATs) are the rate-limiting enzymes for the de novo pathway of triacylglycerols (TAG) synthesis. Although AGPATs have been extensively explored by evolution, expression and functional studies, little is known on functional characterization of how many members of the AGPAT family are involved in TAG synthesis and their impact on the cell proliferation and apoptosis. Here, 13 AGPAT genes in buffalo were identified, of which 12 AGPAT gene pairs were orthologous between buffalo and cattle. Comparative transcriptomic analysis and real-time quantitative reverse transcription PCR (qRT-PCR) further showed that both AGPAT1 and AGPAT6 were highly expressed in milk samples of buffalo and cattle during lactation. Knockdown of AGPAT1 or AGPAT6 significantly decreased the TAG content of buffalo mammary epithelial cells (BuMECs) and bovine mammary epithelial cells (BoMECs) by regulating lipogenic gene expression (p < 0.05). Knockdown of AGPAT1 or AGPAT6 inhibited proliferation and apoptosis of BuMECs through the expression of marker genes associated with the proliferation and apoptosis (p < 0.05). Our data confirmed that both AGPAT1 and AGPAT6 could regulate TAG synthesis and growth of mammary epithelial cells in buffalo. These findings will have important implications for understanding the role of the AGPAT gene in buffalo milk performance.
Assuntos
Aciltransferases , Búfalos , Animais , Bovinos , Feminino , Aciltransferases/genética , Aciltransferases/metabolismo , Búfalos/genética , Búfalos/metabolismo , Células Epiteliais/metabolismo , Lactação/genética , Glândulas Mamárias Animais/metabolismo , Leite/metabolismo , Triglicerídeos/metabolismoRESUMO
AIMS: Granulosa cells (GCs) are the major cellular component in a follicular microenvironment and play an indispensable role in ovarian function. This study was conducted to investigate the effects of ethanol exposure on the cellular and transcriptional changes of ovarian GCs. METHODS: For this purpose, bovine GCs were exposed to different concentrations of ethanol (0, 50, 100, 200, 500 and 1000) to mimic the effects of alcohol in in vitro. Subsequently, 100 and 1000 mM concentrations were discarded from further experiments, as 100 mM was not different from 50 mM, and 1000 mM was supertoxic to the cells. RESULTS: The results showed that there was a gradual loss of cell viability with the increase of the ethanol concentration, i.e. lowest viability was observed at the highest concentration (1000 mM), which is further supported by cell proliferation assay. Mitochondrial activity decreased significantly at higher concentrations. The expression of NRF2 decreased significantly (P < 0.05) in ethanol-exposed cells compared with the cells in the control group at the 6-h time point, whereas the expression was increased in 500 mM concentration at the 24-h time point. The expression of antioxidant genes, downstream to Nrf2-pathway activation, showed that overall expression pattern similar to NRF2. CONCLUSION: The result of this study prompted us to postulate that ethanol exposure decreases the ability of GCs to handle stress by downregulating the expression of genes involved in Nrf2-pathway.
Assuntos
Etanol/toxicidade , Células da Granulosa/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/genética , Animais , Apoptose/efeitos dos fármacos , Bovinos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dano ao DNA , Relação Dose-Resposta a Droga , Regulação para Baixo , Etanol/administração & dosagem , Feminino , Expressão Gênica , Técnicas In Vitro , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacosRESUMO
Maternal nutrition during pregnancy is one of the major intrauterine environmental factors that influence fetal development by significantly altering the expression of genes that might have a consequence on the physiological, morphological, and metabolic performance of the offspring in the postnatal period. The impact of maternal dietary protein on the expression of genes in sheep fetal skeletal muscle development is not well understood. The current study aims to investigate the impact of high and low maternal dietary protein on the holistic mRNA expression in the sheep fetal skeletal muscle. Dams were exposed to an isoenergetic high-protein diet (HP, 160-270 g/day), low-protein diet (LP, 73-112 g/day), and standard protein (SP, 119-198 g/day) diets during pregnancy. Fetal skeletal muscles were obtained at the 105th day of pregnancy and mRNA expression profiles were evaluated using Affymetrix GeneChip™ Ovine Gene 1.0 ST Array. The transcriptional analysis revealed a total of 323, 354, and 14 genes were differentially regulated (fold change > 2 and false discovery rate ≤ 0.05) in HP vs. SP, LP vs. HP, and SP vs. LP, respectively. Several myogenic genes, including MYOD1, MYH2, MYH1, are significantly upregulated, while genes related to the immune system, such as CXCL11, HLA-E, CXCL10, CXCL9, TLRs, are significantly downregulated in the fetal muscle of the HP group compared to those of SP and LP group. Bioinformatic analysis revealed that the majority of these genes are involved in pathways related to the immune system and diseases. The results of our study demonstrate that both augmented and restricted dietary proteins in maternal diet during pregnancy alter the expression of genes as well as the offspring's genetic marks.
Assuntos
Ração Animal , Proteínas Alimentares , Feto , Exposição Materna , Músculo Esquelético/metabolismo , Transcriptoma , Animais , Biologia Computacional/métodos , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Ontologia Genética , Anotação de Sequência Molecular , Gravidez , Mapeamento de Interação de Proteínas , Mapas de Interação de ProteínasRESUMO
Despite its essential role in ovulation, oxidative stress (OS) has been found to be cytotoxic to cells, while microRNAs (miRNAs) are known as a major regulator of genes involved in cellular defense against cytotoxicity. However, a functional link between OS and miRNA expression changes in granulosa cells (GCs) remains to be investigated. Here, we investigate the OS modulation of apoptosis-associated miRNAs and their biological relevance in bovine GCs. Following the evaluation of cell viability, accumulation of reactive oxygen species (ROS), cytotoxicity and mitochondrial activity, we used a ready-to-use miRNA PCR array to identify differentially regulated miRNAs. The results showed that exposure to 150 µM H2O2 for 4 h creates remarkable signs of OS in GCs characterized by more than 50% loss of cell viability, higher nuclear factor erythroid 2-related factor 2 (NRF2) nuclear translocation, significantly (p < 0.05) higher abundance of antioxidant genes, significantly (p < 0.001) higher accumulation of ROS, lower mitochondrial activity and a higher (p < 0.001) number of apoptotic nuclei compared to that of the control group. miRNA expression analysis revealed that a total of 69 miRNAs were differentially regulated in which 47 and 22 miRNAs were up- and downregulated, respectively, in stressed GCs. By applying the 2-fold and p < 0.05 criteria, we found 16 miRNAs were upregulated and 10 miRNAs were downregulated. Target prediction revealed that up- and downregulated miRNAs potentially targeted a total of 6210 and 3575 genes, respectively. Pathway analysis showed that upregulated miRNAs are targeting the genes involved mostly in cell survival, intracellular communication and homeostasis, cellular migration and growth control and disease pathways. Our results showed that OS modulates the expression of apoptosis-associated miRNAs that might have effects on cellular or molecular damages.
Assuntos
Células da Granulosa/metabolismo , MicroRNAs/metabolismo , Estresse Oxidativo , Animais , Apoptose , Bovinos , Sobrevivência Celular , Células Cultivadas , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Células da Granulosa/citologia , Peróxido de Hidrogênio/química , MicroRNAs/genética , Mitocôndrias/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Regulação para CimaRESUMO
Since the first evidence for the involvement of microRNAs (miRNAs) in various reproductive processes through conditional knockout of DICER, several studies have been conducted to investigate the expression pattern and role of miRNAs in ovarian follicular development, oocyte maturation, embryo development, embryo-maternal communication, pregnancy establishment and various reproductive diseases. Although advances in sequencing technology have fuelled miRNA studies in mammalian species, the presence of extracellular miRNAs in various biological fluids, including follicular fluid, blood plasma, urine and milk among others, has opened a new door in miRNA research for their use as diagnostic markers. This review presents data related to the identification and expression analysis of cellular miRNA in mammalian female fertility associated with ovarian folliculogenesis, oocyte maturation, preimplantation embryo development and embryo implantation. In addition, the relevance of miRNAs to female reproductive disorders, including polycystic ovary syndrome (PCOS), endometritis and abnormal pregnancies, is discussed for various mammalian species. Most importantly, the mechanism of release and the role of extracellular miRNAs in cell-cell communication and their potential role as non-invasive markers in female fertility are discussed in detail. Understanding this layer of regulation in female reproduction processes will pave the way to understanding the genetic regulation of female fertility in mammalian species.
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Prenatal maternal feeding plays an important role in fetal development and has the potential to induce long-lasting epigenetic modifications. MicroRNAs (miRNAs) are non-coding, single-stranded RNAs that serve as one epigenetic mechanism. Though miRNAs have crucial roles in fetal programming, growth, and development, there is limited data regarding the maternal diet and miRNA expression in sheep. Therefore, we analyzed high and low maternal dietary protein for miRNA expression in fetal longissimus dorsi. Pregnant ewes were fed an isoenergetic high-protein (HP, 160-270 g/day), low-protein (LP, 73-112 g/day), or standard-protein diet (SP, 119-198 g/day) during pregnancy. miRNA expression profiles were evaluated using the Affymetrix GeneChip miRNA 4.0 Array. Twelve up-regulated, differentially expressed miRNAs (DE miRNAs) were identified which are targeting 65 genes. The oar-3957-5p miRNA was highly up-regulated in the LP and SP compared to the HP. Previous transcriptome analysis identified that integrin and non-receptor protein tyrosine phosphatase genes targeted by miRNAs were detected in the current experiment. A total of 28 GO terms and 10 pathway-based gene sets were significantly (padj < 0.05) enriched in the target genes. Most genes targeted by the identified miRNAs are involved in immune and muscle disease pathways. Our study demonstrated that dietary protein intake during pregnancy affected fetal skeletal muscle epigenetics via miRNA expression.
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Small ruminants, especially sheep, are essential for sustainable agricultural production systems, future food/nutrition security, and poverty reduction in developing countries. Within developed countries, the ability of sheep to survive on low-quality forage intake could act as buffer against climate change. Besides sheep's importance in sustainable agricultural production, there has been less ongoing work in terms of sheep genetics in Near East, Middle East and in Africa. For lamb meat production, body weight and average daily gain (ADG) until weaning are critical economic traits that affects the profitability of the industry. The current study aims to identify single nucleotide polymorphisms (SNPs) that are significantly associated with pre-weaning growth traits in fat tail Akkaraman lambs using a genome-wide association study (GWAS). A total of 196 Akkaraman lambs were selected for analysis. After quality control, a total of 31,936 SNPs and 146 lambs were used for subsequent analyses. PLINK 1.9 beta software was used for the analyses. Based on Bonferroni-adjusted p-values, one SNP (rs427117280) on chromosome 2 (OAR2) had significant associations with weaning weight at day 90 and ADG from day 0 to day 90, which jointly explains a 0.8% and 0.9% of total genetic variation respectively. The Ovis aries natriuretic peptide C (NPPC) could be considered as a candidate gene for the defined significant associations. The results of the current study will help to increase understanding of the variation in weaning weight and ADG until weaning of Akkaraman lambs and help enhance selection for lambs with improved weaning weight and ADG. However, further investigations are required for the identification of causal variants within the identified genomic regions.
Assuntos
Estudo de Associação Genômica Ampla , Ovinos , Animais , Peso Corporal/genética , Estudo de Associação Genômica Ampla/veterinária , Ovinos/genética , DesmameRESUMO
MicroRNAs (miRNAs) are a group of tiny molecules of 18-22 nucleotide long noncoding RNA that regulate the post-transcriptional gene expression through translational inhibition and/or mRNA destabilization. Because of their involvement in important developmental processes, it is highly likely that the altered expression of miRNAs could be associated with abnormal conditions like suboptimal growth or diseases. Thus, the expression of miRNAs can be used as biomarkers in pathophysiological conditions. Recently, a handful of miRNAs are detected in cell-free conditions including biofluids and cell culture media and they exhibit specific expression patterns that are associated with altered physiological conditions. Extracellular miRNAs are not only extremely stable outside cells in a variety of biofluids but also they are easy to acquire. These characteristics led to the idea of using extracellular miRNAs as a potential biomarker for the onset and prognosis of cancer. Although miRNAs have been proposed as a potential diagnostic tool for cancer detection, their application in the routine clinical investigation is yet to come. First, this review will provide an insight into the extracellular miRNAs, particularly, their release mechanisms and characteristics, and the potential of extracellular miRNAs as a biomarker in cancer detection. Finally, it will discuss the potential of using extracellular miRNAs in different cancer diagnoses and challenges associated with the clinical application of extracellular miRNAs as noninvasive biomarkers.
Assuntos
Biomarcadores Tumorais/genética , MicroRNA Circulante/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias/diagnóstico , Neoplasias/genética , RNA Neoplásico/genética , Biomarcadores Tumorais/sangue , MicroRNA Circulante/sangue , Detecção Precoce de Câncer/métodos , Exossomos/química , Feminino , Humanos , Masculino , Neoplasias/sangue , Neoplasias/patologia , Especificidade de Órgãos , Prognóstico , RNA Neoplásico/sangueRESUMO
Dietary macronutrients have been regarded as a basic source of energy and amino acids that are necessary for the maintenance of cellular homeostasis, metabolic programming as well as protein synthesis. Due to the emergence of "nutrigenomics", a unique discipline that combines nutritional and omics technologies to study the impacts of nutrition on genomics, it is increasingly evident that macronutrients also have a significant role in the gene expression regulation. Gene expression is a complex phenomenon controlled by several signaling pathways and could be influenced by a wide variety of environmental and physiological factors. Dietary macronutrients are the most important environmental factor influencing the expression of both genes and microRNAs (miRNA). miRNA are tiny molecules of 18 to 22 nucleotides long that regulate the expression of genes. Therefore, dietary macronutrients can influence the expression of genes in both direct and indirect manners. Recent advancements in the state-of-the-art technologies regarding molecular genetics, such as next-generation sequencing, quantitative PCR array, and microarray, allowed us to investigate the occurrence of genome-wide changes in the expression of genes in relation to augmented or reduced dietary macronutrient intake. The purpose of this review is to accumulate the current knowledge focusing on macronutrient mediated changes in the gene function. This review will discuss the impact of altered dietary carbohydrate, protein, and fat intake on the expression of coding genes and their functions. In addition, it will also summarize the regulation of miRNA, both cellular and extracellular miRNA, expression modulated by dietary macronutrients.
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Sulforaphane (SFN) has received a great deal of research attention because of its ability to induce the production of a battery of antioxidant enzymes in certain concentrations through the activation of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling pathway, which may effectively neutralize reactive oxygen species (ROS) induced oxidative stress. This study was conducted to investigate the potential of different concentrations of SFN in inducing antioxidative and apoptotic effects in granulosa cells (GCs). For this purpose, bovine GCs were collected from preovulatory antral follicles and cultured with different concentrations of SFN (0-80 µM) and based on phenotypic evaluation three concentrations were selected: 2 µM (low), 10 µM (medium), and 20 µM (high) for further investigations. The results showed that there was a dramatic loss of cell viability and higher cytotoxic effects of SFN on GCs at higher concentrations (>15 µM). The expression of NRF2 increased significantly (p < 0.05) with fold change ranged 3-8 in SFN treated GCs, whereas Kelch Like ECH Associated Protein 1 (KEAP1) expression was either downregulated or similar as control group under the same conditions. Moreover, the relative expression of the genes (PRDX1, CAT, TXN1and SOD1) downstream to NRF2 activation was found to be highly expressed (fold change ranged from 2 to 5, p < 0.05) in SFN treated GCs compared to the untreated control. In addition, ROS accumulation was higher in GCs treated with 20 µM SFN which in turn results in a higher accumulation of lipid droplets. Compared to control, no changes in the mitochondrial activity was observed at 2 and 10 µM SFN concentrations; however, significantly lower mitochondrial activity was found at high concentration (20 µM). The results of this study clearly showed that 10 µM SFN concentration played a crucial role in activating Nrf2 pathway without inducing apoptotic characteristics and this concentration may have beneficial effects in boosting the production of phase II antioxidant enzymes in GCs. However, at high concentration (20 µM), SFN may generate excessive ROS that causes mitochondrial dysfunction and induces cellular stress and eventually leads to apoptosis. These data strongly suggest a concentration dependent antioxidative and apoptotic effects of SFN on GCs.
Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Bovinos , Células da Granulosa/efeitos dos fármacos , Isotiocianatos/farmacologia , Animais , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Estresse Oxidativo , Espécies Reativas de Oxigênio , SulfóxidosRESUMO
BACKGROUND: Despite its role in increasing the number of offspring during the lifetime of an individual animal, controlled ovarian hyperstimulation (COH) may have detrimental effects on oocyte development, embryo quality and endometrial receptivity. Circulating miRNAs in bio-fluids have been shown to be associated with various pathological conditions including cancers. Here we aimed to investigate the effect of COH on the level of extracellular miRNAs in bovine follicular fluid and blood plasma and elucidate their mode of circulation and potential molecular mechanisms to be affected in the reproductive tract. METHOD: Twelve simmental heifers were estrous synchronized and six of them were hyperstimulated using FSH. Follicular fluid samples from experimental animals were collected using ovum pick up technique at day 0 of the estrous cycle and blood samples were collected at day 0, 3 and 7 of post ovulation. The expression profile of circulatory miRNAs in follicular fluid and blood plasma were performed using the human miRCURY LNA™ Universal RT miRNA PCR array system. A comparative threshold cycle method was used to determine the relative abundance of the miRNAs. RESULTS: A total of 504 and 402 miRNAs were detected in both bovine follicular fluid and blood plasma, respectively. Of these 57 and 21 miRNAs were found to be differentially expressed in follicular fluid and blood plasma, respectively derived from hyperstimulated versus unstimulated heifers. Bioinformatics analysis of those circulating miRNAs indicated that their potential target genes are involved in several pathways including TGF-beta signaling pathway, MAPK signaling pathway, pathways in cancer and Oocyte meiosis. Moreover, detail analysis of the mode of circulation of some candidates showed that most of the miRNA were found to be detected in both exosomal and Ago2 protein complex fraction of both follicular fluid and blood plasma. CONCLUSION: Our data provide the consequence of hyperstimulation induced changes of extracellular miRNAs in bovine follicular fluid and blood plasma, which may have a potential role in regulating genes associated not only with bovine ovarian function but also involved in altering various physiological in bovine oocytes, embryos and modulating reproductive tract environment.
Assuntos
Líquido Folicular/metabolismo , MicroRNAs/metabolismo , Indução da Ovulação/veterinária , Animais , Proteínas Argonautas/metabolismo , Bovinos , Estro/fisiologia , Exossomos/metabolismo , Feminino , Indução da Ovulação/métodos , Plasma/metabolismo , Progesterona/metabolismoRESUMO
Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue (BCB) staining of the corresponding oocytes. Both Exoquick precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal fraction of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNA™ Universal RT miRNA PCR array system was used to profile miRNA expression. This analysis found that miRNAs were present in both exosomal and non-exosomal fraction of bovine follicular fluid. We found 25 miRNAs differentially expressed (16 up and 9 down) in exosomes and 30 miRNAs differentially expressed (21 up and 9 down) in non-exosomal fraction of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus oocyte complex. To further explore the potential roles of these follicular fluid derived extra-cellular miRNAs, the potential target genes were predicted, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. This study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment.