RESUMO
This study aimed to evaluate the effect of including soybean molasses (SM) on performance, blood parameters, carcass traits, meat quality, fatty acid, and muscle (longissimus thoracis) transcriptomic profiles of castrated lambs. Twenty Dorperâ ×â Santa Inês lambs (20.06â ±â 0.76 kg body weight [BW]) were assigned to a randomized block design, stratified by BW, with the following treatments: CON: 0 g/kg of SM and SM20: 200 g/kg of SM on dry matter basis, allocated in individual pens. The diet consisted of 840 g/kg concentrate and 160 g/kg corn silage for 76 d, with the first 12 d as an adaptation period and the remaining 64 d on the finishing diet. The SM20 diet increased blood urea concentration (Pâ =â 0.03) while reduced glucose concentration (Pâ =â 0.04). Lambs fed SM showed higher subcutaneous fat deposition (Pâ =â 0.04) and higher subcutaneous adipocyte diameter (Pâ <â 0.01), in addition to reduced meat lipid oxidation (Pâ <â 0.01). SM reduced the quantity of branched-chain fatty acids in longissimus thoracis (Pâ =â 0.05) and increased the quantity of saturated fatty acids (Pâ =â 0.01). In the transcriptomic analysis, 294 genes were identified as differentially expressed, which belong to pathways such as oxidative phosphorylation, citric acid cycle, and monosaccharide metabolic process. In conclusion, diet with SM increased carcass fat deposition, reduced lipid oxidation, and changed the energy metabolism, supporting its use in ruminant nutrition.
This study investigated the effects of incorporating soybean molasses (SM) into the diet of castrated lambs on various aspects of their performance and meat quality. Twenty lambs were divided into two groups: one was fed a control diet without SM whereas the other was fed a similar diet but containing 20% of SM. The feeding trial lasted for 76 d. Results showed that the SM inclusion in the diet led to increased blood urea levels and decreased glucose concentrations. SM inclusion also resulted in lambs with higher levels of subcutaneous fat and larger adipocytes, while reducing meat lipid oxidation. Moreover, SM altered fatty acid composition in the meat, decreasing branched-chain fatty acids and increasing saturated fatty acids. In agreement with these findings, transcriptomic analysis revealed a significant change in the expression of genes related to energy metabolism in the muscle of lambs fed SM. In conclusion, incorporating SM in lamb's diet increased fat deposition, improved meat quality, and induced a transcriptomic change in the muscle energy metabolism, supporting its potential use in ruminant nutrition.
Assuntos
Ração Animal , Dieta , Glycine max , Metabolismo dos Lipídeos , Carne , Melaço , Gordura Subcutânea , Animais , Ração Animal/análise , Dieta/veterinária , Glycine max/química , Gordura Subcutânea/metabolismo , Gordura Subcutânea/efeitos dos fármacos , Masculino , Carne/análise , Metabolismo dos Lipídeos/efeitos dos fármacos , Ovinos , Fenômenos Fisiológicos da Nutrição Animal , Ácidos Graxos/metabolismo , Distribuição Aleatória , Músculo Esquelético/metabolismo , Músculo Esquelético/efeitos dos fármacos , Oxirredução , Carneiro Doméstico , Suplementos Nutricionais/análiseRESUMO
BACKGROUND: The worldwide growing demand for human insulin for treating diabetes could be supplied by transgenic animals producing insulin in their milk. METHODS AND RESULTS: Pseudo-lentivirus containing the bovine ß-casein promoter and human insulin sequences was used to produce modified adult fibroblasts, and the cells were used for nuclear transfer. Transgenic embryos were transferred to recipient cows, and one pregnancy was produced. Recombinant protein in milk was evaluated using western blotting and mass spectrometry. One transgenic cow was generated, and in milk analysis, two bands were observed in western blotting with a molecular mass corresponding to the proinsulin and insulin. The mass spectrometry analysis showed the presence of human insulin more than proinsulin in the milk, and it identified proteases in the transgenic milk that could convert proinsulin into insulin and insulin-degrading enzyme that could degrade the recombinant protein. CONCLUSION: The methodologies used for generating the transgenic cow allowed the detection of the production of recombinant protein in the milk at low relative expression compared to milk proteins, using mass spectrometry, which was efficient for detecting recombinant protein with low expression in milk. Milk proteases could act on protein processing converting recombinant protein to functional protein. On the other hand, some milk proteases could act in degrading the recombinant protein.
Assuntos
Leite , Proinsulina , Feminino , Gravidez , Animais , Bovinos , Humanos , Animais Geneticamente Modificados/metabolismo , Proinsulina/análise , Proinsulina/metabolismo , Leite/química , Proteínas Recombinantes/metabolismo , Insulina/análise , Peptídeo Hidrolases/metabolismoRESUMO
Much effort has been employed to improve the quality of embryos obtained by in vitro production (IVP) given the relevance of this technology to current livestock systems. In this context, dynamic IVP systems have proved beneficial to the embryo once they mimic fluid flows and mechanical forces resulting from the movement of ciliated cells and muscle contraction in the reproductive tract. In the present study, we sought to confirm these initial findings as well as assess potential molecular consequences to the embryo by applying micro-vibration (45 Hz for 5 s once per 60 min) during both oocyte maturation and embryo culture in cattle. As a result, micro-vibration led to lower incidence of apoptosis in blastocysts following vitrification-thawing. Further analyses revealed epigenetic and transcriptional changes in blastocysts derived from the micro-vibration treatment, with a total of 502 differentially expressed genes. Enrichment analyses linked differentially expressed genes to 'Oxidative phosphorylation', 'Cytokine-cytokine receptor interaction', and 'Signaling pathways regulating pluripotency of stem cells'. Yet, a meta-analysis indicated that the transcriptional changes induced by micro-vibration were not toward that of in vivo-derived embryos. In conclusion, micro-vibration increases the cryoresistance of bovine embryos, but caution should be taken given the unclear consequences of epigenetic and transcriptional abnormalities induced by the treatment.
Assuntos
Epigenômica , Transdução de Sinais , Animais , Bovinos/genética , Células-TroncoRESUMO
Besides their canonical roles as energy sources, short-chain fatty acids act as metabolic regulators of gene expression through histone posttranslational modifications. Ketone body ß-hydroxybutyrate (BHB) causes a novel epigenetic modification, histone lysine ß-hydroxybutyrylation (Kbhb), which is associated with genes upregulated in starvation-responsive metabolic pathways. Dairy cows increase BHB in early lactation, and the effects of this increase on cellular epigenomes are unknown. We searched for and identified that Kbhb is present in bovine tissues in vivo and confirmed that this epigenetic mark is responsive to BHB in bovine and human fibroblasts cultured in vitro in a dose-dependent manner. Maturation of cumulus-oocyte complexes with high concentrations of BHB did not affect the competence to complete meiotic maturation or to develop until the blastocyst stage. BHB treatment strongly induced H3K9bhb in cumulus cells, but faintly in oocytes. RNA-seq analysis in cumulus cells indicated that BHB treatment altered the expression of 345 genes. The downregulated genes were mainly involved in glycolysis and ribosome assembly pathways, while the upregulated genes were involved in mitochondrial metabolism and oocyte development. The genes and pathways altered by BHB will provide entry points to carry out functional experiments aiming to mitigate metabolic disorders and improve fertility in cattle.
Assuntos
Ácido 3-Hidroxibutírico , Células do Cúmulo , Epigênese Genética , Histonas , Lisina , Oócitos , Ácido 3-Hidroxibutírico/metabolismo , Ácido 3-Hidroxibutírico/farmacologia , Animais , Bovinos , Células do Cúmulo/metabolismo , Feminino , Histonas/metabolismo , Humanos , Lisina/metabolismo , Oócitos/metabolismoRESUMO
In vivo- and in vitro-produced bovine embryos have different metabolic profiles and differences in gene transcription patterns. These embryos also have a distinct ability to establish and sustain early pregnancies. Small extracellular vesicles (sEVs) are secreted by embryos and carry bioactive molecules, such as miRNAs. We hypothesize that in vivo or in vitro-produced bovine hatched blastocysts on Day 9 and the sEVs secreted by them have different miRNA profiles. To address this hypothesis, embryos of both groups were placed in in vitro culture on Day 7. After 48 h, hatched embryos and hatched embryo-conditioned media (eCM) of both groups were collected. A total of 210 miRNAs were detected in embryos of both groups, of these 6 miRNAs were downregulated, while 7 miRNAs were upregulated in vitro group when compared to in vivo group. sEVs were isolated from eCM to determine miRNA profile. A total of 106 miRNAs were detected in both groups, including 14 miRNAs upregulated in sEVs from in vivo-eCM, and 2 miRNAs upregulated in sEVs from in vitro-eCM. These miRNAs express in embryos and sEVs secreted by them regulate early embryonic developmental and endometrial pathways, which can modify embryo-maternal communication during early pregnancy and consequently affect pregnancy establishment.
Assuntos
Vesículas Extracelulares , MicroRNAs , Animais , Blastocisto/metabolismo , Bovinos , Técnicas de Cultura Embrionária , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Vesículas Extracelulares/metabolismo , Feminino , MicroRNAs/genética , MicroRNAs/metabolismo , GravidezRESUMO
Offspring born to obese and diabetic mothers are prone to metabolic diseases, a phenotype that has been linked to mitochondrial dysfunction and endoplasmic reticulum (ER) stress in oocytes. In addition, metabolic diseases impact the architecture and function of mitochondria-ER contact sites (MERCs), changes which associate with mitofusin 2 (MFN2) repression in muscle, liver and hypothalamic neurons. MFN2 is a potent modulator of mitochondrial metabolism and insulin signaling, with a key role in mitochondrial dynamics and tethering with the ER. Here, we investigated whether offspring born to mice with MFN2-deficient oocytes are prone to obesity and diabetes. Deletion of Mfn2 in oocytes resulted in a profound transcriptomic change, with evidence of impaired mitochondrial and ER function. Moreover, offspring born to females with oocyte-specific deletion of Mfn2 presented increased weight gain and glucose intolerance. This abnormal phenotype was linked to decreased insulinemia and defective insulin signaling, but not mitochondrial and ER defects in offspring liver and skeletal muscle. In conclusion, this study suggests a link between disrupted mitochondrial/ER function in oocytes and increased risk of metabolic diseases in the progeny. Future studies should determine whether MERC architecture and function are altered in oocytes from obese females, which might contribute toward transgenerational transmission of metabolic diseases.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Oócitos/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Feminino , GTP Fosfo-Hidrolases/genética , Homeostase/fisiologia , Camundongos , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Músculo Esquelético/metabolismo , Transdução de SinaisRESUMO
There is evidence of a purifying filter acting in the female germline to prevent the expansion of deleterious mutations in the mitochondrial DNA (mtDNA). Given our poor understanding of this filter, here we investigate the competence of the mouse embryo to eliminate dysfunctional mitochondria. Toward that, mitochondria were damaged by photoirradiation of NZB/BINJ zygotes loaded with chloromethyl-X-rosamine (CMXRos). The resultant cytoplasm was then injected into C57BL/6J zygotes to track the levels of NZB/BINJ mtDNA during the preimplantation development. About 30% of NZB/BINJ mtDNA was present after injection, regardless of using photoirradiated or non-photoirradiated cytoplasmic donors. Moreover, injection of photoirradiated-derived cytoplasm did not impact development into blastocysts. However, lower levels of NZB/BINJ mtDNA were present in blastocysts when comparing injection of photoirradiated (24.7% ± 1.43) versus non-photoirradiated (31.4% ± 1.43) cytoplasm. Given that total mtDNA content remained stable between stages (zygotes vs. blastocysts) and treatments (photoirradiated vs. non-photoirradiated), these results indicate that the photoirradiated-derived mtDNA was replaced by recipient mtDNA in blastocysts. Unexpectedly, treatment with rapamycin prevented the drop in NZB/BINJ mtDNA levels associated with injection of photoirradiated cytoplasm. Additionally, analysis of mitochondria-autophagosome colocalization provided no evidence that photoirradiated mitochondria were eliminated by autophagy. In conclusion, our findings give evidence that the mouse embryo is competent to mitigate the levels of damaged mitochondria, which might have implications to the transmission of mtDNA-encoded disease.
RESUMO
Mitochondrial function, largely regulated by the dynamics of this organelle, is inextricably linked to the oocyte health. In comparison with most somatic cells, mitochondria in oocytes are smaller and rounder in appearance, suggesting limited fusion. The functional implications of this distinct morphology, and how changes in the mitochondrial shape translate to mitochondrial function in oogenesis is little understood. We, therefore, asked whether the pro-fusion proteins mitofusins 1 (MFN1) and 2 (MFN2) are required for the oocyte development. Here we show that oocyte-specific deletion of Mfn1, but not Mfn2, prevents the oocyte growth and ovulation due to a block in folliculogenesis. We pinpoint the loss of oocyte growth and ovulation to impaired PI3K-Akt signaling and disrupted oocyte-somatic cell communication. In support, the double loss of Mfn1 and Mfn2 partially rescues the impaired PI3K-Akt signaling and defects in oocyte development secondary to the single loss of Mfn1. Together, this work demonstrates that the mitochondrial function influences the cellular signaling during the oocyte development, and highlights the importance of distinct, nonredundant roles of MFN1 and MFN2 in oogenesis.
Assuntos
Comunicação Celular/fisiologia , GTP Fosfo-Hidrolases/metabolismo , Oócitos/metabolismo , Folículo Ovariano/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Oócitos/fisiologia , Oogênese/fisiologia , Ovulação/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Induced pluripotent stem cells (iPSCs) provide a promising means of creating custom-tailored cell lines for cellular therapies. Their application in regenerative medicine, however, depends on the possibility that the maintenance and differentiation of cells and organs occur under defined conditions. One major component of stem cell culture systems is the substrate, where the cells must attach and proliferate. The present study aimed to investigate the putative cytotoxic effects of poly(vinyl alcohol) (PVA)-based matrices on the in vitro culture of mouse fetal fibroblasts. In addition, the PVA-based hydrogels were used to determine the capacity of bovine induced pluripotent stem cells (biPSCs) to adhere and proliferate on synthetic substrates. Our results show that both cell types interacted with the substrate and presented proliferation during culture. The biPSCs formed new colonies when cell suspensions were placed onto the hydrogel surface for culture. These results may represent a new characterized xeno-free clinical grade culture system to be widely applied in cell-based therapies.
Assuntos
Fibroblastos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Álcool de Polivinil/química , Animais , Bovinos , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Meios de Cultura , Hidrogéis/química , Teste de Materiais , Camundongos , Microscopia Eletrônica de Varredura , Medicina Regenerativa , Solubilidade , Espectroscopia de Infravermelho com Transformada de Fourier , Células-Tronco/citologiaRESUMO
The cellular reprogramming into pluripotency is influenced by external and internal cellular factors, such as in vitro culture conditions (e.g., environmental oxygen concentration), and the aging process. Herein, we aimed to generate and maintain equine iPSCs (eiPSCs) derived from fibroblasts of a horse older than 20 years and to evaluate the effect of different levels of oxygen tension (atmospheric 20% O2, 5% O2, or 20% to 5% O2) on these cells. Fibroblasts were reprogrammed, and putative eiPSCs were positive for positive alkaline phosphatase detection; they were positive for pluripotency-related genes OCT4, REX1, and NANOG; immunofluorescence-positive staining was presented for OCT4 and NANOG (all groups), SOX2 (groups 5% O2 and 20% to 5% O2), and TRA-1-60, TRA-1-81, and SSEA-1 (only in 20% O2); they formed embryoid bodies; and there is spontaneous differentiation in mesoderm, endoderm, and ectoderm embryonic germ layers. In addition to the differences in immunofluorescence analysis results, the eiPSC colonies generated at 20% O2 presented a more compact morphology with a well-defined border than cells cultured in 5% O2 and 20% to 5% O2. Significant differences were also observed in the expression of genes related to glucose metabolism, mitochondrial fission, and hypoxia (GAPDH, GLUT3, MFN1, HIF1α, and HIF2α), after reprogramming. Our results show that the derivation of eiPSCs was not impaired by aging. Additionally, this study is the first to compare high and low oxygen cultures of eiPSCs, showing the generation of pluripotent cells with different profiles. Under the tested conditions, the lower oxygen tension did not favor the pluripotency of eiPSCs. This study shows that the impact of oxygen atmosphere has to be considered when culturing eiPSCs, as this condition influences the pluripotency characteristics.
RESUMO
Oocyte mitochondria are increased in number, smaller, and rounder in appearance than mitochondria insomatic cells. Moreover, mitochondrial numbers and activity are narrowly tied with oocyte quality because of the key role of mitochondria to oocyte maturation. During oocyte maturation, mitochondria display great mobility and cluster at specific sites to meet the high energy demand. Conversely, oocyte mitochondria are not required during early oogenesis as coupling with granulosa cells is sufficient to support gametes needs. In part, this might be explained by the importance of protecting mitochondria from oxidative damage that result in mutations in mitochondrial DNA (mtDNA). Considering mitochondria are transmitted exclusively by the mother, oocytes with mtDNA mutations may lead to diseases in offspring. Thus, to counterbalance mutation expansion, the oocyte has developed specific mechanisms to filter out deleterious mtDNA molecules. Herein, we discuss the role of mitochondria on oocyte developmental potential and recent evidence supporting a purifying filter against deleterious mtDNA mutations in oocytes.(AU)
Assuntos
Oócitos/fisiologia , Fertilidade , Transmissão de Doença Infecciosa , DNA Mitocondrial/efeitos adversos , DNA Mitocondrial/genética , Mitocôndrias/químicaRESUMO
Oocyte mitochondria are increased in number, smaller, and rounder in appearance than mitochondria insomatic cells. Moreover, mitochondrial numbers and activity are narrowly tied with oocyte quality because of the key role of mitochondria to oocyte maturation. During oocyte maturation, mitochondria display great mobility and cluster at specific sites to meet the high energy demand. Conversely, oocyte mitochondria are not required during early oogenesis as coupling with granulosa cells is sufficient to support gametes needs. In part, this might be explained by the importance of protecting mitochondria from oxidative damage that result in mutations in mitochondrial DNA (mtDNA). Considering mitochondria are transmitted exclusively by the mother, oocytes with mtDNA mutations may lead to diseases in offspring. Thus, to counterbalance mutation expansion, the oocyte has developed specific mechanisms to filter out deleterious mtDNA molecules. Herein, we discuss the role of mitochondria on oocyte developmental potential and recent evidence supporting a purifying filter against deleterious mtDNA mutations in oocytes.
Assuntos
DNA Mitocondrial/efeitos adversos , DNA Mitocondrial/genética , Fertilidade , Oócitos/fisiologia , Transmissão de Doença Infecciosa , Mitocôndrias/químicaRESUMO
Oocyte mitochondria are increased in number, smaller, and rounder in appearance than mitochondria in somatic cells. Moreover, mitochondrial numbers and activity are narrowly tied with oocyte quality because of the key role of mitochondria to oocyte maturation. During oocyte maturation, mitochondria display great mobility and cluster at specific sites to meet the high energy demand. Conversely, oocyte mitochondria are not required during early oogenesis as coupling with granulosa cells is sufficient to support gamete's needs. In part, this might be explained by the importance of protecting mitochondria from oxidative damage that result in mutations in mitochondrial DNA (mtDNA). Considering mitochondria are transmitted exclusively by the mother, oocytes with mtDNA mutations may lead to diseases in offspring. Thus, to counterbalance mutation expansion, the oocyte has developed specific mechanisms to filter out deleterious mtDNA molecules. Herein, we discuss the role of mitochondria on oocyte developmental potential and recent evidence supporting a purifying filter against deleterious mtDNA mutations in oocytes.
RESUMO
Embryonic and placental development is highly orchestrated by epigenetic processes. Disruptions in normal placental development, commonly observed in pregnancies produced by nuclear transfer, are associated with abnormal gene expression and altered epigenetic regulation of imprinted and vital placental genes. The objective of this study was to evaluate expression and epigenetic regulation of the imprinted gene TSSC4 in cotyledonary and intercotyledonary tissues from day 60 pregnancies produced by embryo transfer (ET), in vitro fertilization (IVF) and nuclear transfer (NT) in cattle. TSSC4 expression was reduced by 30% in cotyledons at 60days of gestation in the NT group. The proximal promoter region of TSSC4 showed an increase in the permissive histone mark (H3K4me2) and a reduction in the inhibitory histone mark (H3K9me2) in the cotyledons produced by NT, in relation to cotyledons produced by embryo transfer. Interestingly, H3K9me2 was also significantly reduced in cotyledons produced by IVF, compared to the ET controls. DNA methylation, in CpG-rich regions located at the proximal promoter region and the coding region of TSSC4 did not differ. These results suggest that the reduction in TSSC4 expression, observed following NT, can not be explained by the histone changes investigated in the proximal promoter region of the gene, or by changes in methylation in three regions evaluated. Also, a decrease in the levels of H3K9 dimethylation in IVF samples, indicate that in vitro culturing could corroborate with the alterations seen in the NT group.
Assuntos
Bovinos/genética , Transferência Embrionária/métodos , Fertilização in vitro/métodos , Técnicas de Transferência Nuclear , Placenta/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Epigênese Genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Impressão Genômica , Especificidade de Órgãos , Gravidez , Proteínas Supressoras de Tumor/genéticaRESUMO
Early mammalian embryos derived from in vitro fertilization are exposed to conditions distinct from the native oviduct-uterine environment, including atmospheric oxygen that promotes cellular oxidative stress and alters gene expression. High oxygen partial pressure during embryo development is associated with low pregnancy rates and increased embryonic apoptosis. We investigated how bovine embryos responded to high (20%) or low (5%) oxygen partial pressure during in vitro culture, evaluating levels of reactive oxygen species (ROS) as well as changes in the expression of oxidative stress- and epigenetic-related transcripts and miRNAs in blastocysts. Additionally, we determined the global DNA methylation levels in the resulting embryos. Our data indicated that bovine blastocysts produced in vitro under high oxygen partial pressure possessed elevated ROS abundance and exhibited increased expression of CAT, GLRX2, KEAP1, NFR2, PRDX1, PRDX3, SOD1, TXN, and TXNRD1, versus reduced levels of the oxidative stress-related bta-miR-210. These stressed embryos also presented altered expression of the epigenetic-associated transcripts DNMT3A, H2AFZ, H3F3B, HDAC2, MORF4L2, REST, and PAF1. In addition, we demonstrated that embryos cultured under high oxygen partial pressure have increased global DNA methylation, suggesting that DNA hypermethylation is mediated by the deregulation of epigenetic-related enzymes due to oxidative stress.
Assuntos
Antioxidantes/metabolismo , Blastocisto/metabolismo , Metilação de DNA , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Estresse Oxidativo , Animais , Blastocisto/citologia , BovinosRESUMO
The influence of in vitro maturation (IVM) in oocytes is still not totally understood. The aim of this study was to determine the influence of IVM on the metabolism and homeostasis of bovine cumulus-oocyte complexes. In the present study, we demonstrated that IVM leads to accumulation of neutral lipids associated with differential levels of the mono-, di- and triacylglycerols in both cumulus cells and oocytes. We observed that in vitro-matured oocytes exhibited decreased glutathione and reactive oxygen species levels and a lower ATP/ADP ratio when compared to in vivo-matured oocytes, with no significant differences in metabolism and stress-related mRNA or miRNA levels. Moreover, in addition to an increase in lipids in in vitro-matured cumulus cells, fatty acid synthesis and accumulation as well as glycolysis pathway genes were upregulated, whereas those affiliated with the ß-oxidation pathway were decreased. Our gene expression data in cumulus cells suggest the disruption of endoplasmic reticulum stress, apoptosis and cellular stress response pathways during IVM. Furthermore, a total of 19 miRNAs were significantly altered by the maturation process in cumulus cells. These results indicate some new negative influences of the in vitro system in cumulus-oocyte complexes, demonstrating the occurrence of functional disruption in lipid metabolism and stress pathways and showing evidences suggesting the occurrence of altered mitochondrial activity and energy metabolism during IVM, with a massive dysregulation of the corresponding transcripts in the surrounding cumulus cells.
Assuntos
Células do Cúmulo/metabolismo , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/metabolismo , Estresse Oxidativo , Animais , Bovinos , Células Cultivadas , Células do Cúmulo/citologia , Metabolismo Energético , Feminino , Oócitos/citologia , Oxirredução , Espécies Reativas de Oxigênio/metabolismoRESUMO
Pregnancy success results from the interaction of multiple factors, among them are folliculogenesis and early embryonic development. Failure during these different processes can lead to difficulties in conception. Alternatives to overcome these problems are based on assisted reproductive techniques. Extracellular vesicles are cell-secreted vesicles present in different body fluids and contain bioactive materials, such as messenger RNA, microRNAs (miRNAs), and proteins. Thus, our hypothesis is that extracellular vesicles from follicular fluid from 3-6 mm ovarian follicles can modulate bovine embryo development in vitro. To test our hypothesis follicular fluid from bovine ovaries was aspirated and small-extracellular vesicles (<200 nm) were isolated for further analysis. Additionally, small-extracellular vesicles (EVs) were utilized for functional experiments investigating their role in modulating messenger RNA, microRNA as well as global DNA methylation and hydroxymethylation levels of bovine blastocysts. EVs from 3-6 mm follicles were used for RNA-seq and miRNA analysis. Functional annotation analysis of the EVs transcripts revealed messages related to chromatin remodeling and transcriptional regulation. EVs treatment during oocyte maturation and embryo development causes changes in blastocyst rates, as well as changes in the transcription levels of genes related to embryonic metabolism and development. Supplementation with EVs from 3-6 mm follicles during oocyte maturation and early embryo development (until the 4-cell stage) increased the levels of bta-miR-631 (enriched in EVs from 3-6 mm follicles) in embryos. Interestingly, the addition of EVs from 3-6 mm follicles induced changes in global DNA methylation and hydroxymethylation levels compared to embryos produced by the standard in vitro production system. Our results indicate that the supplementation of culture media with EVs isolated from the follicular fluid of 3-6 mm follicles during oocyte maturation and early embryo development can partially modify metabolic and developmental related genes as well as miRNA and global DNA methylation and hydroxymethylation, suggesting that EVs play an important role during oocyte maturation and early embryo development in vitro.
Assuntos
Micropartículas Derivadas de Células , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário/efeitos dos fármacos , Líquido Folicular , Oócitos/metabolismo , Animais , Bovinos , Metilação de DNA/efeitos dos fármacos , Técnicas de Cultura Embrionária , Embrião de Mamíferos/citologia , Feminino , MicroRNAs/metabolismo , Oócitos/citologia , RNA Mensageiro/metabolismoRESUMO
Abnormal placental development is frequent in nuclear transfer (NT) pregnancies and is likely to be associated with altered epigenetic reprogramming. In the present study, fetal and placental measurements were taken on Day 60 of gestation in cows with pregnancies produced by AI, IVF and NT. Placentas were collected and subjected to histological evaluation, the expression of genes important in trophoblast differentiation and expression of the placental imprinted gene pleckstrin homology-like domain, family A, member 2 (PHLDA2), as well as chromatin immunoprecipitation (ChIP) for histone marks within the promoter of PHLDA2. Fewer binucleated cells were observed in NT cotyledons, followed by IVF and AI cotyledons (P<0.05). Expression of heart and neural crest derivatives expressed 1 (HAND1), placental lactogen (PL), pregnancy-associated glycoprotein 9 (PAG-9) and PHLDA2 was elevated in NT cotyledons compared with AI cotyledons. Expression of PHLDA2 was higher in IVF than AI samples (P<0.05). ChIP revealed an increase in the permissive mark dimethylation of lysine 4 on histone H3 (H3K4me2), surprisingly associated with the silent allele of PHLDA2, and a decrease in the inhibitory mark H3K9me2 in NT samples. Thus, genes critical for placental development were altered in NT placentas, including an imprinted gene. Allele-specific changes in the permissive histone mark in the PHLDA2 promoter indicate misregulation of imprinting in clones. Abnormal trophoblast differentiation could have resulted in lower numbers of binucleated cells following NT. These results suggest that the altered expression of imprinted genes associated with NT are also caused by changes in histone modifications.
Assuntos
Expressão Gênica , Código das Histonas , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Técnicas de Transferência Nuclear/veterinária , Placenta/metabolismo , Alelos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Bovinos , Feminino , Histonas/genética , Proteínas Nucleares/genética , Lactogênio Placentário/genética , Lactogênio Placentário/metabolismo , Placentação/fisiologia , Gravidez , Proteínas da Gravidez/genética , Proteínas da Gravidez/metabolismo , Trofoblastos/metabolismoRESUMO
The yolk sac is an extra-embryonic membrane that plays an important role in early embryonic survival. It is the production site for blood cells during embryonic mammalian development and is a likely source of stem cells. The aim of this study was to identify and characterize the putative haematopoietic cells from the yolk sac of bovine embryos at different stages of gestation. The yolk sac regresses according to gestational age and embryos are characterized into groups (I-V) according to the crown-rump measurement. Groups I-III survived in culture longer and exhibited the formation of cell clusters, whereas groups IV and V could not be maintained in culture for an extended period of time. Flow-cytometry analysis revealed that groups I-III had similar characteristics, including high expression levels of the haematopoietic markers CD34, CD90 and CD117. In groups IV and V, decreases were observed in the expression levels of CD117 and CD34. Cells were found to be capable of survival post-cryopreservation and exhibited varying abilities to form colonies in a methylcellulose matrix, depending on gestational age. Cytological analysis revealed the presence of blood cells (lymphocytes and monocytes). Quantitative PCR analysis demonstrated the presence of the haematopoietic progenitor genes GATA3 and LMO2, but not RUNX1. Thus, we have successfully isolated and characterized haematopoietic cells from the bovine embryo yolk sac at varying gestational ages. This study is crucial for the understanding of the development of the haematopoietic system and the embryonic function of this organ. Copyright © 2015 John Wiley & Sons, Ltd.
Assuntos
Células-Tronco Hematopoéticas/citologia , Saco Vitelino/citologia , Animais , Bovinos , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Embrião de Mamíferos/citologia , Feminino , Citometria de Fluxo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/ultraestrutura , Metilcelulose/farmacologia , Reação em Cadeia da Polimerase , GravidezRESUMO
The use of recombinant proteins has increased in diverse commercial sectors. Various systems for protein production have been used for the optimization of production and functional protein expression. The mammary gland is considered to be a very interesting system for the production of recombinant proteins due to its high level of expression and its ability to perform post-translational modifications. Cows produce large quantities of milk over a long period of lactation, and therefore this species is an important candidate for recombinant protein expression in milk. However, transgenic cows are more difficult to generate due to the inefficiency of transgenic methodologies, the long periods for transgene detection, recombinant protein expression and the fact that only a single calf is obtained at the end of each pregnancy. An increase in efficiency for transgenic methodologies for cattle is a big challenge to overcome. Promising methodologies have been proposed that can help to overcome this obstacle, enabling the use of transgenic cattle as bioreactors for protein production in milk for industry.