RESUMO
Multi-locus methylation defects (MLMDs) in imprinted loci have been reported in Beckwith-Wiedemann Syndrome (BWS). Large offspring syndrome (LOS), a phenotypic subgroup of abnormal offspring syndrome (AOS), is considered a molecular and phenotypic model for BWS. Both LOS and BWS have presented epigenetic defects in some common imprinted loci. In this study, methylation-specific restriction digestion assay - quantitative PCR was used to analyze the DNA methylation pattern in differentially methylated regions (DMRs) of the H19 (H19-DMR), KCNQ1OT1 (KvDMR1) and PEG1/MEST (PEG1-DMR) genes in bovine clone tissues from calves that did not survive after birth. Individual and tissue-specific changes in DNA methylation levels in the bovine KvDMR1, H19-DMR, and PEG1-DMR were observed. In contrast to what has been reported in the literature on BWS and AOS/LOS, the KvDMR1 showed gain (GOM) and loss (LOM) of DNA methylation. LOM and GOM events were found in the DMRs studied in animals produced by the same nucleus donor cell line. This is the first report of epimutations in the PEG1-DMR and GOM at the KvDMR1 found in bovine clones. The findings showed that epigenetic modification in imprinted loci in cloned cattle occurred in a multi-locus pattern similar to that seen in human imprinting disorders. Other multi-locus analyzes must be done to elucidate the MLMD pattern in AOS in bovine clones.
Assuntos
Síndrome de Beckwith-Wiedemann , Doenças dos Bovinos , Animais , Síndrome de Beckwith-Wiedemann/genética , Síndrome de Beckwith-Wiedemann/veterinária , Bovinos/genética , Doenças dos Bovinos/genética , Metilação de DNA , Epigênese Genética , Impressão Genômica , Técnicas de Transferência Nuclear/veterináriaRESUMO
In vitro fertilization and somatic cell nuclear transfer are assisted reproduction technologies commonly used in humans and cattle, respectively. Despite advances in these technologies, molecular failures can occur, increasing the chance of the onset of imprinting disorders in the offspring. Large offspring syndrome/abnormal offspring syndrome (LOS/AOS) has been described in cattle and has features such as hypergrowth, malformation of organs, and skeletal and placental defects. In humans, Beckwith-Wiedemann syndrome (BWS) has phenotypic characteristics similar to those found in LOS/AOS. In both syndromes, disruption of genomic imprinting associated with loss of parental-specific expression and parental-specific epigenetic marks is involved in the molecular etiology. Changes in the imprinting pattern of these genes lead to loss of imprinting (LOI) due to gain or loss of methylation, inducing the emergence of these syndromes. Several studies have reported locus-specific alterations in these syndromes, such as hypomethylation in imprinting control region 2 (KvDMR1) in BWS and LOS/AOS. These LOI events can occur at multiple imprinted loci in the same affected individual, which are called multi-locus methylation defect (MLMD) events. Although the bovine species has been proposed as a developmental model for human imprinting disorders, there is little information on bovine imprinted genes in the literature, even the correlation of epimutation data with clinical characteristics. In this study, we performed a systematic review of all the multi-locus LOI events described in human BWS and LOS/AOS, in order to determine in which imprinted genes the largest changes in the pattern of DNA methylation and expression occur, helping to fill gaps for a better understanding of the etiology of both syndromes.
Assuntos
Síndrome de Beckwith-Wiedemann , Doenças dos Bovinos , Animais , Síndrome de Beckwith-Wiedemann/genética , Síndrome de Beckwith-Wiedemann/veterinária , Bovinos , Doenças dos Bovinos/genética , Metilação de DNA , Feminino , Impressão Genômica , Placenta , Gravidez , Técnicas de Reprodução Assistida/veterináriaRESUMO
Insulin is present in the seminal plasma and is involved in sperm activities like motility and capacitation. However, the effects of insulin on the viability of cooled ram sperm are not fully understood. Therefore, the objective of the current study was to evaluate the effect of insulin addition on ram sperm maintained at 5ºC. Sperm samples were collected from six healthy, mature Santa Inês rams. The ejaculates were divided into two aliquots with (insulin group) or without (control group) insulin (3 IU mL-1) in the semen extender, and then cooled at 5°C for 48 hours. Subsequently, the sperm cells were evaluated for motility and kinetics using computer-assisted semen analysis. The samples were evaluated for acrosomal integrity by fluorescein using isothiocyanate combined with peanut agglutinin (FITC-PNA) and membrane functionality by the hypoosmotic swelling test. The semen analysis was performed after 24 or 48 hours of cooling. There was an increased percentage of progressive sperm motility (%), straightness (%), linearity (%) and beat caudal frequency (Hz) in the insulin group after 24 and 48 hours of cooling (p < 0.05). However, insulin did not affect total sperm motility, sperm velocities (VSL, VAP and VCL) (µm seg-1), acrosomal integrity and membrane functionality during cooling (p > 0.05). In conclusion, the addition of 3 IU mL-1 insulin to ram semen extender improved the quality of sperm motility after cooling.(AU)
A insulina está presente no plasma seminal e participa de atividades espermáticas, como a motilidade e capacitação. Entretanto, os efeitos da insulina sobre a viabilidade do espermatozoide ovino resfriado ainda não estão elucidadas. Desta forma, o objetivo do presente estudo foi avaliar os efeitos da adição de insulina sobre o espermatozoide ovino durante o tempo de armazenamento à 5º C. Amostras espermáticas de seis carneiros da raça Santa Inês foram utilizadas. Os ejaculados foram divididos em duas aliquotas, com (grupo insulina) ou sem (grupo controle) adição de insulina (3 UI mL-1) no diluidor seminal e, posteriormente, resfriados até 5oC e mantidos armazenados por 48 horas. Em seguida, os espermatozoides foram avaliados quanto a motilidade e cinética utilizando um Sistema Computadorizado de Análise de Sêmen (CASA). Adicionalmente, as amostras espermáticas foram analizadas quanto a integridade acrosomal por meio de sondas fluorescentes (FITC-PNA) e, funcionalidade de membrana pelo teste hiposmótico. As análises seminais foram realizadas após 24 ou 48 horas de resfriamento. Foram verificados aumentos de espermatozoides com motilidade progressiva (%), retilinearidade (%), linearidade (%) e frequência de batimento caudal (BCF) (Hz) no grupo insulina após 24 ou 48 horas de resfriamento (p < 0.05). Entretanto, não houve efeito da adição de insulina sobre a porcentagem de espermatozoides moveis (%) e das velocidades espermáticas (VSL, VAP e VCL) (µm seg-1), integridade acrossomal e funcionalidade de membrana durante o resfriamento (p > 0.05). Conclui-se que adição de insulina (3 UI mL-1) no diluidor seminal melhora a qualidade da motilidade espermática durante o resfriamento.(AU)
Assuntos
Animais , Sêmen , Ovinos , Criopreservação , Análise do Sêmen , Insulina , DiluiçãoRESUMO
This study aimed to evaluate the effect of regulating phosphatidylinositol 3-kinase (PI3K) activity on the kinetics of oocyte nuclear maturation and the blastocyst rate. To evaluate oocyte viability, nuclear maturation rate and in vitro embryo production, cumulus-oocyte complexes (COCs) were maintained for 0, 10 min, 6 h or 22 h in TCM 199 medium supplemented with 20 nM wortmannin, an inhibitor of PI3K. After each period, COCs were transferred to the same medium without wortmannin and kept under the same conditions until completion of 22 h of in vitro maturation (IVM). To evaluate the effect of time on progression of nuclear maturation, COCs cultivated with 20 nM wortmannin was maintained for 22, 28 or 34 h of IVM. To determine the effect of wortmannin on the activity of maturation-promoting factor (MPF), COCs were kept under IVM conditions in the presence of the inhibitor for 0, 1, 3, 6, or 8 h. Exposure of COCs to wortmannin decreased (P < 0.05) the percentage of oocytes that reached metaphase II (MII) up to 22 h, MPF activity and reduced PI3K activity by 30%. However, after 28 and 34 h, 70% of oocytes reached the MII stage in the presence of inhibitor Moreover, COCs matured in the presence of wortmannin showed an increase (P < 0.05) in the blastocyst rate. These findings suggested that the regulation of the PI3K activity during IVM of bovine COCs interfered with the meiotic progression due to control of MPF activity, positively affecting the blastocyst rate.
RESUMO
A enzima glicogênio sintase quinase-3 (GSK3) atua em várias vias de sinalização pela fosforilação e desfosforilação de proteínas, participando de várias funções celulares. Poucos estudos descrevem sua participação na maturação in vitro (MIV) de oócitos bovinos, mas sabe-se que sua inibição inespecífica tem um impacto negativo nesse processo. O objetivo deste trabalho foi avaliar o efeito de CHIR99021, inibidor específico da GSK3, em diferentes aspectos da MIV de complexos de cumulusoócito (CCOs) bovino e seu impacto na produção in vitro. Os CCOs foram aspirados de ovários de vacas abatidas e maturados em meio suplementado com 0; 1,5; 3,0 e 6,0 μM de CHIR99021. A análise estatística dos resultados por regressão linear (p≤0,01) mostrou que após 22 h de MIV, o tratamento causou redução dose-dependente no grau de expansão das células cumulus; na viabilidade de oócitos avaliada por coloração com calceína-AM e iodeto de propídio; nas taxas de maturação nuclear, e migração de grânulos corticais avaliadas por marcação com orceína acética a 2% e com lectina Lens culinaris-FITC (LCA), respectivamente, além de uma redução na produção de blastocistos. Assim, conclui-se que o CHIR99021 interfere negativamente, de forma dose-dependente na MIV de oócitos bovinos, sugerindo a importância da GSK3 na maturação nuclear e citoplasmática, com consequente impacto para a produção in vitro de embriões bovinos.(AU)
The enzyme glycogen synthase kinase-3 (GSK3) acts in several signaling pathway through phosphorylation and protein dephosphorylation, participating in various cellular functions. Few studies describe its participation in the in vitro maturation (IVM) of bovine oocytes, but its nonspecific inhibition is known to have a negative impact on this process. The objective of this work was to evaluate the effect of CHIR99021, specific inhibition of GSK3, on different aspects of the in vitro maturation of bovine cumulus-oocyte (COCs) complexes. COCs were aspirated from ovaries of slaughtered cows and matured in medium supplemented with0; 1.5; 3.0 and 6.0 μM CHIR99021. Statistical analysis of the results by linear regression (p≤0.01) showed that after 22 hours of IVM the treatment caused dose-dependent reduction in the degree of cumulus cell expansion; oocyte viability evaluated by staining with calcein-AM and propidium iodide; rates of nuclear maturation and migration of cortical granules evaluated by labeling with 2% acetic orcein and Lens culinaris-FITC (LCA), respectively; and a reduction in the of blastocyst rate. It is concluded that CHIR99021 interferes negatively, in a dose-dependent manner in the IVM of bovine oocytes, suggesting the importance of GSK3 in nuclear and cytoplasmic maturation, with a consequent impact on the in vitro bovine embryos production.(AU)
Assuntos
Animais , Feminino , Bovinos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Bovinos/embriologia , Glicogênio Sintase Quinase 3 beta/análise , Glicogênio Sintase Quinase 3 beta/química , Quinase 3 da Glicogênio Sintase , BlastocistoRESUMO
A enzima glicogênio sintase quinase-3 (GSK3) atua em várias vias de sinalização pela fosforilação e desfosforilação de proteínas, participando de várias funções celulares. Poucos estudos descrevem sua participação na maturação in vitro (MIV) de oócitos bovinos, mas sabe-se que sua inibição inespecífica tem um impacto negativo nesse processo. O objetivo deste trabalho foi avaliar o efeito de CHIR99021, inibidor específico da GSK3, em diferentes aspectos da MIV de complexos de cumulusoócito (CCOs) bovino e seu impacto na produção in vitro. Os CCOs foram aspirados de ovários de vacas abatidas e maturados em meio suplementado com 0; 1,5; 3,0 e 6,0 μM de CHIR99021. A análise estatística dos resultados por regressão linear (p≤0,01) mostrou que após 22 h de MIV, o tratamento causou redução dose-dependente no grau de expansão das células cumulus; na viabilidade de oócitos avaliada por coloração com calceína-AM e iodeto de propídio; nas taxas de maturação nuclear, e migração de grânulos corticais avaliadas por marcação com orceína acética a 2% e com lectina Lens culinaris-FITC (LCA), respectivamente, além de uma redução na produção de blastocistos. Assim, conclui-se que o CHIR99021 interfere negativamente, de forma dose-dependente na MIV de oócitos bovinos, sugerindo a importância da GSK3 na maturação nuclear e citoplasmática, com consequente impacto para a produção in vitro de embriões bovinos.
The enzyme glycogen synthase kinase-3 (GSK3) acts in several signaling pathway through phosphorylation and protein dephosphorylation, participating in various cellular functions. Few studies describe its participation in the in vitro maturation (IVM) of bovine oocytes, but its nonspecific inhibition is known to have a negative impact on this process. The objective of this work was to evaluate the effect of CHIR99021, specific inhibition of GSK3, on different aspects of the in vitro maturation of bovine cumulus-oocyte (COCs) complexes. COCs were aspirated from ovaries of slaughtered cows and matured in medium supplemented with0; 1.5; 3.0 and 6.0 μM CHIR99021. Statistical analysis of the results by linear regression (p≤0.01) showed that after 22 hours of IVM the treatment caused dose-dependent reduction in the degree of cumulus cell expansion; oocyte viability evaluated by staining with calcein-AM and propidium iodide; rates of nuclear maturation and migration of cortical granules evaluated by labeling with 2% acetic orcein and Lens culinaris-FITC (LCA), respectively; and a reduction in the of blastocyst rate. It is concluded that CHIR99021 interferes negatively, in a dose-dependent manner in the IVM of bovine oocytes, suggesting the importance of GSK3 in nuclear and cytoplasmic maturation, with a consequent impact on the in vitro bovine embryos production.
Assuntos
Feminino , Animais , Bovinos , Blastocisto , Bovinos/embriologia , /análise , /química , Técnicas de Maturação in Vitro de Oócitos/veterináriaRESUMO
This study evaluated the effect of the addition of conjugated linoleic acid (CLA) to in vitro culture on viability, lipid content, and cryoresistance of bovine embryos at different in vitro culture times. Cumulus oocyte complexes (N = 974) were maturated in vitro for 22 h. In vitro fecundation ensued for 18 h. Viable zygotes were cultivated in vitro in medium supplemented with CLA (100 mM) in the first 72 h (CLA-F), last 72 h (CLA-L), or throughout the culture period (CLA-T). Control embryos (control) were cultivated with no CLA. Embryos were cryopreserved by vitrification for subsequent analysis after devitrification. Effect of CLA on cryoresistance was assessed by cultivating embryos in synthetic oviductal fluid containing 5% fetal bovine serum. Lipid content was quantified using Nile Red staining. No significant difference was observed in cleavage rate, blastocyst:total oocyte ratio, and blastocyst:cleaved oocyte ratio. Culture in CLA-L reduced survival rate 24 h after devitrification compared with CLA-F and CLA-T, although with no statistically significant difference compared with control group. However, CLA-T improved embryo hatching rate and affected lipid content of embryos. Cultures CLA-F and CLA-L increased lipid content compared with control. Yet, lipid content values decreased in embryos treated with CLA-T, but they did not differ significantly from the values observed for oocytes at the germinal vesicle stage. Treatment of bovine embryos with CLA during in vitro cultivation did not affect the production of blastocysts, reducing lipid content and improving cryoresistance. However, the effects of CLA on cryoresistance and lipid content is significant only when embryos are exposed to the compound throughout the cultivation period.(AU)
Assuntos
Animais , Bovinos/embriologia , Criopreservação/veterinária , Ácidos Linoleicos Conjugados/análise , Técnicas de Cultura Embrionária/métodosRESUMO
Genetically modified cattle production is motivated by many factors, including recombinant protein production for therapeutic purposes, disease models and animals presenting improved production traits. Nuclear transfer (NT), combined with efficient cultivation methods, genetic modification and donor cell selection is important for transgenic cattle production. Studies have found that adult cells (such as fibroblasts and cumulus cells, among others) used as nuclear donors achieved results similar to those of fetal cells, with the advantages of easier collection and a known genotype/phenotype. However, no consensus has been reached on the influence of cell type on transgene expression levels and post-reprogramming capacity after nuclear transfer, and these factors appear to be related to epigenetic factors. The development of new strategies, such as chromatin-modifying agents (CMAs), in vitro generation of induced pluripotent cells (iPS cells) and precise genome editing techniques are being explored and may influence nuclear reprogramming success for efficiently producing genetically modified bovine clones.
Assuntos
Animais , Bovinos , Bovinos/crescimento & desenvolvimento , Bovinos/genética , Clonagem de Organismos/tendências , Clonagem de Organismos/veterinária , Epigênese Genética/genética , Melhoramento Genético , TecnologiaRESUMO
Genetically modified cattle production is motivated by many factors, including recombinant protein production for therapeutic purposes, disease models and animals presenting improved production traits. Nuclear transfer (NT), combined with efficient cultivation methods, genetic modification and donor cell selection is important for transgenic cattle production. Studies have found that adult cells (such as fibroblasts and cumulus cells, among others) used as nuclear donors achieved results similar to those of fetal cells, with the advantages of easier collection and a known genotype/phenotype. However, no consensus has been reached on the influence of cell type on transgene expression levels and post-reprogramming capacity after nuclear transfer, and these factors appear to be related to epigenetic factors. The development of new strategies, such as chromatin-modifying agents (CMAs), in vitro generation of induced pluripotent cells (iPS cells) and precise genome editing techniques are being explored and may influence nuclear reprogramming success for efficiently producing genetically modified bovine clones.(AU)
Assuntos
Animais , Bovinos , Bovinos/crescimento & desenvolvimento , Bovinos/genética , Clonagem de Organismos/tendências , Clonagem de Organismos/veterinária , Epigênese Genética/genética , Tecnologia , Melhoramento GenéticoRESUMO
The oviduct is a dynamic organ which facilitates gamete function, fertilization and embryo development. This organ is covered by an epithelium containing ciliated and non-ciliated cells. Secretions of non-ciliated cells compose the oviduct fluid, which will nourish the early embryo. During the period of ovulation, the oviduct exhibits an active role, where the lumen provides an environment suitable for fertilization and the muscle layer contracts rhythmically to move the egg toward the uterus. In this study we aimed to investigate the content of fuel metabolites and enzyme activity assays related to the glycolytic metabolism in bovine oviduct cells such as Glucose-6-phosphate, Glycogen, Pyruvate, Hexokinase, Pyruvate, kinase, Phosphoenolpyruvate carboxykinase and Glucose-6-phosphate dehydrogenase.(AU)