RESUMO
The cryosurvival of embryos is a complex process involving dynamic and integrated morphological, functional, and molecular changes. Here, we evaluated the transcriptional profiling of bovine embryos possessing high and low cryotolerance (HC and LC, respectively) by assessing the resumption of development. Embryos were produced in vitro (N = 1137) and cryopreserved (N = 894). Blastocysts samples possessed pronounced group individualization at RNA sequencing. A total of 114 genes were differentially expressed, and 27 and 84 genes were upregulated in HC and LC, respectively. Among the over-represented biological functions, cellular growth and proliferation, cell death and survival, and organismal survival were predicted to be activated, while cellular movement and cell-to-cell signaling were predicted to be inhibited in HC embryos. Enriched canonical pathways and upstream regulators related to cellular proliferation and survival (HC), inflammatory processes, and cell death (LC) were predicted to represent two embryonic molecular profiles present during the resumption of development after cryopreservation. The marked contrast in transcriptional profiles between HC and LC strongly suggests the influence of embryonic competence after cryopreservation on its respective transcriptome and indicated that HC and LC presented two different molecular strategies to overcome cryopreservation-related stress and resume postcryopreservation development.
Assuntos
Criopreservação/métodos , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Fertilização in vitro/métodos , Transcriptoma , Regulação para Cima/genética , Animais , Apoptose/genética , Blastocisto/metabolismo , Bovinos , Proliferação de Células/genética , Sobrevivência Celular/genética , Técnicas de Cultura Embrionária/métodos , Feminino , RNA-Seq/métodos , Transdução de Sinais/genéticaRESUMO
Global cattle genetic market is experiencing a change of strategy, large genetic companies, traditionally recognized in the artificial insemination field, have also begun to operate in the embryo market. Consequently, the demand for in vitro produced (IVP) embryos has grown. However, the overall efficiency of the biotechnology process remains low. Additionally, the lack of homogeneity of post-cryopreservation survival results of IVP embryos still impairing a massive dissemination of this biotechnology in the field. A great challenge for in vitro production labs is to increase the amount of embryos produced with exceptional quality after each round of in vitro fertilization. Herein, we discuss the molecular and cellular features associated with the competence and cryosurvival of IVP embryos. First, morphofunctional, cellular and molecular competence of the embryos were addressed and a relationship between embryo developmental ability and quality were established with cryosurvival and pregnancy success. Additionally, determinant factors of embryo competence and cryosurvival were discussed including the following effects: genotype, oocyte quality and follicular microenvironment, in vitro production conditions, and lipids and other determining molecules. Finally, embryo cryopreservation aspects were addressed and an embryo-focused approach to improve cryosurvival was presented.
RESUMO
This study assessed the lipid composition of oocytes from different follicle sizes and compared the expression of lipid-related genes and follicular fluid (FF) molecules between groups. We also investigated the functional consequences of differences on embryo development and blastocyst lipid deposits. Oocytes and FF were recovered from different follicle sizes. Oocytes from small (≤5mm) and large (≥6mm) bovine follicles were used to produce Day 7 expanded blastocysts (Day7Ex) and blastocysts that only became expanded at Day 8 (Day8Ex) after insemination. Oocytes from >8mm follicles had the highest lipid content. Few oocyte phospholipid variations were identified between groups. Very long chain fatty acid elongase 6 (ELOVL6) mRNA abundance was reduced in larger follicle-derived oocytes compared with the ≤2mm group. Increased levels of glucose, reactive oxygen species, glutathione and superoxide dismutase activity were also identified in FF from larger follicles. Large follicle-derived embryo development and lipid content of Day7Ex were greater than those derived from small follicles. Day8Ex had greater lipid deposition than Day7Ex. Oocytes and blastocysts exhibited follicle size-specific lipids. Large-follicle oocytes had increased lipid content and became Day7Ex with greater lipid deposition whereas delayed blastocoel expansion associated with a prolonged period of culture determined the lipid accumulation of Day8Ex. The FF microenvironment of large follicles seems to favour embryo development.
Assuntos
Blastocisto/química , Desenvolvimento Embrionário/fisiologia , Lipídeos/análise , Oócitos/química , Folículo Ovariano/metabolismo , Animais , Bovinos , Feminino , Líquido Folicular/metabolismo , Oócitos/crescimento & desenvolvimentoRESUMO
Global cattle genetic market is experiencing a change of strategy, large genetic companies, traditionally recognized in the artificial insemination field, have also begun to operate in the embryo market. Consequently, the demand for in vitro produced (IVP) embryos has grown. However, the overall efficiency of the biotechnology process remains low. Additionally, the lack of homogeneity of post-cryopreservation survival results of IVP embryos still impairing a massive dissemination of this biotechnology in the field. A great challenge for in vitro production labs is to increase the amount of embryos produced with exceptional quality after each round of in vitro fertilization. Herein, we discuss the molecular and cellular features associated with the competence and cryosurvival of IVP embryos. First, morphofunctional, cellular and molecular competence of the embryos were addressed and a relationship between embryo developmental ability and quality were established with cryosurvival and pregnancy success. Additionally, determinant factors of embryo competence and cryosurvival were discussed including the following effects: genotype, oocyte quality and follicular microenvironment, in vitro production conditions, and lipids and other determining molecules. Finally, embryo cryopreservation aspects were addressed and an embryofocused approach to improve cryosurvival was presented.(AU)
Assuntos
Animais , Feminino , Bovinos , Criopreservação/métodos , Criopreservação/veterinária , Transferência Embrionária/classificação , Transferência Embrionária/tendências , Transferência Embrionária/veterináriaRESUMO
Global cattle genetic market is experiencing a change of strategy, large genetic companies, traditionally recognized in the artificial insemination field, have also begun to operate in the embryo market. Consequently, the demand for in vitro produced (IVP) embryos has grown. However, the overall efficiency of the biotechnology process remains low. Additionally, the lack of homogeneity of post-cryopreservation survival results of IVP embryos still impairing a massive dissemination of this biotechnology in the field. A great challenge for in vitro production labs is to increase the amount of embryos produced with exceptional quality after each round of in vitro fertilization. Herein, we discuss the molecular and cellular features associated with the competence and cryosurvival of IVP embryos. First, morphofunctional, cellular and molecular competence of the embryos were addressed and a relationship between embryo developmental ability and quality were established with cryosurvival and pregnancy success. Additionally, determinant factors of embryo competence and cryosurvival were discussed including the following effects: genotype, oocyte quality and follicular microenvironment, in vitro production conditions, and lipids and other determining molecules. Finally, embryo cryopreservation aspects were addressed and an embryofocused approach to improve cryosurvival was presented.
Assuntos
Feminino , Animais , Bovinos , Criopreservação/métodos , Criopreservação/veterinária , Transferência Embrionária/classificação , Transferência Embrionária/tendências , Transferência Embrionária/veterináriaRESUMO
Mammalian oocytes resume meiosis spontaneously after removal from the ovarian follicle. We tested the effects of a 2-h prematuration treatment (Pre-IVM) with forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX) in bovine cumulus-oocyte complexes (COCs) on the lipid content of oocytes and blastocysts, on the membrane lipid composition of blastocysts and on the transcriptional profiling of cumulus cells and blastocysts in a high-throughput platform. Embryonic development rates to the morula (mean 56.1%) or blastocyst (mean 26.3%) stages were unaffected by treatment. Lipid content was not affected after Pre-IVM, but was increased after IVM in treated oocytes. Conversely, the lipid content was reduced in Pre-IVM blastocysts. Pre-IVM COCs generated blastocysts containing blastomeres with more unsaturated lipids in their membranes. Pre-IVM also altered the relative abundance of 31 gene transcripts after 2h and 16 transcripts after 24h in cumulus cells, while seven transcripts were altered in blastocysts. Our results suggest that the Pre-IVM treatment affected the lipid composition and transcriptional profiles of COCs and blastocysts. Therefore, Pre-IVM with FSK and IBMX could be used either to prevent spontaneous meiotic resumption during IVM or to modulate lipid composition in the membrane and cytoplasm of blastocysts, potentially improving bovine embryos.
Assuntos
Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/metabolismo , AMP Cíclico/agonistas , AMP Cíclico/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Blastocisto/citologia , Bovinos , Colforsina/farmacologia , Células do Cúmulo/citologia , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Feminino , Técnicas de Maturação in Vitro de Oócitos , Oócitos/crescimento & desenvolvimento , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma/efeitos dos fármacosRESUMO
O Herpesvirus bovino tipo 5 (BoHV-5) tem sido recentemente descrito no sêmen contaminado. Apesar de ter sido identificado no sêmen bovino, sua influência na produção de embriões in vitro não foi ainda relatada. O presente estudo teve como objetivo verificar a susceptibilidade de oócitos bovinos maturados e fertilizados invitro, com sêmen congelado, infectado experimentalmente com BoHV-5, antes da congelação, avaliando-se o desenvolvimento embrionário subsequente e o possível carreamento do vírus para o embrião via espermatozoide. Ejaculados de touro foram misturados e divididos em dois grupos: Grupo I, sêmen não exposto ao vírus e, Grupo II, sêmen exposto ao vírus. A presença do BoHV-5 foi investigada através de PCR e teste de hibridização in situ (ISH). Os dados foram analisados através de Teste-t de Student com P< 0,05 considerado como significativo. O vírus foi detectado em embriões avaliados com 168 h pós-inseminação. Não houve diferença significativa entre os diferentes grupos de tratamento para as taxas de produção in vitro de embriões para os respectivos estágios de desenvolvimento. Assim, conclui-se que, o sêmen contaminado com o BoHV-5 pode infectar os oócitos durante a fertilização in vitro, com carreamento do vírus para os embriões, não afetando, porém, o seu desenvolvimento in vitro.
Bovine herpesvirus type 5 (BoHV-5) has been recently described from contaminated semen. Despite having been identified in bovine semen, its transmission through use of infected fresh semen, just before freezing, on the in vitro embryo production has not yet been reported so far. Thus, this study was carried out to investigate the susceptibility of matured bovine oocytes to BoHV-5 infection after using frozen semen previously exposed to the virus, and their ability to develop for advanced blastocyst stages following in vitro fertilization (IVF). Ejaculates from bulls were mixed and divided into two groups, as follows: Group 1, semen not exposed to the virus, and Group 2, infected sperm. The presence of BoHV-5 was investigated by PCR and in situ hybridization assays. Results were analyzed through use of Student t - test with P < 0.05 considered as significant. The virus was detected in blastocysts evaluated at 168 h post-insemination. There was no significant difference between groups for the in vitro embryo production at any developmental stage. Thus, it is concluded that semen infected with BoHV-5 can infect oocytes during IVF, with the virus transmission to embryos via sperm; however, embryonic development was not impaired, as evidenced by the subsequent blastocyst formation.
Assuntos
Animais , Bovinos , Desenvolvimento Embrionário , Embrião de Mamíferos , Preservação do Sêmen , Preservação do Sêmen/veterináriaRESUMO
O Herpesvirus bovino tipo 5 (BoHV-5) tem sido recentemente descrito no sêmen contaminado. Apesar de ter sido identificado no sêmen bovino, sua influência na produção de embriões in vitro não foi ainda relatada. O presente estudo teve como objetivo verificar a susceptibilidade de oócitos bovinos maturados e fertilizados invitro, com sêmen congelado, infectado experimentalmente com BoHV-5, antes da congelação, avaliando-se o desenvolvimento embrionário subsequente e o possível carreamento do vírus para o embrião via espermatozoide. Ejaculados de touro foram misturados e divididos em dois grupos: Grupo I, sêmen não exposto ao vírus e, Grupo II, sêmen exposto ao vírus. A presença do BoHV-5 foi investigada através de PCR e teste de hibridização in situ (ISH). Os dados foram analisados através de Teste-t de Student com P< 0,05 considerado como significativo. O vírus foi detectado em embriões avaliados com 168 h pós-inseminação. Não houve diferença significativa entre os diferentes grupos de tratamento para as taxas de produção in vitro de embriões para os respectivos estágios de desenvolvimento. Assim, conclui-se que, o sêmen contaminado com o BoHV-5 pode infectar os oócitos durante a fertilização in vitro, com carreamento do vírus para os embriões, não afetando, porém, o seu desenvolvimento in vitro.(AU)
Bovine herpesvirus type 5 (BoHV-5) has been recently described from contaminated semen. Despite having been identified in bovine semen, its transmission through use of infected fresh semen, just before freezing, on the in vitro embryo production has not yet been reported so far. Thus, this study was carried out to investigate the susceptibility of matured bovine oocytes to BoHV-5 infection after using frozen semen previously exposed to the virus, and their ability to develop for advanced blastocyst stages following in vitro fertilization (IVF). Ejaculates from bulls were mixed and divided into two groups, as follows: Group 1, semen not exposed to the virus, and Group 2, infected sperm. The presence of BoHV-5 was investigated by PCR and in situ hybridization assays. Results were analyzed through use of Student t - test with P < 0.05 considered as significant. The virus was detected in blastocysts evaluated at 168 h post-insemination. There was no significant difference between groups for the in vitro embryo production at any developmental stage. Thus, it is concluded that semen infected with BoHV-5 can infect oocytes during IVF, with the virus transmission to embryos via sperm; however, embryonic development was not impaired, as evidenced by the subsequent blastocyst formation.(AU)
Assuntos
Animais , Bovinos , Embrião de Mamíferos , Preservação do Sêmen , Preservação do Sêmen/veterinária , Desenvolvimento Embrionário , Herpesvirus Bovino 5RESUMO
The veterinary cardiology has growing importance in equine medicine. There are studies of standardization of electrocardiographic parameters of many races, according to their stature and ability. However, no studies are in the literature with the American Miniature Horse. To evaluate the electrocardiogram (ECG) tracing configuration of this breed at rest and to verify the influence of age and sex on ECG parameters, 203 horses including 143 females and 60 males were divided into four age groups (foals, yearlings, adults and elderly). Electrocardiographic parameters were performed by computerized electrocardiogram (TEB), and the parameters were evaluated in six leads of frontal plane (Lead I, II, III, aVR, aVL and aVF) and base-apex (BA). Heart rates (HR) decreased with increasing age were higher in males than in females. Sinus tachycardia followed by sinus arrhythmia was dominant in both sexes. The cardiac axis was higher in males and ranged between 120° and 150° for foals, 30° and 60° for yearlings and adults, and 60° and 90° for the elderly. The P wave was bifid in several animals. The P-wave amplitude and T-wave duration from lead II and BA were larger in males than in females. The majority of the animals exhibited ST segment depression and a negative T-wave. The most common QRS complex morphology was Qr. Differences were observed between the electrocardiographic tracings of males and females, and age influenced the ECG parameters. Therefore, this study established the ECG patterns for the American Miniature Horse breed and could be used to determine the influence of age and sex on several of the studied variables.(AU)
A cardiologia veterinária possui crescente importância na medicina equina. Existem estudos de padronização dos parâmetros eletrocardiográficos de muitas raças, de acordo com sua estatura e aptidão. No entanto, não há na literatura trabalhos com os equinos da raça Miniature Horse. Os objetivos deste trabalho foram avaliar a configuração do traçado do eletrocardiograma (ECG) em repouso, de equinos desta raça, além de verificar a influência do sexo e da idade sobre os parâmetros eletrocardiográficos desses animais. Foram utilizados 203 equinos desta raça, hígidos, sendo 143 fêmeas e 60 machos, classificados em quatro faixas etárias (potros, sobreanos, adultos e idosos). Os exames eletrocardiográficos foram realizados por eletrocardiograma computadorizado (TEB), e os parâmetros foram avaliados em seis derivações do plano frontal (DI, DII, DIII, aVR, aVL e aVF) e base-ápice (BA). A frequência cardíaca (FC) diminuiu com a progressão da idade, e foi maior nos machos do que nas fêmeas. O ritmo mais comum em ambos os sexos foi taquicardia sinusal, seguido de arritmia sinusal. O eixo cardíaco foi maior nos machos do que nas fêmeas, e nos potros ficou entre 120 e 150o, nos sobreano e adultos permaneceu entre 30 e 60o, e nos idosos entre 60 e 90o. A amplitude da onda P e a duração da onda T foram maiores nos machos do que nas fêmeas na DII e BA. A maioria dos animais apresentou segmento ST infradesnivelado e onda T negativa. A morfologia do complexo QRS mais encontrada em todos os casos foi Qr. Este estudo permitiu estabelecer os padrões eletrocardiográficos para a raça Mini Horse e conseguiu verificar a influência da progressão da idade nas diversas variáveis estudadas, bem como a diferença entre os sexos.(AU)
Assuntos
Animais , Arritmia Sinusal/veterinária , Eletrocardiografia/veterinária , Cavalos , Taquicardia Sinusal/veterinária , Técnicas de Diagnóstico Cardiovascular/veterinária , Frequência Cardíaca , Padrões de ReferênciaRESUMO
The veterinary cardiology has growing importance in equine medicine. There are studies of standardization of electrocardiographic parameters of many races, according to their stature and ability. However, no studies are in the literature with the American Miniature Horse. To evaluate the electrocardiogram (ECG) tracing configuration of this breed at rest and to verify the influence of age and sex on ECG parameters, 203 horses including 143 females and 60 males were divided into four age groups (foals, yearlings, adults and elderly). Electrocardiographic parameters were performed by computerized electrocardiogram (TEB), and the parameters were evaluated in six leads of frontal plane (Lead I, II, III, aVR, aVL and aVF) and base-apex (BA). Heart rates (HR) decreased with increasing age were higher in males than in females. Sinus tachycardia followed by sinus arrhythmia was dominant in both sexes. The cardiac axis was higher in males and ranged between 120° and 150° for foals, 30° and 60° for yearlings and adults, and 60° and 90° for the elderly. The P wave was bifid in several animals. The P-wave amplitude and T-wave duration from lead II and BA were larger in males than in females. The majority of the animals exhibited ST segment depression and a negative T-wave. The most common QRS complex morphology was Qr. Differences were observed between the electrocardiographic tracings of males and females, and age influenced the ECG parameters. Therefore, this study established the ECG patterns for the American Miniature Horse breed and could be used to determine the influence of age and sex on several of the studied variables.(AU)
A cardiologia veterinária possui crescente importância na medicina equina. Existem estudos de padronização dos parâmetros eletrocardiográficos de muitas raças, de acordo com sua estatura e aptidão. No entanto, não há na literatura trabalhos com os equinos da raça Miniature Horse. Os objetivos deste trabalho foram avaliar a configuração do traçado do eletrocardiograma (ECG) em repouso, de equinos desta raça, além de verificar a influência do sexo e da idade sobre os parâmetros eletrocardiográficos desses animais. Foram utilizados 203 equinos desta raça, hígidos, sendo 143 fêmeas e 60 machos, classificados em quatro faixas etárias (potros, sobreanos, adultos e idosos). Os exames eletrocardiográficos foram realizados por eletrocardiograma computadorizado (TEB), e os parâmetros foram avaliados em seis derivações do plano frontal (DI, DII, DIII, aVR, aVL e aVF) e base-ápice (BA). A frequência cardíaca (FC) diminuiu com a progressão da idade, e foi maior nos machos do que nas fêmeas. O ritmo mais comum em ambos os sexos foi taquicardia sinusal, seguido de arritmia sinusal. O eixo cardíaco foi maior nos machos do que nas fêmeas, e nos potros ficou entre 120 e 150o, nos sobreano e adultos permaneceu entre 30 e 60o, e nos idosos entre 60 e 90o. A amplitude da onda P e a duração da onda T foram maiores nos machos do que nas fêmeas na DII e BA. A maioria dos animais apresentou segmento ST infradesnivelado e onda T negativa. A morfologia do complexo QRS mais encontrada em todos os casos foi Qr. Este estudo permitiu estabelecer os padrões eletrocardiográficos para a raça Mini Horse e conseguiu verificar a influência da progressão da idade nas diversas variáveis estudadas, bem como a diferença entre os sexos.(AU)
Assuntos
Animais , Cavalos , Eletrocardiografia/veterinária , Taquicardia Sinusal/veterinária , Arritmia Sinusal/veterinária , Padrões de Referência , Técnicas de Diagnóstico Cardiovascular/veterinária , Frequência CardíacaRESUMO
In a 2×2 factorial experimental design, embryo development, cryotolerance and global gene expression of Nellore (Bos taurus indicus) and Simmental (Bos taurus taurus) blastocysts produced in vitro (IVP) and in vivo (multiple ovulation derived embryo, MODE) were assessed. Blastocyst production was higher in Nellore than in Simmental (47.7±2.0% vs 27.0±2.0%) cows. The total numbers of ova or embryos recovered (5.5±0.9 vs 3.7±0.8) and transferable embryos (3.8±1.0 vs 2.3±0.8) per cow were not different between breeds. Simmental and MODE (34.6% and 38.5%, n=75 and 70) blastocysts had higher survival rates after cryopreservation compared with Nellore and IVP (20.2% and 18.1%, n=89 and 94) embryos, respectively. Differences between transcriptomes were addressed by principal-component analysis, which indicated that gene expression was affected by subspecies (158 genes), origin (532 genes) and interaction between both subspecies and origin (53 genes). Several functional processes and pathways relevant to lipid metabolism and embryo viability involving differentially expressed genes were identified. The lipid metabolism-related genes were upregulated in Simmental (AUH and ELOVL6) and IVP (ACSL3 and ACSL6) blastocysts. The expression profiles of genes related to mitochondrial metabolism (ATP5B), oxidative stress (GPX4), apoptosis (DAD1, DAP, PRDX2), heat shock (HSPA5), pregnancy (IFNT2, PAG2) and cell differentiation (KRT18) varied between experimental groups.
Assuntos
Blastocisto/fisiologia , Criopreservação/veterinária , Fertilização in vitro/veterinária , Fertilização , Perfilação da Expressão Gênica/veterinária , Regulação da Expressão Gênica no Desenvolvimento , Inseminação Artificial/veterinária , Animais , Blastocisto/metabolismo , Bovinos , Sobrevivência Celular , Técnicas de Cultura Embrionária/veterinária , Transferência Embrionária/veterinária , Feminino , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Técnicas de Maturação in Vitro de Oócitos/veterinária , Masculino , Análise de Sequência com Séries de Oligonucleotídeos/veterinária , Gravidez , Análise de Componente Principal , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Especificidade da EspécieRESUMO
In pre-implantation embryos, lipids play key roles in determining viability, cryopreservation and implantation properties, but often their analysis is analytically challenging because of the few picograms of analytes present in each of them. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) allows obtaining individual phospholipid profiles of these microscopic organisms. This technique is sensitive enough to enable analysis of individual intact embryos and monitoring the changes in membrane lipid composition in the early stages of development serving as screening method for studies of biology and biotechnologies of reproduction. This article introduces an improved, more comprehensive MALDI-MS lipid fingerprinting approach that considerably increases the lipid information obtained from a single embryo. Using bovine embryos as a biological model, we have also tested optimal sample storage and handling conditions before the MALDI-MS analysis. Improved information at the molecular level is provided by the use of a binary matrix that enables phosphatidylcholines, sphingomyelins, phosphatidylserines, phosphatidylinositols and phosphoethanolamines to be detected via MALDI(±)-MS in both the positive and negative ion modes. An optimal MALDI-MS protocol for lipidomic monitoring of a single intact embryo is therefore reported with potential applications in human and animal reproduction, cell development and stem cell research.
Assuntos
Blastocisto/química , Lipídeos de Membrana/análise , Fosfolipídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Bovinos , Feminino , Masculino , Lipídeos de Membrana/química , Fosfolipídeos/química , Fosfolipídeos/classificaçãoRESUMO
O interesse nas pesquisas com células-tronco derivadas de anexos fetais de diversas espécies cresceu exponencialmente nas últimas décadas em virtude de serem fontes de células-tronco adultas com potencial de diferenciação em diversas linhagens celulares que apresentam pouca ou nenhuma imunogenicidade, apresentando-se assim como alternativa de grande importância para a formação de bancos celulares. Apesar do crescente interesse, os estudos para espécie equina ainda são escassos. O objetivo deste trabalho foi isolar, caracterizar e diferenciar células-tronco mesenquimais (CTMs) derivadas do líquido amniótico equino obtidas do terço inicial, médio e final da gestação (LA-CTMs), comparando suas características. Foram colhidas 23 amostras de líquido amniótico as quais foram submetidas às análises morfológica, imunocitoquímica, imunofenotípica por citometria de fluxo e às diferenciações osteogênica, adipogênica e condrogênica in vitro. Todas as amostras demonstraram adesão ao plástico e morfologia fibroblastóide. No ensaio imunocitoquímico as células de todos os grupos foram imunomarcadas para CD44, PCNA e vimentina com ausência de marcação para citoqueratina e Oct-4. Na citometria de fluxo observou-se a expressão de CD44 e CD90 e ausência de expressão de CD34, sendo que os marcadores CD44 e CD90 mostraram padrão de expressão decrescente em relação ao desenvolvimento gestacional. As amostras obtidas de todas as fases da gestação foram capazes de diferenciação nas linhagens osteogênica, condrogênica e adipogênica. Portanto, as células obtidas do líquido amniótico apresentaram características morfológicas, imunofenotípicas e potencial de diferenciação típicos das CTMs, demonstrando que a colheita pode ser realizada em qualquer fase gestacional. No entanto, mais pesquisas devem ser realizadas principalmente quanto à expressão de marcadores de pluripotencialidade (como o Oct-4) e ao seu potencial de diferenciação em linhagens extra mesodermais já relatados na literatura.
The interest in stem cells derived from fetal annexes of many species has exponentially increased during the last decades, because they are adult stem cell sources with potential of differentiation in several cell lineages; which present little or no immunogenicity and are an alternative with great importance for storage cell banks. Despite the rising interest, studies for the equine species are still rare. The aim of this study was to isolate, characterize and differentiate mesenchymal stem cells derived from equine amniotic fluid obtained from initial, middle and late third of gestation (AF-MSCs), and compare their results. Twenty three samples from equine amniotic fluid were evaluated by morphological, immunocytochemical and immunophenotypical (Flow cytometer) assays and osteogenic, adipogenic and chondrogenic in vitro differentiation. All samples demonstrated plastic adhesion and fibroblastoid morphology. The immunocytochemical assay demonstrated cells from all the studied groups were positive for CD44, PCNA and vimentin and negative for cytokeratin and Oct-4. Flow cytometry demonstrated expression of CD44 and CD90 and no expression of CD34, where CD44 and CD90 markers presented decreasing pattern of expression in relation to the gestational development. All samples collected from all gestational phases were capable to differentiate in osteogenic, chondrogenic and adipogenic lineages. Thus, cells obtained from equine amniotic fluid presented morphological and immunophenotypical characteristics and potential of differentiation typical of MSCs showing that the collection can be performed at any stage of pregnancy. However, more studies should be performed about the expression of pluripotent markers as Oct-4 and the differentiation potential for extra mesodermal lineages prior demonstrated in the literature.
Assuntos
Animais , Feminino , Cavalos/fisiologia , Células-Tronco/fisiologia , Fibroblastos/fisiologia , Imuno-Histoquímica , Líquido Amniótico/química , Separação Celular/veterinária , Transplante de Células-Tronco Mesenquimais/veterináriaRESUMO
O interesse nas pesquisas com células-tronco derivadas de anexos fetais de diversas espécies cresceu exponencialmente nas últimas décadas em virtude de serem fontes de células-tronco adultas com potencial de diferenciação em diversas linhagens celulares que apresentam pouca ou nenhuma imunogenicidade, apresentando-se assim como alternativa de grande importância para a formação de bancos celulares. Apesar do crescente interesse, os estudos para espécie equina ainda são escassos. O objetivo deste trabalho foi isolar, caracterizar e diferenciar células-tronco mesenquimais (CTMs) derivadas do líquido amniótico equino obtidas do terço inicial, médio e final da gestação (LA-CTMs), comparando suas características. Foram colhidas 23 amostras de líquido amniótico as quais foram submetidas às análises morfológica, imunocitoquímica, imunofenotípica por citometria de fluxo e às diferenciações osteogênica, adipogênica e condrogênica in vitro. Todas as amostras demonstraram adesão ao plástico e morfologia fibroblastóide. No ensaio imunocitoquímico as células de todos os grupos foram imunomarcadas para CD44, PCNA e vimentina com ausência de marcação para citoqueratina e Oct-4. Na citometria de fluxo observou-se a expressão de CD44 e CD90 e ausência de expressão de CD34, sendo que os marcadores CD44 e CD90 mostraram padrão de expressão decrescente em relação ao desenvolvimento gestacional. As amostras obtidas de todas as fases da gestação foram capazes de diferenciação nas linhagens osteogênica, condrogênica e adipogênica. Portanto, as células obtidas do líquido amniótico apresentaram características morfológicas, imunofenotípicas e potencial de diferenciação típicos das CTMs, demonstrando que a colheita pode ser realizada em qualquer fase gestacional. No entanto, mais pesquisas devem ser realizadas principalmente quanto à expressão de marcadores de pluripotencialidade (como o Oct-4) e ao seu potencial de diferenciação em linhagens extra mesodermais já relatados na literatura.(AU)
The interest in stem cells derived from fetal annexes of many species has exponentially increased during the last decades, because they are adult stem cell sources with potential of differentiation in several cell lineages; which present little or no immunogenicity and are an alternative with great importance for storage cell banks. Despite the rising interest, studies for the equine species are still rare. The aim of this study was to isolate, characterize and differentiate mesenchymal stem cells derived from equine amniotic fluid obtained from initial, middle and late third of gestation (AF-MSCs), and compare their results. Twenty three samples from equine amniotic fluid were evaluated by morphological, immunocytochemical and immunophenotypical (Flow cytometer) assays and osteogenic, adipogenic and chondrogenic in vitro differentiation. All samples demonstrated plastic adhesion and fibroblastoid morphology. The immunocytochemical assay demonstrated cells from all the studied groups were positive for CD44, PCNA and vimentin and negative for cytokeratin and Oct-4. Flow cytometry demonstrated expression of CD44 and CD90 and no expression of CD34, where CD44 and CD90 markers presented decreasing pattern of expression in relation to the gestational development. All samples collected from all gestational phases were capable to differentiate in osteogenic, chondrogenic and adipogenic lineages. Thus, cells obtained from equine amniotic fluid presented morphological and immunophenotypical characteristics and potential of differentiation typical of MSCs showing that the collection can be performed at any stage of pregnancy. However, more studies should be performed about the expression of pluripotent markers as Oct-4 and the differentiation potential for extra mesodermal lineages prior demonstrated in the literature.(AU)
Assuntos
Animais , Feminino , Cavalos/fisiologia , Líquido Amniótico/química , Fibroblastos/fisiologia , Células-Tronco/fisiologia , Imuno-Histoquímica/veterinária , Transplante de Células-Tronco Mesenquimais/veterinária , Separação Celular/veterináriaRESUMO
Lipid droplets, subspecies (Bos taurus indicus vs. Bos taurus taurus), and in vitro culture are known to influence cryopreservation of bovine embryos. Limited information is available regarding differences in membrane lipids in embryo, such as phosphatidylcholines (PC) and sphingomyelins (SM). The objective of the present study was to compare the profiles of several PC and SM species and relate this information to cytoplasmic lipid levels present in Nellore (B. taurus indicus) and Simmental (B. taurus taurus) blastocysts produced in vitro (IVP) or in vivo (ET). Simmental and IVP embryos had more cytoplasmic lipid content than Nellore and ET embryos (n = 30). Blastocysts were submitted to matrix-assisted laser desorption/ionization mass spectrometry. Differences in the PC profile were addressed by principal component analysis. The lipid species with PC (32:1) and PC (34:1) had higher ion abundances in Nellore embryos, whereas PC (34:2) was higher in Simmental embryos. IVP embryos had less abundant ions of PC (32:1), PC (34:2), and PC (36:5) compared to ET embryos. Moreover, ion abundance of PC (32:0) was higher in both Nellore and Simmental IVP embryos compared to ET embryos. Therefore, mass spectrometry profiles of PC and SM species significantly differ with regard to unsaturation level and carbon chain composition in bovine blastocysts due to subspecies and in vitro culture conditions. Because PC abundances of Nellore and Simmental embryos were distinct (34:1 vs. 34:2), as were those of IVP and ET embryos (32:0 vs. 36:5), they are potential markers of postcryopreservation embryonic survival.