Investigação do efeito da atmosfera gasosa na maturação e fecundação in vitro sobre o metabolismo celular e epigenético de embriões bovinos
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2019-10-14
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Miziara, Carolina
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Universidade Estadual Paulista (Unesp)
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As tecnologias de reprodução assistida ou ARTs (“Assisted Reproductive Technology”) são amplamente utilizadas na reprodução humana e animal. Para estes procedimentos, gametas e embriões são expostos a um ambiente artificial que mimetiza o trato reprodutivo. Um dos fatores mais discrepantes neste contexto é a atmosfera gasosa, visto que in vivo a concentração de O2 encontrada no trato reprodutivo é menor do que a utilizada no ambiente in vitro, sendo comumente utilizada a concentração de 20% de oxigênio neste sistema artificial. Altas concentrações deste gás provocam estresse oxidativo e aumento das concentrações de espécies reativas de oxigênio (EROs) e, consequente maior proporção de células apoptóticas em oócitos e embriões. Entretanto, pouco se sabe sobre as consequências da mudança de atmosfera gasosa durante as fases iniciais da produção in vitro de embriões e as possíveis consequências no remodelamento epigenético. Para validar a hipótese de que diferentes concentrações de oxigênio durante a maturação e a fecundação in vitro modulam a transcrição de genes envolvidos no estresse oxidativo e modeladores epigenéticos em oócitos, células do cumulus e embriões bovinos. Além disso, o estresse oxidativo gerado nos embriões exerce efeito sobre os níveis globais de metilação do DNA e marcas de histonas, cinco experimentos foram realizados com objetivo de investigar o papel da atmosfera gasosa nos passos iniciais da produção in vitro de embriões bovinos. Para diferenciar os efeitos da tensão de oxigênio nas etapas iniciais da PIVE o desenvolvimento embrionário foi analisado, testando a tensão de oxigênio atmosférica (20% O2) e a baixa tensão de oxigênio (5% O2) na maturação e fecundação in vitro de embriões bovinos. Os grupos formados foram: baixa tensão de O2 na maturação e fecundação in vitro (M5F5), baixa tensão de O2 na maturação e alta tensão na fecundação in vitro (M5F20), alta tensão de O2 na maturação e fecundação in vitro (M20F20) e alta tensão de O2 na maturação e baixa tensão na fecundação in vitro (M20F5). No primeiro experimento, analisou-se o efeito da tensão de oxigênio na maturação in vitro por meio da análise da metáfase II, quantificação da concentração intracelular de EROs e glutationa reduzida (GSH), assim como expressão de mRNAs relacionados a vias de resposta ao estresse oxidativo e remodelamento epigenético em oócitos e células do cumulus. Os resultados iniciais indicaram que oócitos maturados em alta e baixa tensão de O2 não apresentam diferença na maturação nuclear. Oócitos maturados em alta tensão de O2 geram maior concentração de EROs e GSH comparado ao ambiente de baixa tensão de oxigênio. As células do cumulus de oócitos submetidos a alta tensão de oxigênio na maturação in vitro, apresentaram níveis de transcritos maiores para o gene SOD1 relacionado a via de resposta ao estresse oxidativo celular. No segundo experimento, analisou-se o efeito da tensão de oxigênio na fecundação in vitro por meio da análise da taxa de fecundação. A partir dos dados obtidos neste experimento, foi constatado que zigotos expostos a um ambiente de baixa tensão de O2 na FIV não resultam em maior taxa de polispermia. Todos os presumíveis zigotos foram cultivados em baixa tensão de oxigênio até atingirem o estágio de blastocisto. No terceiro experimento, avaliou-se o efeito da tensão de oxigênio na maturação e fecundação in vitro e sua influência no desenvolvimento embrionário. Na taxa de blastocisto, quando houve interação entre ambos os fatores (tensão de oxigênio na MIV e FIV) (one way ANOVA, p≥0.05), a produção de embriões demonstrou que o grupo M20F20 apresentou uma maior produção (50.63 ± 4.55) em relação aos grupos M20F5 (29.66 ± 2.81), M5F5 (32.00 ± 3.27) e M5F20 (31.88 ± 3.99). No entanto, na análise dos efeitos dos fatores (two way ANOVA, p≤0.05), a alta tensão de oxigênio mostrou um efeito benéfico na MIV e FIV através das taxas de desenvolvimento. Na análise de expressão gênica dos blastocistos não houve interação (p>0.05), de modo que os efeitos principais de MIV (p<0.05) diferiram. Alguns genes analisados apresentaram um aumento nos transcritos de embriões que foram maturados em baixa tensão de oxigênio e relacionados a via de resposta ao estresse oxidativo (NRF2, SOD1, SOD2) e remodelamento epigenético (KDM5A e TET1). No quarto experimento deste estudo, demonstrou-se o efeito da tensão de oxigênio a nível intracelular de GSH e EROs em blastocistos. Embriões coletados no dia 7 do desenvolvimento embrionário demonstraram que os níveis intracelulares de GSH foram maiores nos grupos M20F20 e M5F5, em relação aos grupos M20F5 e M5F20. As concentrações intracelulares de EROs foram menores nos grupos M20F20 e M5F5, não existindo diferença entre esses grupos. Já os grupos M20F5 e M5F20 tiveram maior acúmulo de EROs e diferiram entre si. No último experimento deste estudo (V), observou-se o efeito da tensão de oxigênio nos padrões globais de metilação do DNA e marcas de histonas específicas em blastocistos. Na análise dos níveis globais de metilação do DNA, foi encontrado maiores níveis de 5mC nos grupos M5F20 e M5F5, em relação aos grupos M20F5 e M20F20, todos os grupos diferiram entre si. Os níveis de 5hmC foram analisados e a média da intensidade de fluorescência foi superior em embriões do grupo M20F5 e M5F5 comparado aos grupos M5F20 e M20F20, existindo diferença entre os grupos. Na marcação das histonas ambas se comportaram de maneira semelhante, sendo encontrada maiores níveis de H3K9me2/me3 em embriões dos grupos M5F5 e M20F20, em relação a M5F20 e M20F5, existindo diferença entre os grupos. Em conclusão, este estudo elucidou as diferenças que a alta e a baixa tensão de oxigênio podem provocar nas fases iniciais da produção in vitro de embriões bovinos e possíveis consequências na homeostase celular. Contribuiu para o entendimento dos mecanismos básicos da interação ambiente e genoma, além da aplicação dos conhecimentos adquiridos para a melhoria de biotecnologias nas etapas iniciais da produção in vitro de embriões bovinos.
Assisted Reproductive Technology (ARTs) are widely used in human and animal reproduction. For these procedures, gametes and embryos are exposed to an artificial environment that mimics the reproductive tract. One of the most discrepant factors in this context is the gaseous atmosphere, since in vivo the O2 concentration found in the reproductive tract is lower than that used in the in vitro environment, and the concentration of 20% oxygen in this artificial system is commonly used. High concentrations of this gas cause oxidative stress and increased concentrations of reactive oxygen species (ROS) and, consequently, higher proportion of apoptotic cells in oocytes and embryos. However, little is known about the consequences of gaseous atmosphere change during the early stages of in vitro embryo production and the possible consequences on epigenetic remodeling. To validate the hypothesis that different oxygen concentrations during maturation and in vitro fertilization modulate the transcription of genes involved in oxidative stress and epigenetic modelers in oocytes, cumulus cells and bovine embryos. In addition, oxidative stress generated in embryos has an effect on global DNA methylation and histone tagging levels. Five experiments were carried out to investigate the role of the gaseous atmosphere in the initial steps of in vitro production of bovine embryos. To differentiate the effects of oxygen tension in the early stages of IVPE, embryonic development was analyzed by testing atmospheric oxygen tension (20% O2) and low oxygen tension (5% O2) on in vitro maturation and fertilization of bovine embryos. The groups formed were: low tension O2 at in vitro maturation and fertilization (M5F5), low tension O2 at maturation and high in vitro fertilization (M5F20), high tension O2 at in vitro maturation and fertilization (M20F20) and high tension O2 at maturation and low tension at in vitro fertilization (M20F5). In the first experiment, the effect of oxygen tension on in vitro maturation was analyzed by metaphase II analysis, quantification of intracellular concentration of ROS and reduced glutathione (GSH), as well as expression of stress response pathwayrelated mRNAs. Oxidative and epigenetic remodeling in oocytes and cumulus cells. Initial results indicated that oocytes matured at high and low O2 tension do not differ in nuclear maturation. Oocytes matured at high tension O2 generate higher concentration of ROS and GSH compared to the low oxygen tension environment. Cumulus cells from oocytes subjected to high oxygen tension at in vitro maturation showed higher transcript levels for the SOD1 gene related to the cellular oxidative stress response pathway. In the second experiment, the effect of oxygen tension on in vitro fertilization was analyzed by analyzing the fertilization rate. From the data obtained in this experiment, it was found that zygotes exposed to a low tension O2 environment in IVF do not result in higher polyspermia rate. All presumed zygotes were cultured at low oxygen tension until they reached the blastocyst stage. In the third experiment, the effect of oxygen tension on in vitro maturation and fertilization and its influence on embryonic development was evaluated. In the blastocyst rate, when there was interaction between both factors (oxygen tension in IVM and IVF) (one way ANOVA, vi p≥0.05), embryo production showed that the M20F20 group had a higher production (50.63 ± 4.55) in for the groups M20F5 (29.66 ± 2.81), M5F5 (32.00 ± 3.27) and M5F20 (31.88 ± 3.99). However, in the analysis of factor effects (two way ANOVA, p≤0.05), high oxygen tension showed a beneficial effect on IVM and IVF through developmental rates. In the gene expression analysis of blastocysts there was no interaction (p> 0.05), so that the main effects of IVM (p <0.05) differed. Some genes analyzed showed an increase in embryo transcripts that were matured at low oxygen tension and related to the oxidative stress response pathway (NRF2, SOD1, SOD2) and epigenetic remodeling (KDM5A and TET1). In the fourth experiment of this study, the effect of intracellular oxygen tension of GSH and ROS on blastocysts was demonstrated. Embryos collected on day 7 of embryonic development showed that intracellular GSH levels were higher in groups M20F20 and M5F5 than in groups M20F5 and M5F20. Intracellular ROS concentrations were lower in groups M20F20 and M5F5, with no difference between these groups. The M20F5 and M5F20 groups had higher ROS accumulation and differed among themselves. In the last experiment of this study (V), the effect of oxygen tension on global DNA methylation patterns and specific histone marks in blastoc was observed. In the last experiment of this study (V), we observed the effect of oxygen tension on global DNA methylation patterns and specific histone markings in blastocysts. In the analysis of global DNA methylation levels, higher levels of 5mC were found in groups M5F20 and M5F5, compared to groups M20F5 and M20F20, all groups differed. The 5hmC levels were analyzed and the mean fluorescence intensity was higher in M20F5 and M5F5 embryos compared to M5F20 and M20F20, with differences between groups. In histone marking both behaved similarly, with higher levels of H3K9me2 / me3 found in embryos of groups M5F5 and M20F20, compared to M5F20 and M20F5, with differences between groups. In conclusion, this study elucidated the differences that high and low oxygen tension may cause in the early stages of in vitro production of bovine embryos and possible consequences on cellular homeostasis. It contributed to the understanding of the basic mechanisms of environment and genome interaction, as well as the application of the knowledge acquired to the improvement of biotechnologies in the early stages of in vitro production of bovine embryos. Keywords: oxygen tension, oxidative stress, in vitro production of embryos (IVPE), methylation, histones.
Assisted Reproductive Technology (ARTs) are widely used in human and animal reproduction. For these procedures, gametes and embryos are exposed to an artificial environment that mimics the reproductive tract. One of the most discrepant factors in this context is the gaseous atmosphere, since in vivo the O2 concentration found in the reproductive tract is lower than that used in the in vitro environment, and the concentration of 20% oxygen in this artificial system is commonly used. High concentrations of this gas cause oxidative stress and increased concentrations of reactive oxygen species (ROS) and, consequently, higher proportion of apoptotic cells in oocytes and embryos. However, little is known about the consequences of gaseous atmosphere change during the early stages of in vitro embryo production and the possible consequences on epigenetic remodeling. To validate the hypothesis that different oxygen concentrations during maturation and in vitro fertilization modulate the transcription of genes involved in oxidative stress and epigenetic modelers in oocytes, cumulus cells and bovine embryos. In addition, oxidative stress generated in embryos has an effect on global DNA methylation and histone tagging levels. Five experiments were carried out to investigate the role of the gaseous atmosphere in the initial steps of in vitro production of bovine embryos. To differentiate the effects of oxygen tension in the early stages of IVPE, embryonic development was analyzed by testing atmospheric oxygen tension (20% O2) and low oxygen tension (5% O2) on in vitro maturation and fertilization of bovine embryos. The groups formed were: low tension O2 at in vitro maturation and fertilization (M5F5), low tension O2 at maturation and high in vitro fertilization (M5F20), high tension O2 at in vitro maturation and fertilization (M20F20) and high tension O2 at maturation and low tension at in vitro fertilization (M20F5). In the first experiment, the effect of oxygen tension on in vitro maturation was analyzed by metaphase II analysis, quantification of intracellular concentration of ROS and reduced glutathione (GSH), as well as expression of stress response pathwayrelated mRNAs. Oxidative and epigenetic remodeling in oocytes and cumulus cells. Initial results indicated that oocytes matured at high and low O2 tension do not differ in nuclear maturation. Oocytes matured at high tension O2 generate higher concentration of ROS and GSH compared to the low oxygen tension environment. Cumulus cells from oocytes subjected to high oxygen tension at in vitro maturation showed higher transcript levels for the SOD1 gene related to the cellular oxidative stress response pathway. In the second experiment, the effect of oxygen tension on in vitro fertilization was analyzed by analyzing the fertilization rate. From the data obtained in this experiment, it was found that zygotes exposed to a low tension O2 environment in IVF do not result in higher polyspermia rate. All presumed zygotes were cultured at low oxygen tension until they reached the blastocyst stage. In the third experiment, the effect of oxygen tension on in vitro maturation and fertilization and its influence on embryonic development was evaluated. In the blastocyst rate, when there was interaction between both factors (oxygen tension in IVM and IVF) (one way ANOVA, vi p≥0.05), embryo production showed that the M20F20 group had a higher production (50.63 ± 4.55) in for the groups M20F5 (29.66 ± 2.81), M5F5 (32.00 ± 3.27) and M5F20 (31.88 ± 3.99). However, in the analysis of factor effects (two way ANOVA, p≤0.05), high oxygen tension showed a beneficial effect on IVM and IVF through developmental rates. In the gene expression analysis of blastocysts there was no interaction (p> 0.05), so that the main effects of IVM (p <0.05) differed. Some genes analyzed showed an increase in embryo transcripts that were matured at low oxygen tension and related to the oxidative stress response pathway (NRF2, SOD1, SOD2) and epigenetic remodeling (KDM5A and TET1). In the fourth experiment of this study, the effect of intracellular oxygen tension of GSH and ROS on blastocysts was demonstrated. Embryos collected on day 7 of embryonic development showed that intracellular GSH levels were higher in groups M20F20 and M5F5 than in groups M20F5 and M5F20. Intracellular ROS concentrations were lower in groups M20F20 and M5F5, with no difference between these groups. The M20F5 and M5F20 groups had higher ROS accumulation and differed among themselves. In the last experiment of this study (V), the effect of oxygen tension on global DNA methylation patterns and specific histone marks in blastoc was observed. In the last experiment of this study (V), we observed the effect of oxygen tension on global DNA methylation patterns and specific histone markings in blastocysts. In the analysis of global DNA methylation levels, higher levels of 5mC were found in groups M5F20 and M5F5, compared to groups M20F5 and M20F20, all groups differed. The 5hmC levels were analyzed and the mean fluorescence intensity was higher in M20F5 and M5F5 embryos compared to M5F20 and M20F20, with differences between groups. In histone marking both behaved similarly, with higher levels of H3K9me2 / me3 found in embryos of groups M5F5 and M20F20, compared to M5F20 and M20F5, with differences between groups. In conclusion, this study elucidated the differences that high and low oxygen tension may cause in the early stages of in vitro production of bovine embryos and possible consequences on cellular homeostasis. It contributed to the understanding of the basic mechanisms of environment and genome interaction, as well as the application of the knowledge acquired to the improvement of biotechnologies in the early stages of in vitro production of bovine embryos. Keywords: oxygen tension, oxidative stress, in vitro production of embryos (IVPE), methylation, histones.
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Palavras-chave
Tensão de oxigênio, Estresse oxidativo, Produção in vitro de embriões (PIVE), Metilação, Histonas