Efeito do genótipo de camundongo sobre a metilação de DNA e competência de desenvolvimento de oócitos expostos ao estresse térmico in vivo
Data
2022-01-26
Autores
Carvalho, Caroline Alencar Imaeda de 
Orientadores
Lopes, Fabíola Freitas de Paula
Tipo
Trabalho de conclusão de curso
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A exposição de oócitos a condições ambientais adversas compromete eventos celulares e moleculares durante a oogênese. Embora os efeitos celulares causados pelo estresse térmico sobre os gametas de mamíferos já tenham sido bem estudados, pouco se sabe sobre a susceptibilidade de diferentes genótipos sobre o perfil epigenético do oócito induzidas pelo estresse térmico. Portanto, o modelo de estresse térmico em câmara climática foi estabelecido em camundongos das linhagens C57BL/6 e Suíça para determinar o impacto sobre o padrão de metilação de DNA e a competência oocitária. Para tanto, seis casais de cada linhagem foram acasalados e no dia 10 pós-natal, as fêmeas (matrizes) e as ninhadas foram alocadas em câmaras climáticas com temperatura controlada: controle (21°C/24h) ou estresse térmico (35°C/12h durante o período de luz e 21°C/12h durante o período escuro) por 11 dias. Nesta janela do dia 10 ao dia 21 pós-natal ocorre uma onda majoritária de metilação de DNA de novo durante a oogênese de camundongos. Após o dia 21, as ninhadas foram desmamadas e mantidas na temperatura controle até o final do experimento. Ao atingirem a puberdade as fêmeas foram superovuladas e eutanasiadas para coleta de oócitos visando a determinação do nível de metilação de DNA por imunofluorescência e submetidos à ativação partenogenética para determinação da competência oocitária. Uma análise adicional da competência oocitária contou com a produção in vivo de embriões que foram classificados conforme o estádio do desenvolvimento. Houve um efeito marcante do genótipo sobre a massa corporal dos animais, sendo significativamente menor na linhagem C57BL/6 nas matrizes (P< 0,0001), filhotes antes da sexagem (P=0,0003) e fêmeas (P<0,0001). O estresse térmico reduziu a massa corporal das matrizes (P= 0,0053) e a variação da massa corporal da ninhada (P=0,029) na linhagem Suíça. A porcentagem de oócitos que atingiu os estádios de mórula e blastocisto após a ativação partenogenética foi reduzida (P = 0,0003) pela temperatura na linhagem Suíça. No entanto, não houve efeito do estresse térmico na produção in vivo de embriões, sugerindo uma contribuição paterna e/ou das condições de desenvolvimento embrionário in vivo na mitigação dos danos causados pelo estresse térmico. A linhagem C57BL/6 não respondeu ao protocolo de ativação partenogenética padrão e apresentou falhas nos acasalamentos e cuidado parental reduzindo o número de animais para experimentação. Por isso, não foi possível mensurar o impacto do estresse térmico sobre a competência oocitária nessa linhagem.
The exposure of oocytes to adverse environmental conditions compromises cellular and molecular events during oogenesis. Although the cellular effects caused by heat stress on mammalian gametes have already been well studied, still don’t know about the susceptibility of different genotypes on the epigenetic profile of the oocyte induced by heat stress. Therefore, a climate chamber heat stress model was established in C57BL/6 and Swiss mice to determine the impact on DNA methylation pattern and oocyte competence. For that, six couples of each lineage were mated and on the 10th postnatal day, the females (matrices) and the litters were placed in climatic chambers with controlled temperature: control (21°C/24h) or heat stress (35°C). /12h during the light period and 21°C/12h during the dark period) for 11 days. In this window from day 10 to day 21 postnatal a majority wave of de novo DNA methylation occurs during mouse oogenesis. After day 21, the litters were weaned and kept at the control temperature until the end of the experiment. Upon reaching puberty the females were superovulated and euthanized for oocyte collection to determine the level of DNA methylation by immunofluorescence and submitted to parthenogenetic activation to determine oocyte competence. An additional analysis of oocyte competence included in vivo production of embryos that were classified according to developmental stage. There was a marked effect of the genotype on the body mass of the animals, being significantly lower in the C57BL/6 strain in the sows (P<0.0001), pups before sexing (P=0.0003) and females (P<0.0001 ). Heat stress reduced the body mass of the sows (P= 0.0053) and the variation of the body mass of the litter (P=0.029) in the Swiss strain. The percentage of oocytes that reached the morula and blastocyst stages after parthenogenetic activation was reduced (P = 0.0003) by temperature in the Swiss strain. However, there was no effect of heat stress on in vivo embryo production, suggesting a contribution of paternal and/or in vivo embryonic development conditions in mitigating the damage caused by heat stress. The C57BL/6 strain did not respond to the standard parthenogenetic activation protocol and presented failures in mating and parental care, reducing the number of animals for experimentation. Therefore, it was not possible to measure the impact of heat stress on oocyte competence in this strain.
The exposure of oocytes to adverse environmental conditions compromises cellular and molecular events during oogenesis. Although the cellular effects caused by heat stress on mammalian gametes have already been well studied, still don’t know about the susceptibility of different genotypes on the epigenetic profile of the oocyte induced by heat stress. Therefore, a climate chamber heat stress model was established in C57BL/6 and Swiss mice to determine the impact on DNA methylation pattern and oocyte competence. For that, six couples of each lineage were mated and on the 10th postnatal day, the females (matrices) and the litters were placed in climatic chambers with controlled temperature: control (21°C/24h) or heat stress (35°C). /12h during the light period and 21°C/12h during the dark period) for 11 days. In this window from day 10 to day 21 postnatal a majority wave of de novo DNA methylation occurs during mouse oogenesis. After day 21, the litters were weaned and kept at the control temperature until the end of the experiment. Upon reaching puberty the females were superovulated and euthanized for oocyte collection to determine the level of DNA methylation by immunofluorescence and submitted to parthenogenetic activation to determine oocyte competence. An additional analysis of oocyte competence included in vivo production of embryos that were classified according to developmental stage. There was a marked effect of the genotype on the body mass of the animals, being significantly lower in the C57BL/6 strain in the sows (P<0.0001), pups before sexing (P=0.0003) and females (P<0.0001 ). Heat stress reduced the body mass of the sows (P= 0.0053) and the variation of the body mass of the litter (P=0.029) in the Swiss strain. The percentage of oocytes that reached the morula and blastocyst stages after parthenogenetic activation was reduced (P = 0.0003) by temperature in the Swiss strain. However, there was no effect of heat stress on in vivo embryo production, suggesting a contribution of paternal and/or in vivo embryonic development conditions in mitigating the damage caused by heat stress. The C57BL/6 strain did not respond to the standard parthenogenetic activation protocol and presented failures in mating and parental care, reducing the number of animals for experimentation. Therefore, it was not possible to measure the impact of heat stress on oocyte competence in this strain.