Bloqueio meiótico em oócitos de equinos como alternativa para melhora da viabilidade
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Data
2023-01-27
Autores
Jorge, Mariana Lemos Nagib
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A manutenção dos oócitos equinos em bloqueio meiótico, seja através do uso de inibidores de meiose ou manutenção deles em temperatura ambiente, é benéfica para o posterior desenvolvimento embrionário. Com o objetivo de testar a viabilidade da manutenção de oócitos em meio de cultivo embrionário comercial disponível no Brasil (Botuembryo), comparando ao meio de cultivo acrescido de inibidor de meiose (Butirolactona), antes da maturação in vitro (MIV), concebeu-se este ensaio. Foram utilizados 149 oócitos equinos, separados em 3 grupos: Controle, Botuembryo e Butirolactona. O grupo Controle seguiu direto para a MIV por 24 horas, os grupos Botuembryo e Butirolactona passaram por um processo de pré-maturação (PM) por 20 horas com os respectivos produtos, e depois seguiram para a MIV por mais 24 horas. No grupo Controle, o momento 1 (M1) corresponde ao momento da obtenção dos oócitos, já nos grupos Botuembryo e Butirolactona, o M1 corresponde ao final da PM. O momento 2 (M2), nos 3 grupos, foi após a MIV. Após a coloração, a maturação nuclear e citoplasmática foi avaliada pela microscopia confocal. A investigação da expressão gênica foi realizada por RT-qPCR. Nos oócitos foram analisados os genes: Proteína Morfogenética Óssea 15 (BMP15), Conexina 37 (Cx37), Fator de Crescimento e Diferenciação 9 (GDF9) e Metaloproteinase de Matriz 2 (MMP2); já nas células do cumulus (CCs) foram avaliados os genes: Ampiregulina (AREG), Conexina 43 (Cx43), Hialurona Sintetase 2 (HAS2), Receptor de FSH (FSHR) e Receptor de LH (LHR). Na avaliação da maturação nuclear foram encontrados resultados semelhantes entre os grupos tratados e na análise da maturação citoplasmática foi encontrado no grupo Butirolactona o maior percentual de oócitos imaturos no M1, maior percentual de oócitos maduros no M2, e menor percentual de degeneração. Por outro lado, os dados da expressão gênica, demonstraram que houve falha do bloqueio da maturação molecular no grupo Butirolactona, com aumento da abundância de AREG e HAS2 no M1, e diminuição no M2. Além disso, observou-se superioridade do grupo Botuembryo, entre os grupos tratados, no M1, em relação a menor abundância de AREG, LHR e HAS2 e maior abundância de FSHR; já no M2, apresentou maior abundância de GDF9, indicando melhor qualidade tanto dos oócitos como das CCs. Embora os resultados obtidos com o uso da Butirolactona ou do Botuembryo sejam semelhantes em relação a maturação nuclear e citoplasmática, os resultados de maturação molecular demonstraram a superioridade do Botuembryo para manter a qualidade de oócitos e CCs antes da realização de técnicas de fertilização assistida em equinos.
The maintenance of equine oocytes in meiotic block, either using meiosis inhibitors or keeping them at room temperature, is beneficial for the later embryonic development. With the objective of testing the viability of maintaining oocytes in commercial embryonic culture medium available in Brazil (Botuembryo), comparing to the culture medium added with a meiosis inhibitor (Butyrolactone), before in vitro maturation (IVM), this assay was conceived. One hundred and forty-nine equine oocytes were used, separated into 3 groups: Control, Botuembryo and Butyrolactone. The Control group went straight to IVM for 24 hours, the Botuembryo and Butirolactone groups went through a pre-maturation (PM) process for 20 hours with the respective products, and then went to IVM for another 24 hours. In the Control group, moment 1 (M1) corresponds to the moment of obtaining the oocytes, while in the Botuembryo and Butyrolactone groups, M1 corresponds to the end of PM. Moment 2 (M2), in the 3 groups, was after IVM. After staining, nuclear and cytoplasmic maturation was assessed by confocal microscopy. The investigation of gene expression was performed by RT-qPCR. The following genes were analyzed in the oocytes: Bone Morphogenetic Protein 15 (BMP15), Connexin 37 (Cx37), Growth and Differentiation Factor 9 (GDF9) and Matrix Metalloproteinase 2 (MMP2); in the cumulus cells (CCs), the following genes were evaluated: Ampiregulin (AREG), Connexin 43 (Cx43), Hyalurone Synthetase 2 (HAS2), FSH Receptor (FSHR) and LH Receptor (LHR). In the evaluation of nuclear maturation, similar results were found between the treated groups and in the assessment of cytoplasmic maturation, the highest percentage of immature oocytes in M1, the highest percentage of mature oocytes in M2, and the lowest percentage of degeneration were found in the Butyrolactone group. On the other hand, gene expression data showed that there was a failure to block molecular maturation in the Butyrolactone group, with an increase in the abundance of AREG and HAS2 in M1, and a decrease in M2. In addition, superiority was observed for the Botuembryo group, between treated groups, in M1, in relation to the lower abundance of AREG, LHR and HAS2 and the higher abundance of FSHR, in M2, it presented a greater abundance of GDF9, indicating better quality of both oocytes and CCs. Although the results obtained with the use of Butyrolactone or Botuembryo are similar in relation to nuclear and cytoplasmic maturation, the molecular maturation results demonstrated the superiority of Botuembryo to maintain the quality of oocytes and cumulus cells before carrying out assisted fertilization techniques in horses.
The maintenance of equine oocytes in meiotic block, either using meiosis inhibitors or keeping them at room temperature, is beneficial for the later embryonic development. With the objective of testing the viability of maintaining oocytes in commercial embryonic culture medium available in Brazil (Botuembryo), comparing to the culture medium added with a meiosis inhibitor (Butyrolactone), before in vitro maturation (IVM), this assay was conceived. One hundred and forty-nine equine oocytes were used, separated into 3 groups: Control, Botuembryo and Butyrolactone. The Control group went straight to IVM for 24 hours, the Botuembryo and Butirolactone groups went through a pre-maturation (PM) process for 20 hours with the respective products, and then went to IVM for another 24 hours. In the Control group, moment 1 (M1) corresponds to the moment of obtaining the oocytes, while in the Botuembryo and Butyrolactone groups, M1 corresponds to the end of PM. Moment 2 (M2), in the 3 groups, was after IVM. After staining, nuclear and cytoplasmic maturation was assessed by confocal microscopy. The investigation of gene expression was performed by RT-qPCR. The following genes were analyzed in the oocytes: Bone Morphogenetic Protein 15 (BMP15), Connexin 37 (Cx37), Growth and Differentiation Factor 9 (GDF9) and Matrix Metalloproteinase 2 (MMP2); in the cumulus cells (CCs), the following genes were evaluated: Ampiregulin (AREG), Connexin 43 (Cx43), Hyalurone Synthetase 2 (HAS2), FSH Receptor (FSHR) and LH Receptor (LHR). In the evaluation of nuclear maturation, similar results were found between the treated groups and in the assessment of cytoplasmic maturation, the highest percentage of immature oocytes in M1, the highest percentage of mature oocytes in M2, and the lowest percentage of degeneration were found in the Butyrolactone group. On the other hand, gene expression data showed that there was a failure to block molecular maturation in the Butyrolactone group, with an increase in the abundance of AREG and HAS2 in M1, and a decrease in M2. In addition, superiority was observed for the Botuembryo group, between treated groups, in M1, in relation to the lower abundance of AREG, LHR and HAS2 and the higher abundance of FSHR, in M2, it presented a greater abundance of GDF9, indicating better quality of both oocytes and CCs. Although the results obtained with the use of Butyrolactone or Botuembryo are similar in relation to nuclear and cytoplasmic maturation, the molecular maturation results demonstrated the superiority of Botuembryo to maintain the quality of oocytes and cumulus cells before carrying out assisted fertilization techniques in horses.
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Palavras-chave
Reprodução assistida, Fertilidade, ICSI, Maturação oocitária, Equino, Reprodução assistida, Tecnologias reprodutivas, Técnicas de maturação in vitro de oócitos, Fecundidade