Efeitos da substituição de sfb por igf-i sobre os aspectos celulares e moleculares da produção in vitro de embriões bovinos
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Data
2018-05-18
Autores
Serrano-Recalde, Elena Carolina
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Universidade Estadual Paulista (Unesp)
Resumo
O objetivo do presente estudo foi avaliar o benefício da substituição do soro fetal bovino (SFB) pelo fator de crescimento semelhante à insulina tipo I (IGF-1) durante a maturação in vitro (MIV) ou cultivo in vitro (CIV), sobre qualidade embrionária e expressão de genes em embriões pré- e pós-compactação. Delineamento experimental foi fatorial 3 x 3 (três suplementos de MIV e três de CIV), com 9 grupos experimentais. Foram realizadas 20 réplicas (oócitos ≈ 400/grupo). Complexos cúmulus-oócito (COCs) graus I e II foram maturados in vitro com a adição de 10% de SFB (SFB), ou 3 mg/mL polivinil-álcool (PVA), ou PVA + 100 ng/mL de IGF-1 (IGF) a 38,5 ºC em 5% de CO2 em ar por 22 a 24 horas. Foram fertilizados e incubados durante 18 horas. Os zigotos foram cultivados com a adição de: 2,5% de SFB (SFB), ou 3 mg/mL de PVA (PVA), ou PVA + 100 ng/mL de IGF-1 (IGF) por sete dias a 38,5ºC em 5% de CO2 em ar. As taxas de clivagem e blastocisto foram avaliadas após 48 e 168 horas de cultivo, respectivamente. A técnica simplificada de coloração diferencial de células da massa celular interna (MCI) e trofoectoderma (TE) foi utilizada para avaliar a distribuição celular de blastocistos (n = 155) e a técnica de TUNEL para avaliação do índice de apoptose (n = 207). Concentrações de glicose e lactato obtidas do meio MIV utilizaram-se para analisar o metabolismo de glicose. mRNA foi extraído de embriões de 6 – 8 células colhidos após 66 horas post-inseminação (4 pools de 15 embriões por grupo) e de blastocistos expandidos de 7 dias (4 pools de 5 embriões por grupo). A expressão gênica foi realizada no sistema BioMark HD® de microfluídica, pelo arranjo 96.96 Dynamic Array. Os dados foram analisados ANOVA do PROC GLIMMIX do SAS. Foi utilizado o teste Tukey para comparação de médias. Valores de p ≤ 0.05 foram considerados significativos. A clivagem foi maior (p < 0,05) nos grupos maturados em SFB. MIV e CIV com SFB presentou maior (p < 0.05) taxa de produção de blastocisto e maior quantidade de blastocistos expandidos que PVA e IGF. MIV com IGF-I aumentou o consumo de glicose e síntese de lactato dos COCs e levou à produção de blastocistos com maior (p < 0.05) número total de células. Embriões cultivados em IGF-I tiveram maior (p < 0.05) quantidade de células na MCI e embriões cultivados em PVA tiveram maior (p < 0.05) apoptose do que SFB. Os genes NANOG, OTX2, POU5F1 e IFNT2 foram mais expressos em blastocistos cultivados em PVA e IGF do que em SFB. O IGF-I regula TP53, BAX, CASP3, CASP9, HSPA1A e IGF1R para prevenir apoptose. IGF-I durante a MIV em meio semi-definido estimula o metabolismo de glicose de COCs, melhora a qualidade embrionária, aumentando o número total de células de blastocistos. IGF-I durante a CIV aumenta o numero de células na MCI, melhora a expressão de biomarcadores importantes de qualidade embrionária com a possibilidade de melhorar o desenvolvimento embrionário.
The aim of the present study was to analyze the benefits of fetal bovine serum (FBS) replacement by insulin-like growth factor I (IGF-I) during in vitro maturation (IVM) and in vitro culture (IVC), on embryo quality and temporal gene expression in embryos pre- and post-compaction. A 3 x 3 factorial design was performed (three supplements for IVM and three for IVC), with a total of 9 experimental groups. A total of 20 replicates (oocytes ≈ 400/group) were performed. Grade I and II cumulus-oocyte complexes (COCs) matured in vitro with the addition of 10% of FBS (FBS), or 3 mg/mL of polyvinyl-alcohol (PVA), or PVA + 100 ng/mL IGF-1 (IGF) at 38.5 ºC in an atmosphere of 5% CO2 for 22 to 24 hours. After IVM, oocytes were fertilized and incubated for 18 hours. Possible zygotes were cultured with the respective addition of: 2.5% of FBS (FBS), or 3 mg/mL of PVA (PVA), or PVA + 100 ng/mL of IGF-1 (IGF) for seven days at 38.5 ºC in an atmosphere of 5% CO2. Cleavage and blastocyst rates were analyzed at 48 and 168 hours of culture respectively. Simplified technique for differential staining of inner cell mass (ICM) and trophectoderm (TE) cells was performed to analyze cell allocation of blastocysts (n = 155) and TUNEL assay for apoptosis rate analysis (n = 207). Glucose and lactate concentrations were measured in IVM spent media to analyze glucose metabolism. mRNA was extracted from 6 – 8 cells embryos collected after 66 hours post insemination (4 pools of 15 embryos per group) and 7 day expanded blastocysts (4 pools of 5 embryos per group). Gene expression analysis was performed with BioMark HD® system with microfluidic chip 96.96 Dynamic Array. Data were analyzed by ANOVA from PROC GLIMMIX model from SAS. Tuckey test was used to compare means. P value ≤ 0.05 was considered to be significant. Cleavage rate was higher (p < 0.05) for groups matured in FBS. IVM and IVC in FBS presented higher (p < 0.05) total blastocyst yield and greater quantity of expanded blastocysts than PVA and IGF. IVM with IGF-I increased glucose uptake and lactate synthesis of COCs and produced blastocysts with increased (p < 0.05) total cell number. Embryos cultured in IGF-I had greater (p < 0.05) amount of cells in the ICM and embryos cultured in PVA had higher (p < 0.05) apoptosis rate than FBS. NANOG, OTX2, POU5F1 and IFNT2 genes were more expressed in blastocysts cultured in PVA and IGF than in FBS. IGF-I regulates TP53, BAX, CASP3, CASP9, HSPA1A and IGF1R genes to prevent apoptosis. The addition of IGF-I during IVM in chemically semi-defined media stimulates glucose metabolism of COCs and improves embryo quality, increasing total cell number of blastocysts. The addition of IGF-I during IVC increases the amount of cells of the ICM, improves expression of important embryo quality biomarkers with the possibility to enhance embryo development.
The aim of the present study was to analyze the benefits of fetal bovine serum (FBS) replacement by insulin-like growth factor I (IGF-I) during in vitro maturation (IVM) and in vitro culture (IVC), on embryo quality and temporal gene expression in embryos pre- and post-compaction. A 3 x 3 factorial design was performed (three supplements for IVM and three for IVC), with a total of 9 experimental groups. A total of 20 replicates (oocytes ≈ 400/group) were performed. Grade I and II cumulus-oocyte complexes (COCs) matured in vitro with the addition of 10% of FBS (FBS), or 3 mg/mL of polyvinyl-alcohol (PVA), or PVA + 100 ng/mL IGF-1 (IGF) at 38.5 ºC in an atmosphere of 5% CO2 for 22 to 24 hours. After IVM, oocytes were fertilized and incubated for 18 hours. Possible zygotes were cultured with the respective addition of: 2.5% of FBS (FBS), or 3 mg/mL of PVA (PVA), or PVA + 100 ng/mL of IGF-1 (IGF) for seven days at 38.5 ºC in an atmosphere of 5% CO2. Cleavage and blastocyst rates were analyzed at 48 and 168 hours of culture respectively. Simplified technique for differential staining of inner cell mass (ICM) and trophectoderm (TE) cells was performed to analyze cell allocation of blastocysts (n = 155) and TUNEL assay for apoptosis rate analysis (n = 207). Glucose and lactate concentrations were measured in IVM spent media to analyze glucose metabolism. mRNA was extracted from 6 – 8 cells embryos collected after 66 hours post insemination (4 pools of 15 embryos per group) and 7 day expanded blastocysts (4 pools of 5 embryos per group). Gene expression analysis was performed with BioMark HD® system with microfluidic chip 96.96 Dynamic Array. Data were analyzed by ANOVA from PROC GLIMMIX model from SAS. Tuckey test was used to compare means. P value ≤ 0.05 was considered to be significant. Cleavage rate was higher (p < 0.05) for groups matured in FBS. IVM and IVC in FBS presented higher (p < 0.05) total blastocyst yield and greater quantity of expanded blastocysts than PVA and IGF. IVM with IGF-I increased glucose uptake and lactate synthesis of COCs and produced blastocysts with increased (p < 0.05) total cell number. Embryos cultured in IGF-I had greater (p < 0.05) amount of cells in the ICM and embryos cultured in PVA had higher (p < 0.05) apoptosis rate than FBS. NANOG, OTX2, POU5F1 and IFNT2 genes were more expressed in blastocysts cultured in PVA and IGF than in FBS. IGF-I regulates TP53, BAX, CASP3, CASP9, HSPA1A and IGF1R genes to prevent apoptosis. The addition of IGF-I during IVM in chemically semi-defined media stimulates glucose metabolism of COCs and improves embryo quality, increasing total cell number of blastocysts. The addition of IGF-I during IVC increases the amount of cells of the ICM, improves expression of important embryo quality biomarkers with the possibility to enhance embryo development.
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Palavras-chave
fertilização in vitro, blastocisto, fator de crescimento, expressão gênica, desenvolvimento embrionário