ABSTRACT
The pellucid zone (PZ) is a protective embryonic cells barrier against chemical, physical or biological substances. This put, usual transfection methods are not efficient for mammal oocytes and embryos as they are exclusively for somatic cells. Carbon nanotubes have emerged as a new method for gene delivery, and they can be an alternative for embryos transfection, however its ability to cross the PZ and mediated gene transfer is unknown. Our data confirm that multiwall carbon nanotubes (MWNTs) can cross the PZ and delivery of pDNA into in vitro-fertilized bovine embryos. The degeneration rate and the expression of genes associated to cell viability were not affected in embryos exposed to MWNTs. Those embryos, however, had lower cell number and higher apoptotic cell index, but this did not impair the embryonic development. This study shows the potential utility of the MWNT for the development of new method for delivery of DNA into bovine embryos.
Subject(s)
Blastocyst/metabolism , DNA/administration & dosage , Gene Transfer Techniques , Nanotubes, Carbon , Animals , Cattle , Cell Culture Techniques , Cells, Cultured , Female , Genes, Reporter , Nanotubes, Carbon/chemistry , Nanotubes, Carbon/ultrastructure , Nuclear Transfer Techniques , Plasmids/administration & dosage , Plasmids/genetics , PregnancyABSTRACT
Background: candidate genes and their polymorphisms have been associated with traits of economic interest in cattle, representing an important strategy for genetic improvement of complex traits. Bone morphogenetic protein 4 (BMP4) is a member of the transforming growth factor beta (TGFβ) superfamily, which is involved with several events of embryonic, fetal, and adult development in vertebrates. Objective: the aim of this study was to evaluate the degree of association between polymorphism in the BMP4 gene (guanine for thymine- G>T, SNP rs109778173) and the performance of Gyr oocyte donors, including the rate of Cumulus-oophorus complex obtained in each session of follicular aspiration (OPU), embryo development, and pregnancy rates. Methods: DNA was extracted from hair follicles from 50 oocyte donors and genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Data from 212 OPU-IVP sessions was collected, and the following traits were associated with BMP4 polymorphism (SNP rs109778173): number and ratio of viable Cumulus-oophorus complexes; number of cleaved embryos at day 4 of culture; number of transferable embryos at day 7 of culture, and pregnancies on days 30 and 60 after embryo transfer. Results: the studied BMP4 polymorphism was significantly associated (p<0.01) with the number and ratio of viable cumulus-oocyte complexes, and the ratio of pregnancies at 30 days. Conclusion: the GT genotype (SNP rs109778173) was associated with inferior performance regarding OPU-IVP traits. This finding suggests possible genetic effects of BMP4 on performance of Gyr oocyte donors. Studies are needed on the BMP4 gene regarding subsequent changes in embryonic development of said mutation.
Antecedentes: genes candidatos y sus polimorfismos han sido asociados a características de interés económico en ganado, representando una excelente estrategia para el mejoramiento genético de características complejas. La proteína morfogénica ósea 4 (BMP4) es un miembro de la superfamilia del factor de crecimiento transformante beta (TGFβ); que controla innumerables eventos del desarrollo embrionario, fetal y de adultos en vertebrados. Objetivo: el objetivo de este estudio fue evaluar el grado de asociación entre un polimorfismo en el gen BMP4 (guanina por timina-G>T-, SNP rs109778173) y el desempeño de donantes de ovocitos de la raza Gyr, incluyendo la tasa de complejos cummulus-oophorus obtenidos en cada sesión de aspiración folicular (OPU), el desarrollo embrionario y la tasa de preñez. Métodos: el ADN fue extraído del folículo piloso de 50 vacas Gyr donantes de ovocitos, el genotipado fue realizado por la técnica de reacción en cadena de polimerasa-polimorfismos de longitud de fragmentos de restricción (PCR-RFLP). Datos de 212 sesiones de OPU-IVP fueron colectados y las características a seguir fueron asociadas con el polimorfismo en el gen BMP4 (SNP rs109778173): número y tasa de complejos de Cumulus-oophorus viables, de embriones clivados al día 4 de cultivo, de embriones transferibles al día 7 de cultivo, y de preñeces a los días 30 y 60 posterior a la transferencia de los embriones. Resultados: el polimorfismo fue significativamente asociado (p<0,01) con número y tasa de complejos Cumulus-oophorus viables y tasa de preñez al día 30. Conclusión: el genotipo GT (SNP rs109778173) fue asociado a resultados inferiores relacionados con características de OPU-IVP. Este resultado sugiere posibles efectos genéticos del gen BMP4 sobre el desempeño de donadoras de oocitos de la raza Gyr, siendo necesarios estudios del gen BMP4 en relación a las alteraciones en el desarrollo embrionario subsecuentes a la mutación citada.
Antecedentes: genes candidatos e seus polimorfismos têm sido associados a características de interesse econômico no gado bovino, representando uma importante estratégia para o melhoramento genético de características complexas. A proteína morfogênica óssea 4 (BMP4) é um membro da superfamília do fator de crescimento transformante beta (TGFβ); que controla diversos eventos do desenvolvimento embrionário, tanto fetal quanto adulto em vertebrados. Objetivo: avaliar o grau de associação entre um polimorfismo no gene BMP4 (guanina por timina-G>T-, SNP rs109778173) e o desempenho de doadoras de oócitos da raça Gyr, incluindo a taxa de complexos Cumulus-oophorus obtidos em cada sessão de aspiração folicular (OPU), o desenvolvimento embrionário e a taxa de prenhez dos embriões. Métodos: o DNA foi extraído do folículo piloso de 50 vacas Gyr doadoras de oócitos, e a genotipagem foi realizada pela técnica de reação em cadeia da polimerase-polimorfismo no comprimento de fragmentos de restrição (PCR-RFLP). Dados de 212 sessões de OPU-IVP foram coletados, e as características a seguir foram associadas com o polimorfismo no gene BMP4 (SNP rs109778173): número e taxa de complexos Cumulus-oophorus viáveis dos embriões clivados no dia 4 de cultivo, dos embriões transferíveis no dia 7 de cultivo e de prenhezes no dia 30 e 60 após a transferência dos embriões. Resultados: o polimorfismo estudado foi significativamente associado (p<0,01) com o número e a taxa dos complexos Cumulus-oophorus viáveis e a taxa de prenhezes ao dia 30. Conclusão: o genótipo GT (SNP rs109778173) foi associado a resultados inferiores relacionados a características OPU-IVP. Este resultado sugere possíveis efeitos genéticos do gene BMP4 sobre o desempenho de doadoras de oócitos da raça Gyr, fazendo-se necessários estudos no gene BMP4 em relação às alterações no desenvolvimento embrionário subsequentes ao polimorfismo citado.
ABSTRACT
With an aim to improve the in vitro production of bovine embryos, the present study investigated the effect of serum and oxygen tension during IVM on oocyte developmental competence. Four experimental groups were evaluated: G1, 10% oestrus cow serum (OCS) with 20% O(2); G2, 0.1% polyvinyl alcohol (PVA) with 20% O(2); G3, 10% OCS with 5% O(2); and G4, 0.1% PVA with 5% O(2). The proportion of MII oocytes, blastocyst rates and total cell number were not affected (P > 0.05) when the OCS was replaced with PVA under 5% O(2), whereas a higher (P < 0.05) blastocyst rate and total cell number were found with OCS compared with PVA under 20% O(2). The apoptosis index was lower in blastocysts from oocytes matured with PVA under 5% O(2) (G4) compared with other groups (G1, G2 and G3), but no differences (P > 0.05) were found in maturation and blastocyst rates. Significant differences were found in the amount of specific transcripts in oocytes matured under different conditions. In conclusion maturation with PVA and 5% O(2) provides an efficient in vitro culture condition for the maturation of bovine oocytes.
