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Doctoral Thesis
Full name
Paula Ripamonte Figueiredo
Knowledge Area
Date of Defense
Pirassununga, 2005
Meirelles, Flavio Vieira (President)
Buratini Júnior, José
Caetano, Alexandre Rodrigues
Ferraz, Jose Bento Sterman
Papa, Paula de Carvalho
Title in Portuguese
Genômica funcional da ativação do genoma e do bloqueio embrionário em bovinos
Keywords in Portuguese
ativação do genoma
bloqueio embrionário
embriões bovinos
Abstract in Portuguese
Apesar da grande melhora nos resultados de desenvolvimento embrionário in vitro, cerca de 40% dos oócitos bovinos fecundados não completam o desenvolvimento na fase de pré-implantação. Diversos fatores estão relacionados a este fenômeno, conhecido como bloqueio do desenvolvimento embrionário. Partindo da premissa que o bloqueio no desenvolvimento ocorre normalmente, durante a ativação do genoma embrionário, aproximadamente, no 4º ciclo celular em bovinos, formulou-se a hipótese de que os genes transcritos no momento da ativação do genoma embrionário estão relacionados ao bloqueio. Nesta tese, um sistema fluorescente de Differential Display PCR (DDPCR) foi desenvolvido para isolar e identificar fragmentos de mRNAs expressos diferencialmente entre embriões que se desenvolvem mais rápido e com melhor taxa de desenvolvimento e aqueles que apresentam desenvolvimento mais lento e com maior taxa de bloqueio. Dentre 176 fragmentos recuperados, 27 foram clonados, seqüenciados e 30 genes identificados. Dois genes, PI3K e ITM2B foram quantificados pela PCR em tempo real. Os resultados sugerem que duas diferentes ativações do genoma podem estar ocorrendo: o grupo de desenvolvimento rápido ativa genes ligados ao desenvolvimento embrionário e, o grupo lento ativa os genes ligados à sobrevivência ou morte celular.
Title in English
Functional genome of genome actication and bovine developmental block
Keywords in English
bovine embryo
developmental block
Differential Display PCR
genome activation
Abstract in English
The embryonic developmental block occurs at the 8-cell stage in bovine and is characterized for a lengthening of the cell cycle. At the same stage, also takes place the maternal-embryonic transition (i.e. the activation of the embryonic genome). These events are highly correlated and many genes are activated at the 4th cell cycle however, their functions are mostly unknown. The study of gene expression during this stage will help understand the mechanisms involved in the maternal-embryonic transition and ultimately lead to improvements of in vitro embryo production rates. The aim of this study was to identify genes differentially expressed between bovine embryos with or without developmental competence to reach the blastocyst stage, using Differential Display PCR methodology. Embryos with fast cleavage divisions showing 8 cells at 48 hpi and high potential of development (R8), and embryos with slow cleavage divisions showing 4 cells at 48hpi (L4) and 8 cells at 80 hpi (L8), both with reduced rates of development to blastocyst, were analyzed. We developed an alternative protocol for amplification and recovery of differentially expressed genes from extremely small initial amounts of RNA (10 to 25 pg of mRNA) from preimplantation bovine embryos without need of radio-isotopes. A total of 176 differentially expressed bands were recovered, 27 isolated-fragments were cloned and sequenced confirming the expected primer sequences and allowing the recognition identification of 30 gene transcripts related to bovine embryonic physiology. Two genes, PI3K and ITM2B were chosen for relative quantification of mRNA using Real-Time PCR. Results suggest two different embryonic genome activation mechanisms: fast-developing embryos activate genes related to embryonic development, and slow-developing embryos activate genes related to cellular survival and/or death.
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