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Regular Article |
a Department of Animal Science and Production, University College Dublin, Lyons Research Farm, Newcastle, County Dublin, Ireland
b Dpto. de Mejora Genética y Biotecnologíaand
c Dpto. de Reproducción Animal y Conservación de Recursos Zoogenéticos, INIA, Madrid 28040, Spain
Using reverse transcriptase-amplified fragment length polymorphism (RT-AFLP) analysis of differential mRNA expression and semiquantitative reverse transcriptase-polymerase chain reaction, we compared mRNA expression in bovine blastocysts from 4 sources, known to differ in quality in terms of their ability to withstand cryopreservation: 1) in vitro culture in synthetic oviduct fluid of in vitro-matured (IVM)/in vitro fertilized (IVF) zygotes; 2) in vitro culture in TCM-199 supplemented with granulosa cells (coculture) of IVM/IVF zygotes; 3) in vivo culture in the ewe oviduct of IVM/IVF zygotes; or 4) superovulation, artificial insemination, and nonsurgical embryo recovery. Total mRNA was isolated from pools of blastocysts and reverse transcription was performed. Triplicate reactions from each sample were displayed, and only consistent banding variations were recorded. Using AFLP-differential display assay, we found that cDNA banding patterns are highly conserved between the 4 groups of blastocysts studied; however, there was a difference of 7% in bands either missing or expressed across the groups. Fifty bands were reamplified, and a sequence comparison search revealed similarity of 14 isolated fragments to ribosomal and mitochondrial genes, 16 matched to described cDNA, and 20 corresponded to unknown sequences that may represent novel genes. The study of 7 differentially expressed mRNAs known to be involved in developmental process in the embryo suggests roles for apoptosis, oxidative stress, gap junctions, and differentiation in the determination of embryo quality. The aberrant transcription patterns detected in in vitro-produced bovine embryos compared with those produced in vivo may explain their reduced quality in terms of viability after cryopreservation.
1 This work was financed by the European Union Commission (QLK3-CT-1999-00104) and by Ministerio de Ciencia y Tecnología (Spain) (RTA01-064 and RTA01-063-C2-1). D.R. was supported by a grant from the Greek State Scholarships Foundation.
2 Correspondence: Alfonso Gutiérrez-Adán, Dpto. de Reproducción Animal y Conservación de Recursos Zoogenéticos, INIA, Ctra de la Coruña Km 5.9, Madrid 28040, Spain. FAX: 91 347 4014; agutierr{at}inia.es
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