The main objective of this study was to identify mRNA transcripts associated with embryonic developmental competence. In cattle, mRNA transcripts, ribosomes, and proteins accumulated during the growth phase are drawn on to sustain maturation, fertilization, and the initial cell cycle divisions up to the 8- to 16-cell stage. Early cleaving mammalian zygotes are more likely to develop to the blastocyst stage than their later cleaving counterparts, thus reflecting the intrinsic quality of the oocytes from which they originated. We describe the combination of this well-established model for the retrospective determination of developmental competence in mammalian oocytes with a technique for wide screening of differential gene expression in different biological populations. Immature cumulus oocyte complexes were recovered from surface visible follicles on abattoir ovaries, washed, and submitted to routine in vitro maturation and fertilization. Two-cell embryos were removed from culture at 3-hr intervals from 24 to 42 hr post insemination (pi). Two populations of two-cell embryos were identified; those that cleaved early (before 27 hpi) and those that cleaved late (after 33 hpi). Suppressive subtractive hybridization was carried out on cDNA from the two populations, following which, differentially expressed amplicons were subcloned and sequenced. The sequences were submitted to the nonredundant and expressed sequence tag (EST) databases at NCBI using the BLAST algorithm. The differential expression of three selected candidate genes that were identified as putatively upregulated in the early cleaving zygotes were chosen for further investigations; histone H3, cyclin B1, and GDF-9B. Using quantitative real time PCR we have shown that histone H3A is significantly more abundant in embryos that cleave earliest.
Copyright 2004 Wiley-Liss, Inc.