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Gamete Biology |
AgResearch Crown Research Institute, Ruakura Campus, Hamilton 2001, New Zealand
We have performed suppressive subtraction hybridization (SSH) of populations of developmentally competent and incompetent bovine oocytes from large (
5-mm) and small (
2-mm) follicles to isolate messenger RNA associated with the attainment of developmental competency. RNA was amplified in a linear fashion and then subjected to the SSH procedure to produce a library enriched for genes associated with competency. One thousand clones of this library were subjected to a differential screening approach to identify 31 potentially upregulated isolates. Sequencing revealed these to represent 21 genes. To rigorously identify the degree of upregulation and reproducibility thereof, we examined the expression of these genes in three separate pools of developmentally competent and incompetent oocytes by quantitative real-time PCR. Results indicated that upregulation varied from zero to threefold, showing that accurate quantification is essential for the interpretation of such differential screening experiments. Furthermore, it appears that the molecular causes for poor developmental capacity may be highly complex and be reliant on many small changes. We further characterized a selection of these novel and known maternally expressed genes for their absolute expression levels during maturation in the presence or absence of an inhibitor of transcription and during preattachment development. Last, the effect of nuclear transfer on the levels of these genes was assayed. Nuclear transfer was found to differentially affect transcript levels of genes expressed after embryonic genome activation but did not prevent the degradation of maternal transcripts or result in activation of maternal genes that are silent at blastocyst stages.
2 Correspondence: Peter L. Pfeffer, AgResearch Crown Research Institute, Ruakura Campus, East Street, Hamilton 2001, New Zealand. FAX: +64 7 8385628; peter.pfeffer{at}agresearch.co.nz
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