109 Differential gene expression in dominant and subordinate bovine ovarian follicles analysed using DDRT-PCR C. O'Meara 1 , D. Egan 2 , F.M. Doohan 2 and A.C.O. Evans 1 1 Department of Animal Science and Production, 2 Department of Environmental Resource Management, Faculty of Agriculture,University College Dublin, Belfield, Dublin 4, Ireland. Introduction Follicular atresia is a term, which refers to the degeneration of non-ovulatory follicles within the ovaries. Greater than 99% of follicles present at birth become atretic with only less than 1% achieving ovulation (Van Voorhis, 1999). All the follicles that do not progress to ovulate die by apoptosis (Van Wezel et al., 1999). The dominant follicle destined for survival is promoted and apoptosis is prevented by endocrine and local paracrine factors. The mechanisms controlling apoptosis within ovarian follicles and the mechanisms by which a single follicle escapes cell death are not clear and most likely involves the expression of genes that have not yet been described. The aim of this study was to examine the differences between proliferating and regressing tissue from dominant and subordinate follicles from bovine ovaries for the expression of novel genes using DDRT-PCR. Materials and Methods Animals and follicles Ovaries were selected from heifers (n=4) based on corpus luteum (CL) size between day 3 to 5 of the oestrous cycle. Follicles were dissected, measured and categorised as dominant or subordinate. Follicular fluid was aspirated and the concentrations of oestradiol were determined by radioimmunoassay. Total ribonucleic acid (RNA) was extracted from follicle walls (theca and granulosa cells) by guanidinium thiocyanate (GTC) extraction method (Chomczynski and Sacchi, 1987) and quantified using a spectrophotometer. Differential Display- Reverse Transcriptase Polymerase Chain Reaction (DD-RTPCR) Analysis was performed on RNA extracts to compare gene expression patterns in dominant and subordinate follicles. Two 10-mer primers (from 16 primers screened) SC10-58 and OPE-3 were selected for DDRT-PCR, cDNA was amplified by PCR and electrophoresed through a 6% polyacrylamide gel (Oto et al., 1993). DNA was then visualised by silver staining (Doyle, 1996) and differentially expressed (banding) patterns were analysed. Bands of DNA were excised and amplified using PCR reaction components as previously described by Liang and Pardee (1992). DNA products were cloned using pGEM-T Vector System II (Promega corporation, USA) and bacterial cultures were harvested by a Nucleic Acid Purification kit (Clontech labs., USA). Purified PCR products were then sequenced (MWG-biotech, Germany) and subjected to BLAST analysis. Statistics Comparisons were made using a students t-test and are presented as means ± SEM. Results and Discussion Follicles and follicular fluid oestradiol concentrations Dominant and subordinate follicles were discriminated on the basis of diameter (9.5 ± 0.54 mm and 7.0 ± 0.4 mm, respectively; P < 0.05) and oestradiol concentration (157.5 ± 65.0 ng ml -1 and 10.3 ± 5.4 ng ml -1 , respectively; P < 0.05). Following RNA extraction there was 181.5 ± 22.4 μg and 135.4 ± 14.2 μg follicle -1 RNA recovered from dominant and subordinate follicles, respectively (P > 0.05). Recovery of RNA was not different among follicle types. Analysis of DDRT – PCR, selection, cloning and analysis Approximately 590 cDNA bands were identified from polyacrylamide gel screening and 26 appeared to be differentially expressed between dominant and subordinate follicle samples. Of the 10 differentially expressed bands 1 (at 134 bp) of 3 bands and 4 (at 243 bp, 243 bp, 243 bp and 281 bp) of 7 bands using primers SC10-58 and OPE-03, respectively, were reamplified by PCR. All 5 bands were over-expressed in dominant compared to subordinate follicles. All 5 bands were successfully sequenced. However, a multiple alignment revealed 99% similarity between 3 bands from primer OPE-03 (at 243 bp). The BLAST analysis of DNA sequences for these 3 bands collectively and for the remaining 2 bands showed no significant homology to any sequence in the nucleic acid or protein sequence databases and may represent genes that are unique and significant for follicle development. Genes found to be specific to dominant follicles need to be identified and analysed further. Their relevance to follicle survival could have exciting implications and are subject to further investigations. Conclusion This research identified differences in gene expression between dominant and subordinate follicles in bovine ovaries using differential DDRT-PCR. Three sequences were identiifed and were found not similar to any previously reported genes and are currently under further investigation. Acknowledgements This work was funded by the Faculty of Agriculture Interdisciplinary Research Fund, UCD. References Chomczynski, P., Sacchi, N. (1987) Annals of bioch. 162, 156- 159 Doyle, K. (1996) In; Doyle, K. (ed), Promega Co. USA pp 147-162 Liang, P., and Pardee A.B. (1992) Science, 257, 967-97 Oto, M., Miyake, S., Yuasa, Y. (1993) Anal Biochem, 213, 19-22 Van Voorhis B.J. (1999) Encyclopedia of reproduction. Academic press, California, pp 376-389 Van Wezel, I.L., Dharmarajan, A.M., Lavranos, T.C., Rodgers, R.J. (1999) Endocrin.140, 2602-2612 View publication stats View publication stats