SAGE in Cultured Murine COC During Early Maturation. Mus musculus

In cultured cumulus oocyte complexes (COC) FSH induces gene transcription required for germinal vesicle breakdown (GVBD). Experiments were performed to determine the critical period when gene transcription is required for GVBD and identify candidate mRNAs involved. Experiment I: murine COC were cultured 4h in the presence of FSH with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) added at different intervals after the start of culture. COC cultured with FSH underwent GVBD (82+/-7%). When DRB was added at 0, 5 or 10 min after culture initiation, oocyte maturation was blocked (17+/-7%, 14+/-6% and 21+/-6% GVBD, respectively). When DRB was added after 15, 20 or 30 min, progressively more COC underwent GVBD (37+/-6%, 39+/-6% and 66+/-6%, respectively). The critical period of transcription required for GVBD occurred between 15 and 30 min after culture initiation. Experiment II: COC were cultured for 25 min in the presence (plusDRB) or absence (minusDRB) of DRB. SAGE libraries were generated from COC RNA of each treatment group. A total of 48,431 and 45,367 tags were sequenced for the plusDRB and minusDRB libraries, respectively. Criteria used to identify transcripts of interest included a total tag count of at least 10 across both libraries and a 3-fold or greater difference in expression between libraries. Using these criteria, 39 and 27 transcripts were identified as differentially expressed at the P