Submitted on December 15, 2005
Accepted on August 3, 2006
Identification of Candidate mRNAs Associated with Gonadotropin-Induced Maturation of Murine Cumulus Oocyte Complexes using Serial Analysis of Gene Expression
Karina F. Rodriguez1, Le Ann Blomberg2, Kurt A. Zuelke2, Jeremy R Miles1, Joseph Eric Alexander1, and Charlotte E. Farin1*
1 Department of Animal Science, North Carolina State University, Raleigh, North Carolina, United States
2 USDA-ARS Biotechnology and Germplasm Laboratory, Beltsville, Maryland, United States
* To whom correspondence should be addressed. E-mail: char_farin{at}ncsu.edu.
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-
-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
0.01 and P0.001 levels, respectively. Differentially expressed transcripts were classed into major categories that included cell growth, development and regulation of gene expression. Differentially expressed transcripts represent candidates potentially involved in regulating maturation of murine COC.
