PARENT SESSION
GAMETE BIOLOGY AND GAMETOGENESIS - A

Monday, August 2, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(157) USE OF SERIAL ANALYSIS OF GENE EXPRESSION (SAGE) FOR IDENTIFICATION OF CANDIDATE GENES ASSOCIATED WITH GONADOTROPIN-INDUCED OOCYTE MATURATION IN THE MOUSE.

Rodriguez, Karina¹, Blomberg, Le Ann², Zuelke, Kurt², Miles, Jeremy ³, Alexander, Joseph¹, Farin, Charlotte¹, ¹ North Carolina State University, Raleigh, NC² USDA-ARS, Beltsville, MD³ North Carolina State University, Raleigh, NC

ABSTRACT- In cultured cumulus oocyte complexes (COC) FSH induces gene transcription required for germinal vesicle breakdown (GVBD). Culture of murine COC in the presence of FSH and the transcriptional inhibitor, 5,6-dichloro-1--D-ribofuranosylbenzimidazole (DRB), prevents gene transcription and the resumption of meiosis. The identity of genes involved in meiotic resumption is unknown. Two experiments were performed to determine the critical period in which gene transcription is required for GVBD and the identity of candidate genes involved in this process. Exp. I: murine COC were cultured for 4h in Waymouth medium supplemented with 0.5 g/ml FSH and 120 M DRB added at either 0, 5, 10, 15, 20, 30 or 40 min after the start of culture (28 per treatment per replicate; 4 replicates). COC cultured with FSH but not DRB underwent GVBD (82±7%). In contrast, 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 greater proportions of COC underwent GVBD (37±6%, 39±6% and 66±6%, respectively). Thus, the critical period for transcription required for GVBD was between 15 and 30 min after culture initiation. Exp. II: a total of 3,540 COC were cultured for 25 min in Waymouth medium supplemented with 5% fetal bovine serum and 0.5 g/ml FSH in the presence or absence of 120 M DRB (1,770 COC per treatment). SAGE libraries were generated using 9-10 g COC whole cell RNA from each treatment group. Data were analyzed using SAGE 2000 4.5 software (Invitrogen Inc). A total of 53,278 tags were produced and represented 5,280 unique putative mRNA transcripts. Two criteria were used to identify transcripts of interest: a total tag count of at least 15 across both libraries and at least a 3-fold difference in expression between libraries. Using these criteria, 16 tags were identified as differentially expressed between the 2 libraries. These tags were assigned identities to genes involved in cell growth/signaling pathways (n=4), oncogenes (n=4), receptors (n=2), gap junctional proteins (n=1), cell cycle (n=1), metabolic processes (n=2) and novel transcripts (n=2). These genes represent candidates potentially involved in the transcriptional regulation of murine oocyte maturation.

KEY WORDS: transcription, meiosis, oocyte, FSH