PARENT SESSION
SLIDE SESSION 22: OOCYTE DEVELOPMENT
Chairs: Hugh Clarke, David Albertini, Maria Viveiros (Trainee)
Univ Ottawa-Morisset 218
1:30 PM-3:30 PM

QUANTITATIVE EVALUATION OF THE mRNA LEVELS OF BOVINE GRANULOSA CELL GENE MARKERS ASSOCIATED WITH OOCYTE DEVELOPMENTAL COMPETENCE.

Gagné, Dominic¹, Robert, Claude¹, Bousquet, Daniel², Barnes, Frank³, Sirard, Marc-André¹, ^{1 2 3}

ABSTRACT- When oocytes are obtained for in vitro maturation and fertilization (IVM-IVF), it is known that not all of them have the ability to develop into an embryo. The follicular environment is an important factor contributing to the capacity of the oocyte to sustain embryonic development. We hypothesized that the granulosa cells surrounding the oocyte should reflect the oocyte maturation status. The differential display, suppressive subtractive hybridization and the nylon based array techniques were previously used in our laboratory to isolate candidate granulosa cells gene markers associated with bovine oocyte developmental competence. To validate their specificity, 7 candidates were evaluated by real time quantitative RT-PCR. Granulosa cells pools were collected during transvaginal guided ovum pick up and were classified upon the capacity of the respective oocyte pools to develop into a blastocyst after IVM-IVF. Three classes were done: 0% embryo development (e.d.), 0% < e.d. < 100% and 100% e.d.. Eight pools of each class were used to assess the specificity of the candidates. The granulosa cells total RNA was extracted using Trizol Reagent (Gibco BRL, Burlington, ON), the reverse transcription reactions were done in 20l and 2l was used for each PCR reaction. The PCR reactions were conducted in a Light Cycler apparatus (Roche, Laval, QC) and the detection was done using SYBR Green (Roche, Laval, QC). For GABA receptor, prostaglandin receptor and matrix metalloproteinase, the mRNA levels were very low and inconsistent across all the classes. The mRNA levels for protein kinase inhibitor and metalloproteinase inducer were not significantly different between the classes whereas URF-4 and progesterone receptor mRNA levels were higher in granulosa cells surrounding a competent oocyte (P<0.05). Variation between samples suggest that several markers may be needed for proper oocyte diagnostic purposes. Overall, these techniques are efficient to isolate differentially expressed genes. This work was supported by the Natural Sciences and Engineering Research Concil of Canada and Semex Canada.

KEY WORDS: granulosa cells, oocyte developmental competence , gene marker, real time PCR