Differential Display and Suppressive Subtractive Hybridization Used to Identify Granulosa Cell Messenger RNA Associated with Bovine Oocyte Developmental Competence1

The main objective of this study was to identify mRNA expressed in the granulosa cells characterizing differentiated follicles bearing developmentally competent bovine oocytes. Analytical comparisons were made on mRNA pools of granulosa cells using differential display reverse transcription polymerase chain reaction (DDRT) analysis and suppressive subtractive hybridization (SSH). With DDRT, mRNA patterns of granulosa cells from small (< 4 mm) and large (> 8 mm) follicles cultured in the presence or absence of LH were compared to identify mRNA associated with follicular size or with the LH response. Nine clones were sequenced, and two were identified. One of the clones, DRAK 1, was associated with the presence of LH in the medium. Other comparisons directed toward the identification of mRNA associated with the presence of a competent oocyte were done on granulosa cells collected in vivo from superstimulated heifers. With the DDRT analysis, four clones associated with the oocyte developmental competence status were identified. With the SSH analysis, four clones specific to the presence of an incompetent oocyte were sequenced and none were identified, whereas 49 clones specific to the presence of a competent oocyte were sequenced and 18 were identified. Among these clones, early growth response 1, sprouty 2, cytochrome C oxidase, matrix metalloproteinase inducer, matrix metalloproteinase, epiregulin, prostaglandin receptor, and progesterone receptor were the most relevant to the ovarian physiology being examined.