IN-VITRO PRODUCTION OF ESTRADIOL BY BOVINE GRANULOSA-CELLS - EVALUATION OF CULTURE CONDITION, STAGE OF FOLLICULAR DEVELOPMENT, AND LOCATIONOF CELLS WITHIN FOLLICLES

In vitro estradiol (E(2)) production by bovine granulosa cells was eva luated under several culture conditions, which included the presence o r absence of fetal bovine serum (FBS; 2.5 and 10%), serum substitutes (1% Nutridoma [Boehringer-Mannheim, Indianapolis, IN], 2% UltroSer G [ IBF Biotechnics, Villenue-la Garenne, France]), selenium (Se; 10 ng/ml ), lipoprotein (0.25% Excyte/ml), O-2 concentration (5 and 20%), and t wo attachment factors (Pronectin F and PepTite-2000). Dulbecco's Modif ied Eagle's medium:Ham's F-12 medium (1:1 mixture) containing 1 mu M a ndrostenedione, 1 mu g/ml insulin, and 0.1% BSA was the basal medium e valuated. The optimum conditions determined were the basal medium in 5 % O-2. These conditions were then used to ascertain whether or not E(2 ) production by granulosa cells varied with respect to location of cel ls within a follicle. Follicular fluid was aspirated and centrifuged t o obtain granulosa cells expected to be primarily luminal and cumulus cells. Follicles were then bisected, and remaining mural granulosa cel ls were removed by scraping the follicle wall with a fine plastic loop . Aspirated granulosa cells secreted more (p < 0.01) E(2) than scraped cells. Production of E, during Days 0 to 2 of culture by aspirated (0 .15 +/- 0.05 ng/mu g DNA) and scraped (0.02 +/- 0.01 ng/mu g DNA) gran ulosa cells from small follicles (< 8 mm) was less than that by aspira ted (6.30 +/- 2.20 ng/mu g DNA) and scraped cells (1.90 +/- 1.00 ng/mu g DNA) from large follicles (greater than or equal to 8 mm). During D ays 2 to 4 of culture when compared to Days 0 to 2, E(2) production in creased for aspirated (but not scraped) granulosa cells from small fol licles (0.66 +/- 0.23 ng/mu g DNA)). In contrast, E(2) production decr eased (p < 0.05) over time in culture for aspirated (2.10 +/- 0.50 ng/ mu g DNA) and scraped (0.16 +/- 0.07 ng/mu g DNA) granulosa cells from large follicles. Thus, granulosa cells proximal to the basement membr ane may be less differentiated with regard to E(2) production than cel ls distal to the basement membrane. In addition, aspirated granulosa c ells from small follicles appear to continue to differentiate toward E (2)-producing cells during culture, a characteristic difficult to obta in with bovine granulosa cells.