In vitro production of estradiol by bovine granulosa cells: Evaluation of culture condition, stage of follicular development, and location of cells within follicles

Abstract

In vitro estradiol (E2) production by bovine granulosa cells was evaluated under several culture conditions, which included the presence or 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), O2 concentration (5 and 20%), and two attachment factors (Pronectin F and PepTite-2000). Dulbecco's Modified Eagle's medium:Ham's F-12 medium (1:1 mixture) containing 1 μM androstenedione, 1 μg/ml insulin, and 0.1% BSA was the basal medium evaluated. The optimum conditions determined were the basal medium in 5% O2. These conditions were then used to ascertain whether or not E2 production by granulosa cells varied with respect to location of cells within a follicle. Follicular fluid was aspirated and centrifuged to obtain granulosa cells expected to be primarily luminal and cumulus cells. Follicles were then bisected, and remaining mural granulosa cells were removed by scraping the follicle wall with a fine plastic loop. Aspirated granulosa cells secreted more (p < 0.01) E2 than scraped cells. Production of E2 during Days 0 to 2 of culture by aspirated (0.15 ± 0.05 ng/μg DNA) and scraped (0.02 ± 0.01 ng/μg DNA) granulosa cells from small follicles (< 8 mm) was less than that by aspirated (6.30 ± 2.20 ng/μg DNA) and scraped cells (1.90 ± 1.00 ng/μg DNA) from large follicles (≥ 8 mm). During Days 2 to 4 of culture when compared to Days 0 to 2, E2 production increased for aspirated (but not scraped) granulosa cells from small follicles (0.66 ± 0.23 ng/μg DNA)). In contrast, E2 production decreased (p < 0.05) over time in culture for aspirated (2.10 ± 0.50 ng/μg DNA) and scraped (0.16 ± 0.07 ng/μg DNA) granulosa cells from large follicles. Thus, granulosa cells proximal to the basement membrane may be less differentiated with regard to E2 production than cells distal to the basement membrane. In addition, aspirated granulosa cells from small follicles appear to continue to differentiate toward E2- producing cells during culture, a characteristic difficult to obtain with bovine granulosa cells.