Development and Validation of a Short-Term, Serum-Free Culture System for Bovine Granulosa Cells: Evaluation of the Effects of Somatotropin and Growth Hormone-Releasing Factor on Estradiol Production

• Published in: Journal of dairy science 2002-01, Vol.85 (1), p.68-78
• Main Authors: Jimenez-Krassel, F., Ireland, J.J.
• Format: Article
• Language: English
• Subjects: Androstenedione - analysis; Animal cells; Animals; Biological and medical sciences; Biotechnology; Cattle; Cells, Cultured; Culture Media, Serum-Free; Dihydrotestosterone - analysis; Dose-Response Relationship, Drug; Establishment of new cell lines, improvement of cultural methods, mass cultures; estradiol; Estradiol - biosynthesis; Estrous Cycle - physiology; Eukaryotic cell cultures; Female; first-wave follicles; Follicle Stimulating Hormone - pharmacology; Fundamental and applied biological sciences. Psychology; Granulosa Cells - cytology; Granulosa Cells - drug effects; Granulosa Cells - metabolism; Growth Hormone - pharmacology; Growth Hormone-Releasing Hormone - pharmacology; Methods. Procedures. Technologies; Ovarian Follicle - physiology; ovaries from an abattoir; Progesterone - biosynthesis; Reproducibility of Results; short-term granulosa cell culture
• ISSN: 0022-0302; 1525-3198
• DOI: 10.3168/jds.S0022-0302(02)74054-X

The objective of this study was to develop and validate a short-term, serum-free culture system to determine whether recombinant bovine somatotropin (rbST) or recombinant bovine growth hormone-releasing factor (rbGRF) altered the estradiol-producing capacity of bovine granulosa cells isolated from dominant or subordinate follicles of the first follicular wave. Thus, ovaries were obtained at an abattoir from cows that were between d 2 to 5 or 6 to 10 of the estrous cycle. Three size classes of follicles were isolated from each cow's ovaries: small (2 to 5mm in diameter), medium (6 to 14mm), or the largest (6 to 19mm). In vivo steroid-producing capacity of follicles was assessed by measuring concentration of estradiol, progesterone, androstenedione and 5α-dihydrotestosterone in each follicle. In vitro steroid-producing capacity was assessed by culturing granulosa cells from the different follicle sizes for 48h in serum-free media with 19-OH androstenedione and measuring the estradiol and progesterone concentrations in media at the end of culture. The effect of different doses of FSH, rbST, or rbGRF on estradiol and progesterone production by granulosa cells from each follicle size class during d 2 to 5 or 6 to 10 was also evaluated. A high percentage (91.7%) of the largest follicles obtained on d 2 to 5 was estrogen-active (estradiol>progesterone) compared with other follicle classifications (d 2 to 5, small = 0%, medium = 13.8%; d 6 to 10, small = 0%, medium = 3.3%, largest = 33.3%). Estradiol was highest (P