INTRAOVARIAN REGULATION OF FOLLICULAR DEVELOPMENT

Pituitary gonadotrophins are required for normal follicular growth and development; the proliferation of granulosa cells, however, cannot be attributed to the direct actions of these hormones. Since polypeptide growth factors are known to have mitogenic actions on granulosa cells, the possibility that the surrounding thecal cells may be a source of growth factors that are physiologically important in regulating granulosa cell proliferation was examined in the bovine ovary. In support of this hypothesis bovine thecal cells in culture were shown to secrete products that stimulated [H-3]thymidine incorporation into bovine granulosa cell DNA, and led to an increase in cell number. To characterize the growth-promoting factor thecal cell conditioned medium was concentrated and the peptides fractionated on a Bio-Gel P-60 column in 1.0 M acetic acid. Fractions were tested for their ability to stimulate [H-3]thymidine incorporation into bovine granulosa cell DNA. Growth-promoting activity was located in the 6-9 K and the 16 K molecular weight fractions. The peak fractions inhibited FSH-induced aromatase activity in rat granulosa cells, indicating the presence of EGF-like or TGF-alpha-like peptides. A specific radioimmunoassay for TGF-alpha indicated that the active fractions that promoted both growth and aromatase activity contained TGF-alpha. An immunohistochemical approach was also used to localize TGF-alpha in the bovine ovary throughout the various stages of follicular development. Immunoperoxidase staining was most intense in the theca of follicles at the discrete physiological stages known to show rapid granulosa cell growth. Staining intensity for TGF-alpha declined in large pre-ovulatory follicles, coincident with the known decline in granulosa cell mitosis. Northern analysis of bovine theca mRNA showed the presence of the TGF-alpha mRNA transcript. A growth inhibitory activity was observed in the fractions with a molecular weight of approx. 25 K. The inhibitory activity had the molecular weight of TGF-beta, and TGF-beta was found to inhibit [H-3]thymidine incorporation into bovine granulosa cell DNA. The presence of TGF-beta-like activity was also indicated by the stimulation of FSH-induced aromatase activity in rat granulosa cells. In summary, bovine thecal cells secrete both TGF-alpha and TGF-beta in vitro. As shown in the bioassay, the TGF-alpha-like and the TGF-beta-like factors have pronounced effects on the proliferation of bovine granulosa cells; TGF-alpha promotes cell growth whereas TGF-beta inhibits cell growth. The relative concentrations of TGF-alpha and TGF-beta may determine the rates of proliferation of granulosa cells during follicular development.