Role of growth factors and estrogen as modulators of growth, differentiation, and expression of gonadotropin subunit genes in primary cultured sheep pituitary cells.

Abstract

Although the pituitary is known to produce several growth factors, their effects on pituitary cell growth and differentiation are still unclear, particularly in normal tissue. Using primary cultures of aged ewe pituitaries cultured in serum-free conditions, we studied the effects of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor-beta (TGF beta 1), insulin, and 17 beta-estradiol (E2) on the growth, differentiation, and expression of gonadotropin subunit genes. After 72-h incubation of the monolayer (day 5) with optimal concentrations of each factor, [3H]thymidine incorporation was increased significantly (P < 0.01) over the control values by 33 +/- 8% (mean +/- SEM; n = 3; 10 nM E2), 36 +/- 10% (1 ng/ml TGF beta 1), 83 +/- 12% (10 ng/ml bFGF), and 118 +/- 12% (1 nM EGF). Insulin showed a two-phase dose-response curve, increasing [3H]thymidine uptake by 34 +/- 9% at 10 ng/ml and by 63 +/- 13% at 10 micrograms/ml. Cell counting using a Coulter counter confirmed these results. Characterization of cell types by immunocytochemistry (avidin-biotin-peroxidase complex technique) revealed that the cell cultures were predominantly gonadotrophs. However, the cultures contained cells that did not stain with any specific ovine or human antiserum against LH beta, FSH, TSH beta, PRL, GH, ACTH, or glial fibrillary acidic protein, but were of epithelial cell lineage, as shown by positive keratin staining. Treatment with EGF and bFGF increased the proportion of these undifferentiated pituitary cells and induced changes in their morphology to large cuboidal cells containing large nuclei. After treatment with E2, insulin, and TGF beta 1, pituitary cells remained differentiated, although with E2, staining for gonadotrophs was much reduced. Northern blot analysis revealed that E2 treatment for 0-48 h progressively reduced the mRNA for FSH beta (16 +/- 4.5% of control values) and LH beta (12.4 +/- 2.5%), but had little effect on the common alpha-subunit (88.4 +/- 4.6%). TGF beta 1, however, stimulated the expression of FSH beta subunit gene by 142 +/- 4.6% (P < 0.01) of the control value, but had no significant effect on LH beta and common alpha-subunit genes. Insulin, EGF, and bFGF showed no significant effect on the expression of these three subunit genes. The data define the direct effects of growth factors and E2 on the growth and differentiation of normal sheep pituitary cells and gonadotrophs in particular, which may be of relevance to the pathophysiology of the pituitary and in the multistage process of pituitary tumorigenesis.