Epidermal growth factor and gonadotropin-releasing hormone stimulate proliferation of enriched population of gonadotropes

Recent studies of epidermal growth factor (EGF) receptors on gonadotropes show that they appear early in the estrous cycle on immature gonadotropes, most of which could be identified by LH messenger RNA only. As diestrous gonadotropes translate the messenger RNAs, the percentages of LH and FSH cells with EGF receptors increase to reach a peak during proestrus. To learn more about the function of EGF in gonadotrope regulation, parallel studies of its mitogenic potential were conducted. To test this in a cell growth assay, we initially developed a protocol for enrichment of gonadotropes by counterflow centrifugation (elutriation). Analysis of immunolabeled cells in the enriched fraction showed that the population contained 90-95% cells with LH and/or FSH antigens. Less than 4% have TSH or PRL antigens, and less than 7% have ACTH antigens. About 15% of the enriched population expressed GH antigens in male rats and nearly 30% of the population express GH in females. This agrees with the known hormone storage overlap between these cells, especially in proestrous female rats. The MTT cell growth/cell death assay was then used to test the mitogenic potential of EGF, GnRH, and activin. This assay showed a linear relationship between plated cell numbers and optical density of the media after the MTT reaction was run. The enriched gonadotropes were plated in 96-microwell trays and grown for 3-4 days in the presence of defined media alone (no serum), or defined media containing 0.5-10 ng/ml EGF, 0.5-1 nM GnRH, 60 ng/ml activin or two of these factors. In all of the 12 experiments, each of the factors stimulated a 3- to 10-fold increase in optical density values, depending on the dose of the stimulating factor. The effects of any two factors were not additive. Because the MTT assays do not discriminate between mitogenic effects and enhanced cell survival, a second group of tests was run with mixed cultures of pituitary cells from diestrous female rats. These cells were cultured in the same combinations of EGF with and without GnRH for 3 h. During the last hour of culture, they were exposed to bromodeoxyuridine (BrDU) to identify cells that were synthesizing DNA. Cells in the S phase were thereby detected with dual immunocytochemical labeling for nuclear BrDU and gonadotropins. The analysis of dual labeled cells showed a 3-fold increase in percentage of LH or FSH cells with BrDU labeled nuclei following EGF or GnRH stimulation. The effects of the two growth factors were not additive. Collectively, these data confirm previous studies showing mitogenic functions for activin and now add EGF and GnRH as mitogens for gonadotropes.