Heterogeneity in mammotrophs prepared from diethylstilbestrol-induced prolactinomas

Pituitary glands from ovariectomized F-344 rats bearing diethylstilbestrol-containing capsules for 70-100 days were dissociated and separated into three size classes by unit gravity sedimentation. The average percentage of large mammotrophs in fraction I was 86 +/- (SE) 2%, the percentage of intermediate-sized mammotrophs in fraction II was 86 +/- 4%, and the percentage of small mammotrophs in fraction III was 72 +/- 3%. By electron microscopy, cells in fractions I and II contained cytoplasmic secretory granules, whereas those in fraction III were either sparsely granulated or agranular. Cells retained their morphology after culture. Biological (B) and immunological (I) assays of PRL released into the medium after 24-h culture both showed that fraction I and II cells released more hormone than fraction III cells. The B/I ratios of released PRL were consistently greater than 1. Analysis of the separated mammotrophs by flow cytometry indicated major differences in light scatter profiles, both before and after culture. Since these differences have been shown to reflect secretory granule content, changes in perpendicular light scatter after culture were interpreted to indicate that small mammotrophs accumulated granules, whereas some of the large mammotrophs lost granules. Results from a cell blot assay indicated that fraction I and II cells secreted more hormone than fraction III cells. This same rank order was found when live cells were stained and analyzed for surface PRL. However, the reverse plaque assay yielded different information, viz. more plaques were found around fraction III cells than in fraction I or II cells. Finally, PRL contained in alkaline extracts of fraction I, II, and III cells was analyzed by Western blotting after electrophoresis under nondenaturing, denaturing, or denaturing and reducing conditions. Fraction III cells contained a prominent and unique PRL variant that had low mobility in native gels and an apparent mol wt in the range of 10-14K. After 24 h the culture medium from fraction III cells also contained this low mol wt variant. Our data indicate considerable heterogeneity in form and function within the mammotroph population of diethylstilbestrol-induced prolactinomas.