Functional Maturation of Somatotropes in Fetal Rat Pituitaries: Analysis by Reverse Hemolytic Plaque Assay*

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Abstract
Immunocytochemistry (ICC) and a reverse hemolytic plaque assay for GH [growth hormone] were used to investigate the temporal relationships between the initiation of hormone storage and release by developing somatotropes and the onset of responsiveness of these cells to stimulatory and inhibitory secretagogues. Anterior pituitaries obtained from rats on days 18-21 of fetal development (pups were generally delivered on fetal day 22, which is equivalent to day 0 of neonatal life) were monodispersed with trypsin, cultured for 24 h, and then subjected to reverse hemolytic plaque assay and/or ICC for GH. GH-containing cells (determined by ICC) were extremely rare (< 1%) in cultures derived from day 18 fetuses, but accounted for 22.4%, 25.2%, and 24.5% of all cells in cultures from day 19-21 fetuses, respectively. The proportion of GH-releasing cells, as determined in a long term (120-min incubation with antibody) plaque assay, was < 1%, 22.4%, and 22.9% for days 18, 20, and 21, respectively, but only 13.6% for day 19 cells. Thus, many pituitary cells from day 19 fetuses contained, but did not release, GH. While GH-releasing factor (1-44) (1 .times. 10-7 M) had no effect on the percentage of GH plaque-forming cells in long term incubations, it enhanced (by approximately the same degree in day 19-21 groups) the percentage of cells that formed plaques and the size of the plaques in short term (45-min) incubations with antibody. Somatostatin (1 .times. 10-7 M) exerted inhibitory effects on these variables when tested in long term incubations, and age of the donor rats did not influence pituitary responsiveness to this secretagogue. These results suggest that the capacities of fetal somatotropes to store GH and release it under basal and regulated conditions are attained, in large part, within an extremely narrow time frame between days 18 and 19 of fetal development.