Differentiation of the melanotrophic cells of rat pituitary primordium in organotypic culture in defined medium

Abstract

Organotypic cultures, in defined medium, of pituitary primordia obtained from 15-day-old rat fetuses were performed in order to study the in vitro differentiation of melanotrophic cells. The morphological and ultrastructural features of the transplants resembled those of the gland developing in vivo. In situ hybridization on semi-thin sections, using a 35S-labelled oligonucleotide probe, revealed pro-opiomelanocortin-mRNA-containing cells on the first day of culture in the anterior lobe and after 2-3 days in the intermediate lobe. Immunoperoxidase labelling of adjacent sections showed that the same cells reacted with antibodies against alpha-melanocyte-stimulating hormone (alpha MSH), gamma 3MSH and adrenocorticotropic hormone in both lobes. The pro-opiomelanocortin-mRNA-containing cells formed progressively conspicuous areas in the intermediate lobe, which was almost uniformly labelled after 6 days. In the anterior lobe, these cells remained scattered in small cell groups, and colloidal gold immunolabelling showed the progressive disappearance of alpha MSH labelling from the secretory vesicles in cells exhibiting morphological features of adult corticotrophic cells. Both the alpha MSH content of the explants and alpha MSH release into the culture medium increased with time. Treatment with the dopamine agonist bromocriptine induced a strong dose-dependent decrease in alpha MSH secretion, which was significant after 3 days in culture, indicating that dopamine D2 receptors are able to regulate hormonal release of melanotrophic cells at early stages. This system constitutes a suitable model for further studies of factors controlling cell differentiation and cellular interactions involved in histogenesis.