ULTRASTRUCTURAL STUDIES OF THE PARS INTERMEDIA

Abstract

Various ultrastructural studies were performed on the pars intermedia of the vertebrate pituitary gland. These involved the histochemical localization of adenosine triphosphatase, examination of neurointermediate lobes maintained in long term organ culture, and freeze-fracture studies to locate morphological evidence of exocytosis and intercellular communication. ATPase localizations were carried out with the Wachstein-Meisel technique in the pars intermedia of the frog (Rana berlandieri forreri), rat (Sprague-Dawley), lizard (Anolis carolinensis), and snake (Thamnophis sp.). In all tissues examined, reaction product was almost always localized between membranes of non-parenchymal cells and parenchymal cells, between two non-parenchymal cell membranes, or in other intermembrane spaces bounded on at least one side by a non-parenchymal cell. In the frog, non-parenchymal cells are scattered throughout the pars intermedia and have glial-like processes that may partially encase or separate the secretory cells. In the rat pars intermedia, non-parenchymal cells surround the individual lobules and comprise the posterior epithelial lining. Since the reaction product could be partially inhibited by ouabain, it is suggested that non-parenchymal cells contain an ATPase, possibly a Na⁺K⁺ATPase, which might then allow these cells to serve as regulators of the extracellular environment. Furthermore, since ouabain or removal of potassium is inhibitory to melanophore stimulating hormone (MSH) secretion from isolated pars intermedia in vitro, it is also possible that these cells may play a functional role in the control of MSH secretion. Additional preliminary studies with the more specific potassium dependent paranitrophenyl phosphatase (K⁺-pNPPase) localization were carried out on the frog pars intermedia. However, the procedure had to be modified to obtain a reaction product and the resulting localizations were inconclusive. In the organ culture studies, neurointermediate lobes from the frog pituitary were maintained in media with and without serum for up to six months. The cultured tissue was examined periodically by light microscopy and transmission electron microscopy, and the culture medium was bioassayed for the presence of MSH. Microscopic observations revealed a high degree of preservation of the pars intermedia at four weeks, with isolated aresa of some tissue maintaining histological integrity over the six-month time course. Bioassays showed the glands to be continuously secreting MSH; however, larger yields of hormone were obtained in media lacking serum. Thus, organ culture of the vertebrate neurointermediate lobe may provide a unique method for the production of large quantities of MSH and for studying other melanotropic and opiate peptides as they may be synthesized and secreted by the pars intermedia. The possibility of exocytosis occurring in the pars intermedia was studied by preparing freeze-fracture replicas of the frog neurointermediate lobe. Examination indicated the presence of clearly defined morphological structures characteristic of such a secretory process. These structures include cleavage planes through plasma membrances of parenchymal cells, with frequent bulges devoid of intermembrane particles or regions of continuity between corresponding E or P faces of the plasma and granule membrances where fusion has occurred. The question of intercellular communication in the pars intermedia was also studied by examining the freeze-fracture replicas of the neurointermediate lobe. Initial attempts to find ultrastructural evidence of intercellular communication in thin sectioned material both with and without lanthanum hydroxide had proven unsuccessful. However, cleavage planes through plasma membranes revealed single strands of closely packed intermembrane particles similar to the "diminutive" gap junction previously described in the endocrine pancreas. Thus, the presence of such structures along with the available electrophysiological data indicates coupling between cells of the pars intermedia. Replicas also contained morphological structures characteristic of tight junctions and desmosomes along with small aggregations of intermembrane particles whose functional significance remains unclear.