Evelyne Vila-Porcile

The problem of the folliculo-stellate cells in the pituitary gland

In 1953, in one of the first papers dealing with the ultrastructure of the rat anterior pituitary, Rinehart and Farquhar (1) noticed the presence of peculiar stellate cells. In a second report (2), they described ‘certain agranular stellate cells with processes extending between the parenchymal cells’ and they concluded: ‘it may be that they represent an additional structural and functional unit’. Two years later, Farquhar (3) introduced the notion of ‘follicular cell’ in a now famous abstract from Anatomical Record, as ‘a sixth cell type’ located ‘throughout the anterior lobe in groups around follicles or ductules which contain colloid of low density’. Following this short paper, many electron microscopy works have been published, describing either ‘stellate’ cells, or ‘follicular’ cells (i.e. cells lining a follicular cavity) in the pituitary of various species.

Angiotensinogen, Prorenin, and Renin Are Co-localized in the Secretory Granules of All Glandular Cells of the Rat Anterior Pituitary: An Immunoultrastructural Study

In addition to the circulating renin–angiotensin system (RAS), a local system has been postulated in the anterior pituitary because immunodetection of its components in various mammalian species. However, different cell types appear to be involved in different species, and there is no general consensus on the subcellular localization of prorenin, renin and angiotensinogen. In this ultrastructural study, we investigated and quantified the presence of these components using double or triple immunogold labeling methods, in all the immunologically identified glandular cell types of the rat anterior pituitary. In contrast to previous reports, all these components were identified not only in lactotropes and gonadotropes but also in somatotropes, corticotropes, and thyrotropes. The highest levels were detected in lactotropes and gonadotropes, and renin gave the greatest signal. Angio-tensinogen, prorenin, and renin were co-localized in the secretory granules of all rat pituitary glandular cell types. The simultaneous detection of the substrate (angiotensinogen) and both its specific cleavage enzyme and its proenzyme within the same granule suggests intragranular processing of this component. Moreover, the localization of these three constituents in the secretory granules also suggests that, in the rat anterior pituitary, they follow the regulated secretory pathway.

Aggregation of a subpopulation of vimentin filaments in cultured human skin fibroblasts derived from patients with giant axonal neuropathy.

Giant axonal neuropathy (GAN) is a generalized disorder of intermediate filament networks which results in the formation of an ovoid aggregate in a large variety of cell types. We investigated the cytoskeletal organization of cultured skin fibroblasts derived from three GAN patients by indirect immunofluorescence, confocal, and electron microscopy. Whereas the organization of microfilaments seemed normal, the microtubule network appeared disorganized and tangled. The organization of the intermediate filament network, composed of vimentin, was probed with three antibodies directed against different epitopes: two vimentin-specific antibodies, a monoclonal antibody (mAb V9) and a polyclonal antibody, and a serum specific for all type III IFPs (PI serum). These experiments showed that 20% of cultured skin fibroblasts from GAN patients have a vimentin aggregate composed of densely packed filaments which coexists with a well-organized vimentin network. After depolymerization of microtubules with nocodazole, all fibroblasts from GAN patients contained a vimentin aggregate which seemed to arise from a subpopulation of vimentin filaments normally integrated in the vimentin network. Such aggregates were never observed in any condition in control fibroblasts. Moreover, the ultrastructural analysis of GAN cells revealed the presence of swollen mitochondria. We suggest that GAN may be due to a defect in a factor which stabilizes cytoplasmic intermediate filament networks, and we speculate on its identification and properties.

Subcellular distribution of laminin and prolactin in stimulated and blocked prolactin cells in the pituitary of lactating rats

SummaryLaminin (LAM), a glycoprotein component of basement membranes, has been previously detected within several subcellular compartments of prolactin (PRL) cells in the pituitary gland. The present work was aimed at comparing the subcellular localization of PRL, a specific secretory product, with that of LAM, in relation to the secretory activity of PRL cells. LAM and PRL were located in parallel, by ultrastructural immunocytochemistry, in PRL cells of lactating female Wistar rats, either stimulated by suckling, or blocked by weaning, or reactivated by suckle following short-term weaning. Variations in physiological conditions were correlated with a redistribution of PRL immunoreactivity within morphologically modified compartments. The Golgi apparatus became hypertrophied, and PRL impressively accumulated within saccules of the Golgi stacks of blocked cells. On the contrary, no apparent changes occurred in LAM distribution, at least at the Golgi level. Only a slight increase of LAM immunoreactivity was observed in rough endoplasmic reticulum after a long weaning period. PRL could be detected in most of the secretory granules and particularly in forming elements, whereas LAM was observable at the peripheral edge of some mature granules. Such a labeling was not markedly influenced by the physiological state. The prominent structures, indicative of crinophagic activity, characteristic of blocked cells, contained masses of dense material, which were always immunopositive with antibodies to PRL, but never to LAM. These observations could suggest that, in PRL cells, intracellular transport and exportation of LAM are controlled by mechanisms independent from those involved in the regulation of PRL secretion.

STRUCTURAL AND FUNCTIONAL DIFFERENCES BETWEEN PROLACTIN CELLS FROM THE INNER AND OUTER ZONES OF THE MALE RAT ANTERIOR PITUITARY

Abstract.The aim of this study was to compare the ultrastructure of prolactin cells in the inner and outer zones of the male rat anterior pituitary, and to relate their morphological features to their secretory activity, by means of standard and ultrastructural reverse hemolytic plaque assays (RHPA). The immuno-ultrastructural study showed that in the inner pituitary small-granulated cells represented 52% of the prolactin cells, there being only 5% with large granules, whereas the prolactin cells with large granules accounted for 52% in the outer zone, with only 7% being small-granulated. Percentages of cells with intermediate-sized granules were 43% and 41%, respectively. Analysis of RHPA data revealed that, under basal conditions, prolactin cells secreted more actively in the inner zone than in the outer zone. Stimulation with thyrotropin-releasing hormone or KCl treatment increased the percentage of secretors and the sizes of hemolytic plaques in both zones. However, in response to thyrotropin-releasing hormone, the increase in number of secretors was always higher in the outer zone, whereas the enlargement of plaque sizes was greater for the ”inner” cells. These findings are in favor of the small-granulated cells, which predominate in the inner zone, being in a stage of active secretion and responsiveness.

Adaptation of the reverse hemolytic plaque assay to electron microscopy : a study of the individual secretory activity in prolactin cell subpopulations

We used the reverse hemolytic plaque assay (RHPA), a method for detecting secretion by single cells, to demonstrate functional heterogeneity among prolactin (PRL) cells of rat anterior pituitary at the light microscopic (LM) level. We attempted to adapt RHPA for electron microscopy (EM) to define the relationships between fine structure and secretory activity in individual PRL cells. A major modification of the technique, intended to improve preservation of ultrastructure, was allowing the cells to recover after their enzymatic dispersion from the pituitary tissue, through a brief (3-4 hr) culture step before the assay. Adaptation for EM was achieved by the use of plastic slides for construction of specialized Cunningham chambers, permitting application of all the EM procedures (flat embedding, punching of selected areas) usually employed for cultured pituitary monolayers. Moreover, immunocytochemical pre- and post-embedding methods were also applied for cell identification and study of subcellular hormone distribution. Such a modified RHPA enabled us to analyze the ultrastructure of plaque-forming cells surrounded by their companion red blood cell ghosts. The first results with EM RHPA showed that under basal conditions a subpopulation of PRL cells containing small granules (150-200 nm) was actively secreting, whereas PRL cells with large (300-600 nm) and irregular granules appeared to be stimulated by thyrotropin-releasing hormone or KCl. The EM RHPA technique described here might receive more general application and could be utilized for study of many other secretory systems.