Scanning electron microscopy (SEM) observation showed that fully differentiated spherical adipocytes were embraced by a network of collagens and fibroblastic preadipocytes. The properties of both the collagen networks and the preadipocytes allow the adipocytes to be interconnected, forming a fat-cell cluster, which can anchor to the bottom of a culture dish. In this network structure, collagen fibrils and fibrillar bundles were closely arranged and stratified. We found that immunostained collagens appeared to form extracellular network structures, which can be observed by SEM. The extracellular network of fibronectin was the first to develop among the extracellular matrix (ECM) components, though it became degraded with the progress of adipocyte differentiation. The type I collagen network was the last to develop and remained well organized through the late stage of adipocyte differentiation. The extracellular networks of type III, V, and VI collagen developed by the mid-stage and remained in the late stage of adipocyte differentiation. The network structures of type IV collagen and laminin became degraded during the differentiation process and localized at the surface of spherical cells. In addition to these basement membrane components, types III, V, and VI collagens also showed pericellular spherical staining patterns. These results demonstrated that the constitution and distribution of the ECM are altered during adipocyte differentiation, suggesting that the organization of each ECM component into a suitable structure is a requirement for the differentiation and maintenance of unilocular adipocytes.

We examined the topographic cell affinity of different types of hormone-producing cells in the rat anterior pituitary gland. Pituitaries were removed from 8-10 week-old male SD rats, and were fixed in sublimate-formalin. The 40 mm sections were obtained with a Microslicer, then double-immunolabeled (FITC and TexasRed methods) using antibodies against five types of pituitary hormones. Topographic affinity was examined threedimensionally using a confocal microscope, and was expressed as attachment rate. Statistical analysis revealed that the different types of hormoneproducing cells did not exist at random locations in the gland, but rather showed specific topographic affinities. The following pairs of cell types were observed to have significantly high affinities: LH and PRL cells, ACTH and GH cells, and TSH and GH cells. In contrast, the following pairs of cell types were observed to have no significant affinities: GH and PRL cells, ACTH and LH cells, TSH and LH cells, ACTH and TSH cells, and cells of the same type. These specific topographic affinities between different types of anterior pituitary cells may be intimately related to cell-to-cell interactions in the gland.

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Hormone-producing cells in the rat anterior pituitary gland are not randomly distributed; rather, there are specific topographic affinities among five cell types (Noda et al., Acta Histochem. Cytochem. 2001;34:313-319). In this study we reconstructed these affinities, at least partially, in primary monolayer culture. Pituitary cells collected from adult male rats were enzymatically dispersed and cultured for 72 hr at a density of 1 ϫ 10 5 cells/cm 2 . We double-immunostained cells using antibodies against hormones, and then used confocal laser microscopy to examine the ability of the cells to attach to each other. We also statistically analyzed the affinity of all combinations of the five types of hormone-producing cells. We observed clusters by electron microscopy to identify junctional complexes between the cells. Confocal laser microscopy indicated that the features and attachment patterns of hormoneproducing cells in vivo were similar to those in vitro. Statistical analyses revealed that the rates at which the five types of hormone-producing cells attached to growth hormone (GH)-, prolactin (PRL), and luteinizing hormone (LH)-producing cells were unequal, which suggests there are specific topographic affinities. The specific rates of adrenocorticotropic hormone (ACTH)-producing cell attachment to GH cells, LH to PRL cells, and PRL to LH cells were high, whereas that of PRL attachment to PRL cells was low. In addition, the rates correlated with the data from our previous in vivo study. Ultrastructural observations revealed few junctional complexes between hormone-producing cells. These results indicate that anterior pituitary hormone-producing cells can attach to specific types of cells by means of specific and/or nonspecific adhesion factors, and can reconstruct the topographic nature of the pituitary gland. Anat Rec Part A 272A: 548 -555, 2003.