RESUMO
In cell cultures of dispersed rat anterior pituitary, the specific identification of each cell type based on their staining properties and the ultrastructural features of secretory granules has proved to be unreliable. The existence of pituitary cell subtypes and the striking remodeling of the cell surface and intracellular organelles, further complicate the specific identification of pituitary cell populations. An immunocytochemical study of dissociated pituitary cells in culture was carried out to identify the cellular hormonal content by applying specific antibodies against prolactin (PRL), and growth (GH), luteinizing (LH beta), adrenocorticotrophic (ACTH) and thyrotrophic (TSH) hormones. Specifically bound IgG was exposed by the electron microscope with protein A-gold complex. Typical lactotrophs, somatotrophs and gonadotrophs are easily recognized because they retain the main features described in the pituitary tissue in situ. Other undefined groups of cells bearing small or medium round secretory granules can be identified by immunocytochemistry as PRL, GH or TSH producing cells. The latter technique was critical for the characterization of the hormonal content of secretory granules, the shape, size, electron density and cytoplasmic distribution of which differ substantially from those described in the intact gland. Cells displaying rare small oval or sharp pointed secretory granules were identified as gonadotrophs with anti-LH beta, while corticotrophs showed granules with irregular profiles not previously reported in the gland. These remarkable morphological changes appear to be related to the interruption of the flow of hypothalamic hormones and the disruption of structural and paracrine interrelationships. This investigation reveals that immunocytochemistry is essential for the specific recognition of the various pituitary cell types, and particularly of atypical cells exhibiting morphological features not found in the pituitary gland in situ.
Assuntos
Adeno-Hipófise/citologia , Hormônios Adeno-Hipofisários/análise , Hormônio Adrenocorticotrópico/análise , Animais , Células Cultivadas , Feminino , Hormônio do Crescimento/análise , Hormônio Luteinizante/análise , Masculino , Microscopia Imunoeletrônica/métodos , Organelas/ultraestrutura , Prolactina/análise , Ratos , Ratos Wistar , Tireotropina/análiseRESUMO
The development and dynamics of thyrotropin (TSH), adrenocorticotropic hormone (ACTH), prolactin (PRL), and growth hormone (GH) cells have been studied using immunocytochemical techniques and rabbit antisera, raised against the relevant human hormone, in the pars distalis of Bufo arenarum larvae at different stages of development. The four types of cells studied were identified in different zones of the pars distalis: TSH cells occurred mainly in the centro-ventral zone, ACTH cells in the rostral and dorsal zones, GH cells in the central and caudal zones, and PRL cells in the anterior two-thirds of the gland. This distribution pattern does not show significant changes with development. Morphometry and stereology were used to evaluate the changes observed in the volume of the pars distalis and the immunoreactive cells during development. The former increased during larval growth and decreased throughout the metamorphic climax. The results obtained on cell number, volume density, and total volume suggest that, during larval growth (pre-prometamorphosis) of B. arenarum, TSH, PRL, GH and ACTH cells show a proliferative period with storage of their hormones; a second period involving hormone release occurs at the metamorphic climax.
Assuntos
Adeno-Hipófise/citologia , Hormônios Adeno-Hipofisários/análise , Hormônio Adrenocorticotrópico/análise , Animais , Bufo arenarum , Feminino , Hormônio do Crescimento/análise , Imuno-Histoquímica , Metamorfose Biológica/fisiologia , Adeno-Hipófise/química , Adeno-Hipófise/crescimento & desenvolvimento , Prolactina/análise , Tireotropina/análiseRESUMO
A simple technique for determination of the molecular (Stokes) radius of radioiodinated proteins was developed using the same column and chromatographic conditions employed in routine radioimmunoassay tracer purification. The calibration curve for five radioiodinated standard proteins presented a highly significant correlation (r = -0.996; P less than 0.001) and allowed precise molecular radius determination for labeled human growth hormone (hGH), luteotropin (hLH), follicle-stimulating hormone (hFSH), thyrotropin (hTSH), prolactin (hPRL), and corticotropin (hACTH), enabling detection of differences of the order of +/- 3%. The validity of the method was verified by determining the molecular radius of hGH in both "cold" (unlabeled standards and unknowns) and "hot" (radioiodinated standards and unknowns) systems. The technique can be applied in a very simple manner, requiring just one simple additional calibration run before Sephadex G-100 tracer purification. Furthermore, it can be applied to any protein, even when only extremely limited amounts are available. Since the standards and unknowns are labeled and chromatographed under identical conditions, potential common alterations of the molecule due to oxidation, iodine incorporation, tracer-carrier interactions, etc., are automatically corrected for.