RESUMO
In the present report, the prolactin secretory pathways were re-examined in cultured lactotrophs submitted to various experimental conditions of stimulation, inhibition and/or alteration of the intracellular flow of the synthesis and release of prolactin. Primary cultures of rat pituitary cells stimulated with thyrotropin-releasing hormone, or inhibited with either cycloheximide or dopamine in the presence or absence of 0.1 microg/ml brefeldin A, were used. The radioimmunoassay quantification of released and intracellular prolactin was correlated with ultrastructural and immunocytochemical studies. Brefeldin A diminished significantly the secretion and the intracellular content of prolactin 4 h after application, while morphological effects were seen starting from 30 min. The drug did not modify the response to thyrotropin-releasing hormone (120% increment). The simultaneous incubation of brefeldin A with cycloheximide or dopamine diminished the released prolactin concomitant with a lower (cycloheximide) or greater (dopamine) hormonal intracellular prolactin content with respect to brefeldin A. The combined treatment cycloheximide-dopamine inhibited prolactin secretion. The ultrastructural and immunocytochemical features of lactotrophs supported these radioimmunoassay data. These results revealed that prolactin release in vitro in the presence or not of brefeldin A is dependent on either: the neosynthesized hormone that can be inhibited by cycloheximide, and the hormone stored in granules, the exocytosis of which was blocked by dopamine, indicates the contribution of both constitutive and regulated pathways in the secretory process. The brefeldin A blockade of the intracellular transport also disclosed morphological evidence of an alternative pathway of prolactin secretion through vesicles originated in the rough endoplasmic reticulum bypassing the Golgi complex.
Assuntos
Adeno-Hipófise/metabolismo , Prolactina/metabolismo , Animais , Brefeldina A/farmacologia , Células Cultivadas , Feminino , Técnicas Imunoenzimáticas , Microscopia Eletrônica , Adeno-Hipófise/efeitos dos fármacos , Adeno-Hipófise/ultraestrutura , Inibidores da Síntese de Proteínas/farmacologia , Radioimunoensaio , Ratos , Ratos WistarRESUMO
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
In the pituitary glands of lactating rats there is a striking proliferation of lactotropes during pregnancy and lactation. After the cessation of lactation, the population of lactotropes experiences an extensive programmed cell death of redundant cells until it reaches prepregnancy levels. The regressive changes occurring in lactotropes are autolytic in nature and usually these cells are engulfed by stellate cells. Other involuting lactotropes are mobilized toward the blood capillaries and some go into the capillary lumen for final disposal, presumably in the mononuclear phagocyte system. In this study we report a sequence of events occurring in regressing lactotropes, investigated by a systematic electron microscope analysis of the rat pituitary gland at different periods after interruption of lactation. Direct disposal of degenerating lactotropes and their cell remnants appears to be an alternative pathway for the clearance of the regressing cells that occur extensively among surplus lactotropes following ablactation.
Assuntos
Lactação/fisiologia , Adeno-Hipófise/citologia , Animais , Morte Celular/fisiologia , Retículo Endoplasmático Rugoso/fisiologia , Retículo Endoplasmático Rugoso/ultraestrutura , Feminino , Complexo de Golgi/fisiologia , Complexo de Golgi/ultraestrutura , Masculino , Microscopia Eletrônica , Fagocitose/fisiologia , Adeno-Hipófise/ultraestrutura , Gravidez , Ratos , Ratos WistarRESUMO
In the pituitary gland of pregnant and lactating rats a striking proliferation of lactotrophs occurs to meet the increased demands for prolactin. Following interruption of lactation the redundant lactotrophs undergo a massive degeneration until pre-pregnant proportions are re-established. Immunocytochemical detection of prolactin allows the recognition of degenerating lactotrophs until advanced stages of degeneration and leads to the conclusion that this process is autolytic in nature. Histochemistry of acid phosphatase reveals a remarkable accumulation of this enzyme in Golgi cisternae and lysosomes. At later stages of degeneration the acid phosphatase spreads throughout the entire cell. The presence of increased numbers of necrotic cells appears to activate phagocytosis of stellate cells and, to a lesser extent, of follicular cells. Stellate cells responsible for the secondary processing of cell residues are isolated cells characterized by a prominent oval nucleus and an electron-lucent cytoplasm with scarce organelles and extensive cytoplasmic processes. They appear as scavenger cells engulfing cell remnants and debris. Immunocytochemistry of S-100 protein discloses differential staining of two types of cell, one forming clusters of 2-4 cells with faint immunoreactivity, while the other type consists of isolated cells with a stellate profile and stronger labelling to S-100 protein.
Assuntos
Lactação/fisiologia , Hipófise/fisiologia , Fosfatase Ácida/metabolismo , Aldeídos , Animais , Citoplasma/enzimologia , Feminino , Imuno-Histoquímica , Lisossomos/enzimologia , Hipófise/citologia , Gravidez , Ratos , Ratos Wistar , Proteínas S100/imunologia , Proteínas S100/metabolismo , Fixação de TecidosRESUMO
The permeability of the materno-fetal barrier in the chorioallantoic placenta was studied in guinea pigs with gestation at term applying a variety of electron-opaque tracers via maternal circulation. None of the tracers tested was seen permeating the syncytiotrophoblast in the materno-fetal direction up to a 20 min interval when the fine structure of the placenta was satisfactorily preserved. The lanthanum chloride, cationized ferritin and horseradish peroxidase bound to the trophoblast surface, apparently due to electrostatic forces more than to specific receptors, and no uptake of these probes was detected in the cytoplasm. Albumin-colloidal gold complex, also used as a tracer, yielded similar results. As reported in other species with more complex syncytiotrophoblastic organization, this layer investing maternal lacunae is a highly selective permeability barrier.