Two types of mammosomatotropes in mouse adenohypophysis

Summary Two types of mammosomatotropes (MS), the small-granule and vesicle-granule MS, were detected in mouse adenohypophysis by electron microscopy and immunohistochemistry. Both cell-types were immunoreactive to prolactin (PRL) and growth hormone (GH) antisera. The small-granule MS contained small, round, solid secretory granules about 100 nm in diameter, and were smaller than the classical GH and PRL cell-types. The vesicle-granule MS contained secretory granules like cored vesicles, and were larger than classical GH and PRL cells. Small-granule MS were immunoreactive to both PRL and GH antisera in the same region of the cell cytoplasm; the vesicle-granule MS, however, were immunoreactive to only PRL antiserum in most cytoplasmic areas, and a positive response to both PRL and GH antisera was confined to only certain small areas.     

Adenohypophysial cell types in the lamprey pituitary: current state of the art

Adenohypophysial cell types in the pituitary of adult sea lampreys, Petromyzon marinus, was localized by means of immunocytochemical and lectin cytochemical techniques. At least four types of adenohypophysial hormone cells are present in the pituitary of adult sea lampreys. The first type of cell is ACTH-like and occupies most parts of the rostral pars distalis (RPD), but a few scattered ACTH-like cells are also present in the proximal pars distalis (PPD). The second type of cell is MSH-like and occupies the whole pars intermedia. The third type of cell is GH/PRL-like and occupies the dorsal half of the PPD. These GH/PRL-like cells were initially detected by heterologous immunocytochemistry using antibodies to salmon GH, salmon PRL and blue shark GH, after hydrated autoclave pretreatment of sections. Later, by use of an antiserum raised against a synthetic peptide corresponding to the partial sequence of lamprey GH/PRL, the same cells as those containing GH/PRL-like immunoreactivity were stained positively. Similarity of the topographic distributions between lamprey GH/PRL-like cells and gnathostome fish GH cells in the pituitary suggests that GH/PRL-like cells in the lamprey may be GH cells. The last type of cell is GTH-like and occupies the ventral half of the PPD. Although GTH has not yet been isolated from the lamprey pituitary, our immunocytochemical data suggest that GTH-like material in the sea lamprey pituitary is more closely related to mammalian-like LH, rather than to FSH or TSH. These four types of adenohypophysial cells occupy most parts of the lamprey adenohypophysis and indeed there is little room for TSH or PRL cells. Thus, the present study further suggests that GH and LH-like GTH are ancestral forms of GH/PRL/SL family and glycoprotein hormones, respectively.     

Immunocytochemical identification and ontogeny of adenohypophyseal cells in a cave fish,Phreatichthys andruzzii (Cypriniformes: Cyprinidae)

The morphogenesis of the pituitary gland and the chronological appearance of adenohypophyseal cells were investigated for the first time in the Somalian cave fish Phreatichthys andruzzii by immunocytochemistry. The adult adenohypophysis contained: a rostral pars distalis, with prolactin (PRL) cells arranged in follicles and adrenocorticotropic (ACTH) cells, a proximal pars distalis with somatotropic (GH), β‐thyrotropic (TSH), β‐gonadotropic type I (FSH) and type II (LH) cells and a pars intermedia with α‐somatolactin (SL), α‐melanotropic (MSH) and β‐endorphin (END) cells. All regions were deeply penetrated by neurohypophyseal branches. At hatching (24 h post‐fertilization) the pituitary was an oval cell mass, close to the ventral margin of diencephalon. The first immunoreactive cells appeared as follows: PRL at 0·5 days after hatching (dah), GH and SL at 1·5 dah, END at 2 dah, TSH, ACTH and MSH at 2·5 dah, FSH at 28 dah and LH at 90 dah. The neurohypophysis appeared at 5 dah and branched extensively inside the adenohypophysis at 130 dah, but there was no boundary between rostral pars distalis and proximal pars distalis at this stage. The potential indices of prolactin and growth hormone production increased until 28 and 60 dah, respectively. The potential index of growth hormone production correlated positively with total length. Activity of PRL and GH cells, measured as ratio of cell area to nucleus area, was significantly higher in juveniles than in larvae.     

Immunocytochemical characterization of adenohypophyseal cells in the greater weever fish (Trachinus draco)

The adenohypophysis of the greater weever fish (Trachinus draco) was studied using histochemical and immunocytochemical methods. The adenohypophysis comprised the rostral pars distalis (RPD), the proximal pars distalis (PPD), and the pars intermedia (PI). Neurohypophysis showed a patent hypophyseal stalk which was divided into several branches intermingled with the adenohypophysis. Salmon prolactin (PRL)-immunoreactive (ir) cells, arranged in follicles, resided in the RPD and the most rostral part of the ventral PPD. Human adrenocorticotropin (ACTH)-ir cells were located in the RPD between PRL-ir cells and the neurohypophyseal processes. Salmon and seabream somatotropin (GH)-ir cells were located in both the dorsal and the ventral PPD. Some GH-ir cells were seen in surrounding and in contact with neurohypophyseal branches, whereas other isolated or clustered GH-ir cells were embedded in adenohypophyseal cells of the PPD. In addition, isolated or clustered GH-ir cells were also detected in the tissue of the PPD covering the most rostral part of PI. Only one class of salmon and carp gonadotropin (GTH)-ir cells was detected. Isolated or clustered GTH-ir cells resided in both the dorsal and the ventral PPD and were seen surrounding the PI and in the tissue of the PPD covering the most rostral part of PI. In addition, a few scattered GTH-ir cells were observed in the ventral RPD. Scattered groups of thyrotropin (TSH)-ir cells were present in the anteroventral PPD. Salmon and seabream somatolactin (SL)-ir and bovine melanotropin (MSH)-ir cells were intermingled surrounding the neurohypophyseal tissue. SL-ir cells were negative to periodic acid-Schiff technique. MSH-ir cells showed a very weak immunoreactivity to anti-human ACTH((1-24)) serum. In addition to the PI location, few isolated or clustered SL- and MSH-ir cells were observed in the dorsal PPD.     

Immunocytochemical identification of growth hormone cells in the adenohypophysis of the golden hamster

Somatotrophs or growth hormone (GH) cells in the adenohypophysis of golden hamsters were identified by immunocytochemical staining with polyclonal rabbit anti-human GH. They were oval or columnar in shape, and had secretory granules of two size ranges, 90-150 nm and 280-320 nm, which were present in the same cells; no subtypes of GH cells were observed. Secretory granules were located in the peripheral portion of the cytoplasm or concentrated at the vascular pole of the cell. Flattened cisternae of the rough endoplasmic reticulum in parallel array and a moderately developed Golgi apparatus were often found in the cytoplasm. No sex difference was noticed in the population ratio of GH cells. Immunocytochemical staining with anti-GH or anti-prolactin (PRL) antibodies on separate adjacent sections revealed that the GH and PRL were stored in two different cell types.     

Epidermal growth factor, insulin, and estrogen stimulate development of prolactin-secreting cells in cultures of GH3 cells

Pituitary tumor GH3 cells synthesize and secrete both growth hormone (GH) and prolactin (PRL). Morphological and functional changes of GH3 cells induced by epidermal growth factor (EGF, 10 nM), insulin (300 nM), and estradiol-17beta (E2, 1 nM) were studied. Treatment of cultures of GH3 cells for 6 days with EGF, insulin, or E2 alone, and with EGF plus E2 did not affect the total number of GH3 cells, but a combination of EGF, insulin, and E2 decreased the total number of GH3 cells compared with control treatment. DNA-synthesizing cells were detected by monitoring 5-bromo-2'-deoxyuridine (BrdU) uptake. EGF, E2, or a combination of EGF, insulin, and E2 significantly decreased the proportion of BrdU-labeled cells (21.1+/-1.7%, 21.0+/-1.4%, 18.2+/-1.3%; P<0.05, P<0.05, P<0.01, respectively) compared with control treatment (28.6+/-1.5%), but insulin did not (31.4+/-2.4%). Immunocytochemical analysis of GH3 cells cultured in 5% fetal calf serum-supplemented medium (control) showed that about 70% of all GH3 cells were GH-immunoreactive cells (GH-ir cells), apparently containing only GH, and 14% were mammosomatotrophs (MS cells), containing both GH and PRL, while PRL-immunoreactive cells (PRL-ir cells), containing only PRL, were not detected. No GH or PRL immunoreactivity could be detected in the remaining cells (15%). EGF decreased the proportion of GH-ir cells. The effects of EGF were enhanced by simultaneous exposure to insulin and E2; this decreased the proportion of GH-ir cells to about 20% of the total GH3 cells and significantly increased the proportion of MS cells to 300% of controls. Treatment with EGF plus insulin, EGF plus E2, or a combination of EGF, insulin, and E2 all stimulated the appearance of PRL-ir cells. Exposure to EGF caused a significant decrease in GH mRNA (P<0.01) and a significant increase in PRL mRNA (P<0.05). These observations suggest that EGF is closely involved in differentiation of PRL-ir cells from GH-ir cells via MS cells in GH3 cell cultures. Cytosine arabinoside (10(-7) M), an inhibitor of cell division, did not affect the changes in proportion of the three cell types induced by treatment with a combination of EGF, insulin, and E2. It is therefore probable that the transdifferentiation does not require mitosis of the GH3 cells.     

Immunocytochemical and ultrastructural characterization of mammosomatotrope-, growth hormone-, and prolactin-cells from the gilthead sea bream (Sparus aurata l., Teleostei): an ontogenic study

Growth hormone (GH), prolactin (PRL), and mammosomatotrope (MS) cells of gilthead sea bream, Sparus aurata, a teleost fish, were studied in specimens from hatching to 15 months (adults) using conventional electron microscopy and an immunogold method using anti-tilapia GH sera and anti-chum salmon PRL serum. MS cells, immunoreactive to both anti-GH sera and anti-PRL sera, had been first identified in fish in a previous study in newly hatched larvae and in older larvae and juvenile specimens of Sparus aurata by light microscopic immunocytochemistry. In the present work, MS cells reacted positively to immunogold label only in older larvae and juveniles and their secretory granules immunoreacted with both GH and PRL antisera or with only one of them. MS cells were ultrastructurally similar to the PRL cells, with which they coincided in time. This is the first report on the ultrastructural characterization of MS cells in fish. In adults, the secretory granules of GH cells (immunoreactive to anti-GH serum) were mainly round, of variable size, and had a homogeneous, highly electron-dense content. Irregularly shaped secretory granules were also present. PRL cells (immunoreactive to anti-PRL serum) were usually observed in a follicular arrangement; they showed few, small, and mainly round secretory granules with a homogeneous and high or medium electron-dense content. Some oval or elongated secretory granules were also observed. GH and PRL cells that showed involutive features were also found. In newly hatched larvae, GH, PRL, and MS cells could not be distinguished either by their ultrastructure or by the immunogold labeling of the secretory granules. In 1-day-old larvae, presumptive GH and PRL cells were observed according to their position in the pituitary gland. In 2-day-old larvae, a few cells showed some of the ultrastructural features described for GH and PRL cells of adults. During development, the number, size, and shape of the secretory granules in both cell types clearly increased and the organelles developed gradually. Some GH cells were found undergoing mitosis.     

Detection of mammosomatotroph cells and identification of the coexistence of growth hormone and prolactin within the same secretory granules in these cells using confocal laser scanning microscopy

This study was designed to examine whether mammosomatotroph cells (MS cells) can be easily detected using confocal laser scanning microscopy (CLSM) and whether the coexistence of growth hormone (GH) and prolactin (PRL) within the same secretory granule can be identified in the MS cell using CLSM. Conventional epoxy resin-embedded tissues of mixed GH- and PRL-secreting human pituitary adenomas were used for this double-labelling immunofluorescent study by CLSM. A semithin section of the tissue after plastic removal and bleaching was immunohistochemically double-stained with primary antibodies against GH and PRL, followed by secondary antibodies conjugated with Rhodamine (GH) and FITC (PRL). MS cells simultaneously showing fluorescence of both Rhodamine and FITC were easily detected by CLSM at lower magnification. At higher magnification, the coexistence of Rhodamine and FITC on the same secretory granule was identified by using a superimposed display. This finding was confirmed by immunoelectron microscopy. The CLSM technique may be useful for the study of MS cells.     

Subcellular distribution of secretogranins I and II in GH3 rat tumoral prolactin (PRL) cells as revealed by electron microscopic immunocytochemistry

The GH3 rat pituitary cell line which secretes prolactin (PRL) is characterized by the paucity and small size of secretory granules. We looked for the presence, in these cells and in normal PRL cells, of two acidic tyrosine-sulfated proteins which are widely distributed in dense-core secretory granules of endocrine and neuronal cells, secretogranins I and II, using immunofluorescence and electron microscope immunoperoxidase techniques. Both secretogranins were detected in secretory granules of GH3 cells and of normal cells. Moreover, with our pre-embedding approach, secretogranins were localized within some RER cisternae and within all sacules of the Golgi stacks in both PRL cell models. A few small vesicles, large dilated vacuolar or multivesicular structures, and some lysosome-like structures were also immunoreactive. Double localization of secretogranins and PRL performed on GH3 cells by immunofluorescence indicated that all cells contained secretogranins I and II, whereas only 50-70% of the cells contained PRL. Moreover, in the case of hormone treatment known to increase the number of secretory granules, most if not all mature secretory granules were immunoreactive for secretogranins, whereas in certain cells some of the granules were apparently not immunoreactive for PRL. These immunocytochemical observations show that GH3 cells, which under normal conditions form only a small number of secretory granules, produce secretogranins and package them into these granules.     

Induction of mammotroph development by a combination of epidermal growth factor,insulin, and estradiol-17beta in rat pituitary tumor GH3 cells

Several reports have indicated that prolactin-secreting cells (PRL cells) are generated from growth hormone-secreting cells (GH cells). We have shown that treatment with a combination of epidermal growth factor (EGF), insulin, and estradiol-17beta (E (2)) induces the appearance of PRL cells in pituitary tumor GH3 cells. The aim of the present study was to clarify the involvement of mitosis in the cytogenesis of PRL cells in rat pituitary and GH3 cells. The effects of the treatment with EGF, insulin and E(2) on DNA-replication were studied by detecting the uptake of bromodeoxyuridine (BrdU) into the nucleus. In cultured rat pituitary cells, BrdU-labeled PRL cells were observed irrespective of the hormone treatment. In GH3 cells, BrdU-labeled GH cells and mammosomatotrophs (MS cells) were detected; BrdU-labeled PRL cells were not detected, however, when GH3 cells were treated with BrdU for 3 hr and then immediately examined for BrdU-labeling. BrdU-labeled PRL cells were found only when GH3 cells treated with BrdU were allowed to grow for another 3 days. This finding suggests that during the additional 3-day culture, BrdU-labeled PRL cells were generated from BrdU-labeled cells other than PRL cells. These results indicate that PRL cells are transdifferentiated from GH cells or MS cells in GH3 cells by a combined treatment with EGF, insulin and E(2), while PRL cells in rat pituitaries are able to proliferate in response to the hormone treatment. Thus, there may be two pathways for cytogenesis of PRL cells: the transdifferentiation of GH cells or MS cells, and a self-duplication of PRL cells.     

Development and retention of phenotypically specialized cells in pituitary allografts in the hamster (Mesocricetus auratus)

Summary We used immunohistochemistry to identify cells present in pituitary allografts in the hamster. Hypophyses removed from neonatal hamsters or adenohypophyses removed from adult females were placed beneath renal capsules of hypophysectomized adult females. Serum PRL, LH, and GH concentrations were measured at two, five, and eight weeks after placement of allografts. Allografts were removed after eight weeks and stained for cells containing PRL, LH, FSH, GH, or ACTH. Allografts did not release LH or GH. Those of adult adenohypophyseal tissue released significantly more PRL. The morphology of allografts of neonatal hypophyseal tissue resembled that of the adult adenohypophysis in situ. Lactotrophs, corticotrophs, somatotrophs and LH-cells were observed; very few FSH-cells were present. Allografts of adult adenohypophyseal tissue contained pituitary cells, numerous cavities, often enclosing lymphoid cells, and fibrous tissue. Atypical lactotrophs were the numerically dominant cells in these allografts; all other cells were present. The LH-cells outnumbered FSH-cells. These observations suggest that: (a) development of normal adenohypophyseal morphology can occur in an ectopic position; (b) intracellular hormones are present in cells in an ectopic site; (c) development and retention of intracellular FSH is more dependent on occupation of the normal position of the adenohypophysis than is retention of intracellular LH; and (d) release of PRL occurs from atypical cells in allografts of adult adenohypophyseal tissue.     

The mammosomatotroph: a transitional cell between growth hormone and prolactin producing cells? An immunocytochemical study

In this report the occurrence of mammosomatotroph (MS) cells was correlated with changes in the somatotroph population of adult rat pituitary gland submitted to various experimental conditions (ovariectomized, orchidectomized and intact males, and after treatment with oestradiol benzoate). Cell and volume density of somatotrophs were assessed in sections stained with the immunogold-silver enhancement technique. Mammosomatotrophs were identified by double immunogold labelling at the electron microscopic level. Colocalization of prolactin (PRL) and growth hormone (GH) in the same cell was rarely observed. Only a few MS cells (0.1–0.2% of all parenchymal cells) were found in some experimental models. Oestrogen treatment decreased both cell and volume density of somatotrophs in ovariectomized rats. In this model, serum GH increased significantly but no changes in the pituitary content of the hormone were observed. Our results demonstrate that MS cells are an uncommon cell type in the pituitary of adult ovariectomized, orchidectomized and intact male rats. The oestrogen treatment, which is well known to induce proliferation of lactotrophs, has no effects on the MS population. Data presented in this report do not support the suggested role for mammosomatotrophs as transitional cells in the presumptive interconversion of PRL and GH producing cells.     

Immunocytochemical studies on the pituitary pars distalis of the Japanese long-fingered bat,Miniopterus schreibersii fuliginosus

Summary Immunocytochemical studies were performed to describe the characteristics of cell types and their distribution in the pars distalis of Japanese long-fingered bat, Miniopterus schreibersii fuliginosus, collected at various stages of the reproductive cycle. Six distinct cell types have been identified in the pars distalis by the unlabeled immunoperoxidase technique and by the ABC method. Growth hormone (GH) and prolactin (PRL) cells were immunostained with antisera against chicken GH and ovine PRL. The GH-immunoreactive cells were round or oval orangeophilic cells distributed throughout the pars distalis with prominent aggregation in the posterolateral region. The PRL cells were pleomorphic carminophilic cells that occurred in small groups within the central and dorsocaudal regions of the pars distalis. They were sparsely distributed in the central region of the pars distalis in the hibernating bats, but increased significantly in the pregnant and lactating bats. The adrenocorticotropic (ACTH) cells were large round or polygonal amphophilic cells in the rostroventral and ventrolateral regions of the pars distalis. The thyrotropic (TSH) cells were small rounded or polygonal and distributed mainly in the ventrolateral region of the pars distalis. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) cells were identified immunocytochemically with antisera against the specific beta subunits of ovine LH and rat FSH. There were two populations of LH and FSH cells, one aggregated in the zona tuberalis and the other scattered singly throughout the rest of the pars distalis. The aggregated cells were immunoreactive with both antisera directed to LH and FSH, while scattered cells were reactive solely with antiserum to either LH or FSH and exhibited seasonal variations. In females, the proportional volume of the pars distalis occupied by LH cells was significantly reduced during pregnancy and lactation. No evidence of involution was observed in pars distalis cells except for PRL cells in males or females during hibernation.     

Pituitary lactotrophs and somatotrophs in pregnancy: a correlative in situ hybridization and immunocytochemical study.

Lactotroph hyperplasia is a prominent finding in the adenohypophyses of pregnant women. In order to elucidate the morphogenesis of this change, pituitaries from 16 women in various phases of pregnancy were collected at autopsy and studied by histology, immunocytochemistry and in situ hybridization. The results showed that the increase in the amount of prolactin (PRL) mRNA paralleled the progressive lactotroph hyperplasia. The presence of mitoses in PRL-immunoreactive cells provided evidence that proliferation of preexisting lactotrophs contribute to lactotroph accumulation. Growth hormone (GH) immunoreactive cells showed a marked reduction in GH mRNA indicating that GH synthesis was inhibited. In many GH-immunoreactive cells, PRL mRNA became apparent. These findings demonstrate that GH is stored following discontinuation of GH synthesis. It appears that, when PRL is secreted in excess during pregnancy, somatotrophs are recruited to produce PRL. These somatotrophs begin to express PRL mRNA, transform to bihormonal mammosomatotrophs and possibly later to lactotrophs, contributing to PRL production. Mature somatotrophs may be regarded as reserve cells in the adenohypophysis, having the potential to switch hormone synthesis and to convert to mammosomatotrophs and possibly lactotrophs.     

Distribution of immunoreactivities for adenohypophysial hormones in the pituitary gland of the polypteriform fish, Polypterus endlicheri

Polypteriform fish constitutes the most primitive living descendent of the ancient bony fish. In polypteriform fish, only proopiomelanocortin (POMC) has been identified so far in the adenohypophysis, which is surprising in view of their evolutionary importance. In the present study, distribution of immunoreactive adenohypophysial hormones was examined in juvenile individuals of Polypterus endlicheri. Antisera to tetrapod and fish adenohypophysial hormones were used as immunostaining probes. Adrenocorticotropin (ACTH)-like cells were detected by antisera to salmon POMC N-terminal peptide, porcine ACTH and mammalian alpha-melanotropin (MSH), and were distributed in the rostral pars distalis in close proximity to the hypophysial duct. MSH-like cells were found in the pars intermedia, and were stained by anti-salmon N-Ac-beta-endorphin II as well as anti-mammalian alpha-MSH and anti-salmon POMC-N terminal peptide. Prolactin (PRL)-like cells were detected only after application of anti-sturgeon PRL, and were distributed in the rostral pars distalis, where PRL-positive material was found in columnar mucinous cells lining the diverticuli of the hypophysial duct. Growth hormone (GH)-like cells were stained with antisera to sturgeon GH, human GH, salmon GH and blue shark GH, and were distributed in the proximal pars distalis. Somatolactin (SL)-like cells were stained with anti-salmon SL, and were distributed in the pars intermedia. Two types of glycoprotein hormone-positive cells were detected in the proximal pars distalis. Although both types of cells were stained with several antisera to glycoprotein hormones, such as sturgeon LHbeta and salmon LHbeta, it was difficult to know which types of cells produce LH, FSH, or TSH. Thus, the present study revealed seven types of adenohypophysial hormone-like cells in the Polypterus pituitary gland, which may provide the morphological basis for better understanding on evolution of the pituitary gland and the adenohypophysial hormones in vertebrates.     

Regulation of chromogranin B/secretogranin I and secretogranin II storage in GH4C1 cells

GH4C1 cells are a rat pituitary tumor cell strain in which the level of cellular prolactin (PRL) and PRL-containing secretory granules can be regulated by hormone treatment. The chromogranins/secretogranins (Sg) are a family of secretory proteins which are widely distributed in the secretory granules of endocrine and neuronal cells. In the present study, we investigated in GH4C1 cell cultures the regulation of the cell content of the Sg by immunoblotting and the relationship between the storage of Sg I and Sg II and PRL by double immunocytochemistry. GH4C1 cells grown in the presence of gelded horse serum, a condition in which these cells contain a low level of secretory granules, contained low levels of PRL, Sg I, and Sg II. Treatment of GH4C1 cells with a combination of 17 beta-estradiol, insulin, and epidermal growth factor for 3 days, known to induce a marked increase in the number of secretory granules, increased the cell contents of PRL, Sg I, and Sg II. To determine whether the induction of PRL was morphologically associated with that of the Sg, the distribution of PRL and the Sg was determined by double immunofluorescence microscopy. After hormone treatment, 54% of cells showed positive PRL immunoreactivity, fluorescence being extranuclear and consistent with staining of the Golgi zone and secretory granules. Forty-six percent of PRL-positive cells stained coincidently for Sg I, while 72% of the PRL cells were also reactive with anti-Sg II. To determine whether PRL storage was associated with storage of at least one of the Sg, cells were stained with anti-PRL and anti-Sg I and anti-Sg II together. Eighty-six percent of PRL cells stained for one or the other of the Sg. Therefore, PRL storage in GH4C1 cell cultures is closely but not completely associated with the storage of Sg I and/or II.     

A continuous lineage of rat adenohypophysis stromal cells: characterisation and effects on GH(3)B(6) prolactin-secreting cell behaviour

Although some studies have shown a possible modulation of the stroma on the hormonal secretion, it is not clear as to what are the requirements for these cellular interactions. In the present work, a homogeneous and continuous lineage of rat adenohypophysis stromal cells (APS9 cells) obtained from rat adenohypophysis primary culture was established. Using immunocytochemical methods and electron microscopy, we have characterised APS9 cells as elongated fibroblastoid-like cells with intercellular contacts, expressing alpha-smooth muscle actin, type IV collagen and laminin. By biochemical procedures, higher amounts of chondroitin sulphate and heparan sulphate were found in the pericellular and extracellular compartments of APS9 cell culture. In order to evaluate the possible effects of APS9 cell on GH(3)B(6) prolactin-secreting cell survival and/or proliferation, we established co-culture and proliferation assays. When GH(3)B(6) cells were cultivated on APS9 cell substrate, they displayed an organisation of many cellular cords strongly attached and covering all the stromal cell area, establishing punctual interactions or extensive surface associations between adjacent cells. Prolactin immunoreactivity appeared to be more scattered throughout the cytoplasm and accumulated in its periphery. When plated on glass coverslips, on newborn rat skin fibroblasts, on murine haematopoietic bone marrow stroma cell line or on murine foetal liver stroma cell line, GH(3)B(6) cells changed their organisation and presented a decrease in cell number and adherence to the substrate. Our results showed that the APS9 cell/GH(3)B(6) cell interactions favour cell growth and probably PRL secretion, and raises questions about the specificity of different organs and/or animal species stromas on the hormone secretion.     

Immunocytochemical study of the GH cells in the anterior pituitary gland of human fetus II. Anencephalic fetus

In order to elucidate the effects of hypothalamic regulation on the morphology of GH cells, light and electron microscopic immunocytochemical examinations were carried out comparing GH cells in the anterior pituitary gland of anencephalic fetus with those of normal fetuses. Three types of GH cells were identified in the anterior pituitary gland of anencephalic fetus as well as in the normal fetus. Type-I is a small, round cell containing a few small secretory granules. Type-III is a large, polygonal cell with numerous large secretory granules. Type-II is a polygonal cell with medium-sized secretory granules. The Type-II GH cell was predominant in both anencephalic and normal fetuses. The most striking difference between anencephalic and normal fetuses was the presence of atypical forms of the Type II cell. These were polygonal cells containing secretory granules, which were either immunopositive or immunonegative to anti-human GH (anti-hGH) serum. Furthermore, two other types of GH cells were identified. The somatomammotroph (SM cell) contained GH and PRL in different granules within the same cell. Also, a different type of the GH cell was noted containing two varieties of secretory granules; one was immunolabeled only with anti-hGH and the other was not immunolabeled to either anti-hGH or anti-human PRL (anti-hPRL). From these results, we suggest that an absence of hypothalamic regulation in the anencehpalic does not seriously modify GH cell morphology but induces an altered GH storage pattern in some of the cells.     

Sorting of three secretory proteins to distinct secretory granules in acidophilic cells of cow anterior pituitary

The distribution of three proteins discharged by regulated exocytosis--growth hormone (GH), prolactin (PRL), and secretogranin II (SgII)--was investigated by double immunolabeling of ultrathin frozen sections in the acidophilic cells of the bovine pituitary. In mammotrophs, heavy PRL labeling was observed over secretory granule matrices (including the immature matrices at the trans Golgi surface) and also over Golgi cisternae. In contrast, in somatotrophs heavy GH labeling was restricted to the granule matrices; vesicles and tubules at the trans Golgi region showed some and the Golgi cisternae only sparse labeling. All somatotrophs and mammotrophs were heavily positive for GH and PRL, respectively, and were found to contain small amounts of the other hormone as well, which, however, was almost completely absent from granules, and was more concentrated in the Golgi complex, admixed with the predominant hormone. Mixed somatomammotrophs (approximately 26% of the acidophilic cells) were heavily positive for both GH and PRL. Although admixed within Golgi cisternae, the two hormones were stored separately within distinct granule types. A third type of granule was found to contain SgII. Spillage of small amounts of each of the three secretory proteins into granules containing predominantly another protein was common, but true intermixing (i.e., coexistence within single granules of comparable amounts of two proteins) was very rare. It is concluded that in the regulated pathway of acidophilic pituitary, cell mechanisms exist that cause sorting of the three secretory proteins investigated. Such mechanisms operate beyond the Golgi cisternae, possibly at the sites where condensation of secretion products into granule matrices takes place.