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.     

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.     

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.     

Distribution of growth hormone and prolactin in secretory granules of the normal and neoplastic human adenohypophysis

Growth hormone [GH] and prolactin [PRL] can be demonstrated simultaneously in electron micrographs by means of the double immunocytochemical labeling technique using colloidal gold particles of two different sizes. This method was used to study biopsy specimens obtained from 15 patients suffering from acromegaly, 11 patients suffering from prolactinomas, and eight biopsy specimens obtained during adenomectomy from the normal, paraadenomatous pituitary tissue. Four granule populations with different immunoreactions were found: (1) granules containing GH only, (2) granules containing PRL only, (3) mixed granules containing GH and PRL, and (4) granules displaying no immunoreactivity. The existence of mixed granules indicated that the two hormones are synthesized by the same cell and in communicating compartments of the cells; i.e., the rough-surfaced endoplasmic reticulum. The number of GH-containing granules (pure GH granules and mixed GH-PRL granules) was greater than that of PRL-containing granules (pure PRL granules and mixed PRL-GH granules) in adenomas causing acromegaly and in the normal pituitary tissue, whereas the opposite was true for prolactinomas. The number of PRL-containing granules was larger in biopsy specimens from patients who had acromegaly and hyperprolactinemia than in patients with acromegaly and normal serum PRL levels.     

Conversion of growth hormone-secreting cells into prolactin-secreting cells and its promotion by insulin and insulin-like growth factor-1 in vitro

The newly established rat pituitary cell line, MtT/S, has pituitary somatotroph (growth hormone-producing cell)-like characteristics, i.e., the cells produce growth hormone (GH), possess GH-immunopositive secretory granules, and respond to GH-releasing hormone. When MtT/S cells were cultured in regular medium no prolactin (PRL) cells were observed and PRL was not detected, by radioimmunoassay or Western blot analysis, in the medium or the cells. However, GH production and the GH cell population decreased markedly when the cells were incubated with insulin or insulin-like growth factor-1 (IGF-1). After stimulation with insulin or IGF-1 there was a 2-day lag period, then some PRL was detected in the medium; after 5 days a number of PRL cells appeared. Double immunocytochemistry indicated clearly that no cell contained both PRL and GH. These results show that insulin and IGF-1 stimulate conversion of MtT/S cell line GH cells to PRL cells. This suggests that the MtT/S cell line is an excellent model system which shows the GH-cell/PRL-cell lineage.     

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.     

The mechanisms by which vasoactive intestinal peptide (VIP) and thyrotropin releasing hormone (TRH) stimulate prolactin release from pituitary cells

The effect of vasoactive intestinal peptide (VIP) on prolactin (PRL) secretion from pituitary cells is reviewed and compared to the effect of thyrotropin releasing hormone (TRH). These two peptides induced different secretion profiles from parafused lactotrophs in culture. TRH was found to increase PRL secretion within 4 s and induced a biphasic secretion pattern, while VIP induced a monophasic secretion pattern after a lag time of 45–60 s.The secretion profiles are compared to changes in adenylate cyclase activity, production of inositol polyphosphates, changes in intracellular calcium concentrations and changes in electrophysiological properties of the cell membrane.Abbreviations AC adenylate cyclase - DG diacyglycerol - GH growth hormone - GTP guanosine trisphosphate - G_i GTP binding proteins that mediate inhibition of adenylate cyclase and that are pertussis toxin sensitive - G_s GTP binding protein that mediates stimulation of adenylate cyclase - GH cells clonal rat pituitary tumor cells producing PRL and/or growth hormone - GH₃ GH₄C₁ and GH₄B6 subclones of GH cells - PKA protein kinase A - PKC protein kinase C - PLC phospholipase C - PRL prolactin - TPA 12-O-tetradecanoyl phorbol 13-acetate - TRH thyrotropin releasing hormone - VIP vasoactive intestinal peptide     

Multiple hormone storage by cells of the human pituitary

While immunostaining serial semi-thin sections of acrylic resin-embedded normal human pituitary using antisera to human pituitary hormones, it became clear that several cells were stained by more than one antiserum. The tissue had been surgically excised from a patient with a prolactinoma. The tumor, which was immunoreactive only with antiprolactin antiserum, was distinctly different from the pieces of tissue under study which had normal pituitary architecture and demonstrated immunoreactivity with antisera against all six of the common pituitary hormones. A major immunoelectron microscopic investigation, using immunocolloidal gold and immunoperoxidase methods, revealed cells in which follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL) were co-localized to the same electron-dense granules. Some similar cells also possessed electron-lucent granules immunoreactive only for anti-PRL antiserum. Adrenocorticotrophic hormone (ACTH) and PRL were also found in the same cell but were very largely localized to separate, morphologically different populations of electron-dense and -lucent storage granules. By employing double immunolabeling, a few granules in the ACTH/PRL cells were shown to be immunoreactive to both anti-ACTH and anti-PRL antisera. The possibility that the multipotential stem cells is discussed.     

Participation of voltage-sensitive calcium channels in pituitary hormone release

The role of extracellular Ca2+ in pituitary hormone release was studied in primary cultures of rat anterior pituitary cells. The basal levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyrotropin (TSH), and adrenocorticotropin (ACTH) secretion were independent of extracellular Ca2+ concentration ([Ca2+]e). In contrast, the basal levels of growth hormone (GH) and prolactin (PRL) release showed dose-dependent increases with elevation of [Ca2+]e, and were abolished by Ca2+-channel antagonists. Under Ca2+-deficient conditions, BaCl2 mimicked the effects of calcium on PRL and GH release but with a marked increase in potency, and also increased basal LH and FSH release in a dose-dependent manner. In the presence of normal [Ca2+]e, depolarization with K+ maximally increased cytosolic [Ca2+] ([Ca2+]i) from 100 to 185 nM and elevated LH, FSH, TSH, ACTH, PRL, and GH release by 7-, 5-, 4-, 3-, 2-, and 1.5-fold, respectively. These effects of KCl were abolished in Ca2+-deficient medium or in the presence of the Ca2+-channel antagonist, Co2+, and were diminished by the dihydropyridine Ca2+-channel antagonist, nifedipine. The Ca2+-channel agonist BK 8644 (100 nM) enhanced the hormone-releasing actions of 25 mM KCl upon PRL, LH, FSH, GH, TSH, and ACTH by 2.3-, 2.0-, 1.8-, 1.7-, 1.6-, and 1.4-fold, respectively. The dose- and voltage-dependent actions of BK 8644 were specific for individual cell types; BK 8644 enhanced GH, PRL, TSH, LH, and ACTH secretion in the absence of any depolarizing stimulus, with ED50 values of 8, 10, 150, 200, and 400 nM, respectively. However, in the presence of 50 mM KCl, the ED50 values for BK 8644 were 1.5, 2, 3, 5, and 7 nM for GH, PRL, ACTH, TSH, and LH, respectively. [3H]BK 8644 bound specifically to pituitary membranes with Kd values of 0.8 nM and concentrations of about 900 channels per cell. These observations provide evidence for the presence and participation of voltage-sensitive calcium channels in the secretion of all five populations of anterior pituitary cells.     

Isolation and characterization of rat-mouse somatic cell hybrids secreting growth hormone and prolactin

Interspecific somatic cell hybrid clones have been isolated and characterized in order to study growth hormone (GH) and prolactin (PRL) gene expression. Rat pituitary tumor cells (GH3, 69 chromosomes) secreting rat GH and PRL were grown for 48 h together with nonhormone secreting, aminopterin-sensitive murine fibroblast cells (LMTK-, 55 chromosomes) and fused using polyethylene glycol. Resultant heterokaryons were selected in hypoxanthine-aminopterin-thymidine (HAT) medium and cloned. Five clones produced rat GH and PRL. Hormone-producing hybrids morphologically resembled the mouse parent fibroblast. Hybrids grew in monolayers and contained 80-142 chromosomes, and marker chromosomes for both rat (small submetacentric) and mouse (bi-armed and large true metacentric) were identified. The interspecific nature of the hybrids was further confirmed by the presence of both rat and mouse adenosine deaminase and superoxide dismutase isozymes. Using specific antisera and indirect immunoperoxidase staining, both hybrid clones and GH3 rat parental cells stained positively for rat GH and PRL, while the murine fibroblast parental cells were negative. Hormone production by the hybrids has been sustained for over twenty subcultures; secretion rates were initially 150 ng PRL and 321 ng GH/10(6) cells/24 h and are currently 100 ng PRL and 90 ng GH/10(6) cells/24 h. Parental GH3 rat cells secreted 720 ng PRL and 660 ng GH/10(6) cells/24 h. Exposure of hybrids to KCl (50 mM) resulted in acute stimulation of rat PRL, but not rat GH release, and long-term incubation with thyrotropin-releasing hormone (TRH, 80 nM) stimulated PRL secretion. Hormone-dependent modulation of PRL secretion was transferred to the hybrid cell thus enabling the model to be used in studying regulation of PRL gene expression.     

Specific antisense RNA inhibition of growth hormone production in differentiated rat pituitary tumour cells.

An expression vector that carried an inverted 800 base pair insert of the rat growth hormone (rGH) cDNA downstream of the SV40 promotor was used to transfect two different growth hormone (GH) producing rat pituitary cell strains, GH12C1 and GH3. This resulted in a specific transient inhibition of growth hormone production up to 75 percent in the course of 72 hours. GH synthesis reduction occurred parallel to a decrease of GH cytoplasmic mRNA levels. Levels of beta-actin and guanine nucleotide-binding regulatory protein (G protein) mRNAs were unaltered, but PRL mRNA levels were increased. Transfection with a control vector did not affect GH production.     

Electroporation of rat pituitary (GH) cell lines: optimal parameters and effects on endogenous hormone production

An efficient electroporation procedure was established for the genetic transformation of two clonal strains of hormone producing rat pituitary cells (GH12C1 and GH3). We used the bacterial chloramphenicol acetyltransferase (CAT) gene as reporter gene to determine optimal conditions for electroporation. The conditions found to be optimal, measured as expression of the highest CAT activity, were 240-300 V and a DNA concentration of 30-60 micrograms/ml in sucrose buffer. Cell viability was then about 50 per cent. Maximum CAT activity was seen 24 hours after electroporation. The electroporation procedure, in the presence or absence of DNA, caused a transient decrease in endogenous growth hormone (GH) and prolactin (PRL) production.     

Distribution of growth hormone-like cells in the pituitary of adult sea lampreys,Petromyzon marinus

Growth hormone (GH), prolactin (PRL) and somatolactin (SL) are members of a pituitary hormone family that are believed to have evolved from a common ancestral gene by duplication and subsequent divergence. Since these hormones are found both in bony fish and cartilaginous fish, their ancestral form(s) should be present in the Agnatha. Thus, although there is no convincing evidence that the lamprey pituitary secretes GH or PRL, GH- and/or PRL-like immunoreactivity was examined in the pituitary of adult sea lampreys (Petromyzon marinus), using antibodies to GHs, PRLs and SL of mammalian and/or fish origins. Our initial attempt with ordinary immunohistochemical procedures failed to detect any positive reactions in the lamprey pituitary. Following the hydrated autoclave pretreatment of the sections, anti-salmon GH, anti-salmon PRL and anti-blue shark GH gave positive reactions in most cells distributed in the dorsal half of the proximal pars distalis. These results suggest that the material immunoreactive to those antibodies is related, to some extent, to GH/PRL, but enhancement of immunoreactivity to reveal this by the hydrated autoclave pretreatment of sections is needed due to low crossreactivity. The similarity of the topographic distributions within the pituitary between lampreys and teleosts suggests that lamprey GH/PRL-like cells are GH cells of the lamprey.     

Regulated expression of the prolactin gene in rat pituitary tumor cells

Prolactin (PRL) gene expression in three strains of GH cells (rat pituitary tumor cells) has been quantitated by measurement of: (a) intracellular and extracellular PRL, (b) cytoplasmic translatable PRL-specific mRNA (mRNAPRL), and (c) molecular hybridization of cytoplasmic poly(A) RNA to cDNAPRL (DNA complementary to mRNAPRL). Three GH cell lines utilized in this investigation were a PRL-producing (PRL+) strain, GH4C1, a PRL nonproducing 5-bromo-deoxyuridine resistnat (PRL- BrdUrdr) strain, F1BGH12C1, and a new strain, 928-9b, derived by fusion of PRL+ cells with a nuclear monolayer of the PRL-, BrdUrdr GH cell strain. PRL production is a characteristic of 928-9b cells, but the level of PRL production (2-4 micrograms/mg protein/24 h) is much lower than that of the PRL+ strain, GH4C1 (15-25 micrograms/mg protein/24 h). Levels of cytoplasmic translatable mRNAPRL and cytoplasmic PRL-RNA sequences quantitated with a cDNAPRL probe were also much lower in 928-9b as compared to the PRL+ parent. PRL-RNA sequences could not be detected in the PRL- strain. Thyrotopin-releasing hormone (TRH) stimulates PRL synthesis about threefold and inhibit a growth hormone (GH) synthesis 72% in the PRL+ strain. TRH has no effect on the synthesis of either PRL or GH in the 928-9b strain, although TRH receptors could be detected in these cells. Stimulation of PRL synthesis in the PRL+ strain by TRH could be correlated with increases in levels of cytoplasmic translatable mRNAPRL and increases in cytoplasmic PRL-RNA sequences. These results demonstrate that the graded expression of the PRL gene at the basal level, and in response to TRH, is caused by the regulated production of specific mRNA, i.e., mRNAPRL in these three GH cell strains.     

Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells

A novel neuropeptide which stimulates adenylate cyclase in rat anterior pituitary cell cultures was isolated from ovine hypothalamic tissues. Its amino acid sequence was revealed as: His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln- Met-Ala- Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln-Arg-Val-Lys-Asn-Lys - NH2. The N-terminal sequence shows 68% homology with vasoactive intestinal polypeptide (VIP) but its adenylate cyclase stimulating activity was at least 1000 times greater than that of VIP. It increased release of growth hormone (GH), prolactin (PRL), corticotropin (ACTH) and luteinizing hormone (LH) from superfused rat pituitary cells at as small a dose as 10(-10)M (GH, PRL, ACTH) or 10(-9)M (LH). Whether these hypophysiotropic effects are the primary actions of the peptide or what physiological action in the pituitary is linked with the stimulation of adenylate cyclase by this peptide remains to be determined.     

Rat ghrelin stimulates growth hormone and prolactin release in the tilapia,Oreochromis mossambicus

Recently, ghrelin (Ghr), a new peptide which specifically stimulates growth hormone (GH) release from the pituitary, was identified in the rat and human stomach. Ghrelin has been shown to stimulate GH release by acting through a growth hormone secretagogue (GHS) receptor in the rat. The present study describes the in vitro effect of rat Ghr on the release of GH and two forms of prolactin (PRL(177) and PRL(188)) in the tilapia, Oreochromis mossambicus. Rat Ghr stimulated the release of GH in a dose-related manner after 8 and 24 hr of incubation. Rat Ghr also significantly stimulated the release of PRL(177) and PRL(188) in a dose-related manner after 24 hr. Rat Ghr had no effect on the pituitary content of GH or PRL(188), but significantly increased PRL(177) content. These results show for the first time that rat Ghr significantly stimulates GH and PRL release in teleosts, and suggest that Ghr and a GHS receptor are present in fish.