Immunocytochemical localisation of prolactin and growth hormone in the ovine pituitary

Summary Using the peroxidase-antiperoxidase immunocytochemical staining technique, prolactin and growth hormone cells have been identified and described in the ovine pituitary. The image analysing computer, Quantimet 720, was used to assess accurately the size range of the secretory granules in these cell types. The area size distributions of the prolactin and growth hormone granules are similar. An increased proportion of larger granules was observed in the prolactin cells post-partum. Serial sections stained alternately for prolactin or growth hormone confirmed that the cells contain either prolactin or growth hormone but not both.     

Immunocytochemical demonstration of growth hormone,prolactin and somatostatin-like immunoreactivities in the brain of larval,young adult and upstream migrant adult sea lamprey,Petromyzon marinus

Summary Growth hormone, prolactin and somatostatinlike immunoreactivities were demonstrated in the brains of larval, young adult (parasitic) and upstream migrant adult sea lampreys, Petromyzon marinus, by means of immunoperoxidase techniques. Growth hormone (GH) and prolactin (PRL) were observed within separate perikarya in the nucleus praeopticus, within fibers in the commissura praeinfundibularis, and in nerve endings within the neurohypophysis of larval and adult-stage lampreys. Cell bodies demonstrating immunoreactive growth hormone were more numerous than those reactive for prolactin. Unlike in the upstream migrant adult lamprey, no GH or PRL was demonstrated in the adenohypophysis of larval or parasitic lamprey.Somatostatin (SRIF)-like immunoreactive neurons were demonstrated in the nucleus commissurae praeinfundibularis, anterior and posterior pars ventralis hypothalami, pars dorsalis thalami, and the tegmentum motorium rhombencephali of larval, parasitic and upstream migrant adult lampreys. Many of the SRIF containing neurons within the hypothalamus were cerebrospinal fluid (CSF)-contacting cells. SRIF fibers were found throughout most of the brain predominating within the nucleus praeopticus, pars ventralis hypothalami, and the nucleus interpeduncularis. No SRIF immunoreactivity was found within the neurophyophysis. The possible functions of these peptides within the brain of the lamprey are discussed.     

Binding and internalization of gold-conjugated somatostatin and growth hormone-releasing hormone in cultured rat somatotropes

Summary The synthetic peptides somatostatin (SRIF) and growth hormone-releasing hormone (GRH) were coupled directly to colloidal gold of different particle sizes. Both conjugates were biologically active in displacing the corresponding radiolabeled hormones from high affinity binding sites in pituitary membranes. Release of growth hormone (GH) from cultured anterior pituitary cells was modulated by both conjugates alone or in combination. Ultrastructural studies were performed with cells incubated at 4° C (2 h) and 37° C (2 min-2 h) with one of the labeled peptides or their combination. Somatotropes were identified by immunostaining with anti-rGH followed by protein A-ferritin, thus obtaining a triple labeling. Both hormone conjugates were internalized in different vesicles in the beginning but accumulated during longer incubation times in the same compartment. The secretory vesicles and the nucleus were not labeled by any hormone conjugate. In contrast to SRIF-gold, the uptake of GRH-gold conjugate decreased with longer incubation times. This effect could be neutralized by simulatenous incubation of the somatotropes with both regulating hormones. Hence, whereas the binding and internalization of SRIF by somatotropes do not seem to be influenced by GRH, the corresponding processes for GRH are stimulated by the presence of SRIF.     

Immunocytochemical localization of growth hormone releasing factor (GHRF)-containing structures in the rat brain using anti-rat GHRF serum

The distribution of growth hormone releasing factor (GHRF) immunoreactive structures in the rat hypothalmus was studied after colchicine treatment with PAP immunocytochemistry in vibratome sections using an antiserum directed to rat hypothalamic GHRF. The majority of the GHRF-immunoreactive cell bodies were found in the arcuate nucleus, the medial perifornical region, and the ventral premammillary nuclei of the hypothalamus. Scattered cells were seen in the lateral basal hypothalamus, the medial and lateral portions of the ventromedial nucleus, and the dorsomedial and paraventricular nuclei. Immunoreactive fibers were observed in all the regions mentioned above. GHRF terminals were located in the central region of the median eminence. In addition, GHRF-immunoreactive neuronal processes were seen in the ventral region of the dorsomedial nucleus, the medial preoptic and suprachiasmatic regions, dorsal portion of the suprachiasmatic nucleus, bed nucleus of the stria terminals and the hypothalamic portion of the stria terminals. The localization of GHRF-immunoreactive terminals in the median eminence reinforces the view that GHRF plays a physiological role in the regulation of pituitary function. In addition, the localization of GHRF-immunoreactive structures in areas not usually considered to project to the median eminence suggest that GHRF may act as a neuromodulator or neurotransmitter.     

Immunocytochemical demonstration of the neurosecretory systems containing putative moult-inhibiting hormone and hyperglycemic hormone in the eyestalk of brachyuran crustaceans

Summary By use of antisera raised against purified moultinhibiting (MIH) and crustacean hyperglycemic hormone (CHH) from Carcinus maenas, complete and distinct neurosecretory pathways for both hormones were demonstrated with the PAP and immunofluorescence technique. By double staining, employing a combination of silver-enhanced immunogold labelling and PAP, both antigens could be visualized in the same section. Immunoreactive structures were studied in Carcinus maenas, Liocarcinus puber, Cancer pagurus, Uca pugilator and Maja squinado. They were only observed in the X-organ sinus gland (SG) system of the eyestalks and consisted of MIH-positive perikarya, which were dispersed among the more numerous CHH-positive perikarya of the medulla terminalis X-organ (XO). The MIH-positive neurons form branching collateral plexuses adjacent to the XO and axons that are arranged around the CHH-positive central axon bundle of the principal XO-SG tract. In the SG, MIH-positive axon profiles and terminals, clustered around hemolymph lacunae, are distributed between the more abundant CHH-positive axon profiles and terminals. Colocalisation of MIH and CHH was never observed. The gross morphology of both neurosecretory systems was similar in all species examined, however, in U. pugilator and M. squinado immunostaining for MIH was relatively faint unless higher concentrations of antiserum were used. Possible reasons for this phenomenon as well as observed moult cycle-related differences in immunostaining are discussed.     

Immunocytochemical localisation of prolactin and growth hormone in the perinatal sheep pituitary

Summary The peroxidase anti-peroxidase immunocytochemical staining technique has been used to identify prolactin and growth hormone cells in pituitaries from fetal and neonatal sheep. The size of the secretory granules in these cell types has been measured using the image analysing computer Quantimet 720. The area size distributions of the fetal prolactin and growth hormone granules were compared with those in the neonate and the adult. It appears that the gestational age of the fetus may influence the size range of prolactin secretory granules.     

Immunocytochemical and immuno-electron-microscopical study of growth hormone cells in male and female rats of various ages

Summary Growth hormone (GH) secretory cells were identified by immunogold cytochemistry, and were classified on the basis of the size of secretory granules. Type I cells contained large secretory granules (250\2-350 nm in diameter). Type II cells contained the large secretory granules and small secretory granules (100\2-150 nm in diameter). Type III cells contained the small secretory granules. The percentages of each GH cell type changed with aging in male and female rats of the Wistar/Tw strain. Type I cells predominated throughout development; the proportion of type I cell was highest at 6 months of age, and decreased thereafter. The proportion of type II and type III cells decreased from 1 month to 6 months of age, but then increased at 12 and 18 months of age. The pituitary content of GH was highest at 6 months of age, and decreased thereafter. Estrogen and androgen, which are known to affect GH secretion, caused changes in the proportion of each GH cell type. The results suggest that when GH secretion is more active the proportion of type I GH cell increased, and when GH secretion is less active the proportion of type II and type III cells increased. The type III GH cell may therefore be an immature type of GH cell, and the type I cell the mature type of GH cell. Type II cells may be intermediate between type I and III cells.     

Growth hormone binding and stimulation of amino acid uptake in epithelial cells of rat ventral prostate

The binding of [125I]-human growth hormone (hGH) was studied in epithelial cells isolated from rat ventral prostate. Binding and degradation were dependent on time and temperature. The effect of a lysosomotropic agent suggested internalization and lysosomal degradation of the hormone. Dissociation and stoichiometric studies indicated the existence of a single class of GH receptors with a Kd of 0.7 nM and a binding capacity of 46 fmol hGH bound mg-1 cell protein. The receptor appeared to possess a somatotrophic nature since lactogenic hormones such as human placental lactogen and rat prolactin exhibited a very low degree of competition (whereas a variety of unrelated hormones and neuropeptides showed no effect). GH-stimulated leucine uptake by the cells in a time- and dose-dependent manner, half maximal effect being observed at 0.32 nM GH thus suggesting a direct relationship with the binding step.     

Developmental correlation between hypothalamic somatostatin and hypophysial growth hormone

Summary The objective of the present study was to determine, by means of immunocytochemistry, the age in fetal development at which GH is first detectable in the pituitary gland and somatostatin in the median eminence, and to correlate temporally the development of these two hormones throughout the remainder of pregnancy. Mice were studied at 15–19 days of gestation with the peroxidase-antiperoxidase (PAP) technique of Sternberger. Somatotropes in the pars distalis were initially detected at 16 days of gestation and by 17 days they were a prominent component of the parenchymal cell population of the hypophysis. These cells were ovoid and distributed uniformly throughout the pars distalis; many were located adjacent to sinusoidal capillaries. Their number and staining intensity increased by 19 days. Somatostatin was not consistently observed in the median eminence until 19 days of gestation. Reaction product indicative of the presence of somatostatin in presumptive nerve endings was located on the ventral surface of the median eminence and in the external lamina of the infundibulum in proximity to the superficial portal capillaries. Results of the present investigation support the concept that the potential for neuroendocrine control of GH secretion exists in the mouse by the end of fetal development. Several hypotheses concerning the temporal relationship between the appearance of somatostatin in the hypothalamus and of GH in the anterior pituitary gland are discussed.Supported by a Biomedical Research Support Grant (NIH RR 5417). Appreciation is extended to the National Pituitary Agency, NIAMDD for the following radioiodination-grade hormones: hGH, rPRL, rTSH, rFSH and hCG     

Ultrastructural localization of prolactin,growth hormone and luteinizing hormone by immunocytochemical techniques in the bovine pituitary

Summary The technique of ultrastructural immunocytochemistry involving the unlabeled antibody and the soluble peroxidase-antiperoxidase complex was used to identify and describe the prolactin (P) cells, somatotropic (STH) cells and luteinizing hormone (LH) cells in the bovine anterior pituitary gland. This method was used to localize the three hormones at the electron microscopic level. Staining of varying intensity was found on the secretory granules and on the small granules and vesicles within the Golgi complex. No stain was found in nuclei, on mitochondria or in the endoplasmic reticulum.     

Immunoreactive neuronal pathways of growth hormone-releasing hormone (GRH) in the brain and pituitary of the teleost Gadus morhua

Summary Using an antiserum directed against the C-terminus of hGRH(1–44)NH₂ and another recognizing the mid portion to C-terminal of hGRH(1–40)OH, we identify two immunocytochemically distinct GRH-immunoreactive systems in the brain of the codfish, Gadus morhua. The antiserum directed against GRF(1–44)NH₂ stains cell bodies exclusively in the rostral pars distalis. The other antiserum immunoreactive with GRF(1–40)OH reacts with a population of parvocellular and magnocellular neuronal cell bodies in the hypothalamus and with two major axonal pathways which project toward the median eminence and terminate primarily in the pars nervosa. These results indicate the presence of at least two forms of hGRH-like peptides in the teleost which may have different roles in the regulation of pituitary function.     

Characterization of insulin-like growth factor binding protein-1 kinases from human hepatoma cells

The phosphorylation of insulin-like growth factor binding protein-I (IGFBP-1) alters its binding affinity for insulin-like growth factor I (IGF-I) and thus regulates the bioavailability of IGF-I for binding to the IGF-I receptor. The kinase(s) responsible for the phosphorylation of IGFBP-1 has not been identified. This study was designed to characterize the IGFBP-1 kinase activity in HepG2 human hepatoma cells, a cell line that secretes IGFBP-1 primarily as phosphorylated isoforms. IGFBP-1 kinase activity was partially purified from detergent extracts of the cells by phosphocellulose chromatography and gel filtration. Two kinases of approximate M_r 150,000 (peak I kinase) and M_r 50,000 (peak II kinase) were identified. Each kinase phosphorylated IGFBP-1 at serine residues that were phosphorylated by intact HepG2 cells. The kinases were distinct based on their differential sensitivity to inhibition by heparin (IC₅₀ = 2.5 and 16.5 μg/ml, peak I and II kinase, respectively) and inhibition by the isoquinoline sulfonamide CKI-7 (IC₅₀ = 50 μM and 100 μM, peak I and II kinase, respectively). In addition, a tenfold molar excess of nonradioactive GTP relative to [gamma-³²P]ATP lowered the incorporation of ³²P into IGFBP-1 by 80% when the reaction was catalyzed by the peak I kinase, whereas GTP had no effect on the reaction catalyzed by the peak II kinase. In the presence of polylysine, IGFBP-1 was radiolabeled by the partially purified kinase activity when [gamma-³²P]GTP served as the phosphate donor indicating the presence of casein kinase II activity. Furthermore, IGFBP-1 was phosphorylated by purified casein kinase I and casein kinase II at sites phosphorylated by the peak I and peak II kinases. Our data suggest that at least two kinases could be responsible for the phosphorylation of IGFBP-1 in intact HepG2 cells and that the kinases are related to the casein kinase family of protein kinases. © 1996 Wiley-Liss, Inc.     

Tilapia adropin: the localization and regulation of growth hormone gene expression in pituitary cells

The peptide hormone adropin, encoded by the energy homeostasis-associated (Enho) gene, plays a role in energy homeostasis and the control of vascular function. The aim of this study was to examine the role of adropin in growth hormone (GH) gene expression at the pituitary level in tilapia. As a first step, the antiserum for the tilapia adropin was produced, and its specificity was confirmed by antiserum preabsorption and immunohistochemical staining in the tilapia pituitary. Adropin could be detected immunocytochemically in the proximal pars distalis (PPD) of the tilapia pituitary. In primary cultures of tilapia pituitary cells, tilapia adropin was effective in increasing GH mRNA levels. However, removal of endogenous adropin by immunoneutralization using adropin antiserum inhibited GH gene expression. In parallel experiments, pituitary cells co-treated with ovine pituitary adenylate cyclase activating polypeptide 38 (oPACAP38) and adropin showed a similar increase level compared to those treated with oPACAP38 alone, whereas insulin-like growth factor 1 (IGF1) not only had an inhibitory effect on basal GH mRNA levels, but also could abolish adropin stimulation of GH gene expression. In pituitary cells pretreated with actinomycin D, the half-life of GH mRNA was enhanced by adropin. Taken together, these findings suggest that adropin may serve as a novel local stimulator for GH gene expression in tilapia pituitary.     

Role of growth hormone receptor signaling in osteogenesis from murine bone marrow progenitor cells

Growth hormone (GH) regulates many of the factors responsible for controlling the development of bone marrow progenitor cells (BMPCs). The aim of this study was to elucidate the role of GH in osteogenic differentiation of BMPCs using GH receptor null mice (GHRKO). BMPCs from GHRKO and their wild-type (WT) littermates were quantified by flow cytometry and their osteogenic differentiation in vitro was determined by cell morphology, real-time RT-PCR, and biochemical analyses. We found that freshly harvested GHRKO marrow contains 3% CD34 (hematopoietic lineage), 43.5% CD45 (monocyte/macrophage lineage), and 2.5% CD106 positive (CFU-F/BMPC) cells compared to 11.2%, 45%, and 3.4% positive cells for (WT) marrow cells, respectively. When cultured for 14 days under conditions suitable for CFU-F expansion, GHRKO marrow cells lost CD34 positivity, and were markedly reduced for CD45, but 3- to 4-fold higher for CD106. While WT marrow cells also lost CD34 expression, they maintained CD45 and increased CD106 levels by 16-fold. When BMPCs from GHRKO mice were cultured under osteogenic conditions, they failed to elongate, in contrast to WT cells. Furthermore, GHRKO cultures expressed less alkaline phosphatase, contained less mineralized calcium, and displayed lower osteocalcin expression than WT cells. However, GHRKO cells displayed similar or higher expression of cbfa-1, collagen I, and osteopontin mRNA compared to WT. In conclusion, we show that GH has an effect on the proportions of hematopoietic and mesenchymal progenitor cells in the bone marrow, and that GH is essential for both the induction and later progression of osteogenesis.     

Structure activity studies of the serine-AIB dipeptide domain in 2,3-dihydroisothiazole based growth hormone secretagogues

A series of growth hormone secretagogues (GHSs) based on 2,3-dihydroisothiazole has been synthesized in the search for a potential treatment of growth hormone deficiency or frailty in the elderly. This paper describes the evaluation of the SAR of the benzyl-d-Ser-aminoisobutyric acid dipeptide fragment. Introduction of substituents in the peptide backbone and in the phenyl ring has been investigated, as well as replacements for the benzyl group and for the AIB residue. A number of modifications resulted in enhanced potency over the parent benzyl-d-Ser-AIB derivative.     

Expression of calcium-binding protein regucalcin mRNA in hepatoma cells

Whether the gene expression of hepatic Ca²⁺-binding protein regucalcin is altered in hepatomas was investigated. The change in regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin complementary DNA (0.9 kb). Rat hepatoma was induced by continuous feeding of basal diet containing 0.06% 3-methyl-4-dimethylaminoazobenzene (3-Me-DAB). After 35 weeks feeding, rats were sacrificed, and the non-tumorous and tumorous tissues of the livers were removed. In individual rats, the regucalcin mRNA levels in the tumorous tissues were generally decreased in comparison with that of the non-tumorous tissues of the chemical-fed rats, although the chemical administration might decrease the mRNA expression in normal rat liver, suggesting that the chemical administration causes a suppresive effect on the mRNA expression. When the genomic DNA extracted from the liver tumorous tissues was digested with restriction enzymes (EcoRI, BamHI and HindIII) and analyzed by Southern blotting, no rear-ranged band was found in the regucalcin gene from the hepatoma. Interestingly, in the transplantable Morris hepatoma cells, the regucalcin mRNA was markedly expressed, while the albumin mRNA was expressed only slightly. The present study demonstrates that regucalcin mRNA is clearly expressed in the transformed cells (Morris hepatoma cells).     

Culture of human pituitary prolactin and growth hormone cells

Summary Fragments of pituitary tissue obtained from a total of 37 patients with either breast cancer, diabetic retinopathy, galactorrhea, or acromegaly were dissociated into single cell suspensions prior to cell culture. Release of human growth hormone (hGH) and human prolactin (hPRL) into the culture medium was measured by radioimmunoassay. During a 3-week culture period, prolactin cells released 9–13 times the intracellular levels of hPRL at the time of seeding, whereas hGH release from growth hormone cells was only 1–2 times that of their initial intracellular level during this same time. Both growth hormone and prolactin cells retained distinctive ultrastructural features during culture. The prolactin cells responded to TRH stimulation by elevated release of PRL into the medium. No evidence for mitotic division of prolactin cells in vitro was found.This work was supported by NCI Contract NO 1-CB-23863     

Effect of hypophysectomy and growth hormone replacement on hypothalamic GHRH

Long-term (7 and 14 days) hypophysectomy resulted in a striking decrease in growth hormone releasing hormone-like immunoreactivity (GHRH-LI) in the median eminence (ME) of adult male rats, evaluated by both radioimmunoassay and immunohistochemistry. Treatment with human GH (125 μg/rat, twice daily IP for 14 days) prevented, though partially, depletion of GHRH-LI from the ME, as assessed by both methods. These results demonstrate that circulating GH levels regulate the function of GHRH-producing structures, via a feedback mechanism.     

Immunocytochemical identification of growth hormote (GH) cells in the pituitary of three anuran species using an antiserum against purified bullforg GH

An antiserum was prepared against the recently purified bullfrog (bf) growth hormone (GH); it was applied to sections of brain and pituitary of three urodele (Ambystoma, Pleurodeles and Cynops) and three anuran (Xenopus, Bufo vulgaris and B. japonicus) species. No immunostaining was obtained in the urodele pituitary, being consistent with the results of immunoblot analysis of the pituitary homogenate. In the three anuran species, strong immunoreactivity was observed in GH cells that were concentrated in the posterodorsal region of the pars distalis. No GH-like immunoreactivity was detectable in the brain of any of the species. A comparison using adjacent sections stained with anti-bf prolactin (PRL) confirmed the anteroventral localization of PRL cells. Colocalization of GH and PRL was not apparent. These data suggest that the molecular structure of amphibian GHs is considerably different between anurans and urodeles. The antiserum used in the present work shows a high species specificity, recognizing only anuran GHs. In contrast anti-bfPRLlabeled PRL cells in all the amphibian species studied in the present work, suggesting that PRLs possess common amino acid sequences recognized by the anti-bfPRL.     

Ultrastructural studies on prolactin and growth hormone cells in Anguilla pituitaries in long term cultures

Summary Eel hemi-pituitaries were cultured in vitro on high or low sodium media, previously shown to affect differentially prolactin and growth hormone release. After 6 days culture, there were marked differences in the ultrastructure of both prolactin and growth hormone cells from the two groups. Morphometric data on the prolactin cells from SW-adapted eels showed a greater abundance of RER and paucity of secretory granules in cells from the low sodium medium. The size of the Golgi apparatus and the number of exocytosed secretory granules did not differ markedly between experimental groups, in contrast to previous findings on short-term cultures. Differences in the profile diameters of secretory granules are recorded between the experimental groups and the pattern differs markedly from that previously recorded for short-term cultures. The growth hormone cells from low sodium media were characterised by abundant, vesiculated RER, a prominent Golgi apparatus (in SW-adapted animals) and relatively few secretory granules. The activity of these growth hormone cells is in marked contrast to previous findings relating to short-term cultures. The shape and size of the non-granulated (stellate) cells of the RPD was again affected by the osmotic pressure of the medium.I should like to thank Mr. P.F. Hire for his photographic assistance