Somatotropic Research Articles

Cellular Localization of Orexin Receptors in Human Pituitary*

Orexins-A and -B are hypothalamic peptides derived from a precursor called prepro-orexin and relationated with the stimulation of food intake. They act on G protein receptors named orexin receptor 1 (OX(1)R) and orexin receptor 2 (OX(2)R), respectively. In the present study, we used RT-PCR and immunohistochemical techniques to detect the presence of OX(1)R and OX(2)R in human pituitary. A band of the expected size for both OX(1)R and OX(2)R was shown in human pituitary by RT-PCR. The cellular localization of OX(1)R and OX(2)R was carried out using histological techniques. By consecutive sections we demonstrated that OX(1)R was present in acidophil, diffusely distributed cells, which represent the half of the total adenohypophysis cell population. As was expected, these cells were shown to coexpress GH. OX(2)R was found in the pars intermedia and in clusters of basophil cells of the anterior pituitary, which coexpress ACTH. These results were confirmed by double immunofluorescence techniques. We also found focal positivity in axon terminals of neurohypophysis, more intense for OX(2)R than for OX(1)R. In conclusion, these results demonstrated for the first time that OX(1)R and OX(2)R were expressed by somatotrope and corticotrope cells, respectively.

An immunohistochemical study of adenohypophyseal cells in the viviparous reptile Chalcides chalcides.

The morphology of the hypophysis and the immunocharacteristics of the adenohypophyseal cells in the viviparous reptile Chalcides chalcides were studied by light microscopy, using conventional staining methods and an indirect antibody technique (ABC method), respectively. The general morphology of the C. chalcides hypophysis was comparable to that of other reptiles, showing three main regions: the pars distalis, the pars intermedia and the pars nervosa. The gland appeared as an elongated body in a cephalic-caudal direction and was almost completely enclosed in the sella turcica. For this reason, the hypophysis was studied in toto with the brain in decalcified specimens. The pars distalis accounted for most of the whole organ. The pars intermedia surrounded the pars nervosa as a goblet. The pars tuberalis was lacking. The immunohistochemical identification of the adenohypophyseal cells was performed using rabbit antisera against mammalian/synthetic hypophyseal hormones. Prolactin cells were clustered in small cellular cordons in the rostral pars distalis and in the medial pars distalis in both male and female specimens. Somatotropic cells were found in the caudal pars distalis. Corticotropic cells were observed in the medio-rostral pars distalis, as well as in the pars intermedia, where melanotropic cells were also present. Melanotropic cells were confined to the pars intermedia. Gonadotropic cells were mostly distributed in the ventral and lateral portions of the pars distalis, where they were found isolated or in small clusters. Thyrotropic cells were detected in the pars distalis with a distribution similar to that of the gonadotropic cells; however, atypically, they were also found in the pars intermedia. Therefore, the cytological characteristics of the adenohypophyseal cells appeared mostly conserved.

Light and ultrastructural immunocytochemical study of somatotropic cells (GH cells) in ovine adenohypophysis: lactation and weaning influences.

The influence of lactation period and weaning on the distribution, number, and structure of somatotropic cells (GH cells) in ewes was studied using immunocytochemical procedures for light and electron microscopy, as well as morphometric and stereological techniques. The adenohypophyseal gland of 12 ewes of the Segureña breed in different stages of milk production and weaning was studied, while three ewes in anoestrus served as controls. The size of secretory granules was heterogeneous in all stages, suggesting that this characteristic is not related to functional activity. During lactation and weaning the size of GH cells decreased, while the number of 'synthesis cells' increased. The rough endoplasmic reticulum and Golgi complex appeared more developed and small secretory granules showed lower gold particle labelling. These data suggest that GH cells are more active during lactation, particularly during late lactation.

Expression of leukemia inhibitory factor in human pituitary adenomas: a morphologic and immunocytochemical study.

Leukemia inhibitory factor (LIF) is a pleiotropic, neuropoietic cytokine found in bovine, murine, and human fetal and adult pituitary cells, mostly in corticotrophs and somatotrophs. In addition, it has been found in a few GH- and ACTH-producing human pituitary adenomas. The aim of our study was to investigate the presence of LIF in various morphologic types of human pituitary adenomas. Ninety-eight operated pituitary adenomas diagnosed by light microscopy and classified by pituitary hormone immunocytochemistry and electron microscopy were studied. Sixty-eight tumors were functioning and included 15 densely granulated (DG) and 10 sparsely granulated (SG) somatotroph (SM) adenomas, 5 lactotroph (LT), 7 mixed SM-LT and 31 corticotroph (CRT) adenomas. The remaining 30 nonfunctioning tumors included 11 gonadotroph (GON) and 19 null cell (NULL) adenomas. For immunocytochemical demonstration of LIF, a specific polyclonal antiserum was applied on formalin-fixed, paraffin-embedded tissues. The immunohistoscore ranging from 1 to 64 grades was determined by multiplying the immunostaining grade (1-4) by the staining intensity grade (1-4), and by the heterogeneity grade (1-4). Ninety adenomas (92%) were variably immunopositive for LIF. LIF was expressed in 77.5% of CRT, 81.8% of GON, 93.3% of DG-SM, and in all SG-SM, LT, SM-LT and NULL adenomas. LT adenomas showed the highest immunostaining grade, followed by SG-SM, GON, NULL, SM-LT, DG-SM, LT and CRT adenomas. GON adenomas showed the highest immunohistoscore, followed by NULL, DG-SM, SM-LT, SG-SM, LT and CRT adenomas. Nonfunctioning tumors showed a significantly higher immunohistoscore compared to functioning adenomas (p < 0.01). We conclude that LIF expression is frequent in all types of functioning and nonfunctioning pituitary adenomas.

Administration of ACTH to restrained, pregnant sows alters their pigs' hypothalamic-pituitary-adrenal (HPA) axis.

This study was designed to examine the physiology and behavior of pigs whose dams were snared and then injected with ACTH during gestation. Administration of ACTH to dams during pregnancy has been shown to replicate the effects of prenatal stress in other species. Control sows (n = 8) were given no treatment, whereas the treatment sows (ACTH, n = 8) were immobilized by snaring the snout and then administered an i.v. injection of ACTH (1 IU/kg BW) weekly from 6 to 12 wk of gestation. A pig was killed from each sow at 1, 30, and 60 d of age. The hypothalamus, pituitary gland, adrenal glands, and liver were immediately obtained to determine the amounts of corticotropin-releasing hormone (CRH), beta-endorphin, and mRNA for pro-opiomelanocorticotropin (POMC), the ACTH receptor (ACTH-R), and insulin-like growth factor I (IGF-I). Pituitary corticotrope and somatotrope cell numbers and adrenal cortex-to-medulla area ratios (CORT:MED) were also determined. Pigs' behaviors were recorded at 6 and 8 wk of age. At 75 d of age, a blood sample was taken and a biopsy puncture was created on one pig from each litter, then pigs were stressed by mixing. Blood was sampled every other day for 10 d to determine plasma cortisol concentrations and differential leukocyte counts. Biopsy damage was evaluated for healing. At 1 d of age, control pigs tended to weigh more (P = .09), have a lower expression of ACTH-R mRNA (P = .01) and IGF-I mRNA (P = .01), and a lower CORT:MED (P = .04) than ACTH pigs. At 30 d of age, control pigs had a greater concentration of beta-endorphin (P = .01) and tended to have a lower concentration of CRH (P = .09) and IGF-I mRNA (P = .10) than ACTH pigs. At 60 d of age, control pigs tended to have lighter pituitary glands (P = .08), a lower expression of POMC mRNA (P = .02), and a CORT:MED (P = .003) than ACTH pigs. At 8 wk of age, control pigs performed a higher frequency of belly nosing (P = .07) and oral vice behaviors (P = .01) than ACTH pigs. In response to mixing stress, control pigs had lesser concentrations of plasma cortisol (P = .03) and healed faster (P = .006) than ACTH pigs. Thus, exogenous ACTH and restraint during gestation alters the HPA axis of the sow's offspring, and during stressful situations later in life health, and therefore welfare, may be compromised.

Structure-function correlations of growth hormone or/and prolactin-producing pituitary adenomas: an in vitro study with the reverse hemolytic plaque assay.

The purpose of this study was to detect in vitro growth hormone (GH) and prolactin (PRL) secretion from adenomas clinically associated with GH or PRL hypersecretion. The reverse hemolytic plaque assay (RHPA) was applied in order to reveal possible differences among various morphologic adenoma types, and to examine the inhibitory effects of octreotide on GH release as well. The 20 surgically resected pituitary adenomas studied included 15 from acromegalic patients and 5 from patients with hyperprolactinemia. All adenomas were diagnosed by histology, immunocytochemistry and electron microscopy. Among tumors associated with acromegaly, 5 were densely granulated (DG), 5 were sparsely granulated (SG) somatotroph (SM) adenomas, 2 were mammosomatotroph (MSM) and 3 mixed somatotroph-lactotroph cell (mixed SM-LT) adenomas; tumors causing hyperprolactinemia included 4 lactotroph (LT) adenomas and 1 mixed SM-LT adenoma. GH release assessed by the RHPA corresponded to in vivo hormone secretion and to tissue immunoreactivity. Statistical analysis showed significant differences among all morphologic types of SM adenomas, exclusive of SG-SM adenomas compared to mixed SM-LT adenomas. The mean plaque size in DG-SM and MSM adenomas was significantly greater than that of SG-SM and mixed SM-LT adenomas, indicating higher GH secretion by the former two types during the same incubation time. PRL secretion was documented in 2 mixed SM-LT adenomas. Plaques for PRL, but not for GH were formed in all LT adenomas. In all SM and LT adenomas, cells producing large plaques represented a minority of the plaque-forming cell population, however, they accounted for the largest part of the total plaque area, thus the largest part of hormone secretion. Octreotide effects on GH release were studied in 6 adenomas by the RHPA. Octreotide treatment induced a rapid and significant reduction in GH secretion by SM cells in vitro, with a selective effect on high-secreting cells.

Development and Application of Fish Growth Hormone Gene (Abstract)

Growth hormone is a member of a family composed of prolactin, somatolactin, and placental lactogen. It is synthesized, produced, and stored by pituitary somatotroph cells. The secreted growth hormone stimulates the production of insulin-like growth factors which mediate many of its growth-promoting effects. In this study we cloned growth hormone genes from ayu (Plecoglossus altivelis), grouper (Epinephelus awoara) and zebrafish (Danio rerio). With high homology and similarity, the putative growth hormone polypeptides contain 209, 205 and 211 amino acids respectively. Two functional recombinant GHs were expressed from E coli expression system. By inserting the zebrafish GH gene promoter region to pEGFP-1 vector, we analyzed the promoter activity by introducing the constructs into a rat pituitary cell line and zebrafish embryos. Through the fluorescent microscope, green fluorescent protein expression in the developmental stages of transgenic embryos was observed. To identify the timing and spacing of growth hormone expression during embryogenesis, whole mount in situ hybridization was also performed. As a result, growth hormone mRNA was detected as early as 1-cell and 4-cell stages. In pharyngula period, growth hormone was detected in the otic capsule and forehead region. The result of RT-PCR also showed that growth hormone expression was detected in the mature ovary, somite-stage embryos and hatched larvae. For making growth faster transgenic fish, we transferred a plasmid DNA containing carp b-actin promoter and rainbow rout growth hormone cDNA into farmed ayu by sperm-electroporation method. After five-month culture, the existence of transferred DNA was detected by PCR with specific primers in extracted genomic DNA from caudal fin tissue. The mosaic expressions of transgene were detected from gill, caudal fin, liver, brain and muscle. The growth promotion performance was indicated by 2 fold of body weight and 1.3 fold of body length.

Long-term effects of recombinant human erythropoietin therapy on growth hormone secretion in uremic patients undergoing peritoneal dialysis

Recombinant human erythropoietin (rhEPO) is being successfully used for the treatment of uremic anemia. Short-term studies have proved that correction of anemia with rhEPO therapy is accompanied by several changes in growth hormone (GH) secretion in uremic patients. The present study aimed to assess the influence of long-term rhEPO therapy on baseline and stimulated GH concentrations in a group of uremic patients undergoing continuous ambulatory peritoneal dialysis (CAPD). Seven well-nourished and clinically stable CAPD patients were studied. Ten normal subjects were studied as controls. GH responses to direct pituitary stimulation with GH-releasing hormone (GHRH) (100 microg intravenously [i.v.]) and indirect hypothalamic stimulation with insulin-induced hypoglycemia (0.1 U/kg body weight i.v.) and clonidine (0.15 mg/m2 orally), were assessed before and after 3, 6, and 12 months of subcutaneously administered rhEPO therapy. After rhEPO administration, an increase of the hemoglobin concentration was observed in all patients and maintained at about 12 g/dL throughout the study period. rhEPO therapy did not induce any significant change in baseline concentrations of GH and insulin-like growth factor I. Correction of the anemia was accompanied by a clear increase in the area under the curve (AUC) and the area above the baseline (AAB) of GH secretion in response to GHRH stimulation. These changes were statistically significant after 3 and 6 months of therapy, although at 12 months no significant differences in relation to pretreatment values could be observed. rhEPO treatment was associated with a progressive decrement in the GH AUC and AAB in response to hypoglycemic challenge, reaching statistically significant values at months 6 and 12. On the other hand, compared with the control group, GH responses to clonidine were blunted at the start of the study in CAPD patients, and rhEPO therapy was not accompanied by any modification. In conclusion, long-term treatment with rhEPO in CAPD patients is associated with complex and profound effects on somatotrope cell function, characterized by diverse effects on GH responses to stimuli that release GH through different mechanisms. Some of these rhEPO-induced alterations in somatotrope function are dependent on the duration of treatment.

Distribution, characterization, and growth hormone-releasing activity of pituitary adenylate cyclase-activating polypeptide in the European eel, Anguilla anguilla.

The complementary DNA encoding pituitary adenylate cyclase-activating polypeptide (PACAP) has been cloned from two species of teleost fishes, the Sockeye salmon and the Thai catfish, and the amino acid sequence of PACAP has been determined in another teleost, the stargazer. However, to date, the detailed distribution of PACAP immunoreactivity has never been investigated in the fish brain. In the present study, we have determined the localization of PACAP-immunoreactive neurons in the central nervous system of a primitive teleost fish, the European eel Anguilla anguilla, using an antiserum raised against PACAP27. PACAP-positive perikarya were exclusively observed in the diencephalon, i.e. in the preoptic nucleus of the hypothalamus and in the dorsal and ventral nuclei of the thalamus. PACAP-immunoreactive fibers were detected in various areas of the brain, notably in the ventral telencephalon, the diencephalon, the mesencephalon, the cerebellar valvula, and the medulla oblongata. In addition, a dense accumulation of PACAP-containing nerve terminals was found in the pars distalis of the pituitary. The PACAP-like immunoreactivity contained in the eel brain was characterized by HPLC analysis combined with RIA quantification. The major form of PACAP-immunoreactive material coeluted with mammalian PACAP38. Molecular cloning of the PACAP precursor has previously shown that in fish, PACAP and GH-releasing hormone (GHRH) originate from the same precursor. We have thus investigated the effects of PACAP and GHRH on GH secretion from eel pituitary cells in primary culture. Dose-response experiments revealed that PACAP27 and PACAP38 possessed the same efficacy, but PACAP38 was 12 times more potent than PACAP27 in stimulating GH release (ED50 = 4.3 x 10(-10) and 3.5 x 10(-9) M, respectively). In contrast, GHRH, even at a high concentration (10(-6) M), had no effect on GH release. Taken together, these data indicate that in the eel, PACAP may play a significant role in the regulation of somatotrope cells: 1) PACAP-immunoreactive neurons are exclusively located in the diencephalon and send numerous projections in the pars distalis; and 2) PACAP, but not GHRH, dose dependently stimulates GH secretion from cultured eel pituitary cells.

Cobalt 60 irradiation of pituitary gland tumors in three cats with acromegaly

The most common cause of naturally developing acromegaly in cats is a growth hormone-secreting adenoma of the pituitary pars distalis somatotropic cells. Irradiation of pituitary gland tumors in cats with acromegaly may result in transient or long-term resolution of acromegaly and diabetes mellitus. Diabetic cats with acromegaly may have a history of weight loss instead of weight gain.

Altered growth of mice divergently selected for body weight is associated with complex changes in the growth hormone/insulin-like growth factor system

Mice investigated in this study were generated by selecting a sub-population of the NMRI out-bred stock (C), for high (H) or low (L) 8-week body weight. After 58 generations of selection, 8-week body weights of the sub-populations were markedly different if compared to controls. To investigate endocrine changes causing the altered growth performance in the different lines of mice, we analysed several components of the growth hormone (GH)/insulin-like growth factor (IGF) system. Pituitary weights of male and female L mice were significantly smaller than those of C and H mice. Relative to body weight, pituitary weights of male mice from the three lines did not differ, however pituitary weight-to-body weight-ratios of female L mice were significantly greater than those of H females. Mean volume densities of somatotropic cells were significantly smaller in L mice than in C and H mice. Serum IGF-I concentrations were significantly lower in the L line than in the C and H lines. H mice displayed significantly increased serum insulin levels both after ad libitum feeding and after a 14 hour fasting period. Ligand blot analysis of serum IGF-binding proteins (IGFBPs) revealed a significant reduction of circulating IGFBP-3 in L mice as compared to C and H mice. In contrast, serum IGFBP-2 mRNA levels were significantly increased in male L mice and showed non significant increases in female L mice. Hepatic IGFBP-2 mRNA levels were significantly increased in L mice and decreased in H mice as compared to C mice. Expression of IGFBP-4 mRNA in the liver was significantly decreased in both selection lines (L, H) as compared to the random-bred controls. Our findings demonstrate that altered growth of mice resulting from selection for body weight is associated with complex changes in the endocrine network of the GH/IGF system.

Diagnosis of GH deficiency in adults

Within an appropriate clinical context, growth hormone deficiency (GHD) in adults must be demonstrated biochemically. The assays of insulin-like growth factor-I (IGF-I) and IGF binding protein-3 (IGFBP-3) per se do not establish the diagnosis of adult GHD. Similarly, the evaluation of spontaneous growth hormone (GH) secretion over 24 h has no diagnostic value in adulthood even when an ultra-sensitive GH assay is used. The diagnosis of adult GHD is established by provocative testing of GH secretion, and insulin-induced hypoglycaemia using the insulin tolerance test (ITT) is indicated as the test of choice. Alternative provocative tests of GH secretion have been proposed and have to be used with appropriate cut-off limits. Testing with GH releasing hormone (GHRH) alone has no diagnostic value, but when GHRH is given in combination with arginine or pyridostigmine it becomes the most potent and reproducible provocative test to evaluate the maximal secretory capacity of somatotrope cells. The potentiating effect of arginine on the GHRH-induced GH response is fully preserved while the stimulatory effect of GHRH + pyridostigmine is reduced in ageing. The GHRH + arginine test is well tolerated and reproducibly distinguishes between normal and GHD adult and elderly subjects. Thus, the GHRH + arginine test is the most promising alternative to the ITT provided that cut-off limits appropriate to its potent stimulatory effect are considered.

Immunocytochemical and Ultrastructural Identification of Pituitary Cell Types in the Protogynous Thalassoma duperrey during Adult Sexual Ontogeny

Abstract Protogynous wrasses (Thalassoma duperrey): females (F), primary males (PM) along with a few terminal-phase males (TM) and sex-changed males (SM), were used to characterize the topographical organization of the pituitary. In general, immunocytochemical and ultrastructural features of the adenohypo-physeal cell types of the saddleback wrasse pituitary resemble those of other teleosts. In the rostral pars distalis (RPD), corticotropic cells were found bordering the neurohypophysis (NH) and surrounding the centroventrally located prolactin cells. Thyrotropic cells formed a small group in the anteriodorsal part of the rostral and proximal pars distalis (PPD). The somatotropic cells were distributed in large clusters, mostly organized in cell cords around the interdigitations of the NH of the dorsal PPD. Cells containing gonadotropin Ib subunit were localized in the dorsal parts of the PPD, in close association with somatotropic cells and gonadotropin IIb subunit containing cells were seen in the centr...

Paracrine control of steroid hormone secretion by chromaffin cells in the adrenal gland of lower vertebrates.

The adrenal glands of lower vertebrates display a notable intermingling between steroidogenic and chromaffin tissues, which increases from Pisces to Aves. As in mammals, adrenal chromaffin cells contain and release, in addition to catecholamines, serotonin and several peptides, which may affect the secretory activity of steroidogenic cells in a paracrine manner. Stimulatory molecules include serotonin, arginine-vasotocin, tachykinins, vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide and calcitonin gene-related peptide; inhibitory molecules are dopamine, somatotropic hormone-release inhibiting hormone and galanin. Epinephrine and norepinephrine appear to stimulate steroid secretion in Aves and to inhibit it in Pisces, while their action in Amphibia is controversial. Likewise, atrial natriuretic peptide exerts an anti-secretagogue action in Amphibia and a marked secretagogue effect in Pisces and Aves. The effects of opioids (enkephalins and endorphins) have scarcely been investigated and the findings obtained are highly questionable. Compared with the amazing mass of investigations carried out in mammals, studies in lower vertebrates are few, and in large part performed in Amphibia and Aves. It appears that much further work has to be done by comparative endocrinologists to fully clarify the physiological relevance of the functional interactions between chromaffin and steroidogenic cells in the adrenal glands of lower vertebrates.

Les nouveaux sécrétagogues de l'hormone de croissance De la physiologie aux premières applications thérapeutiques

Growth hormone releasing peptides (GHRP) are synthetic hexapeptides that physiologically stimulate GH release through two different pathways: 1) central and 2) direct action on somatotropic cells. Animal experiments and first clinical trials show that synthetic GHRP and synthetic analogues could be useful substitutes to recombinant GH in the treatment of GH deficiency, and in pathological conditions which may benefit from amplification of the GH-IGF I axis activity.

Reduction of the somatotrope responsiveness to GHRH and Hexarelin but not to arginine plus GHRH in hyperprolactinemic patients.

Aim of the present study was to verify the maximal secretory capacity of somatotrope cells in patients with pathological hyperprolactinemia (HPRL) comparing it with that in normal age-matched women (NW). To this goal in 12 HPRL normal weight patients (age 28.6 +/- 2.6 yr, BMI 23.1 +/- 1.1 kg/m2) and 8 NW (27.2 +/- 0.8 yr, 22.8 +/- 0.8 kg/m2) we studied the GH response to GHRH (1 microgram/kg i.v.), GHRH plus arginine (ARG, 0.5 g/kg i.v.), an amino acid probably acting at the hypothalamic level inhibiting somatostatin release, and Hexarelin (HEX, 2 micrograms/kg i.v.), a synthetic hexapeptide belonging to GHRP family, which acts concomitantly at the pituitary and the hypothalamic level. IGF-I levels in HPRL were similar to those in NW (179.2 +/- 16.5 micrograms/l and 218.5 +/- 30.8 micrograms/l). In NW the GH response to GHRH (AUC: 1299.5 +/- 186.9 micrograms 90 min/l) was lower (p < 0.02) than those to GHRH + ARG (5252.7 +/- 846.3 micrograms 90 min/l) and HEX 3216.6 +/- 462.3 micrograms 90 min/l) which, in turn, were similar. In HPRL the GH response to GHRH (894.7 +/- 242.4 micrograms 90 min/l) was lower (p < 0.03) than that to HEX (1586.5 +/- 251.3 micrograms 90 min/l) and both were lower (p < 0.03) than that to GHRH + ARG (4468.8 +/- 941.7 micrograms 90 min/l). In HPRL the GH responses to GHRH and HEX were lower than those that in NW (p < 0.03) while that to GHRH + ARG was similar in both groups. These results demonstrate that the somatotrope responsiveness to GHRH and HEX is clearly reduced in patients with pathological hyperprolactinemia. On the other hand, in this condition the GH response to GHRH + ARG is normal. As arginine likely acts via inhibition of hypothalamic somatostatin release, these findings show that the maximal secretory capacity of somatotrope cells in hyperprolactinemia is preserved and indicate that partial refractoriness of somatotrope cells to GHRH and HEX could be due to somatostatinergic hyperactivity.

Description of two types of mammosomatotropes in mink (Mustela vison) adenohypophysis: changes in the population of mammosomatotropes under different physiological conditions.

The present study was undertaken to clarify the existence of mammosomatotropes (MS cells) in the mink adenohypophysis and their possible involvement in the interconversion of mammotrope and somatotrope cells under different physiological conditions: prepubertal, pubertal and adulthood. Electron microscope immunocytochemistry was used to detect growth hormone (GH) and prolactin (PRL) immunoreactivities in the anterior pituitary gland of mink. Primary antisera raised in rabbit (human anti-GH, NIDDK-AFP-1613102481; human anti-PRL, NIDDK-AFP-55781789) were localized with appropriate species-specific antisera coupled to colloidal gold particles of different sizes. MS cells were most frequently observed in adult mink. Double labeling for GH and PRL was presented in two types of MS cells. MS1 cells, observed only in adults, showed an irregular morphology, with many cytoplasmic processes. Within their cytoplasm there were numerous rounded secretory granules of approximately 135 nm mean diameter. In MS1 cells three types of secretory granules were identified. The most numerous contained only PRL. The least frequent contained only GH and the third type contained GH and PRL and appeared in an intermediate quantity. MS2 cells, observed in all age groups, presented a rounded morphology. These cells had a voluminous cytoplasm which showed little development of the organelles and contained a large number of rounded and very electron-dense secretory granules which measured about 160 nm mean diameter. Within MS2 cells two types of secretory granules, according to their immunolabeling, were observed. Monohormonal granules containing GH were the more abundant while bihormonal granules, containing GH and PRL, were present to a lesser degree. Data presented in this report suggest that MS cells could play a role in the interconversion of somatotropes into mammotropes in mink.

AP-1 and Oct-1 Transcription Factors Down-regulate the Expression of the Human PIT1/GHF1 Gene

The pituitary-specific transcription factor Pit-1/GHF-1 is a member of the POU domain family of regulatory proteins. It is involved in the commitment and expansion of the somatotropic cell lineage and activates the transcription of a set of anterior pituitary genes. We have cloned the human PIT1/GHF1 gene and characterized the regulatory mechanisms controlling its promoter activation and regulation. A minimal promoter region (-102 to +15) contains the cis-acting elements that confer to the human PIT1/GHF1 gene a high basal transcriptional activity, the tissue-specific expression, and the autoregulation by Pit-1/GHF-1 protein. The upstream promoter region contains a multiplicity of Pit-1/GHF-1 binding sites that do not show any synergistic interaction with the minimal promoter. The transcriptional activity is negatively regulated by Oct-1 and mediated by an octamer-binding site (OTF). In addition, we have also identified a 12-O-tetradecanoylphorbol-13-acetate-responsive element, which overlaps with a Pit-1/GHF-1 binding site. A mutually exclusive binding of the activator protein-1 (AP-1) and Pit-1/GHF-1 has been observed on this composite site, and AP-1 was shown to down-regulate PIT1/GHF1 transcription.

Reliability of provocative tests to assess growth hormone secretory status. Study in 472 normally growing children.

The reliability of provocative stimuli of GH secretion in the diagnosis of GH deficiency is still controversial. Until now, normative values of GH response to various stimuli have not been established properly. In 472 children and adolescents with normal stature (n = 295, height SDS range -1.5 to 1.2) or normal short stature (n = 177, height SDS range -3.7 to -1.8), we studied the GH response to physical exercise, insulin-induced hypoglycemia, arginine (ARG), clonidine, levodopa, glucagon, pyridostigmine (PD), GHRH, PD + GHRH, and ARG + GHRH. The peak GH responses (range) to various stimuli were: 1) physical exercise: 3.0-28.3 micrograms/L; 2) insulin-induced hypoglycemia: 2.7-46.4 micrograms/L; 3) ARG: 0.5-48.4 micrograms/L; 4) clonidine: 3.8-86.0 micrograms/L; 5) levodopa: 1.9-40.0 micrograms/L; 6) glucagon: 1.9-49.5 micrograms/L; 7) PD: 2.5-35.0 micrograms/L; 8) GHRH: 2.7-102.7 micrograms/L; 9)PD + GHRH: 19.6-106.0 micrograms/L; and 10) ARG + GHRH: 19.4-120.0 micrograms/L. Our results show that all conventional stimuli of GH secretion frequently failed to increase GH levels, showing values lower than that arbitrarily assumed, so far, as minimum normal GH peak, i.e. 7 or 10 micrograms/L. When combined with PD or ARG (substances inhibiting hypothalamic somatostatin release), GHRH becomes the most powerful test to explore the secretory capacity of somatotrope cells (the GH response being always higher than 19 micrograms/L). Therefore, only GHRH combined with PD or ARG may be able to clearly differentiate normal children from patients with GH deficiency, though a normal GH response to these tests cannot rule out the existence of GH hyposecretory state because of hypothalamic dysfunction.

Growth hormone response to growth hormone-releasing hormone varies with the hypothalamic-pituitary abnormalities

We determined growth hormone (GH) and insulin-like growth factor I (IGF-I) levels after a 3 h infusion of escalating doses of growth hormone-releasing hormone (GHRH(1-29)) followed by a bolus injection in hypopituitary patients with marked differences in pituitary features at magnetic resonance imaging (MRI) in order to evaluate further the contribution of MRI in the definition of pituitary GH reserve in GH-deficient patients. Twenty-nine patients (mean age 14.5 +/- 4.0 years) were studied. Group I comprised 13 patients: seven with isolated GH deficiency (IGHD) (group Ia) and six with multiple pituitary hormone deficiency (MPHD) (group Ib) who had anterior pituitary hypoplasia, unidentified pituitary stalk and ectopic posterior pituitary at MRI, Group II consisted of eight patients with IGHD and small anterior pituitary/empty sella, while in group III eight had IGHD and normal morphology of the pituitary gland. Growth hormone and IGF-I levels were measured during saline infusion at 08.30-09.00 h, as well as after infusion of GHRH (1-29) at escalating doses for 3h: 0.2 micrograms/kg at 09.00-10.00 h, 0.4 micrograms/kg at 10.00-11.00 h, 0.6 micrograms/kg at 11.00-12.00 h and an intravenous bolus of 2 micrograms/ kg at 12.00 h. In the group I patients, the peak GH response to GHRH(1-29) was delayed (135-180 min) and extremely low (median 2mU/l). In group II it was delayed (135-180 min), high (median 34.8 mU/l) and persistent (median 37.4 mU/l at 185-210 min). In group III the peak response was high (median 30.8 mU/l) and relatively early (75-120 min) but it declined rapidly (median 14.4 mU/l at 185-210 min). In one group I patient, GH response increased to 34.6 mU/l. The mean basal value of IGF-I levels was significantly lower in group I (0.23 +/- 0.05 U/ml) than in groups II (0.39 +/- 0.13U/ ml, p < 0.01) and III (1.54 +/- 0.46 U/ml, p < 0.001) and did not vary significantly during the GHRH(1-29) infusion. The present study demonstrates that the impaired GH response to 3 h of continuous infusion of escalating doses of GHRH(1-29) was strikingly indicative for pituitary stalk abnormality, strengthening the case for use of GHRH in the differential diagnosis of GH deficiency. The low GH response, more severe in MPHD patients, might be dependent on the residual somatotrope cells, while the better response (34.6 mU/l) in the group Ia patients might suggest that prolonged GHRH infusion could help in evaluating the amount of residual GH pituitary tissue. Pituitary GH reserve, given the GH response to GHRH infusion in GH-deficient patients with small anterior pituitary/empty sella, seems to be maintained.