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GH is banned by the World Anti-Doping Agency as a performance-enhancing anabolic agent. Doping with GH likely began in the early 1980s and became more prevalent with the advent of recombinant technology well before any scientific evidence of benefit. The expectation that GH improves physical function stems from its anabolic and lipolytic properties. Athletic performance depends on muscle strength and the energy required to power muscle function. In recreational athletes, GH selectively improves anaerobic sprint capacity but has not been proven to significantly enhance muscle strength, power, or maximum rate of oxygen consumption. GH is secreted as a family of isoform peptides in a pulsatile manner reflecting intermittent secretion and rapid clearance. Its anabolic actions are largely mediated by IGF-I, which stimulates whole-body protein synthesis, including skeletal muscle and collagen proteins. Two methods have been validated for detecting GH abuse in athletes. The first (the isoform method) is based on distinguishing pure recombinant 22-kDa GH from the heterogeneous isoforms secreted from the pituitary. The second (the marker method) is based on measuring blood levels of GH-responsive proteins, specifically IGF-I and the N-terminal propeptide of type III collagen (P-III-NP). Only a handful of athletes have been caught since the implementation of GH doping tests in 2004. The low rate likely reflects the limitation of in-competition testing using current methods. Improved detection rates may be achieved by more out-of-competition testing, introducing athletes' biological passports, and the development of novel methods. Governance, operational, technical, and political factors influence the effectiveness of an anti-doping program., (Copyright © 2019 Endocrine Society.)

Pituitary somatotropes perform the key function of coordinating organismic growth and body composition with metabolic signals. However, the mechanism by which they sense and respond to metabolic signals via the adipokine leptin is unknown. The complex interplay between the heterogeneous cell types of the pituitary confounds the identification of somatotrope-specific mechanisms. Somatotropes represent 30%-40% of the anterior pituitary population and are derived from a lineage of cells that are activated by the Pit-Oct-Unc domain family domain class 1 transcription factor 1 (POU1F1) to produce GH, prolactin (PRL). and TSH. To determine the mechanism by which leptin controls somatotrope function, we used Cre-LoxP technology and fluorescence-activated cell sorting to purify and study control or leptin receptor-deleted (Lepr null) somatotropes. We report that Lepr-null somatotropes show significant reductions in GH protein (GH) and Gh mRNA. By contrast, enzyme immunoassays detected no changes in ACTH, LH, and FSH levels in mutants, indicating that the control of these hormones is independent of leptin signaling to somatotropes. Reduced TSH and PRL levels were also observed, but interestingly, this reduction occurred only in in Lepr-null somatotropes from mutant females and not from males. Consistent with the sex-specific reduction in Gh mRNA, TSH, and PRL, enzyme immunoassays detected a sex-specific reduction in POU1F1 protein levels in adult female Lepr-null somatotropes. Collectively, this study of purified Lepr-null somatotropes has uncovered an unexpected tropic role for leptin in the control of POU1F1 and all POU1F1-dependent hormones. This supports a broader role for somatotropes as metabolic sensors including sex-specific responses to leptin.

The secretion of endocrine hormones from pituitary cells finely regulates a multitude of homeostatic processes. To dynamically adapt to changing physiological status and environmental stimuli, the pituitary gland must undergo marked structural and functional plasticity. Endocrine cell plasticity is thought to primarily rely on variations in cell proliferation and size. However, cell motility, a process commonly observed in a variety of tissues during development, may represent an additional mechanism to promote plasticity within the adult pituitary gland. To investigate this, we used multiphoton time-lapse imaging methods, GH-enhanced green fluorescent protein transgenic mice and sexual dimorphism of the GH axis as a model of divergent tissue demand. Using these methods to acutely (12 h) track cell dynamics, we report that ovariectomy induces a dramatic and dynamic increase in cell motility, which is associated with gross GH-cell network remodeling. These changes can be prevented by estradiol supplementation and are associated with enhanced network connectivity as evidenced by increased coordinated GH-cell activity during multicellular calcium recordings. Furthermore, cell motility appears to be sex-specific, because reciprocal alterations are not detected in males after castration. Therefore, GH-cell motility appears to play an important role in the structural and functional pituitary plasticity, which is evoked in response to changing estradiol concentrations in the female.

This study aimed to test the hypothesis that the tuberoinfundibular dopaminergic neurons of the arcuate nucleus and/or the lactotroph cells of the anterior pituitary gland are key targets for the programming effects of perinatal glucocorticoids (GCs). Dexamethasone was administered noninvasively to fetal or neonatal rats via the mothers' drinking water (1 mug/ml) on embryonic d 16-19 or neonatal d 1-7, and control animals received normal drinking water. At 68 d of age, the numbers of tyrosine hydroxylase-positive (TH+) cells in the arcuate nucleus and morphometric parameters of pituitary lactotrophs were analyzed. In control animals, striking sex differences in TH+ cell numbers, lactotroph cell size, and pituitary prolactin content were observed. Both pre- and neonatal GC treatment regimens were without effect in adult male rats, but in females, the overriding effect was to abolish the sex differences by reducing arcuate TH+ cell numbers (pre- and neonatal treatments) and reducing lactotroph cell size and pituitary prolactin content (prenatal treatment only) without changing lactotroph cell numbers. Changes in circulating prolactin levels represented a net effect of hypothalamic and pituitary alterations that exhibited independent critical windows of susceptibility to perinatal GC treatments. The dopaminergic neurons of the hypothalamic periventricular nucleus and the pituitary somatotroph populations were not significantly affected by either treatment regimen in either sex. These data show that the adult female hypothalamo-lactotroph axis is profoundly affected by perinatal exposure to GCs, which disrupts the tonic inhibitory tuberoinfundibular dopaminergic pathway and changes lactotroph morphology and prolactin levels in the pituitary and circulation. These findings provide new evidence for a long-term disruption in prolactin-dependent homeostasis in females, but not males, after inappropriate GC exposure in perinatal life.

The SMA1-mouse is a novel ethyl-nitroso-urea (ENU)-induced mouse mutant that carries an a-->g missense mutation in exon 5 of the GH gene, which translates to a D167G amino acid exchange in the mature protein. Mice carrying the mutation are characterized by dwarfism, predominantly due to the reduction (sma1/+) or absence (sma1/sma1) of the GH-mediated peripubertal growth spurt, with sma1/+ mice displaying a less pronounced phenotype. All genotypes are viable and fertile, and the mode of inheritance is in accordance with a semidominant Mendelian trait. Adult SMA1 mice accumulate excessive amounts of sc and visceral fat in the presence of elevated plasma ghrelin levels, possibly reflecting altered energy partitioning. Our results suggest impaired storage and/or secretion of pituitary GH in mutants, resulting in reduced pituitary GH and reduced GH-stimulated IGF-1 expression. Generation and identification of the SMA1 mouse exemplifies the power of the combination of random mouse mutagenesis with a highly detailed phenotype-analysis as a successful strategy for the detection and analysis of novel gene-function relationships.

Human placental GH (hGH-V) is a variant of pituitary hGH (hGH-N) synthesized and secreted by syncytiotrophoblasts during pregnancy. It differs from hGH-V by only 13 amino acid residues, which makes difficult a specific measurement of hGH-V without interference from hGH-N. To overcome the analytical difficulties, we produced new high affinity monoclonal antibodies specific for hGH-V. Precise screening and epitope mapping allowed identification of a pair of monoclonal antibodies suitable to establish a highly sensitive assay for hGH-V measurement. In a prospective, longitudinal study involving 84 normal pregnancies, we measured maternal concentrations of hGH-V, leptin, IGF-I, and cord blood IGF-I. hGH-V was detectable as early as gestational week (GW) 7. Mean concentrations of hGH-V increased from 0.9 +/- 0.5 microg/liter (GW 7-13) to 2.8 +/- 0.9 microg/liter (GW 18-22), 7.3 +/- 2.6 microg/liter (GW 28-32), and 13.0 +/- 9.6 (GW 37-41). A negative correlation was found between prepregnancy body mass index and hGH-V concentrations from GW 28 onward. Peak hGH-V levels occurred at wk 36.5 +/- 2.6 and were significantly lower in obese (P = 0.029) and higher in underweight (P = 0.035) mothers compared with those in mothers of normal weight. The increase in hGH-V between GW 18-22 and GW 28-32 was negatively correlated to the increase in maternal leptin during this period (P = 0.027). Maternal IGF-I concentrations were correlated to those of hGH-V from GW 18 onward (P = 0.039). The strongest correlation was found at GW 28-32 (P = 0.001). Furthermore, maternal hGH-V concentrations in late pregnancy correlated with cord blood IGF-I (P = 0.025) and size of the newborn (P = 0.017). These results, obtained by a new, highly sensitive hGH-V-specific immunoassay, highlight the importance of maternal hGH-V in the regulation of maternal and fetal IGF-I. In addition, the results indicate that maternal weight has a major impact on circulating concentrations of hGH-V.

GH was identified in the sea lamprey, an extant representative of a group of the most ancient vertebrates, the Agnatha. A putative GH-cDNA was cloned from the pituitary by RT-PCR. The entire coding region comprised an open-reading frame of 203 amino acids (aa). The mature protein was also isolated from pituitaries, and fractionated by gel filtration and reverse-phase HPLC. A putative GH was monitored by Western blotting with a rabbit antiserum against a synthetic peptide corresponding to pre-GH sequence (aa 29-45). Sequence analysis of the purified protein demonstrated that the prehormone consists of a signal peptide of 22 aa and the mature protein of 181 aa, which shows 25% sequence identity with sturgeon GH. The site of production was identified through immunohistochemistry to be cells of the dorsal half of the proximal pars distalis of the pituitary. Following cDNA cloning of lamprey IGF cDNA, it was shown using RT-PCR that lamprey GH stimulates IGF expression in lamprey liver. This is the first study in which a member of the GH/prolactin/somatolactin family has been identified in an agnathan. In addition, GH appears to be the only member of this hormone family in the sea lamprey. Evidence suggests that GH is the ancestral hormone in the molecular evolution of the GH family and that the endocrine mechanism for growth stimulation was established at an early stage of vertebrate evolution.

Targeted disruption [knockout (KO)] of the mouse prolactin (PRL) gene created an animal model of primary isolated PRL deficiency in which there is no detectable PRL bioactivity. Pituitary glands of young adult female PRLKO mice were hyperplastic, and many cells had expanded cytoplasms with granular accumulations of an N-terminal peptide encoded by the disrupted PRL gene (KO/10 peptide). Confocal imaging showed that the pituitaries in PRL(+/+) and PRL(+/-) females contained dense accumulations of apparently Golgi-associated immunoreactive PRL. PRLKO female mice (15-18 months old) developed hyperemic pituitary adenomas. The pituitary tumors in PRLKO mice synthesized the KO/10 peptide, which implies that the tumors arise from the lactotroph lineage. Anchorage-independent growth was observed among pituitary cells from PRLKO mice, aged 8 months or older, but not in cells from 3-month-old PRLKO mice. GH cells appeared to be normal in PRLKO pituitaries, but were displaced by the hyperplastic and hypertrophic growth of KO/10-positive cells. Bromocriptine suppressed mean pituitary weight in 8-month-old PRLKO mice compared with vehicle-treated PRLKO animals (20 +/- 0.01 and 60 +/- 10 mg; P < 0.01). We infer that pituitary lactotrophs of PRLKO mice suffer from a dual pathology that includes hypertrophy resulting from endoplasmic reticulum expansion and hyperplasia, with adenomatous transformation, in part as a consequence of disrupted dopaminergic feedback regulation.

Rat pituitary malignant tumor cells; mGH3, show hypervascularization in in vivo xenografts and overexpress parathyroid hormone-related peptide (PTHrP) compared to original GH3 cells. To elucidate whether PTHrP is involved in tumor-derived angiogenesis, we examined the effect of PTHrP on vascular endothelial cells both in vitro and in vivo. Results of in vivo diffusion chamber assay showed a clear hypervascularization on the outer surface of diffusion chambers containing mGH3 tumor cell implants but not in those containing GH3 cells. Co-incubation with antisense PTHrP oligonucleotide (10 microM), but not sense or mismatched PTHrP oligonucleotide, suppressed hypervascularization in diffusion chambers. To further examine the role of PTHrP on endothelial cell function, PTHrP(1-34) was added at various concentrations to cultured bovine endothelial cells (BAECs) harvested from the aorta. PTHrP(1-34) did not alter the proliferation or migration of endothelial cells, but rather dose-dependently increased capillary formation by endothelial cells on the collagen gel matrix. Furthermore, 0.1 mM of 8-bromo-cAMP caused a similar increase in tube formation, which was dose-dependently inhibited by H89, a protein kinase A inhibitor. Our results indicate for the first time that PTHrP is a potential paracrine factor acting via the PKA pathway to enhance angiogenesis through capillary tube formation by endothelial cells in malignant pituitary tumors.

Purinergic G protein-coupled receptors (P2YR) and ion-conducting receptor-channels (P2XR) are present in the pituitary. However, their identification, expression within pituitary cell subpopulations, and the ability to elevate intracellular Ca2+ concentration ([Ca2+]i) in response to ATP stimulation were incompletely characterized. Here we show that mixed populations of rat anterior pituitary cells express messenger RNA transcripts for P2Y2R, P2X2aR, P2X2bR, P2X3R, P2X4R, and P2X7R. The transcripts and functional P2Y2R were identified in lactotrophs and GH3 cells, but not in somatotrophs and gonadotrophs, and their activation by ATP led to an extracellular Ca2+-independent rise in [Ca2+]i in about 40% of cells tested. Lactotrophs and GH3 cells, but not somatotrophs, also express transcripts for P2X7R, P2X3R, and P2X4R. Functional P2X7R were identified in 74% of lactotrophs, whereas 50% of these cells expressed P2X3R and 33% expressed P2X4R. Coexpression of these receptor subtypes in single lactotrophs was frequently observed. Purified somatotrophs expressed transcripts for P2X2aR and P2X2bR, and functional receptors were identified in somatotrophs and gonadotrophs, but not in lactotrophs. Consistent with the cell-specific expression of transcripts for P2X2R and P2X3R, the expression of their functional heteromers was not evident in pituitary cells. Receptors differed in their capacities to elevate and sustain Ca2+ influx-dependent rise in [Ca2+]i during the prolonged ATP stimulation. These results indicate that the purinergic system of anterior pituitary is extremely complex and provides an effective mechanism for generating a cell- and receptor-specific Ca2+ signaling pattern in response to a common agonist.

Peptidylglycine alpha-amidating monooxygenase (PAM) is a bifunctional enzyme expressed in each major anterior pituitary cell type. We used primary cultures of adult male rat anterior pituitary to examine PAM expression, processing, and secretion in the different pituitary cell types and to compare these patterns to those observed in transfected AtT-20 corticotrope tumor cells. Immunostaining and subcellular fractionation identified PAM in pituitary secretory granules and additional vesicular compartments; in contrast, in AtT-20 cells, transfected PAM was primarily localized to the trans-Golgi network. PAM expression was highest in gonadotropes, with moderate levels in somatotropes and thyrotropes and lower levels in corticotropes and lactotropes. Under basal conditions, less than 1% of the cell content of monooxygenase activity was secreted per h, a rate comparable to the basal rate of release of individual pituitary hormones. General secretagogues stimulated PAM secretion 3- to 5-fold. Stimulation with specific hypothalamic releasing hormones demonstrated that different pituitary cell types secrete characteristic sets of PAM proteins. Gonadotropes and thyrotropes release primarily monofunctional monooxygenase. Somatotropes secrete primarily bifunctional PAM, whereas corticotropes secrete a mixture of mono- and bifunctional proteins. As observed in transfected AtT-20 cells, pituitary cells rapidly internalize the PAM/PAM-antibody complex from the cell surface. The distinctly different steady-state localizations of endogenous PAM in primary pituitary cells and transfected PAM in AtT-20 cell lines may simply reflect the increased storage capacity of primary pituitary cells.

In a previous study we showed the existence of GH in the ovine placenta. We now supplement the information available on placental GH and describe the presence and distribution of GH receptor (GH-R) messenger RNA (mRNA) in uterine, fetal, and placental tissues during early pregnancy. GH mRNA was not detected in the placenta before day 27 (d27). Its expression peaked between d40 and d45 and fell after d55. GH mRNA was localized in the trophectoderm and syncytium. During the d35-d50 period, concentrations of GH in the maternal circulation were not increased. In umbilical blood, however, GH was detected from d35 and was presumed to be of placental origin, because GH mRNA was not detected in the fetal pituitary gland on d40. We report on GH-R mRNA expression in the placenta between d20-d120. The relative abundance of GH-R transcripts increased significantly between d25-d43. In the endometrium, GH-R mRNA was detected from d8-d120 of pregnancy and from d4-d16 of the cycle. GH-R mRNA was localized in the trophectoderm, fetal mesoderm, and maternal uterine stroma. In the fetal liver, GH-R mRNA was first detectable on d35. The results of this study indicate that between d35-d50 of pregnancy, the endometrium, placenta, and fetus are all potential targets for the placental GH.

Tuberoinfundibular dopamine tonically inhibits PRL expression and secretion from the pituitary gland by the activation of dopamine D2 receptors (D2R) localized on lactotrophs. Mutant female mice that lack D2Rs have persistent hyperprolactinemia but also develop extensive hyperplasia of pituitary lactotrophs and peliosis of the adenohypophysis at 9 to 12 months of age, while age-matched male D2R-deficient mice have no morphologic adenohypophysial lesion. We now report that both female and male D2R-deficient mice 17 to 20 months of age develop pituitary lactotroph adenomas. Of 12 aged female mice examined, all developed monohormonal PRL-immunoreactive neoplasms that had a characteristic juxtanuclear Golgi pattern of PRL staining and loss of the reticulin fiber network. Several of these adenomas were 50-fold larger than normal glands with marked suprasellar extension and invasion of brain but no gross evidence of distant metastases. They also had striking peliosis that was more marked than the lesion seen in the hyperplastic pituitaries of the younger females. These findings demonstrate that a chronic loss of neurohormonal dopamine inhibition promotes the hyperplasia-neoplasia sequence in adenohypophysial lactotrophs. Our results are analogous to previous data indicating that protracted stimulation of adenohypophysial cells by hormones or growth factors results in proliferation with initial hyperplasia followed by the development of neoplasia. Six aged male D2R-deficient mice had slightly enlarged anterior pituitaries similar in size to normal female glands. However, each case exhibited multifocal, microscopic lactotroph adenomas with strong nuclear immunoreactivity for estrogen receptors and Pit-1 transcription factor. The unexpected development of adenomas in males without preexisting or concomitant hyperplasia suggests that prolonged loss of dopamine inhibition may also cause neoplasia by distinct cellular mechanisms in male and female animals.

In a series of 12 patients (eight women and four men, aged between 20 and 62 years), operated on for a pituitary adenoma shown to be thyrotropic by immunocytochemistry, we performed a retrospective and comparative analysis of clinical and biological data, tumor studies including immunocytochemistry with double labeling, and proliferation marker (proliferative cell nuclear antigen (PCNA) and Ki-67) detection, electron microscopy and culture. Our study leads us to confirm that thyrotropic tumors are rare (12 of 1174 pituitary adenomas: 1%). The main points arising were that: (1) high or normal plasma TSH associated with an increase in plasma alpha-subunit and high thyroid hormone levels is the best criterion for diagnosis; (2) the failure of TSH to respond to TRH or Werner's test is not a reliable criterion for diagnosis; (3) thyrotropic adenomas may be 'silent', without clinical signs of hyperthyroidism and with only slight increase in TSH, tri-iodothyronine and thyroxine concentrations; (4) mitoses and nuclear atypies are frequently detected in large tumors, which are invasive in more than 50% of cases - the first analysis of two proliferation markers (PCNA and Ki-67) bears out the relative aggressiveness of thyrotropic adenomas; (5) thyrotropic adenomas are frequently plurihormonal. Immunocytochemical double labeling, complemented by in vitro study, showed that thyrotropic tumor cells sometimes can or sometimes cannot cosecrete TSH, GH or prolactin. The pathological identification of monohormonal and plurihormonal adenomas seems to be supported by clinical and biological differences.

As is the case in other tetrapod species, the chicken gonadotropins LH and FSH consist of a common alpha subunit and a hormone-specific beta subunit. Gonadotrophs containing LH were shown earlier to be distributed throughout both the caudal and cephalic lobes of the chicken anterior pituitary, but the cellular distribution of FSH in avian species is still uncertain. The purpose of this study was to determine the cellular distribution of FSH-containing chicken gonadotrophs by use of FSH-specific monoclonal antibodies (mAbs). Three new mAbs toward chicken FSH were proven hormone specific by immunodetection of purified hormones on dot blots and by dual-label immunohistochemistry (IHC) on sagittal sections of chicken pituitaries. A rabbit antibody was used to detect chicken LH. Results showed that LH-containing gonadotrophs were densely distributed throughout the anterior pituitary, whereas gonadotrophs containing FSH were much less numerous; in addition, while also present in both lobes, FSH-positive cells were largely absent from the outer margin of the gland. Dual-label IHC revealed that LH and FSH reside almost exclusively in separate gonadotrophs. The identity of FSH-containing cells was further confirmed through use of an antibody to the chicken alpha subunit, which showed that FSH immunoreactivity was always colocalized with the alpha subunit. Our results suggest the possibility that production and secretion of LH and FSH may be regulated differently in chickens than in most other species studied to date.

GH secretion declines with age in rats and humans and a reduction in GH gene expression has been demonstrated in aging rats. GH secretion also diminishes in obesity; thus, the aim of this study was to determine whether GH decrease in aging rats is due to body weight gain or to aging. Three groups of male Wistar rats of different ages were studied (young, 3 months; middle-aged, 11 months; old, 27 months). The middle-aged group was established on a statistical analysis and corresponded to the youngest age at which body weight was not significantly different from the old (27 month) group. Thus, by using this group as control for comparison with animals with the same weight and an older age, the effects due to aging itself could be determined. Body weight (g, mean +/- sD) 3 months: 361 +/- 5.6; 11 months: 713 +/- 39; 27 months: 635 +/- 38. In comparison with 3-month-old rats, the 11-month-old animals showed no difference in pituitary GH messenger RNA (mRNA) accumulation and pituitary and serum IR-GH levels. Similarly IGF-I.a, IGF-I.b mRNA transcripts and IG-FBP-3 mRNA accumulation in the liver showed no significant differences between the two groups. On the contrary, when the 27-month-old rats were compared with the 11-month-old animals, lower levels of pituitary GH mRNA and serum and pituitary IR-GH were found. Pituitary GH mRNA decreased 37.5 +/- 7.7% P < 0.001, pituitary IR-GH content diminished (5.2 +/- 3.4 vs. 55 +/- 10.7 ng/mg of protein, P < 0.001) and serum IR-GH decreased (3.5 +/- 1.8 vs. 12.5 +/- 4.2 ng/ml, P < 0.01). Liver IGF-I.a and IGF-I.b mRNA transcripts accumulation and serum IGF-I were significantly diminished. IGF-I.b mRNA accumulation decreased 35.8 +/- 1.2% P < 0.05 and IGF-I.a 36 +/- 5.6% P < 0.05; serum IR-IGF-I levels diminished (759 +/- 152 vs. 1327 +/- 67 ng/ml, P < 0.05). Liver IGFBP-3 mRNA accumulation decreased 79 +/- 4.2% P < 0.001. These results indicate that the decrease in GH gene expression and secretion, as well as the expression of genes induced by GH such as IGF-I and IGFBP-3, is due to aging and not to the increase in body weight that takes place with aging.

Using quantitative double-label fluorescence immunocytochemistry and confocal microscopy, we have analysed the pattern of expression of SSTR1-5 in normal rat pituitary somatotrophes. Antipeptide rabbit polyclonal antibodies were produced against the extracellular domains of SSTR1-5. SSTR antigens were colocalized in GH positive cells using rhodamine conjugated secondary antibody for SSTRs and FITC-conjugated secondary antibody for GH. SSTR5 was the predominant subtype which was expressed in 86 +/- 9.7% of GH cells followed by SSTR2 in 42 +/- 6.4% of GH positive cells. SSTR4 and SSTR3 were modestly expressed in 23 +/- 4.7% and 18 +/- 3.2% of somatotrophes respectively whereas SSTR1 was the least expressed subtype occurring in only 5 +/- 1.2% of somatotrophes. These results demonstrate variable expression of the 5 SSTRs in somatotrophes. The preponderance of the SST-28 preferring SSTR5 subtype correlates with the reported higher potency of SST-28 than SST-14 for inhibiting GH secretion.

GH has been shown to promote the development and function of leukocytes. The expression of both GH and GH-receptors in lymphoid cells has led to the hypothesis that GH acts in an autocrine or paracrine fashion. The described effects of GH on hematopoiesis and B cell development, led us to investigate GH expression in bone marrow cells. By immunocytochemistry, we show that bone marrow-derived granulocytes and macrophages contain immunoreactive GH. We found that 65 +/- 24% of the granulocytes were stained with anti-GH, whereas 5.8 +/- 1.5% of the granulocytes contained detectable amounts of GH mRNA as assessed by in situ hybridization. To address a possible alternative regulation mechanism in bone marrow and to establish whether locally derived GH might still play a role in pituitary-deficient dwarf mice, we also addressed GH expression in bone marrow from hypopituitary Snell dwarf mice. These mice have a mutated gene for the pituitary transcription factor Pit-1 that is deficient in DNA binding. Our finding that GH expression (immunoreactive protein and mRNA) in bone marrow cells from dwarf mice is similar to that in normal mice points to a Pit-1 independent regulation of GH in mouse bone marrow.

A response of growth hormone (GH) to thyrotropin-releasing hormone (TRH) is observed in lower mammals and patients with diseases such as a cromegaly, but not in normal subjects. We have previously demonstrated the existence of intact TRH receptor mRNA in GH-secreting adenoma. To examine whether intact somatotrophs in the anterior pituitary also express TRH receptor, we attempted to localize both TRHR mRNA and GH immunoreactivity simultaneously. In situ hybridization analysis revealed TRHR mRNAs specifically in the anterior pituitary, and 61.1% of the anterior pituitary cells expressed this transcript. Staining for GH and PRL on the same samples showed that the somatotrophs apparently expressed TRHR mRNA and approximately 62.3% and 30.9% of hybridization-positive cells were somatotorophs and mammotrophs, respectively. Moreover, TRHR mRNA level in the somatotrophs expressed as the number of silver grains per cell was equivalent to that in the mammotrophs. These findings demonstrated expression of the TRHR mRNA in somatotrophs in the rat anterior pituitary, and also showed that more than 50% of the TRHR mRNA detected in the anterior pituitary was derived from these cells.

In several species, placenta has been found to express GH-related proteins. In the ovine placenta, such a protein, ovine chorionic somatommamotropin, has been described, but its involvement in the fetal/placental growth process is not clearly established. The aim of this study was to investigate the occurrence of another GH-related peptide in the ovine placenta. Placental extracts (days 30-140 of pregnancy) showed GH immunoreactivity between days 35-70. SDS-PAGE analysis of these extracts indicated that this immunoreactivity corresponded to 22- and 28-kDa proteins. GH-like immunoreactivity was localized on cotyledonary frozen sections in the syncytium and the trophectoderm. Northern blot analysis of placental RNA showed the expression of GH-hybridizing transcripts migrating to the same position as that of GH pituitary messenger RNA (mRNA). Those transcripts were highly expressed between days 40 and 50. Their sequence analysis showed the existence of three GH mRNA (GHP1, GHP2, and GHP3). GHP1 is identical to pituitary GH mRNA and probably codes for the 22-kDa protein. GHP2 and GHP3 encode the same protein, which differs from GHP1 by four amino acids. This study establishes the expression of GH gene and GH-immunoreactive proteins in the ovine placenta.

The long term efficacy and safety of transsphenoidal resection for preoperative diagnosis of PRL-secreting pituitary adenomas in a large series of women have not been described. Four hundred and nine consecutive women at this university tertiary referral center undergoing transsphenoidal resection for preoperative diagnosis of PRL-secreting pituitary adenoma were followed for a minimum of 4 yr. The objective was to determine the efficacy and morbidity of this procedure and to identify features correlating with the resolution of hyperprolactinemia. Outcome measures included referral, preoperative, surgical, postoperative hospitalization, and long-term follow-up information, including recent PRL concentration. Follow-up was ascertained in 83% of patients who were followed for a mean of 9.2 yr. Recurrence of hyperprolactinemia occurred in 47% of total patients, but in only 16% with a single surgical procedure, histological diagnosis of prolactinoma, and postoperative PRL concentration of 5 ng/mL or less. The best single predictor of cure was postoperative day 1 PRL concentration of 5 ng/mL or less. Eighty-eight percent of women desiring conception conceived within 1 yr. Glucocorticoid-dependent hypopituitarism occurred in 23% of patients undergoing postoperative radiotherapy. There was no operative mortality. Operative morbidity was low. Our experience demonstrates that women undergoing transsphenoidal surgery for diagnosis of PRL-secreting adenoma form a heterogeneous patient population. The best long term results are achieved in the pure prolactinoma group, for whom transsphenoidal resection is generally safe and effective.

We have derived and characterized a set of monoclonal antibodies (mAb) specific for the different human GH (hGH) isoforms. The binding characteristics of each antibody to the hGH isoforms (22K and 20K) were analyzed in direct and competitive immunoassays as well as by Western blot. We studied the effects of these mAb on the biological activity of hGH and showed that they specifically block their respective activities. Using these mAb, we developed several immunoassays that have been applied for the quantitation of the different hGH isoforms in body fluids. Therefore, these mAb may help to unravel the biological function of these variants.

The effects of thyroid hormones on GH secretion and the mechanisms underlying their action are very similar in man and the laboratory animal. We feel that it is possible to organize the available data into a unique pathophysiological model explaining these complex interactions (Table 1). In summary, physiological levels of circulating thyroid hormones are necessary to maintain normal pituitary GH secretion owing to their direct stimulatory actions. When the serum concentrations of thyroid hormone increase above the normal range there is an increase in hypothalamic somatostatin tone, which in turn suppresses pituitary GH secretion and overrides any stimulatory effects. The suppression of GH secretion by thyroid hormones may be mediated at the hypothalamic level also by a decrease in GHRH release.

To elucidate the roles of the hypothalamic peptides, GH-releasing hormone (GRH) and somatostatin (SRIH), potentially responsible for altered GH dynamics in diabetes, we studied the time courses of their changes in level associated with altered GH secretion in streptozotocin (STZ)-induced diabetic mice. Diabetic mice were used at 4, 7, and 14 days after STZ injection for analyses of 1) GH secretion in vivo, 2) hypothalamic GRH and SRIH messenger RNA (mRNA) levels, 3) pituitary GH mRNA and protein contents, and 4) pituitary GH response to GRH in vitro. GH secretion was completely suppressed 7 and 14 days after STZ injection. The hypothalamic GRH mRNA level was reduced to 59.8%, 61.2%, and 48.5% of control values at 4, 7, and 14 days, respectively. In contrast, the hypothalamic SRIH mRNA level was not altered at all of these time points. Pituitary GH mRNA and protein contents were significantly reduced to 70.2% and 61.5% of those in controls, respectively, only at 14 days. Pituitary GH responses to GRH at three doses (10, 50, and 250 nM) in vitro were remarkably increased at 4, 7, and 14 days. These findings indicate that the diabetic state rapidly and primarily inhibits hypothalamic GRH gene expression without affecting SRIH. A persistent decrease in hypothalamic GRH tone has been suggested to result in inhibition of GH synthesis in the pituitary. Enhancement of GH responsiveness to GRH may be due to the up-regulation of GRH receptors in the pituitary.

Cultured murine yolk sac cells transfected with the cytomegalovirus immediate early promoter/human growth hormone (CMVIE-hGH) fusion gene, expressing high levels of hGH in culture, and suspended in Matrigel were subcutaneously (s.c.) injected into experimental mice. The injected cells were shown to form discrete vesicular structures within the Matrigel implant, suggesting directed differentiation of the embryonic yolk sac cells into endothelial tissue. Human growth hormone radioimmune assay of these mice showed sustained physiologically significant levels of hGH in their serum for beyond four months. These results confirmed that long-term cultured murine embryonic yolk sac cells can be induced to differentiate into endothelial cells both in vivo and in vitro and suggested a novel approach to the delivery to the circulation of therapeutic proteins for the treatment of inherited and acquired diseases.

Pituitary-specific expression of the GH gene is dependent on a pituitary-specific transcription factor GH factor-1 (GHF-1), a homeodomain protein also known as pituitary-specific transcription factor-1 (Pit-1). The aim of this study was to investigate the regulation of GHF-1 messenger RNA (mRNA) levels in primary monolayer cultures of rat anterior pituitary cells. Specifically, in addition to direct activators of second messenger signaling systems, we studied the effects of different hormones, all of which are known to be involved in the regulation of somatotroph cell function. We found that GH-releasing hormone (GHRH) increased GHF-1 mRNA levels in a time- and dose-dependent fashion. GHF-1 mRNA levels were increased 2.5-fold (P < 0.01) after incubation for 2 h with 10(-8) M GHRH. Longer incubations (6, 12, or 24 h) with GHRH failed to show a similar stimulatory effect. A significant increase in GHF-1 mRNA concentration (1.7-fold, P < 0.01) was observed after a 2-h treatment with physiological concentrations (10(-11) M) of GHRH. The action of GHRH seems to occur at the transcriptional level without the need of protein synthesis. Thus, treatment of cells with actinomycin D (5 micrograms/ml) completely abolished GHRH-induced increase in GHF-1 mRNA levels. Cycloheximide (23 micrograms/ml) alone increased GHF-1 mRNA levels (6-fold increase after treatment for 12 h, P < 0.01), as well as potentiating GHRH-induced increase in GHF-1 mRNA concentration (9-fold increase after treatment with GHRH plus cycloheximide for 12 h, P < 0.01). The effect of GHRH on GHF-1 mRNA levels could be mimicked by direct activators of second messenger signaling systems such as forskolin (10(-5) M) or the phorbol ester tumor promoter tetradecanoyl phorbol acetate (TPA) (10(-6) M). Other peptides such as pituitary adenylate cyclase activating polypeptide-38 (10(-7) M) but not GHRP-6 (10(-10) to 10(-5) M), were also able to increase GHF-1 mRNA levels. Treatment of the cells with somatostatin (10(-6) M) for either 2 or 48 h failed to modify basal or GHRH-induced GHF-1 mRNA levels. In contrast, pretreatment of the cells with insulin-like growth factor-1 (5 nM) inhibited basal GHF-1 mRNA concentration as well as completely blunting the subsequent response to cells exposed to GHRH for 2 h. These data demonstrate that GHRH, acting at the transcriptional level and through a mechanism not dependent on protein synthesis, plays a stimulatory role on GHF-1 mRNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Transgenic mice expressing a tyrosine hydroxylase-human (h) GH fusion gene in the hypothalamus exhibit a dwarf phenotype. The GH feedback mechanism(s) underlying the growth retardation in these animals was investigated by assessing peptide and messenger RNA (mRNA) levels of the hormones of the hypothalamic-GH-IGF-I axis. Pituitary GH content, hypothalamic GH-releasing hormone (GHRH) and somatostatin (SRIH) content, and serum IGF-I levels were measured by RIA. mRNA levels of hypothalamic GHRH and SRIH and of pituitary GH and the GHRH receptor were measured by Northern blot hybridization. Transgenic mice of both sexes and their wild-type littermates were studied at 2-4 months of age. The pituitary GH content was markedly reduced by 85% in male and by 87% in female transgenic mice compared to that in wild-type controls (P < 0.01 for both). The pituitary GH mRNA content was also decreased by 73% (P = 0.002) in transgenic male mice. Circulating IGF-I levels were significantly reduced by 66% and 68% in male and female transgenic mice, respectively (P = 0.001). The hypothalamic GHRH content was significantly reduced by 19% and 33% (P < 0.05) in male and female transgenic mice, respectively. No significant difference was detected, however, in the hypothalamic SRIH content between wild-type and transgenic mice. Hypothalamic GHRH mRNA levels were significantly decreased by 35% (P = 0.002) in transgenic male mice compared to those in wild-type littermates. In contrast, SRIH mRNA was not significantly changed. An even greater reduction (61%; P = 0.003) was observed in pituitary GHRH receptor mRNA in transgenic mice. These data indicate that the GH deficiency and dwarf phenotype of the tyrosine hydroxylase-hGH transgenic mouse can be attributed primarily to impaired hypothalamic GHRH production. The mechanism of GH feedback inhibition appears to involve direct suppression of GHRH gene expression by locally produced hGH in the hypothalamus.

We reported previously that GH-secreting cells differentiate by day 16 of chicken embryonic development. In the present study, primary cultures of anterior pituitary cells from 10-, 12-, 14-, and 16-day-old chicken embryos were incubated for 2 or 6 days in serum-free medium or medium supplemented with serum from day 12 or day 16 embryos (1% serum by volume). After this culture period, the pituitary cells were recovered and subjected to reverse hemolytic plaque assays for chicken GH. GH-secreting cells did not differentiate spontaneously in serum-free cultures derived from any of the embryonic ages tested, indicating that differentiation of somatotrophs does not occur based only on the relative age of the presumptive GH cell. However, we found that treatment for as little as 2 days with serum from day 16 (but not day 12) embryos stimulated somatotroph differentiation in cultures derived from day 12, 14, and 16 embryos. These results indicate that an activity capable of stimulating GH cell differentiation in vitro is present in day 16 embryonic serum and that the presumptive GH cells were responsive to the somatotroph-differentiating effects of day 16 embryonic serum as early as day 12 of development. Next, serum from day 12, 14, and 16 embryos was evaluated over a 25-fold-range in concentration (0.2-5.0% by volume) using pituitary cells from day 12 embryos as an in vitro bioassay. Treatment with day 12 serum at any of the concentrations tested had no significant effect (P > 0.05), relative to that in serum-free cultures, in which 0.8 +/- 0.4% of all pituitary cells released GH. In contrast, treatment with day 14 and day 16 serum increased the percentage of cells that released GH to 8.0 +/- 1.3% and 12.0 +/- 0.8%, respectively (1% serum by volume). Thus, the level of somatotroph-differentiating activity in embryonic serum was undetectable in day 12 embryos, intermediate in day 14 embryos, and increased to high levels by day 16 of development concomitant with the appearance of GH-secreting cells in vivo reported previously. Next, the specificity of this response to day 16 serum was tested further by treating day 12 cells with peptides known to stimulate GH release in adult animals, GH-releasing hormone and TRH, and a GH-releasing hormone-related peptide (vasoactive intestinal peptide). None of these peptides was found to stimulate somatotroph differentiation at the doses tested (10-9 and 10-7 M).(ABSTRACT TRUNCATED AT 250 WORDS)

TSH-secreting pituitary adenomas are rare. The transcriptional expression (messenger ribonucleic acids: mRNAs) of TSH beta, GH, and PRL in five patients with TSH-secreting pituitary adenoma was studied by the in situ hybridization (ISH) method in order to elucidate their multiple hormone production. These patients showed inappropriately elevated serum TSH and alpha-subunit levels as well as pituitary mass lesions. The tissues from pituitary adenomas were obtained at the time of transsphenoidal surgery and revealed immunohistochemically the expression of alpha-subunit and TSH beta in all patients. Four adenomas were immunohistochemically associated with GH or PRL localization. The presence of pituitary-specific transcriptional factor Pit-1 was demonstrated in all adenomas in the nuclei of many cells. By ISH, signals for TSH beta mRNA were present in all five cases in many adenoma cells. Expression of GH mRNA and PRL mRNA were detected not only in four adenomas in which both hormonal products were immunolocalized but also in one adenoma that was immunohistochemically negative for GH and PRL. Combined staining by ISH and immunohistochemistry revealed the expression of GH mRNA and PRL mRNA in TSH beta-immunoreactive cells. Our findings indicate that TSH-secreting adenomas are multihormone-producing and could arise from precursor or stem cells rather than from differentiated TSH-secreting cells. It is suggested that ISH combined with immunohistochemistry may provide additional detailed information concerning the multidirectional histogenesis of this rare type of adenoma.

Glycoprotein hormone alpha-subunit (alpha SU) is a recognized product of clinically non-functioning, glycoprotein hormone-secreting and somatotroph adenomas but has not been studied systematically in corticotroph tumours. We have performed immunohistochemistry for alpha SU in a consecutive series of four corticotroph tumours causing Nelson's syndrome, three corticotroph macroadenomas, 12 corticotroph microadenomas and one adrenocorticotrophin-secreting bronchial carcinoid tumour. In addition we have assessed alpha SU secretion in vitro in corticotroph adenomas from two subjects with Cushing's disease and two subjects with Nelson's syndrome. Immunohistochemistry, performed after microwave treatment of sections to enhance antigen retrieval, demonstrated alpha SU positivity in 3/4 Nelson's tumours, 2/3 corticotroph macroadenomas, 7/12 microadenomas and one bronchial carcinoid. Eight of the 13 tumours positive for alpha SU were also immunostained after microwave pretreatment of sections for thyrotrophin (six positive), follicle-stimulating hormone (four positive), luteinizing hormone (three positive), beta-chorionic gonadotrophin (five positive), growth hormone (three positive) and prolactin (two positive) immunoreactivity. In vitro cell cultures of all four tumours studied secreted adrenocorticotrophin and three secreted alpha SU, with the variable presence of luteinizing hormone, follicle-stimulating hormone, thyrotrophin, growth hormone and prolactin, in basal culture. The alpha SU secretion was augmented by phorbol ester (160 +/- 15%, SEM, n = 3 wells; p < 0.01) and 8-bromo-cAMP (138 +/- 8%; p < 0.05) in one tumour. These data indicate that plurihormonality and, in particular, alpha SU elaboration and secretion by corticotroph tumours is more common than hitherto recognized. Possible mechanisms include abnormal or deregulated gene expression, autocrine or paracrine effects or a stem cell origin of tumour.(ABSTRACT TRUNCATED AT 250 WORDS)

To examine the molecular basis for the decreased pituitary growth hormone (GH) and thyrotropin (TSH) content during restricted feeding, fasting and diabetes, we measured steady-state levels of mRNA for TSH-alpha, TSH-beta and GH in the pituitary from normal rats either fed ad libitum (C), limited to 75%, 50% and 25% (FR75, FR50, FR25, respectively) of ad libitum intake, or deprived of food for 2 and 4 days (F2 and F4, respectively), and also in streptozotocin-diabetic (D) and D insulin-treated animals. The results from these experimental groups were compared with those in thyroidectomized (Tx) rats. Pituitary mRNA was quantified by Northern blot hybridization with cDNA probes specific for rat TSH-alpha, TSH-beta and GH. Although changes in the pituitary GH mRNA during restricted feeding, fasting and diabetes were similar qualitatively to those induced by hypothyroidism, GH mRNA levels in Tx rats (> 10% of C values) were less than in the other experimental groups (p < 0.001). Pituitaries from FR50, FR25 and D rats also contained less GH mRNA than F2 and F4 animals (p < 0.05). Thyroidectomy resulted in a marked increase in both TSH-beta and TSH-alpha mRNAs, the changes in TSH-beta mRNA being greater than those in TSH-alpha mRNA. In contrast, FR50, FR25, F2, F4 and D rats exhibited a decrease in pituitary TSH-beta mRNA (60%, 50%, 35%, 36% and 33%, respectively, of C values; p < 0.01-0.05) and in TSH-alpha mRNA levels (81%, 64%, 46%, 43% and 36%, respectively, of normal values; p < 0.02-0.05), TSH-beta mRNA showing the greater changes.(ABSTRACT TRUNCATED AT 250 WORDS)

In the present study, a reverse hemolytic plaque assay (RHPA) for chicken GH was established and used to study the ontogeny of somatotroph differentiation and functional responsiveness to GH-releasing hormone (GHRH) during chicken embryonic development. Anterior pituitaries from embryos on days 10, 12, 14, and 16 of incubation were isolated and dissociated into single cells with trypsin. The resulting cells were then subjected to the GH plaque assay under basal and GHRH-stimulated conditions. No GH-releasing cells were detected on day 10 or 12 of embryonic development. In contrast, a few somatotrophs (< 2% of all cells) were consistently found on day 14, and a statistically significant population existed on day 16, when 6.3 +/- 1.4% of all anterior pituitary cells secreted GH. Thus, GH-secreting cells differentiated by embryonic day 16. Treatment of pituitary cells from day 16 embryos with GHRH was found to increase the proportion of GH plaque-forming cells during a shortened assay interval from 1.8 +/- 0.3% under basal conditions to 6.7 +/- 1.2% in the presence of GHRH. This nearly 4-fold increase in the proportion of plaque-forming cells indicates that at least 70% of the initial somatotrophs present on day 16 were responsive to the stimulatory effects of GHRH. To test whether the absence of GH cells on day 12 of embryonic development was due to the presence of cells that produced but did not release GH, pituitary cells from day 12 and day 16 embryos were subjected to immunocytochemistry for GH and to the GH RHPA in parallel. No significant differences were found in the percentage of cells that either contained or released GH on the two embryonic ages tested. On day 12, 1.1 +/- 0.8% of all cells contained GH, as determined by immunocytochemistry, whereas 0.5 +/- 0.5% released GH as determined by RHPA. By day 16, the proportions of cells that contained and released GH had increased to 9.5 +/- 0.6 and 11.2 +/- 2.5%, respectively. Taken together, these results indicate that GH-secreting cells differentiate by day 16 of chicken embryonic development and that these initial somatotrophs are responsive to GHRH. Given that growth and metabolism are regulated in part by GH in chick embryos, these findings suggest that these processes may be under hypothalamic control during late embryonic development in the chicken.

To study the potential role of insulin-like growth factor-I (IGF-I) during early embryogenesis, we have used the amphibian Xenopus laevis, a versatile model of vertebrate development. Using polymerase chain reaction (PCR)-based cloning strategies, we have previously identified two different nonallelic Xenopus IGF-I genes (IGF-I' and IGF-I"). Both are expressed in similar quantities in adult liver. We now report the use of a modification of the reverse transcription-PCR method, designated RNA template-specific PCR (RS-PCR), to detect IGF-I messenger RNA (mRNA) from single oocytes and embryos. The same primer pair was used to amplify both IGF-I' and IGF-I" mRNAs. Slot blot analysis of the RS-PCR products with internal oligonucleotide probes that specifically recognize IGF-I' or IGF-I" sequences revealed that only IGF-I' mRNA was present in follicles surrounding mature (stage VI) oocytes; neither IGF-I mRNA was present in follicles surrounding less mature oocytes (stages I and IV) or within oocytes or unfertilized eggs. After fertilization, IGF-I' mRNA was first detected during early organogenesis (stages 21-23), and increased during subsequent stages of development. To localize early IGF-I' expression, a stage 27 embryo was sliced into 24-microns cross-sections; RS-PCR and slot blot analysis were performed on RNA extracts from consecutive sections. IGF-I' mRNA was expressed in all sections, but was most abundant in the body region from which the visceral organs were developing. In contrast, to the early expression of IGF-I', IGF-I" mRNA was not detected until stage 41, a period corresponding to premetamorphic growth. Reverse PCR for Xenopus GH mRNA demonstrated that the onset of GH gene expression was coincident with the onset of IGF-I" gene expression (stage 41). These data suggest that the early expression of IGF-I' may be GH independent, whereas later expression of IGF-I" is GH dependent. We conclude that the two nonallelic IGF-I genes are expressed differentially in a stage-specific manner and suggest that IGF-I' may play an important role during early organogenesis, whereas IGF-I" may be important for premetamorphic growth.

The factors that determine the hormone and volume responses of pituitary adenomas to the somatostatin analog octreotide are poorly understood. We, therefore, studied the correlation between 111indium-pentetreotide somatostatin receptor scintigraphy (SRS) and the clinical and immunohistochemical classification of pituitary adenomas, on the one hand, and hormone and volume responses, on the other hand. Ten patients with GH-secreting (6 females and 4 males; age, 31-67 yr) and 14 patients with clinically nonfunctioning (NF) macroadenomas (5 females and 9 males; age, 22-79 yr) were preoperatively treated with 300 micrograms/day octreotide, which was increased to 600 and 1500 micrograms/day at weekly intervals and then continued for at least 3 months until surgery. SRS was performed before therapy. A sellar magnetic resonance imaging scan was performed before therapy; 1, 2, and 3 weeks and 3 months after start of therapy; and after surgery. Acromegalics also had an 8-h GH profile, insulin-like growth factor-I determination, and a 100-g oral glucose load at these time points. An attempt was made to identify NF adenomas as gonadotroph adenomas using their LH, FSH, and alpha-subunit responses to TRH. In acromegalic patients, octreotide suppressed mean GH (8-h profile) and insulin-like growth factor-I concentrations from 34.9 +/- 9.7 to 8.1 +/- 3.6 micrograms/L and from 2122 +/- 1025 to 701 +/- 208 micrograms/L, respectively, after 3 months. Significant (26-85% decline) tumor shrinkage occurred in 5 of 10 patients, mainly within the first week. Tumor shrinkage and GH suppression were not correlated. Four of 7 patients had increased pituitary 111indium-pentetreotide uptake, but this did not predict GH suppression or tumor shrinkage. Of the NF adenomas, 2 responded with shrinkage (57% and 96% decline). Four of 12 adenomas had increased 111indium-pentetreotide uptake, but this did not correlate with tumor shrinkage (2 adenomas; 1 gonadotroph and 1 null cell adenoma), immunohistochemistry, or clinical classification. We conclude that preoperative octreotide therapy suppresses GH in most patients and reduces tumor volume in up to 50% of acromegalic patients. It also induces shrinkage in some NF adenomas, although less frequently. SRS does not predict shrinkage of either tumor type. Shrinkage does not correlate with clinical classification or immunohistological characteristics. Further studies are needed to identify the factors that determine the hormone and volume responses of pituitary adenomas to octreotide therapy.

Two families with familial isolated GH deficiency (IGHD) were studied, type II (autosomal dominant) and type I (autosomal recessive), whose GH1 genes exhibit cosegregation with IGHD. DNA sequencing of the GH1 genes of the first family (IGHD II) demonstrated heterozygosity for a T-->C transition in the sixth base of the donor splice site of intron III. The GH1 gene mutation in the second family (IGHD I) was found, in a previous study, to be a G-->C transversion altering the first base of the donor splice site of intron IV. Interestingly, analysis of the transcripts derived from the mutant IGHD II allele revealed that the sequences corresponding to exon III were absent due to an exon skip that causes the loss of amino acids 32-71 from the mature GH protein. In contrast, the IGHD I mutation activates a cryptic donor splice site 73 bases upstream of the normal exon IV donor splice site causing loss of amino acids 103-126 of exon IV followed by a reading frameshift and synthesis of 94 novel amino acids before chain termination 88 nucleotides downstream of the normal GH stop codon. It is hypothesized that, because of the loss of protein sequences derived from exons IV and V, the IGHD I mutation products are not transported to secretory granules and thus cannot perturb secretion of the normal monomeric GH protein. In contrast the T-->C IGHD II mutant allele product retains these sequences and is transported to secretory granules where it can interact with the normal allele product producing a dominant-negative effect at the protein level.

To evaluate the presence of different GH isoforms in serum of girls with Turner's syndrome, we measured the serum GH content using RIAs with three different site-specific monoclonal antibodies (MAbs). We compared the results to those obtained with authentic GH and GH isoforms. Compared to pituitary GH (mol wt, 22K daltons) as the standard for all three MAbs, serum from girls with Turner's syndrome did not displace tracer [125I]GH equally with all three MAbs. The relative amounts of GH-immunoreactive material found in Turner's syndrome were different from the amounts observed in normal adults and most children with idiopathic short stature. The presence of GH, other than 22K GH, in serum from girls with Turner's syndrome was confirmed by affinity chromatography. The existence of different isoforms of GH, as shown by different cross-reactivity patterns with different MAbs to GH, may explain the conflicting results reported for GH secretion in girls with Turner's syndrome.

Forty-one human pituitary adenoma specimens were examined for the presence of estrogen receptor (ER) messenger ribonucleic acid and protein using a combination of ribonuclease protection assay, [3H] estradiol ([3H]E2) binding, and ER immunohistochemistry. ER messenger ribonucleic acid prevalence was high in PRL-immunoreactive tumors (2 of 2), moderate in GH/PRL tumors (2 of 5), and low or absent (0 of 4) in GH tumors. In the GH/PRL-immunostaining tumors, the presence of the ER was uniformly associated with elevated serum PRL levels. Among the gonadotropin-immunostaining tumors, 10 of 17 were ER positive; within this group, those with gonadotroph adenoma characteristics were ER positive, whereas those with null cell/oncocytic characteristics were ER negative. Of the tumors that did not immunostain for any known anterior pituitary hormones, 3 of 11 were ER positive. ER immunohistochemistry in 14 tumors revealed a 100% correlation with ribonuclease protection assay results, whereas [3H]E2 binding, determined in 9 tumors, showed an 87% correlation. In summary, it appears that PRL and a specific class of gonadotropin-immunostaining tumors (identifiable by specific characteristics on electron microscope) contain ER, whereas GH-immunostaining tumors are ER negative. ER expression in normal pituitary paralleled that in macroadenomas (GH, 2.3%; PRL, 50%; FSH, 70%; LH, 83%; TSH, 4%; ACTH, 1%). The ER-positive tumors represent a subset whose growth and secretory profiles may be influenced by the gonadal steroidal milieu or by pharmacological agents that affect E2 levels or ER function.

Attempts to assess human placental GH variant (hGH-V) and chorionic somatomammotropin (hCS) RNA in choriocarcinoma cell lines have been hampered by low levels of expression and limited sensitivity of RNA blotting analysis. We examined human choriocarcinoma BeWo, JAR, and JEG-3 cell lines as well as samples of complete hydatidiform moles for expression of members of the human GH (hGH) gene family using reverse transcriptase-polymerase chain reaction. A single and common set of primers was designed and used to detect products of the hGH/hCS genes as well as distinguish processed RNA from any contaminating DNA. Transcripts from the hCS genes hCS-A and -B were distinguished from placental hGH variant (hGH-V) and hCS-like (hCS-L) gene RNA by diagnostic restriction digestion of the polymerase chain reaction products. The expected pattern of hGH/hCS RNA expression was detected in term placenta, where hCS and hGH-V/hCS-L transcripts represented approximately 95% and approximately 5% of the total hGH/hCS RNA, respectively. The level of hCS RNA varied from 22-99% of the total hGH/hCS RNA in the neoplastic trophoblast samples, and variable levels of hGH-V and hCS-L RNA were also observed. In choriocarcinoma JAR cells, hGH-V RNA represented approximately 78% of the total hGH/hCS RNA compared to approximately 22% for hCS. Further, although low hCS-L RNA levels (< 1%) were found in term placenta and two of the hydatidiform moles, hCS-L transcripts represented 11% of the total hGH/hCS RNA in a third hydatidiform mole. Finally, in contrast to the detection of variable levels of hCS-L RNA in term placenta and hydatidiform mole samples, no hCS-L transcripts were detected in the three choriocarcinoma cell lines examined. These patterns reflect either deregulated hGH/hCS gene expression in neoplastic trophoblasts or differences that accompany the process of differentiation of trophoblast subpopulations. Regardless, this suggests that the control of hGH-V and hCS-L gene expression is distinct from that of the hCS-A and hCS-B genes and raises questions about the possible involvement of hGH/hCS family members in the pathology of placental abnormalities.

The purpose of these experiments was to determine whether a 17-kilodalton (K) fragment of human GH (hGH), hGH-(44-191), a peptide 10 times more potent than intact hGH in causing glucose intolerance in an animal model, circulates in human blood. Analysis of pituitary extracts and sera by Western blotting revealed the presence of a 17K hGH-immunoreactive band in both samples. Monoclonal antibodies to recombinant hGH-(44-191) cross-reacted with the 17K hGH-immunoreactive band of the pituitary. A RIA specific for hGH-(44-191) was developed using recombinant hGH-(44-191) as the tracer and standard, and a mouse anti-hGH-(44-191) serum as the source of antibody. The RIA detected hGH-(44-191) in human sera and pituitary extracts. The concentration of hGH-(44-191) in pituitary glands was 1/100th to 1/500th of the hGH concentration, but in serum its concentration averaged 1-2 times higher than that of hGH. Serum hGH-(44-191) concentrations were higher in pregnant than in nonpregnant women. The data document the existence of hGH-(44-191) in human pituitary gland and serum, and provide initial evidence that it may be physiologically produced. As its concentration is affected by the physiological state of the individual, it may play a role in the expression of the physiological and pathological actions of hGH.

GH receptor immunoreactivity is widely distributed within the rat pituitary gland, although apart from somatotrophs the cell types with GH receptor immunoreactivity have yet to be identified. It is also unknown whether this immunoreactivity reflects the presence of GH binding proteins (GHBPs) or authentic receptors. The possible colocalization of GHBPs and pituitary hormones in somatotrophs, lactotrophs, gonadotrophs, thyrotrophs, and corticotrophs was therefore examined using immunogold electron microscopy. Pituitary sections were indirectly immunostained with antibodies for GH, PRL, LH, FSH, TSH, or ACTH using gold-labeled immunoglobulin G. The same grids were also immunostained with a polyclonal antibody raised against rat GHBP using protein A conjugated to gold particles of a different size. Some sections were gold-labeled using a monoclonal antibody (MAb 4.3) raised against the unique hydrophobic tail of the GHBP, using gold-labeled immunoglobulin G. The cellular and ultrastructural distribution of immunoreactivity within the pituitary gland was similar after labeling with either GHBP antiserum. In all immunoreactive cells the labeling was most intense in secretory granules. Specific staining was not, however, demonstrated in the nucleus, in contrast with earlier findings using MAb 263. Moreover, while staining with MAb 263 appeared to be ubiquitous, some pituitary cells were not labeled by the polyclonal GHBP antiserum or by MAb 4.3. GHBP immunoreactivity was, however, colocalized with hormones in all pituitary cell-types, although in some cells GHBP immunoreactivity was not present in all secretory granules. These results clearly demonstrate the presence of GHBPs in adenohypophyseal cells, in which they appear to be stored or secreted with GH, PRL, LH, FSH, TSH, and ACTH. The function of GHBPs in the pituitary gland is, however, uncertain.

The majority of pituitary tumors are of monoclonal origin; however, the molecular basis for their formation is poorly understood. Somatic mutations in the alpha-subunit of the GTP-binding protein, Gs alpha (gsp oncogene) have been found in about one third of GH-secreting tumors. Mutations in another alpha-subunit of a GTP-binding protein, Gi2 alpha (gip mutations) have been described in other endocrine tumors. In this study, we examined 21 nonfunctioning pituitary tumors and 4 macroprolactinomas for gsp mutations and 27 nonfunctioning tumors and 4 macroprolactinomas for gip mutations. Using the polymerase chain reaction and denaturing gradient gel electrophoresis, 2 nonfunctioning pituitary tumors displayed migration abnormalities when the Gs alpha-gene was analyzed. Sequence analysis of these abnormally migrating polymerase chain reaction products revealed two previously known gsp mutations: arginine at codon 201 altered to cysteine, and glutamine at codon 227 changed to leucine. No gip mutations could be demonstrated. These findings emphasize the monoclonal origin of nonfunctioning pituitary tumors and suggest that cAMP may play a role in tumorigenesis of nonfunctioning pituitary tumors.

This study was designed to investigate whether N-linked glycosylation could account for the presence of glycosylated GH forms (G-GH) in human pituitary extracts. The study was carried out in commercially available pituitary GH preparations (pitGH). Recombinant GHs obtained from eu- or prokaryotic cells were used as controls. Radioiodinated GHs were incubated in tubes containing Concanavalin-A (Con-A) attached to a Sepharose 4B matrix. Pituitary G-GH forms were selectively displaced from Con-A by adding N-acetyl-D-glucosamine or methyl-alpha-D-mannopyranoside and electrophoresed. Autoradiographies of these gels identified a 12-kilodalton (12K) band as the glycosylated form. Due to the fact that this peptide was partially immunoprecipitated with an anti-GH serum and the absence of detectable PRL in the pitGH extracts, it would indicate that such a glycopeptide was GH related. Endoglycosidase-F treatment of pitGH extracts induced a decrease in the mol wt of that 12K peptide, as indicated by the changes observed in its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This demonstrated that the sugar moieties were N-linked. When recombinant GHs were assayed by a similar method, no specific binding to Con-A was detected. NH2-terminal amino acid sequence analysis from the 12K band demonstrated that this band was composed by three peptides. Peptide 1 corresponds to the GH-N 102-119 sequence. Interestingly, peptide 2 exhibits GH-V 1-18 sequence, while peptide 3 seems to be a novel GH-related peptide. Taken together, these data suggest that the pituitary G-GH form found in human pituitary extracts is derived not from the "normal" GH-N gene, but, rather, from the GH-V gene or another unidentified gene. The potentially important pathophysiological implications of this finding need to be investigated.

The effects of dexamethasone (DEX) on the onset of GH expression in the fetal rat pituitary gland were studied. Pregnant rats received oral administration of DEX dissolved in drinking water (25 mg/liter) for 40 h before killing on days 16-19 of pregnancy. The number of immunoreactive GH cells in the control fetus on day 18 was very small, whereas it was remarkably increased by DEX to a compatible value obtained from control fetuses on day 19. The effect of DEX on the induction of immunoreactive GH cells was found to be less potent on day 17 and not evident on day 16. The population of GH cells on day 19 was doubled, and they stained more intensely by GH antiserum after DEX treatment. In situ hybridization revealed that there were many GH messenger RNA (mRNA)-positive cells in the pituitary gland of a DEX-treated fetus at day 18, whereas they were negligible in number in the age-matched control. Northern blot analysis revealed that DEX induced accumulation of small amounts of GH mRNA in the fetal pituitary gland on day 17, whereas it induced a marked elevation of GH mRNA level on day 18. Reducing the levels of endogenous glucocorticoids by the administration of metyrapone (0.5 mg/ml in drinking water) to the pregnant rats from day 17-19 resulted in the significant reduction in the number of GH cells at day 19. Short-term treatment of DEX (duplicate sc injections at 18 h and 6 h before killing) also induced immunoreactive GH cells on day-18 fetuses but not on day-17 fetuses. Immunoreactive GH cells appeared on day 17 only after quadruplicate injections of DEX during last 40 h, suggesting that there is a difference in fetal sensitivity to DEX between days 17 and 18. These results indicate that immunoreactive GH cells and GH mRNA are inducible by DEX at an earlier stage of the fetal life than they normally appear. It is suggested that glucocorticoids play an important role in the development of GH cells in the fetal pituitary gland in vivo.

A human nonpituitary cell line grown under serum-free (sf) conditions (sfRamos Burkitt lymphoma cell line) has been reported to secrete a 29K PRL-like peptide which acts as an autocrine growth factor. Conditioned medium from these cells was examined for lactogenic activity using the Nb2 bioassay and RIAs specific for human GH (hGH) and hPRL. SfRamos conditioned medium stimulated the growth of Nb2 cells. Anti-hGH monoclonal antibodies but not anti-hPRL inhibited the mitogenic effect of sfRamos conditioned medium on Nb2 cells. Immunoreactive hGH but not hPRL was detected by RIA. Immunoprecipitation with anti-hGH polyclonal antibody followed by Western blot analysis with anti-hGH monoclonal antibody revealed a specific 22K band with the same mobility as pituitary hGH. Northern blot analysis with an hGH complementary DNA (cDNA) probe revealed a 1.0-kilobase transcript migrating coincident with pituitary hGH messenger RNA. A less abundant, 1.6-kilobase transcript was also observed. Reverse transcriptase-polymerase chain reaction using specific primers for the hGH cDNA generated the predicted 248-base pair band. Polymerase chain reaction sequencing of this fragment revealed sequence identity to the hGH-N cDNA, demonstrating conclusively the expression of the hGH-N gene in the sfRamos cell line.

The effects of TRH on the primordia pituitary cell differentiation, in from 11-day-old rat embryos cultured in a synthetic medium, were studied by immunocytochemistry. The expression of different pituitary phenotypes were measured as the percentage of immunoreactive area per section of cultured pituitary. Addition of TRH on day 1 of culture induced the thyrotroph and gonadotroph differentiation in a dose-dependent fashion. TRH promoted a fewer differentiation of gonadotrophs than GnRH. Much smaller increase in gonadotropic cell area was observed when 10(-9) M TRH was added on day 4 of culture instead of day 1. This suggests that gonadotroph differentiation can be only transiently induced during early embryonic period. Moreover, addition of TRH on day 1 of culture induces a differentiation of few lactotrophs. Contrary to gonadotrophs, the number of lactotrophs was higher when TRH was supplemented on day 4. As previous reports have demonstrated that the lactotroph differentiation is controlled by the alpha-subunit of glycoproteic hormones, further studies are necessary to determine whether the effect of TRH is direct. TRH had no effects on the differentiation of corticotrophs or somatotrophs. These results in vitro suggest that the availability of TRH in fetal hypothalamus at early embryonic stage may affect the pituitary differentiation in a subpopulation of precursor cells.

It has been shown that mice transgenic for human GH-releasing hormone (GRH) develop hyperplasia of pituitary somatotrophs, lactotrophs, and mammosomatotrophs, cells capable of producing both GH and PRL, by 8 months of age. We now report that GRH transgenic mice 10-24 months of age develop pituitary adenomas, which we characterized by histology, immunohistochemistry, in situ hybridization, and electron microscopy. Of 13 animals examined, all developed GH-immunoreactive neoplasms that had diffuse positivity for GH mRNA by in situ hybridization. Eleven also contained PRL immunoreactivity; in situ hybridization demonstrated focal PRL mRNA in 3 of 5 immunohistochemically positive tumors. Alpha-Subunit was positive by immunohistochemistry in 8 adenomas, and TSH beta was localized in tumor cells of 5 adenomas. The adenomas had variable ultrastructural appearances, ranging from cells that resembled somatotrophs or mammosomatotrophs to cells with features of the glycoprotein hormone cell line. These findings provide conclusive evidence that protracted GRH stimulation of secretory activity can result in proliferation, hyperplasia, and adenoma of adenohypophysial cells.

We have investigated the possibility that local production of GHRH within the adenohypophysis could be an aetiological factor in the development of human pituitary somatotroph and other tumours. We examined 51 human pituitary adenomas for GHRH transcripts using in situ hybridization histochemistry. GHRH transcripts were identified in 13 of 17 somatotroph adenomas, 4 of 10 corticotrophs, 1 of 6 lactotrophs and 1 of 18 endocrinologically inactive adenomas. In 11 GHRH-expressing somatotroph adenomas, SRIH transcripts were also identified. In all cases except one (a corticotroph adenoma) the average number of GHRH transcripts exceeded that found in the arcuate nucleus of simultaneously hybridized rat brain sections. In some pituitary adenomas, GHRH transcripts were clearly localized to a discrete subpopulation of cells and in these, the amount of GHRH mRNA per cell was comparable to that found in the GHRH-expressing cells of the rat arcuate nucleus. Although we have not directly demonstrated an association between the two, the identification of localized, high level GHRH gene expression in somatotroph adenomas suggests that GHRH may have a role in pituitary adenoma formation.

Transgenic mice for the promoter sequence of bovine arginine vasopressin (AVP) gene fused to large SV40 T-antigen coding sequence develop pituitary tumors and insulin-producing pancreatic tumors. In order to establish the cellular composition of the pituitary tumors, histological, immunocytochemical, in situ hybridization, and electron microscopic technics were applied. Pituitary anterior lobe tumors were identified in 10 out of 14 glands examined. In 2 of these cases, intermediate lobe tumors were also found. The anterior lobe tumors contained a variable number of GH immunoreactive cells. In situ hybridization performed in 7 cases revealed a diffuse distribution of GH messenger RNA over all tumor cells. Ultrastructurally, the tumors contained undifferentiated cells with very small secretory granules and rare cells showing some resemblance to somatotrophs. The results indicate that these pituitary tumors are composed of undifferentiated somatotrophs. The presence of a few PRL immunoreactive cells in four tumors and scattered TSH immunoreactive cells in two tumors supports the view that somatotrophs have the potential to produce PRL and TSH. The intermediate lobe tumors were immunoreactive for ACTH and intensely positive for POMC mRNA. In the nontumorous adenohypophyses, no hyperplasia of any cell type was noted. Several GH immunoreactive cells exhibited pleomorphic, giant nuclei and mitoses. In conclusion, the majority of transgenic mice for AVP/large T-antigen develop pituitary tumors originating in and composed of somatotrophs. Less frequently, intermediary lobe tumors were present as well. AVP/SV40 transgenic mice provide a unique experimental model for somatotroph tumors that are neither preceded by, nor associated with somatotroph hyperplasia.

Multiple peptide hormones can be derived from the single human GH gene. In addition to the full-length 191-amino acid 22-kilodalton (kDa) form, a 20-kDa variant can be produced by alternative splicing, and a 5-kDa variant can be produced by posttranslational cleavage. To more fully appreciate the physiological roles of these proteins, we have made a comparison of transgenic mice that constitutively overexpress one or another of these variants. We have found that both the 22-kDa and the 20-kDa forms of human GH stimulate linear growth and liver hypertrophy. The increase in linear growth in 22-kDa transgenic mice does not, however, correlate with an increase in circulating IGF-I; rather, the increase in IGF-I that does finally occur correlates with marked liver pathology. Both groups of mice also develop glomerulosclerosis and suffer from hyperinsulinemia. Although there are histologically obvious lesions in the livers of both the 22-kDa and the 20-kDa transgenic mice, only the former exhibit hyperalbuminemia and hypercholesterolemia. Both forms of GH lead to anemia, which is normocytic in the 20-kDa transgenic mice and macrocytic in the 22-kDa transgenic mice. Despite the presence of high levels of the 5-kDa N-terminal form of human GH, the transgenic mice that express this protein are indistinguishable from their nontransgenic littermates.