GHRH-SST-GH-IGF axis regulates crosstalk between growth and immunity in rainbow trout (Oncorhynchus mykiss) infected with Vibrio anguillarum.

An energy trade-off is existed between immunological competence and growth. The axis of growth hormone releasing hormone, somatostatin, growth hormone, insulin-like growth factor (GHRH-SST-GH-IGF axis) regulates growth performances and immune competences in rainbow trout (Oncorhynchus mykiss). The salmonid-specific whole genome duplication event is known to result in duplicated copies of several key genes in GHRH-SST-GH-IGF axis. In this study, we evaluated the physiological functions of GHRH-SST-GH-IGF axis in regulating crosstalk between growth and immunity. Based on principal components analysis (PCA), we observed the overall expression profiles of GHRH-SST-GH-IGF axis were significantly altered by Vibrio anguillarum infection. Trout challenged with Vibrio anguillarum showed down-regulated igf1s subtypes and up-regulated igfbp1a1. The brain sst genes (sst1a, sst1b, sst3b and sst5) and igfpbs genes (igfbp4s and igfbp5b2) were significantly affected by V. anguillarum infection, while the igfbp4s, igfbp5s, igfbp6s and igf2bps genes showed significant changes in peripheral immune tissues in response to V. anguillarum infection. Gene enrichment analyses showed functional and signaling pathways associated with apoptosis (such as p53, HIF-1 or FoxO signaling) were activated. We further proposed a possible model that describes the IGF and IGFBPs-regulated interaction between cell growth and programmed death. Our study provided new insights into the physiological functions and potentially regulatory mechanisms of the GHRH-SST-GH-IGF axis, indicating the pleiotropic effects of GHRH-SST-GH-IGF axis in regulating crosstalk between growth and immunity in trout.

Thyrotropin-Releasing Hormone (TRH) and Somatostatin (SST), but not Growth Hormone-Releasing Hormone (GHRH) nor Ghrelin (GHRL), Regulate Expression and Release of Immune Growth Hormone (GH) from Chicken Bursal B-Lymphocyte Cultures.

It is known that growth hormone (GH) is expressed in immune cells, where it exerts immunomodulatory effects. However, the mechanisms of expression and release of GH in the immune system remain unclear. We analyzed the effect of growth hormone-releasing hormone (GHRH), thyrotropin-releasing hormone (TRH), ghrelin (GHRL), and somatostatin (SST) upon GH mRNA expression, intracellular and released GH, Ser133-phosphorylation of CREB (pCREBS133), intracellular Ca2+ levels, as well as B-cell activating factor (BAFF) mRNA expression in bursal B-lymphocytes (BBLs) cell cultures since several GH secretagogues, as well as their corresponding receptors (-R), are expressed in B-lymphocytes of several species. The expression of TRH/TRH-R, ghrelin/GHS-R1a, and SST/SST-Rs (Subtypes 1 to 5) was observed in BBLs by RT-PCR and immunocytochemistry (ICC), whereas GHRH/GHRH-R were absent in these cells. We found that TRH treatment significantly increased local GH mRNA expression and CREB phosphorylation. Conversely, SST decreased GH mRNA expression. Additionally, when added together, SST prevented TRH-induced GH mRNA expression, but no changes were observed in pCREBS133 levels. Furthermore, TRH stimulated GH release to the culture media, while SST increased the intracellular content of this hormone. Interestingly, SST inhibited TRH-induced GH release in a dose-dependent manner. The coaddition of TRH and SST decreased the intracellular content of GH. After 10 min. of incubation with either TRH or SST, the intracellular calcium levels significantly decreased, but they were increased at 60 min. However, the combined treatment with both peptides maintained the Ca2+ levels reduced up to 60-min. of incubation. On the other hand, BAFF cytokine mRNA expression was significantly increased by TRH administration. Altogether, our results suggest that TRH and SST are implicated in the regulation of GH expression and release in BBL cultures, which also involve changes in pCREBS133 and intracellular Ca2+ concentration. It is likely that TRH, SST, and GH exert autocrine/paracrine immunomodulatory actions and participate in the maturation of chicken BBLs.

Growth Hormone (GH) Deficient Mice With GHRH Gene Ablation Are Severely Deficient in Vaccine and Immune Responses Against Streptococcus pneumoniae.

The precise impact of the somatotrope axis upon the immune system is still highly debated. We have previously shown that mice with generalized ablation of growth hormone (GH) releasing hormone (GHRH) gene (Ghrh-/-) have normal thymus and T-cell development, but present a marked spleen atrophy and B-cell lymphopenia. Therefore, in this paper we have investigated vaccinal and anti-infectious responses of Ghrh-/- mice against S. pneumoniae, a pathogen carrying T-independent antigens. Ghrh-/- mice were unable to trigger production of specific IgM after vaccination with either native pneumococcal polysaccharides (PPS, PPV23) or protein-PPS conjugate (PCV13). GH supplementation of Ghrh-/- mice restored IgM response to PPV23 vaccine but not to PCV13 suggesting that GH could exert a specific impact on the spleen marginal zone that is strongly implicated in T-independent response against pneumococcal polysaccharides. As expected, after administration of low dose of S. pneumoniae, wild type (WT) completely cleared bacteria after 24 h. In marked contrast, Ghrh-/- mice exhibited a dramatic susceptibility to S. pneumoniae infection with a time-dependent increase in lung bacterial load and a lethal bacteraemia already after 24 h. Lungs of infected Ghrh-/- mice were massively infiltrated by inflammatory macrophages and neutrophils, while lung B cells were markedly decreased. The inflammatory transcripts signature was significantly elevated in Ghrh-/- mice. In this animal model, the somatotrope GHRH/GH/IGF1 axis plays a vital and unsuspected role in vaccine and immunological defense against S. pneumoniae.

Microbial Products and Cytokines in Sleep and Fever Regulation.

Excessive sleepiness and fever are constitutional symptoms associated with systemic infection. Although fevers have been investigated for many years, sleep responses to infectious challenge have only recently been investigated. Inoculation of animals with bacterial, viral, protozoan and fungal organisms result in complex sleep responses dependent upon the microbial agent and route of administration. The general pattern is characterized by an initial robust increase in non-rapid eye movement sleep (NREMS) followed by a period of NREMS inhibition. REMS is inhibited after infectious challenge. The sleep responses are accompanied by fever but the two responses are, in part, independent from each other. Sleep responses, like fevers, may be beneficial to host defense although this area is relatively uninvestigated. Microbial products likely responsible for sleep and fever responses include bacterial muramyl peptides and endotoxin, and viral double stranded RNA. These microbial products induce sleep and fever responses in animal models. The exact mechanism of how these structurally diverse microbial products elicit sleep and fever remain unknown; however these substances share the ability to induce cytokine production. Cytokines such as interleukin-1 (IL-1), tumor necrosis factor, acidic fibroblast growth factor (FGF), and interferon-α (IFN-α) are somnogenic whether given directly into brain or intravenously. Other cytokines lack somnogenic activity, e.g., IL-2, IL-6, IFNß and basic FGF. The somnogenic actions of cytokines probably involve growth hormone-releasing hormone (GHRH) and nitric oxide. Anti-GHRH or inhibition of NO production inhibits normal sleep and inhibits IL-1-induced sleep. In conclusion, cytokines are likely key mediators of fever and sleep responses to infection. The microbial-cytokine altered sleep likely results from an amplification of physiological sleep mechanisms which include cytokines, several neuropeptides and neurotransmitters such as nitric oxide.

Novel neuronal and endocrine autoantibody targets in Autoimmune Polyendocrine Syndrome type 1.

CONTEXT: Although pituitary autoantibodies have frequently been reported in Autoimmune Polyendocrine Syndrome type 1 (APS1), the autoimmune involvement of the hypothalamic-pituitary axis remains to be elucidated. OBJECTIVE: Our aim was to identify in APS1 patients novel autoantibodies, especially against hypothalamic-pituitary targets, and to correlate their presence with clinical features. PATIENTS: We analyzed 14 APS1 patients from Sardinia, compared to other diseases and healthy donors. MEASURE(S): We used immunohistochemistry, on tissues substrates from various neuroendocrine organs, to detect autoantibody targets. Immunoenzymatic assays, as well as absorption with specific antigens were used to reveal autoantibodies against growth hormone (GH), luteinizing hormone (LH) and somatocrinin (GHRH). Clinical evaluations included GH secretory and cardiovascular autonomic neuropathy tests. RESULTS: Sera from 12/14 APS1 patients revealed autoantibodies reacting with the hypothalamic-pituitary axis, cerebellum, substantia nigra, and/or adrenal medulla, as well as with GH, LH and/or GHRH. Of APS1 patients, 5 showed GH deficiency, in association (4/5 cases) with autoantibodies to hypothalamic and/or pituitary targets. Hypogonadotrophic hypogonadism was revealed in one APS1 patient, together with autoantibodies against gonadotropes. Autonomic neuropathy was detected in 5 of 10 patients, associated with autoantibodies to adrenal medulla in 2 cases. Of 5 patients with autoantibodies to cerebellar neurons, 2 reported emotional or memory alterations. CONCLUSIONS: The majority of Sardinian APS1 patients developed autoantibodies to an assortment of neuroendocrine cells. Novel targets of clinical relevance may include pituitary hormones, uncharacterized pituitary targets, and adrenal medullary cells. An high prevalence of GH deficiency, and possibly of autonomic neuropathy, were also revealed.

Catecholaminergic axonal varicosities appear to innervate growth hormone-releasing hormone-immunoreactive neurons in the human hypothalamus: the possible morphological substrate of the stress-suppressed growth.

CONTEXT: Stress is considered to be a major factor in the regulation of growth. Psychosocial dwarfism, characterized with short stature, delayed puberty, and depression, is typically preceded by psychological harassment or stressful environment. It has been observed that stress suppresses GH secretion, possibly via the attenuation of GHRH secretion. However, the exact mechanism of the impact of stress on growth has not been elucidated yet. OBJECTIVE: Our previous studies revealed intimate associations between neuropeptide Y (NPY)-immunoreactive (IR) axonal varicosities and GHRH-IR perikarya in the human hypothalamus. Because NPY is considered to be a stress molecule, NPY-GHRH juxtapositions may represent an important factor of stress-suppressed GHRH release. In addition to NPY, catecholamines are among the major markers of stress. Thus, in the present study, we examined the putative juxtapositions between the catecholaminergic tyrosine hydroxylase (TH)-/dopamine-ß-hydroxylase-/phenylethanolamine N-methyltransferase-IR and GHRH-IR neural elements in the human hypothalamus. To reveal these juxtapositions, double-label immunohistochemistry was used. RESULTS: Our findings revealed that the majority of the GHRH-IR perikarya formed intimate associations with TH-IR fiber varicosities. The majority of these juxtapositions were found in the infundibular nucleus/median eminence. CONCLUSIONS: The lack of phenylethanolamine N-methyltransferase-GHRH associations and the small number of dopamine-ß-hydroxylase-GHRH juxtapositions suggest that the vast majority of the observed TH-GHRH juxtapositions represent dopaminergic associations. The density of the abutting TH-IR fibers on the surface of the GHRH perikarya suggests that these juxtapositions may be functional synapses, and thus, in addition to NPY, catecholamines may regulate GHRH secretion via direct synaptic mechanisms.

A pleiomorphic GH pituitary adenoma from a Carney complex patient displays universal allelic loss at the protein kinase A regulatory subunit 1A (PRKARIA) locus.

Carney complex (CNC) is a familial multiple endocrine neoplasia syndrome associated with GH-producing pituitary tumours and transmitted as an autosomal dominant trait. Mutations of the PRKAR1A gene are responsible for approximately half the known CNC cases but have never found in sporadic pituitary tumours. Pituitary tissue was obtained from an acromegalic CNC patient heterozygote for a common (PRKARIA)i-inactivating mutation. Both immunohistochemistry and electron microscopy showed a highly pleiomorphic pituitary adenoma. The cell culture population appeared morphologically heterogeneous and remained so after more than 30 passages. The mixture was comprised of cells strongly immunostained for GH, spindle-shaped myofibroblast-like cells, and cuboid cells with large axonal projections (negative for GH). The population appeared to have both epithelial and mesenchymal cells. Both at baseline and at passage 30, cytogenetic analysis indicated the presence of normal 46, XY diploid karyotype, whereas losses of the PRKARIA(i) locus were demonstrated in more than 98% of the cells by fluorescent in situ hybridisation, supporting this gene's involvement in pituitary tumorigenesis. Allelic loss may have occurred in a single precursor cell type that differentiated and clonally expanded into several phenotypes. Epithelial-to-mesenchymal transition may also occur in CNC-associated pleiomorphic pituitary adenomas.

Long-term effects on bone of postnatal immunization against GHRH in female and male rats.

The effects of neonatal passive immunization against GHRH on bone was examined in male and female rats. Pups were treated subcutaneously with GHRH-antiserum (GHRH-Ab) from day 1 to day 10 of age. Bone mineral content (BMC) and bone mineral density (BMD) were evaluated at monthly intervals until 7 months. Markers of bone resorption (urinary lysylpyridinoline, LP), bone formation (serum osteocalcin, OC) and serum IGF-I were measured at 2, 3 and 7 months. In male rats, GHRH-Ab did not modify BMC and BMD when compared with controls. In contrast, female rats demonstrated lower whole body and femoral BMC and BMD from 2 to 7 months of age. Reduced bone growth in the females was associated with lower IGF-I levels than controls at 2 and 3 months of age, whereas in males IGF-I titers did not change during the period of the study. LP excretion was higher in GHRH-Ab-treated rats at 2 and 3 months in both sexes. In females, no difference in OC values was recorded, whereas in GHRH-Ab-treated males, there was an increase in OC levels at 2 and 3 months. These data indicate that transient GHRH deprivation induces an osteopenic effect in female rats which is not evident in male rats.

Role for central ghrelin in food intake and secretion profile of stomach ghrelin in rats.

Ghrelin, a 28-amino-acid peptide, has recently been isolated from the rat stomach as an endogenous ligand for the GH secretagogue receptor. We have reported previously that central or peripheral administration of ghrelin stimulates food intake, and the secretion of GH and gastric acid in rats. In the present study, we investigated how much endogenous centrally released ghrelin is involved in the control of food intake and body weight gain. We also examined the profile of ghrelin secretion from the stomach by RIA using two kinds of anti-ghrelin antiserum, one raised against the N-terminal ([Cys(12)]-ghrelin[1-11]) region and one raised against the C-terminal ([Cys(0)]-ghrelin [13-28]) region of the peptide. The former antibody recognizes specifically ghrelin with n- octanoylated Ser 3 (acyl ghrelin), and does not recognize des-acyl ghrelin. The latter also recognizes des-acyl ghrelin (i.e. total ghrelin). Intracerebroventricular treatment with the anti-ghrelin antiserum against the N-terminal region twice a day for 5 days decreased significantly both daily food intake and body weight. Des-acyl ghrelin levels were significantly higher in the gastric vein than in the trunk. Either fasting for 12 h, administration of gastrin or cholecystokinin resulted in increase of both acyl and des-acyl ghrelin levels. The ghrelin levels exhibited a diurnal pattern, with the bimodal peaks occurring before dark and light periods. These two peaks were consistent with maximum and minimum volumes of gastric content respectively. These results suggest that (1) endogenous centrally released ghrelin participates in the regulation of food intake and body weight, (2) acyl ghrelin is secreted from the stomach, (3) intestinal hormones stimulate ghrelin release from the stomach, and (4) regulation of the diurnal rhythm of ghrelin is complex, since ghrelin secretion is augmented under conditions of both gastric emptying and filling.

Regulation of hepatic insulin-like growth factor I leader exonusage in lambs: effect of immunization against growth hormone-releasing factor and subsequent growth hormone treatment.

The establishment of a GH-responsive endocrine IGF-I network is essential for the regulation of postnatal growth. Transcripts of exons 1 and 2 of the mammalian IGF-I gene are alternately spliced onto exon 3, generating class 1 and class 2 mRNA, respectively, each encoding individual signal peptides. The liver is largely responsible for the synthesis of circulating IGF-I and is the main site of expression for class 2 mRNA. The aim of this study was to examine the regulation of hepatic class 1 and 2 mRNA levels in response to changed GH status. Lambs were actively immunized against GRF to suppress GH secretion; hepatic IGF-I mRNA leader exon usage was examined in the presence and absence of GH replacement and in control-immunized lambs. Lambs immunized against GRF exhibited a 17% (P < 0.001) decrease in growth rate as assessed by whole body weight gain, accompanied by decreased circulating IGF-I concentrations (P < 0.001), which were increased by subsequent GH treatment (P < 0.001). Hepatic class 1 and 2 IGF-I mRNA levels decreased in GRF-immunized lambs, although only class 2 transcripts decreased significantly (P < 0.001). Subsequent GH treatment induced increases in class 1 and 2 mRNA levels (P < 0.001) but the increase in class 2 message exceeded that for class 1 (P < 0.001). Thus, the percentage of total IGF-I mRNA accounted for by class 2 mRNA was 45% in control lambs, decreased to less than 20% in GRF-immunized lambs, but increased to 72% in the GRF-immunized lambs treated with GH and correlated with circulating IGF-I concentrations. These data suggest physiological significance for class 1 and 2 IGF-I mRNA species in GH action. Possible functions for such alternative splicing mechanisms are discussed.

Unequal autonegative feedback by GH models the sexual dimorphism in GH secretory dynamics.

Growth hormone (GH) secretion, controlled principally by a GH-releasing hormone (GHRH) and GH release-inhibiting hormone [somatostatin (SRIF)] displays vivid sexual dimorphism in many species. We hypothesized that relatively small differences within a dynamic core GH network driven by regulatory interactions among GH, GHRH, and SRIF explain the gender contrast. To investigate this notion, we implemented a minimal biomathematical model based on two coupled oscillators: time-delayed reciprocal interactions between GH and GHRH, which endow high-frequency (40-60 min) GH oscillations, and time-lagged bidirectional GH-SRIF interactions, which mediate low-frequency (occurring every 3.3 h) GH volleys. We show that this basic formulation, sufficient to explain GH dynamics in the male rat [Farhy LS, Straume M, Johnson ML, Kovatchev BP, and Veldhuis JD. Am J Physiol Regulatory Integrative Comp Physiol 281: R38-R51, 2001], emulates the female pattern of GH release, if autofeedback of GH on SRIF is relaxed. Relief of GH-stimulated SRIF release damps the slower volleylike oscillator, allowing emergence of the underlying high-frequency oscillations that are sustained by the GH-GHRH interactions. Concurrently, increasing variability of basal somatostatin outflow introduces quantifiable, sex-specific disorderliness of the release process typical of female GH dynamics. Accordingly, modulation of GH autofeedback on SRIF within the interactive GH-GHRH-SRIF ensemble and heightened basal SRIF variability are sufficient to transform the well-ordered, 3.3-h-interval, multiphasic, volleylike male GH pattern into a femalelike profile with irregular pulses of higher frequency.

A construct of interactive feedback control of the GH axis in the male.

Growth hormone (GH) secretion is controlled by GH-releasing hormone (GHRH), the GH release-inhibiting hormone somatostatin (SRIF), and autofeedback connections. The ensemble network produces sexually dimorphic patterns of GH secretion. In an effort to formalize this system, we implemented a deterministically based autonomous feedback-driven construct of five principal dose-responsive regulatory interactions: GHRH drive of GH pituitary release, competitive inhibition of GH release by SRIF, GH autofeedback via SRIF with a time delay, delayed GH autonegative feedback on GHRH, and SRIF inhibition of GHRH secretion. This formulation engenders a malelike pattern of successive GH volleys due jointly to positive time-delayed feedback of GH on SRIF and negative feedback of SRIF on GH and GHRH. The multipeak volley is explicated as arising from a reciprocal interaction between GH and GHRH during periods of low SRIF secretion. The applicability of this formalism to neuroendocrine control is explored by initial parameter sensitivity analysis and is illustrated for selected feedback-dependent experimental paradigms. The present construct is not overparameterized and does not require an ad hoc pulse generator to achieve pulsatile GH output. Further evolution of interactive constructs could aid in exploring more complex feedback postulates that confer the vivid sexual dimorphism of female GH profiles.

Interactions of growth hormone secretagogues and growth hormone-releasing hormone/somatostatin.

The class of novel synthetic compounds termed growth hormone secretagogues (GHSs) act in the hypothalamus through, as yet, unknown pathways. We performed physiologic and histochemical studies to further understand how the GHS system interacts with the well-established somatostatin (SRIF)/growth hormone-releasing hormone (GHRH) neuroendocrine system for regulating pulsatile GH secretion. Comparison of the GH-releasing activities of the hexapeptide growth hormone-releasing peptide-6 (GHRP-6) and GHRH administered intravenously to conscious adult male rats showed that the pattern of GH responsiveness to GHRP-6 was markedly time-dependent, similar to that observed with GHRH. Immunoneutralization of endogenous SRIF reversed the blunted GH response to GHRP-6 at trough times, suggesting that GHRP-6 neither disrupts nor inhibits the cyclical release of endogenous hypothalamic SRIF. By striking contrast, passive immunization with anti-GHRH serum virtually obliterated the GH responses to GHRP-6, irrespective of the time of administration. These findings suggest that the GHSs do not act by altering SRIF release but, rather, stimulate GH release via GHRH-dependent pathways. Our dual chromogenic and autoradiographic in situ hybridization experiments revealed that a subpopulation of GHRH mRNA-containing neurons in the arcuate (Arc) nucleus and ventromedial nucleus (VMN) of the hypothalamus expressed the GHS receptor (GHS-R) gene. These results provide strong anatomic evidence that GHSs may directly stimulate GHRH release into hypophyseal portal blood, and thereby influence GH secretion, through interaction with the GHS-R on GHRH- containing neurons. Altogether, these findings support the notion that an additional neuroendocrine pathway may exist to regulate pulsatile GH secretion, possibly through the influence of the newly discovered GHS natural peptide, ghrelin.

Growth hormone-releasing factor affects macronutrient intake during the anabolic phase of zinc repletion: total hypothalamic growth hormone-releasing factor content and growth hormone-releasing factor immunoneutralization during zinc repletion.

Growth hormone-releasing factor (GRF) is thought to perform two distinct functions within the brain. GRF synthesized in the median eminence (ME) stimulates the release of growth hormone (GH) from the pituitary, while GRF in the suprachiasmatic nucleus and median preoptic area (SCN/MPOA) may stimulate selection of dietary protein. These two functions may be coupled to regulate and enhance growth. During zinc repletion, a period characterized by increased protein intake and accelerated growth, we examined this coupling by measuring GRF peptide content in hypothalamic sites and neutralizing GRF function by infusing anti-GRF antibody into the hypothalamus during zinc repletion. Total GRF content and GRF content in the ME and SCN/MPOA were decreased in zinc-deficient (Zn-) rats compared to zinc-adequate (Zn+) rats (P < 0.05). There were no differences in GRF content during zinc repletion in either nuclei. Subsequently, we investigated the macronutrient feeding patterns of rats chronically infused with anti-GRF IgG into the lateral ventricle of the brain during zinc repletion. All Zn- and Zn+ rats administered anti-GRF IgG exhibited a reduction in protein intake during zinc repletion. The Zn- rats receiving anti-GRF-IgG consumed equal amounts of total diet compared to those receiving vehicle during the repletion period however they consumed less carbohydrate (P < 0.05) and considerably more fat (P < 0.02). There were no significant differences in carbohydrate or fat intake in Zn+ rats receiving anti-GRF antibody. These results suggest that GRF likely directs protein intake during normal growth, but may interact with additional appetite-controlling neuropeptides during zinc repletion.

Effects of active immunization against growth-hormone releasing factor on puberty and reproductive development in gilts.

Hormones within the somatotropin cascade influence several physiological traits, including growth and reproduction. Active immunization against growth hormone-releasing factor (GRFi) initiated at 3 or 6 mo of age decreased weight gain, increased deposition of fat, and delayed puberty in heifers. Two experiments were conducted to investigate the effects of GRFi on puberty and subsequent ovulation rate in gilts. Crossbred gilts were actively immunized against GRF-(1-29)-(Gly)2-Cys-NH2 conjugated to human serum albumin (GRFi) or against human serum albumin alone (HSAi). In Exp. 1, gilts were immunized against GRF (n = 12) or HSA (n = 12) at 92 +/- 1 d of age. At 191 d of age, antibody titers against GRF were greater (P < .05) in GRFi (55.5 +/- 1.3%) than in HSAi (.4 +/- 2%) gilts. The GRFi decreased (P < .05) BW (86 +/- 3 vs 104 +/- 3 kg) by 181 d of age and increased (P < .05) backfat depth (15.7 +/- .4 vs 14.8 +/- .4 mm) by 130 d of age. At 181 d of age, GRFi reduced the frequency of ST release (1.0 +/- .5 vs 5.0 +/- .5, peaks/24 h; P < .0001) and decreased (P < .01) ST (1.1 +/- .06 vs 1.7 +/- .06 ng/mL), IGF-I (29 +/- 2 vs 107 +/- 2 ng/mL), and insulin concentrations (3.5 +/- .2 vs 6.3 +/- .2 ng/mL). The GRFi decreased (P < .05) feed conversion efficiency but did not alter age at puberty (GRFi = 199 +/- 5 d vs HSAi = 202 +/- 5 d) or ovulation rate after second estrus (GRFi = 10.7 +/- .4 vs HSAi = 11.8 +/- .5). In Exp. 2, gilts were immunized against GRF (n = 35) or HSA (n = 35) at 35 +/- 1 d of age. The GRFi at 35 d of age did not alter the number of surface follicles or uterine weight between 93 and 102 d of age, but GRFi decreased (P < .05) ovarian weight (.41 +/- .08 vs 1.58 +/- .4 g) and uterine length (17.2 +/- 1.1 vs 25.3 +/- 2.3 cm). Immunization against GRF reduced (P < .05) serum IGF-I (GRFi = 50 +/- 4 vs HSAi = 137 +/- 4 ng/mL) and BW (GRFi = 71 +/- 3 vs HSAi = 105 +/- 3 kg) and increased (P < .05) backfat depth (GRFi = .38 +/- .03 vs HSAi = .25 +/- .02 mm/kg). Age at puberty was similar in GRFi and HSAi gilts, but ovulation rate was lower (P < .05) after third estrus in GRFi (11.3 +/- .8) than in HSAi (13.8 +/- .8) gilts. Thus, GRFi at 92 or 35 d of age decreased serum ST, IGF-I, and BW in prepubertal gilts without altering age of puberty. However, GRFi at 35 d of age, but not 92 d of age, decreased ovulation rate. These results indicate that alterations in the somatotropic axis at 1 mo of age can influence reproductive development in pubertal gilts.

Differential expression of gonadotropin and prolactin antigens by GHRH target cells from male and female rats.

There is a 2- to 3-fold increase in luteinizing hormone-beta (LHbeta) or follicle-stimulating hormone-beta (FSHbeta) antigen-bearing gonadotropes during diestrus in preparation for the peak LH or FSH secretory activity. This coincides with an increase in cells bearing LHbeta or FSHbeta mRNA. Similarly, there is a 3- to 4-fold increase in the percentage of cells that bind GnRH. In 1994, we reported that this augmentation in gonadotropes may come partially from subsets of somatotropes that transitionally express LHbeta or FSHbeta mRNA and GnRH-binding sites. The next phase of the study focused on questions relating to the somatotropes themselves. Do these putative somatogonadotropes retain a somatotrope phenotype? As a part of ongoing studies that address this question, a biotinylated analog of GHRH was produced, separated by HPLC and characterized for its ability to elicit the release of GH as well as bind to pituitary target cells. The biotinylated analog (Bio-GHRH) was detected cytochemically by the avidin-peroxidase complex technique. It could be displaced by competition with 100-1000 nM GHRH but not corticotropin-releasing hormone or GnRH. In cells from male rats exposed to 1 nM Bio-GHRH, 28+/-6% (mean+/-s.d) of pituitary cells exhibited label for Bio-GHRH (compared with 0.8+/-0.6% in the controls). There were no differences in percentages of GHRH target cells in populations from proestrous (28+/-5%) and estrous (25+/-5%) rats. Maximal percentages of labeled cells were seen following addition of 1 nM analog for 10 min. In dual-labeled fields, GHRH target cells contained all major pituitary hormones, but their expression of ACTH and TRH was very low (less than 3% of the pituitary cell population) and the expression of prolactin (PRL) and gonadotropins varied with the sex and stage of the animal. In all experimental groups, 78-80% of Bio-GHRH-reactive cells contained GH (80-91% of GH cells). In male rats, 33+/-6% of GHRH target cells contained PRL (37+/-9% of PRL cells) and less than 20% of these GHRH-receptive cells contained gonadotropins (23+/-1% of LH and 31+/-9% of FSH cells). In contrast, expression of PRL and gonadotropins was found in over half of the GHRH target cells from proestrous female rats (55+/-10% contained PRL; 56+/-8% contained FSHbeta; and 66+/-1% contained LHbeta). This reflected GHRH binding by 71+/-2% PRL cells, 85+/-5% of LH cells and 83+/-9% of FSH cells. In estrous female rats, the hormonal storage patterns in GHRH target cells were similar to those in the male rat. Because the overall percentages of cells with Bio-GHRH or GH label do not vary among the three groups, the differences seen in the proestrous group reflect internal changes within a single group of somatotropes that retain their GHRH receptor phenotype. Hence, these data correlate with earlier findings that showed that somatotropes may be converted to transitional gonadotropes just before proestrus secretory activity. The LH and FSH antigen content of the GHRH target cells from proestrous rats demonstrates that the LHbeta and FSHbeta mRNAs are indeed translated. Furthermore, the increased expression of PRL antigens by these cells signifies that these convertible somatotropes may also be somatomammotropes.

Humoral regulation of physiological sleep: cytokines and GHRH.

Interleukin-1, tumour necrosis factor, and growth hormone releasing hormone form part of the humoral mechanisms regulating physiological sleep. Their injection enhances non-rapid-eye-movement sleep whereas their inhibition reduces spontaneous sleep and sleep rebound after sleep deprivation. Changes in their mRNA levels and changes in their protein levels in the brain are consistent within their proposed role in sleep regulation. Furthermore, results from transgenic and mutant animals also are suggestive of their role in sleep regulation. The sites responsible for the growth hormone releasing hormone somnogenic activity seem to reside in the anterior hypothalamus/basal forebrain. Somnogenic sites for interleukin-1 and tumour necrosis factor likely include the anterior hypothalamus, but also may extend beyond that area. These substances elicit non-rapid-eye-movement sleep via a biochemical cascade that includes other known sleep regulatory substances.

Immune function in transgenic mice overexpressing growth hormone (GH) releasing hormone, GH or GH antagonist.

Effects of life-long exposure to high levels of homologous or heterologous growth hormone (GH) and effects of GH resistance on selected parameters of immune function were studied in adult male transgenic mice overexpressing GH releasing hormone (GHRH), bovine (b) GH or an antagonistic bGH analog. In metallothionein I (MT)-bGH transgenic mice with high peripheral levels of bovine GH, there were significant increases in the absolute weight of the thymus and the spleen and in the mitogenic responses of splenocytes to concanavalin A (ConA), lipopolysaccharide (LPS) and phytohemagglutinin (PHA), as compared to age-matched normal animals. There were no significant differences between MT-bGH transgenic and normal mice in splenocyte viability or in delayed-type hypersensitivity measured by the allergic contact dermatitis response to oxazolone. Similar results, including significant stimulation of splenocyte responses to ConA, LPS, and PHA, were obtained in MT-hGHRH transgenic mice in which overexpression of GHRH leads to striking pituitary enlargement and massive elevation of peripheral levels of homologous (mouse) GH. In MT-bGH-antagonist transgenic mice in which overexpression of an antagonistic bGH analog interferes with the actions of endogenous GH, spleen weight was reduced but proliferative responses of splenocytes to ConA, LPS, and PHA were not affected. It is concluded that overexpression of heterologous or homologous GH in transgenic mice can lead to significant stimulation of some parameters of immune function, whereas antagonism of GH action by expression of an antagonistic GH analog does not affect splenocyte responses to mitogens.

Suppression of growth hormone does not affect ongoing spermatogenesis in rats.

Recent evidence suggests that growth hormone (GH) may enhance physiologic processes, such as spermatogenesis, in addition to causing classical anabolic effects. We have previously shown that testosterone restores spermatogenesis in rats that were made azoospermic by immunization against gonadotropin-releasing hormone (GnRH). In this study, we investigated whether suppression of GH affects spermatogenesis and the ability of testosterone to restore spermatogenesis following immunization against GnRH and/or growth hormone-releasing hormone (GHRH). Twelve rats were actively immunized against GnRH (anti-GnRH), twelve rats were actively immunized against GHRH (anti-GHRH), six rats were immunized against both GnRH and GHRH (anti-GnRH/GHRH), and six rats served as controls. Two weeks after the second booster, six rats each from the anti-GnRH and anti-GHRH groups as well as the six anti-GnRH/GHRH rats received 24-cm testosterone-filled Silastic implants (T), and the remaining six rats from each of these groups received empty Silastic implants. All rats were euthanized 2 months later. Weights of testes and testicular sperm counts were determined. Serum testosterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), growth hormone (GH), and insulin-like growth factor-1 (IGF-1) concentrations were determined by radioimmunoassays. Serum GH and IGF-1 were suppressed in anti-GHRH rats. IGF-1 was partially restored by testosterone in anti-GHRH and in anti-GnRH/GHRH rats, but GH was restored to control value in anti-GnRH/GHRH rats. Serum LH and FSH were suppressed in anti-GnRH and anti-GnRH/GHRH rats, but only FSH was partially restored by testosterone. Suppression of GH did not affect maintenance of spermatogenesis. However, because T partially restored GH and IGF-1 levels in anti-GnRH/GHRH rats and because spermatogenesis was found to be restored in these rats, we conclude that GH does not play a role in the maintenance of spermatogenesis in adult rats, but it may be required for the replenishment of germ cells in experimentally induced regressed rat testes.

Hexarelin exhibits protective activity against cardiac ischaemia in hearts from growth hormone-deficient rats.

Male rats were treated with growth hormone (GH)-releasing hormone antiserum to induce selective GH deficiency. The chronic administration of hexarelin to these GH-deficient rats had a pronounced protective effect against ischaemic and post-ischaemic ventricular dysfunction. Hexarelin prevented hyper-responsiveness of the coronary vascular bed to angiotensin II and also prevented the reduction in generation of 6-keto-prostaglandin F1alpha in perfused hearts from GH-deficient rats. The most plausible interpretation of these findings is that hexarelin acts via stimulation of specific cardiac and vascular receptors, triggering currently unknown cytoprotective mechanisms that are responsible for resistance to ischaemic insults and for the preservation of the integrity of the endothelial vasodilation function.