Isolated deficiency of immunocytochemically detected somatostatin in Snell dwarf, but not in “little,” mice

Immunocytochemical staining of the neuropeptide somatostatin was evaluated in the brains of two growth hormone-deficient mouse mutants, Snell dwarf (dw/dw), and “little” (lit/lit). In Snell dwarf mice, in which GH is undetectable, an isolated somatostatin deficiency was observed in hypothalamic median eminence and in anterior periventricular hypothalamic neurons which are afferent to median eminence, compared to somatostatin staining in normal mice of the same strain (DW/?). Somatostatin staining in all other CNS areas in dwarfs was identical to that in normal mice. In contrast, in little mice, which exhibit 5–10% of normal GH levels, somatostatin expression was comparable between mutants and normal mice (LIT/?) in all CNS areas, including median eminence-afferent neurons in anterior periventricular hypothalamus. The results suggest that expression of somatostatin in hypophysiotropic CNS is dependent upon minimal pituitary GH secretion.     

Nighttime immunoreactive somatostatin content of the median eminence in hypo- and hyperthyroid rats

The median eminence content of immunoreactive somatostatin (IRS) was measured by radioimmunoassay in 107 male albino rats, who were either hypothyroid after surgical thyroidectomy (N = 38), hyperthyroid following a subcutaneous implant of 5 mg of L-thyroxine (N = 36), or otherwise untreated (N = 33). Thyroid function was assessed by determining plasma levels of T4 and TSH from trunk blood obtained at the time of decapitation. Subgroups of animals from the 3 groups were killed either before (1800 hr), during (2200, 0200, 0400 hr), or after the dark portion of their 14:10 LD photoperiod. Although no changes in median eminence IRS content were found throughout the period of study within any of the 3 groups, hypothyroid animals (297.23 +/- 13.47 ng per ME; 620.41 +/- 58 ng IRS/mg protein) had a significantly lower median eminence IRS concentration than untreated rats (355.86 +/- 16.55 ng of IRS per ME, P less than 0.01; 906.86 +/- 96.38 ng IRS/mg protein, P less than 0.05) and hyperthyroid animals (384.12 +/- 14.67 ng per ME, P less than 0.001; 874.1 +/- 104.5 ng IRS@mg protein, P less than 0.05). It is concluded, that the feedback of thyroid hormones on the hypothalamic-pituitary axis during thyroid hormone excess in vivo, contrary to what occurs in hypothyroidism, is probably independent of hypothalamic somatostatin.     

Response of pituitary thyrotrophs to thyrotrophin-releasing hormone in Snell dwarf mice

Summary The effect of thyrotrophin-releasing hormone (TRH) on pituitary thyrotrophs was investigated in Snell dwarf mice (dw/dw) that are genetically deficient in thyrotrophin (TSH) and in normal animals of the same strain. The normal animals were treated with either saline or 10 g TRH per day for 2 weeks, while the dwarf mice were given daily injections of saline, 10 g TRH for 2 weeks or 10 g for 6 weeks. At the end of each experimental period, the pituitary glands were removed and fixed for light-microscopic analysis using immunocytochemistry, or for transmission electron-microscopic study. Compared to thyrotrophs observed in the pituitary glands of untreated normal mice, thyrotrophs in TRH-treated normal mice appeared to be more numerous by immunocytochemistry and showed signs of stimulation by electron microscopy. In contrast, immunostainable thyrotrophs could not be identified in the pituitary glands of untreated or TRH-treated dwarfs. However, a few cells exhibiting ultrastructural features of stimulated thyrotrophs, were noticeable in the dwarfs following TRH administration. Thus, while failing to induce the synthesis of immunoreactive TSH under the applied experimental conditions, exogenous TRH appeared to elicit differentiation of thyrotroph precursors into ultrastructurally recognizable thyrotrophs. The discrepancy between the immunocytochemical and ultrastructural findings remains unresolved; more work is required to clarify the question as to why ultrastructural maturation of thyrotrophs was unaccompanied by the production of immunoreactive TSH.     

Pituitary and serum concentrations of prolactin and GH in Snell dwarf mice.

Secretions of prolactin and GH in dwarf mice were studied using homologous radioimmunoassays. Blood samples from adult male and femal dw/dw mice were collected by orbital puncture and by decapitation. Compared to related normals (+/?), pituitary concentrations of GH and prolactin were very low in dw/dw mice, the concentrations of prolactin being more scarce than those of GH. Prolactin and GH concentrations were also lower in sera of dw/dw mice, but the relative differences appeared sex-dependent: serum GH was more reduced in males than in females while serum prolactin was more depressed in females. The data confirm earlier indications of deficiencies in the circulating levels of prolactin and GH in dwarf mice and suggest that the hypoactivity of these hormones may be crucial to some of the anomalies found in this mutant.     

Alterations in GHRH binding and GHRH receptor mRNA in the pituitary of adult dw/dw rats

Lewis dwarf (dw/dw) rats exhibit growth hormone (GH) deficiency and growth retardation linked to a malfunction of GHRH signaling. In this study, GHRH-receptor (GHRH-R) binding and mRNA in the pituitary of adult male dw/dw and age-matched normal Lewis rats was measured by radioligand binding assay and real-time PCR. Only one of nine pools of dw/dw pituitary membranes revealed detectable binding of [His(1), 125I-Tyr(10), Nle(27)]hGHRH(1-32) amide (B(max); 4.3 fmol/mg protein). In contrast, GHRH-R binding was 22.4 +/- 2.60 fmol/mg protein in normal Lewis rats. mRNA for GHRH-R was detectable in all dw/dw rat pituitaries examined, averaging 21% that of Lewis rats. Low expression of GHRH-R reflects reduced GHRH-R mRNA as well as a possible reduction in translation of the receptor protein.     

Induction of pregnancy-associated murine protein-1 in dwarf mice by human growth hormone

Pregnancy-associated murine protein-1 (PAMP-1) could not be detected in peripheral blood of female dwarf mice (genotype dw/dw of the DW strain). By contrast the normal size females of the DW strain (genotypes +/+ and +/dw) had PAMP-1 serum levels of 18.9 AU +/- 15.7 AU/ml. Following administration of biosynthetic human growth hormone (hGH) every 2 h for 52 h PAMP-1 was detected in all dwarf females at concentrations of 16.0 AU +/- 3.3 AU/ml. The albumin levels in the circulation of DW females of normal size were significantly higher (P less than 0.05) than those of DW dwarfs, and the hGH administration did not change the serum albumin levels. The present experiment adds weight to the suggestion that the PAMP-1 serum level is regulated by GH.     

Stimulation of growth hormone release in dwarf and normal chickens by thyrotrophin releasing hormone (TRH) or human pancreatic growth hormone releasing factor (hpGRF)

The effect of thyrotrophin releasing hormone (TRH) or human pancreatic growth hormone releasing factor (hpGRF) on growth hormone (GH) release was studied in both dwarf and normal Rhode Island Red chickens with a similar genotype except for a sex-linked dw gene. Both TRH (10 micrograms/kg) and hpGRF (20 micrograms/kg) injections stimulated plasma GH release within 15 min in young and adult chickens. The increase in GH release was higher in young cockerels than that in adult chickens. The age-related decline in the response to TRH stimulation was observed in both strains, while hpGRF was a still potent GH-releaser in adult chickens. The maximal and long acting response was observed in young dwarf chickens, suggesting differences in GH pools releasable by TRH and GRF in the anterior pituitary gland. The pituitary gland was stimulated directly by perifusion with hpGRF (1 microgram/ml and 10 micrograms/ml) or TRH (1 microgram/ml). Repeated perifusion of GRF at 40 min intervals blunted further increase in GH release, but successive perifusion with TRH stimulated GH release. The results suggest the possibility that desensitization to the effects of hpGRF occurs in vitro and that the extent of response depends on the number of receptors for hpGRF or TRH and/or the amount of GH stored in the pituitary gland.     

Dominant dwarfism in transgenic rats by targeting human growth hormone (GH) expression to hypothalamic GH-releasing factor neurons.

Expression of human growth hormone (hGH) was targeted to growth hormone-releasing (GRF) neurons in the hypothalamus of transgenic rats. This induced dominant dwarfism by local feedback inhibition of GRF. One line, bearing a single copy of a GRF-hGH transgene, has been characterized in detail, and has been termed Tgr (for Transgenic growth-retarded). hGH was detected by immunocytochemistry in the brain, restricted to the median eminence of the hypothalamus. Low levels were also detected in the anterior pituitary gland by radioimmunoassay. Transgene expression in these sites was confirmed by RT-PCR. Tgr rats had reduced hypothalamic GRF and mRNA, in contrast to the increased GRF expression which accompanies GH deficiency in other dwarf rats. Endogenous GH mRNA, GH content, pituitary size and somatotroph cell number were also reduced significantly in Tgr rats. Pituitary adrenocorticotrophic hormone (ACTH) and thyroid-stimulating hormone (TSH) levels were normal, but prolactin content, mRNA levels and lactotroph cell numbers were also slightly reduced, probably due to feedback inhibition of prolactin by the lactogenic properties of the hGH transgene. This is the first dominant dwarf rat strain to be reported and will provide a valuable model for evaluating the effects of transgene expression on endogenous GH secretion, as well as the use of GH secretagogues for the treatment of dwarfism.     

rdw rats, a new hereditary dwarf model in the rat

A new hereditary dwarf mutation was found in Csk: Wistar-Imamichi rat breeding colony. The mutant rats and control normal rats were studied with clinical, genetical and histological examination. The result showed that the present mutation was inherited in autosomal recessive trait (gene symbol; rdw) and was provoked with the hypoplasia of pars distalis (anterior pituitary), in particular the secretory cells of GH, PRL and TSH. The characteristics of the present mutant (rdw) was very similar to dw mouse, and was usefull for endocrinological research as an animal model of human pituitary dwarfism.     

Hypothalamic catecholamine histofluorescence in dwarf mice

Summary Brains of growth hormone (GH)-and prolactin (PRL)-deficient Ames (df/df) and Snell (dw/dw) dwarf mice and normal mice of the same strains were examined for catecholamine (CA) histofluorescence, with particular emphasis upon the hypothalamic tuberoinfundibular (A12) (arcuate nucleus/median eminence) region, which plays a role in the regulation of both GH and PRL. Dwarfs and normal animals of both types also were treated with a drug regimen to deplete sequentially neuronal CA stores (reserpine), inhibit CA oxidation (nialamide) and load dopaminergic A12 cells with exogenous transmitter (norepinephrine), in order to test viability and axonal transport capacity of A12 neurons. In both types of dwarfs, compared with normals, fluorescence was markedly reduced in the zona externa of the median eminence, which is normally rich in terminals from A12 neurons. Fluorescence in the median eminence was particularly weak in Ames dwarfs, and A12 perikarya were difficult to discern in this group. Snell dwarfs showed reduced fluorescence of A12 perikarya when compared with the brightly fluorescent perikarya seen in normal mice. In supraoptic and paraventricular nuclei, and in the zona interna of the median eminence, CA fluorescence attributable to NE was comparable among dwarfs and normals; fluorescence of dopaminergic perikarya in substantia nigra was also unaffected in dwarfs. Exogenously administered NE effected enhanced fluorescence of A12 Perikarya in normal mice and in Snell dwarfs; NE treatment in the Ames dwarf, however, failed to increase significantly the faint fluorescence of A12 cell bodies. The results indicate that dopaminergic A12 neurons in Snell dwarf mice are present and viable. Reduction in DA in the median eminence in both genetic dwarfs and failure of CA uptake in Ames dwarfs may indicate altered axon morphology or transport capacity, and/or abnormal DA biosynthesis, which may be more severe in Ames than in Snell dwarfs. Thus, genetic alteration in differentiation of pituitary cells may play a significant role in development of the CA systems in the hypothalamus.Supported by PHS Grants HD 18243 (CP), NS15816 and AG00847 (JRS) and HS12671 (AB).     

Cerebroside and Sulfatide Biosynthesis in the Brain of Snell Dwarf Mouse: Effects of Thyroxine and Growth Hormone in the Early Postnatal Period

Snell dwarf mice (dw/dw) and normal mice (+/?) were injected with thyroxine (T₄) (1 μg/animal, four injections) and growth hormone (GH) (20 μg/animal, four injections) from the 5th to the 15th day of life. In the untreated dw/dw mouse brain, the specific activities of UDP-galactose:ceramide galactosyltransferase (CGalT), PAPS:cerebroside sulfotransferase (CST), and 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) were decreased by 28, 25, and 37%, respectively, compared with the control untreated +/? mice. The major effect of T₄ was an increase of the brain CNP in the +/? mice (+40%) and dw/dw mice (+111%). The treatment with T₄ also brought to normal the level of CGalT in dw/dw brain; a somewhat less marked effect on CST was observed. The treatment with GH had a great stimulatory effect on CNP: the specific activity of this enzyme increased by 40 and 69% in +/? and dw/dw mouse brain, respectively. On the contrary, no effect of GH on the CGalT activity was observe in this study. Our results suggest that T₄ and GH may have both independent and complementary actions on the myelin-associated enzymes during the early postnatal period of brain development.     

Role of vasoactive intestinal polypeptide (VIP) in regulating the pituitary function in man

Intramuscular injection of synthetic VIP (200 micrograms) resulted in a rapid increase in plasma prolactin (PRL) concentrations in normal women, which was accompanied by the 4- to 7-fold increase in plasma VIP levels. Mean (+/- SE) peak values of plasma PRL obtained 15 min after the injection of VIP were higher than those of saline control (28.1 +/- 6.7 ng/ml vs. 11.4 +/- 1.6 ng/ml, p less than 0.05). Plasma growth hormone (GH) and cortisol levels were not affected by VIP in normal subjects. VIP injection raised plasma PRL levels (greater than 120% of the basal value) in all of 5 patients with prolactinoma. In 3 of 8 acromegalic patients, plasma GH was increased (greater than 150% of the basal value) by VIP injection. In the in vitro experiments, VIP (10(-8), 10(-7) and 10(-6) M) stimulated PRL release in a dose-related manner from the superfused pituitary adenoma cells obtained from two patients with prolactinoma. VIP-induced GH release from the superfused pituitary adenoma cells was also shown in 5 out of 6 acromegalic patients. VIP concentrations in the CSF were increased in most patients with hyperprolactinemia and a few cases with acromegaly. These findings indicate that VIP may play a role in regulating PRL secretion in man and may affect GH secretion from pituitary adenoma in acromegaly.     

Role of neuroendocrine hormones in murine T cell development. Growth hormone exerts thymopoietic effects in vivo.

Growth hormone (GH) and other neuroendocrine mediators have been implicated previously in T cell development. We therefore examined thymic development in DW/J dwarf mice. DW/J mice lack acidophilic anterior pituitary cells and consequently are totally deficient in the production of GH, as well as other neuroendocrine hormones. DW/J dwarf mice had greatly hypoplastic thymi that continued to decrease in size as the mice aged. Characterization of the different T cell subpopulations within the thymi of dwarf mice indicated a deficiency in CD4+/CD8+ double-positive thymocytes. This deficiency of progenitor cells became more complete as the mice aged culminating in the total disappearance of this subpopulation in some dwarf mice > 3 mo of age. Analysis of the lymph nodes indicated that a population of double-positive (CD4/CD8) T cells appeared in some mice concurrent with the disappearance of double-positive cells in the thymus suggesting that these thymocytes may have migrated to the periphery. However, peripheral T cells from dwarf mice did exhibit Ag-specific responses indicating that these mice have functional T cells. Treatment of the mice with recombinant human GH, which binds both murine growth hormone receptors and murine prolactin receptors, or ovine GH, which binds murine growth hormone receptors but not murine prolactin receptors, resulted in an increase in thymic size and the reappearance of the CD4+/CD8+ double-positive cells within the thymus. Additionally, after GH treatment, the double-positive cells disappeared from the lymph nodes. The thymi of mice treated with GH failed to attain normal size but did develop a normal distribution of T cell progenitors. Thus, GH exerts significant thymopoietic effects in vivo. Neuroendocrine hormones may be important for normal T cell differentiation to occur within the murine thymus.     

Kallikrein-binding protein is induced by growth hormone in the dwarf rat.

Rat kallikrein-binding protein (KBP), a member of the serpin family, is a tissue kallikrein inhibitor. It has been shown to be a potential pathogenic factor of diabetic retinopathy and may play a role in animal development and growth. To determine whether reduced KBP expression is involved in retarded animal growth, we examined the in vivo effect of growth hormone (GH) deficiency on the expression of KBP in the Lewis dwarf (dw/dw). We found that serum levels of functionally active KBP were reduced in the dwarf rat (P < 0.05) as determined by complex formation assay between serum KBP and (125)I-labeled rat tissue kallikrein. Enzyme-linked immunosorbent assay showed that KBP levels were significantly reduced in the serum of the dwarf rat compared to the Lewis rat (213.8 ng/ml vs. 413.8 ng/ml, n = 4, P < 0.01). The decreased KBP levels were confirmed by Western blot analysis. Moreover, treatment of the dwarf rat with recombinant human GH for 4 wk resulted in a significant increase in KBP activity (P < 0.01) and serum KBP levels compared with the untreated dwarf rat (549.8 ng/ml, n = 5, vs. 213.8 ng/ml, n = 4, P < 0.02). Northern blot analysis and densitometry showed that liver KBP mRNA levels were reduced by fivefold in the dwarf rat compared to the Lewis rat and the decrease was reversed by the GH treatment. These results indicate that the KBP levels are regulated at the RNA level. Furthermore, in vitro studies using cultured rat hepatocytes showed that GH may have a direct regulatory effect on KBP expression since KBP levels increased in the conditioned media of cells treated with GH. These results demonstrated that KBP is reduced in the genetic dwarf rat and is restored to normal by GH; therefore, KBP is a GH-dependent protein and may be a new target for studying the mechanism of pathological animal growth.     

Targeted ablation of pituitary gonadotropes in transgenic mice.

LH, FSH, and TSH are heterodimeric glycoprotein hormones composed of a common alpha-subunit and unique beta-subunits. The alpha-subunit is produced in two distinct specialized cell types of the pituitary gland: gonadotropes, which synthesize LH and FSH, and thyrotropes, which synthesize TSH. We have demonstrated that 313 base pairs of the bovine-alpha subunit promoter direct expression of diphtheria toxin A chain specifically to the gonadotropes in transgenic mice. Animals carrying this transgene generally exhibit reproductive failure and lack of gonadal differentiation, consistent with gonadotrope ablation. Lack of gonadotrope activity was verified by RIA and immunohistochemical staining for LH. The phenotype of these transgenic mice is nearly identical to mice homozygous for the spontaneous mutation, hpg, which is due to a deletion in the gene encoding GnRH. Thyrotrope function was judged normal based on overall growth of the animals, appearance of their thyroids, T4 levels measured by RIA, and immunohistochemical staining for TSH. The ablation of gonadotropes but not thyrotropes suggests that separate cis-acting elements are necessary for expression of the alpha-subunit gene in these two cell types. Pituitary content of ACTH and GH was apparently normal, while PRL synthesis and storage were reduced. Thus, in a pituitary almost completely devoid of gonadotropes, most other pituitary functions were normal. This suggests that most pituitary cells are able to differentiate independently of terminal gonadotrope differentiation and can function in the absence of paracrine signaling provided by gonadotropes.     

Fine-structural and immunohistochemical study of anterior pituitary cells of Snell dwarf mice

Summary Snell dwarf mice display remarkable retardation of growth after birth and are known to lack prolactin (PRL), thyroid stimulating hormone (TSH) and growth hormone (GH). The aim of this study was to determine the reason for these hormonal deficiencies. We examined the fine structure of the gland and its immunohistochemical staining pattern with respect to antisera raised against PRL, TSH, GH, adrenocorticotrophic hormone (ACTH) and luteinizing hormone (LH). The gland of control mice reacted immunohistochemically against all antisera used, whereas only ACTH-producing cells (ACTH cells) and LH-producing cells (LH cells) were distinguished in the dwarf mice. ACTH cells in dwarf mice varied in cell shape, although they were similar in size to those of controls. The distribution of secretory granules in the cytoplasm varied from cell to cell. LH cells in the dwarf mice showed immature features, having poorly developed rough endoplasmic reticulum and Golgi apparatus. The cells were about half the size of controls, and secretory granules were smaller. In dwarf mice, non-granulated cells were encountered in addition to granulated ACTH and LH cells. Some of them formed small clusters, characteristic cell junctions being found between the cells; they thus appeared to be follicular cells. The above results suggest that hormone deficiency in Snell dwarf mice is a result of a defect in the hormoneproducing cells in the gland.     

The relationship between age and genotype and circulating concentrations of triiodothyronine (T3), thyroxine (T4), and growth hormone in commercial meat strain chickens

The presence of the sex-linked dwarf gene (dw) in homozygous male (dw/dw) and female (dw/-) meat strain chickens is associated with a significant reduction in circulating levels of triiodothyronine (T3). Heterozygous (Dw/dw) male broiler strain chickens have T3 concentrations similar to those in homozygous (Dw/Dw) male broilers. Genetically normal (Dw/Dw) but significantly slower growing roaster strain male meat chickens had consistently higher T3 than the faster growing broilers at all ages in one experiment but only at 8 weeks in a second experiment. Age and not growth rate appears to have a greater influence on serum T3 concentrations in the slow- and fast-growing normal strains. Growth hormone levels were significantly higher in the dwarf chickens at all ages and in all three experiments. The heterozygous and homozygous broilers had similar GH levels and the slow-growing, genetically normal roasters had intermediate concentrations between the broiler and dwarf lines. GH was influenced to a greater extent by the rate of body weight gain than by increasing age in the genetically normal fast and slow growing strains.     

Pituitary and gonadal function in hypogonadotrophic hypogonadal (hpg) mice bearing hypothalamic implants

GnRH receptor values are 30-50% of normal in pituitaries of hpg male mice, and testicular LH receptors only 8% of normal (160.4 +/- 17.6 and 2013 +/- 208.1 fmol/testis respectively). In male hpg mice bearing fetal preoptic area (POA) hypothalamic implants for 10 days there was no change in pituitary GnRH receptors, pituitary gonadotrophin content, or seminal vesicle weight. However, testicular weights and LH receptors were doubled in 4/10 mice and 2 had increased serum FSH levels. Between 26 and 40 days after implantation pituitary GnRH receptors and pituitary LH increased to normal male levels, although at 40 days serum and pituitary FSH concentrations had reached only 50% of normal values. Testicular and seminal vesicle weights increased more than 10-fold by 40 days after implantation and LH receptors to 70% of normal. In hpg female mice bearing hypothalamic implants for 30-256 days pituitary gonadotrophin concentrations were normal, even though GnRH receptors reached only 60% of normal values (6.18 +/- 0.4 and 9.8 +/- 0.4 fmol/pituitary respectively). Serum FSH was substantially increased from values of less than 30 ng/ml in hpg mice to within the normal female range in hypothalamic implant recipients. Ovarian and uterine weights increased after hypothalamic grafting from only 4-5% to over 74% of normal values. LH receptors increased from 6.5 +/- 1.3 fmol/ovary for hpg mice to 566.9 +/- 39.2 fmol/ovary for implant recipients. Vaginal opening occurred about 23 days after implantation and these animals displayed prolonged periods of oestrus.(ABSTRACT TRUNCATED AT 250 WORDS)