Thymus, peripheral lymphoid tissues and immunological responsiveness of the pituitary dward mouse (author's transl)]

Snell's pituitary dwarf mice (dw) were used for studies on the relationship between hypophysis and lymphoid organs. The age-dependent changes of thymus or spleen weights of dwarf mice were compared with those of normal littermates. The suppression of growth of the thymus or spleen in dwarf mice was recognized at 5th day of age. Although involution of the thymus varied among animals, a strong positive correlation was demonstrated between relative thymus weight and body weight in 30 approximately 40 days old dwarf mice. Lymphoid organs of dwarf mice were reconstituted by injection of growth hormone and or thyroxin. Relative thymus weight significantly increased in dwarf mice when the treatment with growth hormone started at 7 days of age, but the same treatment at 3 months of age did not show any effect on the increment of relative thymus weight. On the other hand, the antibody-forming capacitiy against sheep erythrocytes of dwarf mice was significantly increased even when the treatment with growth hormone was started at 3 months of age. A marked increase in the number of lymphoid cells in dwarf mice was observed by treatment with thyroxin, even if treatment was started either at 7 days or 3 months of age. Similar changes were also obtained in the antibody-forming capacity.     

Immunologic and hematologic effects of neuroendocrine hormones. Studies on DW/J dwarf mice.

DW/J dwarf mice lack acidophilic anterior pituitary cells and are deficient in growth hormone and other neuroendocrine mediators. These mice were examined to determine the effects of these deficiencies on hematopoietic and immune system development. Previous studies have suggested that these mice had immunologic defects primarily involving T cell development. However, we have found that these mice exhibit decreased peripheral blood cell counts affecting all lineages (erythrocytic, leukocytic, and platelets). Examination of lymphoid tissues of dwarf mice indicated that their spleens were hypoplastic. Treatment of these mice with recombinant human growth hormone resulted in a significant improvement of peripheral blood counts and spleen cell number. Analysis of the bone marrow indicated a profound deficiency of B cell progenitors in the dwarf mice. However, in untreated dwarf mice, mature B cells and T cells were observed in the spleens. Although treatment with recombinant human growth hormone could correct the hematopoietic deficiencies in these mice, it did not restore the B cell progenitor populations, suggesting that an absence of growth hormone is not solely responsible for this deficiency. Thus, these mice display significant myeloid and lymphoid deficiencies that have been previously undetected.     

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.     

Heroin self-administration is under control of vasopressin

Heroin self-administration in rats is attenuated by daily intracerebroventricular treatment with desglycinamide⁹, arginine⁸, vasopressin (50 ng/animal). Removal of vasopressin by injection of antivasopressin serum into the cerebrospinal fluid, leads to facilitation of self-administering behavior. Treatment with oxytocin or human growth hormone antiserum fails to influence this behavior, while prolactin antiserum exhibits similar effects as observed with antivasopressin serum. These results indicate that pituitary hormones are physiologically involved in heroin self-administration.     

Electron-microscopic study on the anterior pituitary gland of spontaneous dwarf rats

Summary The spontaneous dwarf rat is a novel experimental model animal on the study of pituitary dwarfism. The fine structure of the anterior pituitary cells was studied in the immature and mature dwarf rats. Pituitary glands were removed from 5-, 10-, 20-day-old immature dwarfs, adult (45 days-16 weeks) dwarfs and normal 3-month-old rats and processed for electron-microscopic observation. In the control animals, growth hormone cells were readily identified by their ultrastructural characteristics, such as the presence of numerous electron-dense secretory granules, 300–350 nm in diameter, well developed rough endoplasmic reticulum and a prominent Golgi complex. In contrast, growth hormone cells were not found in the anterior pituitary gland of the spontaneous dwarf rat at any age examined. Other pituitary cell types, i.e., luteinizing hormone/ follicle stimulating hormone, thyroid stimulating hormone, adrenocorticotropic hormone and prolactin cells, appeared similar in their fine structure to those found in the control rats. In the pituitary gland of dwarf rats, a number of polygonal cells were observed either with no or relatively few secretory granules. The rough endoplasmic reticulum was arranged in parallel cisternae and the Golgi complex was generally prominent in these cells. In addition, many were found to have abundant lysosomes. A few minute secretory granules were occasionally observed; however, the immunogold technique failed to localize growth hormone or prolactin in the granules. The nature of these cells remained obscure in this study. Since their incidence and fine structural features, other than the secretory granules, were quite similar to those of the growth hormone cells in normal rats, we postulate that these cells are dysfunctional growth hormone cells. These results suggest that the cause of the growth impairment in the spontaneous dwarf rat is due to a defect in the functional growth hormone cells in the pituitary gland, and since other pituitary cell types appeared normal, the disorder seems to be analogous to the isolated growth hormone deficiency in the human.     

Aged PROP1 deficient dwarf mice maintain ACTH production

Humans with PROP1 mutations have multiple pituitary hormone deficiencies (MPHD) that typically advance from growth insufficiency diagnosed in infancy to include more severe growth hormone (GH) deficiency and progressive reduction in other anterior pituitary hormones, eventually including adrenocorticotropic hormone (ACTH) deficiency and hypocortisolism. Congenital deficiencies of GH, prolactin, and thyroid stimulating hormone have been reported in the Prop1(null) (Prop1(-/-)) and the Ames dwarf (Prop1(df/df)) mouse models, but corticotroph and pituitary adrenal axis function have not been thoroughly investigated. Here we report that the C57BL6 background sensitizes mutants to a wasting phenotype that causes approximately one third to die precipitously between weaning and adulthood, while remaining homozygotes live with no signs of illness. The wasting phenotype is associated with severe hypoglycemia. Circulating ACTH and corticosterone levels are elevated in juvenile and aged Prop1 mutants, indicating activation of the pituitary-adrenal axis. Despite this, young adult Prop1 deficient mice are capable of responding to restraint stress with further elevation of ACTH and corticosterone. Low blood glucose, an expected side effect of GH deficiency, is likely responsible for the elevated corticosterone level. These studies suggest that the mouse model differs from the human patients who display progressive hormone loss and hypocortisolism.     

Pituitary and plasma levels of growth hormone (GH), follicle stimulating hormone (FSH) and luteinizing hormone (LH) in hereditary dwarf rats (rdw/rdw)]

Koto et al found a new hereditary dwarf mutation from breeding colony of Wistar-Imamichi rat and named 'rdw'. To characterize endocrinological functions in rdw rats, pituitary and plasma levels of pituitary hormones including growth hormone (GH), follicle stimulating hormone (FSH) and luteinizing hormone (LH) were compared between rdw and normal rats. The hormone levels were estimated with radioimmunoassay (RIA). It was found that pituitary and plasma levels of GH of rdw were drastically decreased and those of FSH and LH were inclined to decrease but not remarkable as compared with normal. Rats of rdw were, therefore, considered to be useful as a model animal for endocrinological defects.     

Growth hormone and insulin binding to isolated hepatocytes in the genetically dwarf mouse

The interaction of growth hormone with its specific receptors in dwarf mice was investigated. (1) The interaction of 125I-labeled human growth hormone with isolated mouse liver cells is a specific, time-dependent and saturable process. Hepataocytes of male and female dw/dw mice bound only 10-20% as much growth hormone per unit of cell surface area as those of their litter mates. Scatchard analysis suggested that this decrease in binding was due to a decreased number of receptor sites in th liver cell of the dwarf mouse. (2) In contrast to the marked decrease in growth hormone receptors, the binding of insulin is higher in dwarf mice than in litter mates, at low hormone concentration. (3) Competition and stoichiometric studies indicate that growth hormone and prolactin bind to the same type of binding site in female and male mouse hepatocytes. These results indicate that dwarfism in this animal was associated with a loss in the number of growth hormone binding sites. The decrease in growth hormone receptors and the increase in insulin receptors correlate well with the respective biological activity of these two hormones.     

Alterations of Adrenal Cortex and Thyroid in Mice with Congenital Absence of the Thymus

THE possible role of endocrine factors in the thymus and their control by hormones of the adenohypophysis or its target glands has been investigated extensively in animal models. These include mice whose pituitary function has been inhibited or blocked by anti-pituitary serum^1–3 or anti-growth-hormone serum^4–6, neonatally thymectomized mice^1,3,7–9 and the genetically hypopituitary dwarf mice with thymus atrophy^10–12. Further opportunities have been offered by the observation that genetically hairless “nude” mice (genetic symbol: nu)¹³ have no thymus^14,15.     

Perifusion model system to culture bovine hypothalamic slices in series with dispersed anterior pituitary cells

Summary Dispersed bovine anterior pituitary cells were incubated either in static or perifusion cultures to assess basal growth hormone release as well as stimulatory and inhibitory effects of growth hormone-releasing hormone and somatostatin, respectively, on growth hormone release. Total concentrations of growth hormones over a 12-hour incubation period were fivefold greater in perifused than in static cultures (2034 ± 160 vs. 387 ± 33 ng/12 h). A dose-dependent increase in growth hormone secretion in response to challenge with growth hormone-releasing hormone (10⁻¹² to 10⁻⁸ M) for 1 h was observed in both static and perifusion cultures; however, perifused cells were more responsive to the same concentration of neuropeptide than those in static culture. Concentrations of somatostatin (10⁻¹² to 10⁻⁸ M) for 1 h did not inhibit basal growth hormone secretion in either static or perifusion cultures. To establish model, slices of the hypothalamus, immediately adjacent to the sagittal midline, were perifused in series with anterior pituitary cells, and media effluent was assayed for growth hormone concentrations. Release of growth hormone was pulsatile and seemed to mimic the episodic pattern of bovine secretion. Hypothalamic slices were placed in one chamber of the perifusion system, and basal secretion of growth hormone-releasing hormone and somatostatin was pulsatile in media effluent. Tissue viability of hypothalamic slices and anterior pituitary cells was evaluated by KCl depolarization. Tissues were viable for at least 120 h. Thus, this hypothalamo-pituitary dual chamber perifusion system is a valid in vitro model to study regulation of growth hormone secretion.     

Augmented autophagy pathways and MTOR modulation in fibroblasts from long-lived mutant mice

Fibroblasts from long-lived pituitary dwarf mutants, including Snell dwarf, Ames dwarf and the growth hormone receptor knockout (GHRKO) mice, are resistant in culture to multiple forms of lethal stress. We found that fibroblasts from Snell dwarf and GHRKO mice are more susceptible than control cells to autophagy induced by amino acid withdrawal or by oxidative stress. We also found evidence for lower MTOR function in dwarf cells under conditions that induce autophagy, consistent with the evidence that increased autophagy requires lower TOR activity. Our results provide new hints about the connections between autophagy and aging in long-lived mutants with alterations in GH-IGF1 levels, and suggest a role for hyperactive autophagy in the resistance of cells from these mice to lethal stresses.     

VCAM‐1 upregulation accompanies muscle remodeling following resistance‐type exercise in Snell dwarf (Pit1dw/dw) mice

Snell dwarf mice (Pit1^dw/dw) exhibit deficiencies in growth hormone, prolactin, and thyroid stimulating hormone. Besides being an experimental model of hypopituitarism, these mice are long‐lived (>40% lifespan extension) and utilized as a model of slowed/delayed aging. Whether this longevity is accompanied by a compromised quality of life in terms of muscular performance has not yet been characterized. In this study, we investigated nontrained and trained muscles 1 month following a general validated resistance‐type exercise protocol in 3‐month‐old Snell dwarf mice and control littermates. Nontrained Snell dwarf gastrocnemius muscles exhibited a 1.3‐fold greater muscle mass to body weight ratio than control values although muscle quality, maximum isometric torque normalized to muscle mass, and fatigue recovery were compromised. For control mice, training increased isometric torque (17%) without altering muscle mass. For Snell dwarf mice, isometric torque was unaltered by training despite decreased muscle mass that rendered muscle mass to body weight ratio comparable to control values. Muscle quality and fatigue recovery improved twofold and threefold, respectively, for Snell dwarf mice. This accompanied a fourfold increase in levels of vascular cell adhesion molecule‐1 (VCAM‐1), a mediator of progenitor cell recruitment, and muscle remodeling in the form of increased number of central nuclei, additional muscle fibers per unit area, and altered fiber type distribution. These results reveal a trade‐off between muscle quality and longevity in the context of anterior pituitary hormone deficiency and that resistance‐type training can diminish this trade‐off by improving muscle quality concomitant with VCAM‐1 upregulation and muscle remodeling.     

Activation of CD8 T cells by antigen expressed in the pituitary gland

Ag expressed exclusively in the anterior pituitary gland and secreted locally by pituitary somatotrophs can gain access to the MHC class I presentation pathway and activate CD8 T cells. Influenza nucleoprotein (NP) was expressed as a transgene under the control of the human growth hormone (GH) locus control region. Activation of monoclonal F5 CD8 T cells specific for NP resulted in spontaneous autoimmune pathology of the pituitary gland in mice transgenic for both NP and the F5 TCR. Destruction of somatotrophs resulted in drastically reduced GH levels in adult mice and a dwarf phenotype. Adoptive transfer of F5 T cells into NP-transgenic hosts resulted in full T cell activation, first demonstrable in regional lymph nodes, followed by their migration to the pituitary gland. Despite the presence of activated, IFN-gamma-producing CD8 T cells in the pituitary gland and a slight reduction in pituitary GH levels, no effect on growth was observed. Thus, CD8 T cells have access to the neuroendocrine system and get fully activated in the absence of CD4 help, but Ag recognition in this location causes autoimmune pathology only in the presence of excessive CD8 T cell numbers.     

Fibroblast cell lines from young adult mice of long-lived mutant strains are resistant to multiple forms of stress

Previous studies have shown that dermal fibroblast cell lines derived from young adult mice of the long-lived Snell dwarf mutant stock are resistant, in vitro, to the cytotoxic effects of H(2)O(2), cadmium, UV light, paraquat, and heat. We show here that similar resistance profiles are seen in fibroblast cells derived from a related mutant, the Ames dwarf mouse, and that cells from growth hormone receptor-null mice are resistant to H(2)O(2), paraquat, and UV but not to cadmium. Resistance to UV light, cadmium, and H(2)O(2) are similar in cells derived from 1-wk-old Snell dwarf or normal mice, and thus the resistance of cell lines derived from young adult donors reflects developmental processes, presumably hormone dependent, that take place in the first few months of life. The resistance of cells from Snell dwarf mice to these stresses does not reflect merely antioxidant defenses: dwarf-derived cells are also resistant to the DNA-alkylating agent methyl methanesulfonate. Furthermore, inhibitor studies show that fibroblast resistance to UV light is unaffected by the antioxidants ascorbic acid and N-acetyl-L-cysteine. These data suggest that postnatal exposure to altered levels of pituitary hormones leads to development of cellular resistance to oxidative and nonoxidative stressors, which are stable through many rounds of in vitro cell division and could contribute to the remarkable disease resistance of long-lived mutant mice.     

Development of Immunodeficiency of Pituitary Dwarf Mice

Autosomal recessive pituitary dwarf mutants of the Snell-Baggand Ames mouse strains develop severe immunodeficiency of thethymus-dependent (T cell) system which frequently leads to afatal wasting syndrome. The ontogenetic development of the Tcell system is already subnormal soon after birth as evidencedby diminished responsiveness of thymus and spleen cells to phytohemagglutininand concanavalin A. The immunodeficiency of the dwarf mouseis a consequence of defective pituitary influences which willcause (i) an inadequate production of immunocompetent cellsdue to a central developmental defect primarily affecting thethymus, and (ii) the inability of immunocompetent cells to undergoa rapid and efficient antigen-induced proliferation and differentiationinto antibody-forming cells. The lack of epinephrine-inducedstimulation of adenylate cyclase activity in dwarf spleen andthymus cells suggests that the impaired lymphoid cell proliferationin dwarf mice may be due to inadequate stimulation of cyclicAMP production.     

Hereditary pituitary dwarfism in mice affects skeletal and cardiac myosin isozyme transitions differently

The dwarf mutation in mice interferes with the development of those anterior pituitary cells responsible for production of thyroid stimulating hormone, growth hormone, and prolactin. Myosin isozyme transitions in both cardiac and skeletal muscle were also found to be affected in this mutant. Electrophoresis of native myosins demonstrated that the fetal (V3) to adult (V1) ventricular cardiac isozyme transition was completely blocked in dwarf mice; in contrast, the neonatal to adult fast myosin transition in hind limb skeletal muscle was slowed but not totally inhibited. The persistence of neonatal myosin heavy chain for up to 55-75 d after birth in dwarf mice, as compared with 16 d in normal mice, was directly demonstrated by polypeptide and immunopolypeptide mapping. Morphological examination of 18-36-d-old dwarf skeletal muscles by optical and electron microscopy revealed a relative immaturity, but no signs of gross pathology were evident. Immunocytochemical analysis showed that the abnormal persistence of neonatal myosin occurs in most of the fibers. Multiple injections of thyroxine restored a normal isozyme complement to both cardiac and skeletal muscles within 11-15 d. Therefore, the effects of the dwarf mutation on myosin isozymes can be explained by the lack of thyroid hormone in these animals. Because the synthesis of growth hormone is not stimulated by thyroid hormone in dwarf mice as it would be in normal animals, these results demonstrate that thyroid hormone promotes myosin isozyme transitions independent of growth hormone production.     

A decreased capacity of hepatic growth hormone (GH) receptors and failure of thyrotrophin-releasing hormone to stimulate the peripheral conversion of thyroxine into triiodothyronine in sex-linked dwarf broiler hens

The effect of two different doses of thyrotrophic releasing hormone (TRH) upon the plasma levels of growth (GH) and thyroid hormones in both sex-linked dwarf (dw) and normal (Dw) broiler hens was determined. In normal hens, 1.5 and 24 microg TRH/kg increased the GH plasma concentrations after 15 min. Plasma concentrations of T3 increased significantly 1 h after TRH injection, whereas T4 concentration decreased after 2 following injection of 24 microg/kg TRH. In dwarf hens both doses of TRH increased the plasma concentrations of GH and the GH response lasted longer. However, TRH was ineffective in raising T3 and T4 levels. Saline-injected dwarf birds showed no differences in plasma T4 and T3 levels in comparison with normal hens. A smaller number of hepatic cGH receptors was found in dwarf hens, whereas the affinity of the hepatic GH receptor was not influenced by the genotype. It is concluded that the sex-linked dwarf broiler hen is unable to respond to a TRH-induced GH stimulus probably because of a deficiency in hepatic GH receptors resulting in a failure to stimulate the T4 to T3 converting activity.     

Comparative immunochemical studies with antisera to sheep prolactin and bovine growth hormone on anterior pituitaries of ox,sheep, rats and mice

Summary Rabbit antisera to sheep prolactin and bovine growth hormone were used in the indirect fluorescent antibody technique on cryostat sections of anterior pituitaries of sheep, ox, rats and mice. It is demonstrated that in sheep and ox prolactin and growth hormone are manufactured by different acidophilic pituitary cells. Though the antisera do not precipitate the analogous hormones of rats and mice in gel diffusion tests, evidence is given for the specificity of the cross-reaction of the antisera with the analogous murine hormones in situ as found with the fluorescent antibody technique.We are grateful to Dr. J. D. H. Homan of Organon, Oss (The Netherlands) for the supply of bovine growth hormone and for kindly giving us the information concerning this preparation.We should like to thank Dr. F. J. A. Prop for the prolactin assays, Dr. H. G. Kwa for providing the mouse pituitary tumours and Dr. L. M. Boot for the mouse pituitary isograft in the kidney, Mrs. J. J. Geiger-Koedijk for the conventional staining of pituitary sections, and Mr. J. van der Kamp for the photography.