Hypox Animals Research Articles

The endocrine–immune network during taeniosis by Taenia solium: The role of the pituitary gland

It is well known that sex hormones play an important role during Taenia solium infection; however, to our knowledge no studies exist concerning the immune response following complete or lobe-specific removal of the pituitary gland during T. solium infection. Thus, the aim of this work was to analyze in hamsters, the effects of lack of pituitary hormones on the duodenal immune response, and their impact on T. solium establishment and development. Thus, in order to achieve this goal, we perform anterior pituitary lobectomy (AL, n = 9), neurointermediate pituitary lobectomy (NIL, n = 9) and total hypophysectomy (HYPOX, n = 8), and related to the gut establishment and growth of T. solium, hematoxylin–eosin staining of duodenal tissue and immunofluorescence of duodenal cytokine expression and compared these results to the control intact (n = 8) and control infected group (n = 8). Our results indicate that 15 days post-infection, HYPOX reduces the number and size of intestinally recovered T. solium adults. Using semiquantitative immunofluorescent laser confocal microscopy, we observed that the mean intensity of duodenal IFN-γ and IL-12 Th1 cytokines was mildly expressed in the infected controls, in contrast with the high level of expression of these cytokines in the NIL infected hamsters. Likewise, the duodenum of HYPOX animals showed an increase in the expression of Th2 cytokines IL-5 and IL-6, when compared to control hamsters. Histological analysis of duodenal mucosa from HYPOX hamsters revealed an exacerbated inflammatory infiltrate located along the lamina propria and related to the presence of the parasite. We conclude that lobe-specific pituitary hormones affect differentially the T. solium development and the gut immune response.

Bone and hypopituitarism: not only a mass issue

Bone and hypopituitarism: not only a mass issue

Hypophysectomy and neurointermediate pituitary lobectomy decrease humoral immune responses to T-independent and T-dependent antigens

In rats, hypophysectomy (HYPOX) or neurointermediate pituitary lobectomy (NIL) reduce humoral and cell-mediated immune responses. However, to our knowledge, the differences in the effects of anterior versus posterior pituitary hormones on the immune responses have not been studied to date. We compared in rats, the effects of sham surgery (SHAM), HYPOX, and NIL on humoral immune responses to T cell-independent (TI) type 1 antigen DNP-LPS and to TI type 2 antigen DNP-FICOLL, as well as to T cell-dependent (TD) antigens ovalbumin (OVA) and bovine serum albumin (BSA). The results showed that: (1) both HYPOX and NIL induced a similar and significant decrease in IgM responses towards TI-1 antigens, (2) NIL but not HYPOX induced a decreased IgM response to TI-2 antigens, and (3) both HYPOX and NIL induced similar and significant decrease in IgG responses to TI-2 antigens. Compared with the SHAM group, IgM responses to both TD antigens did not change in HYPOX and NIL animals, whereas the IgG responses to OVA and BSA significantly decreased in HYPOX and NIL animals. These results indicate that hormones of the anterior and posterior pituitary play their own role in the regulation of humoral immune responses.

Differences in spleen mRNA ILs expression in anterior, posterior and total hypophysectomized rats with experimental autoimmune encephalomyelitis (EAE) or adjuvant arthritis (AA); the vasopressin role

Neurointermediate pituitary lobectomy (NIL) in Lewis rats causes mild signs of EAE, while hypophysectomy (HYPOX) and anterior pituitary lobectomy (AL) worse the disease, and desmopressin (vasopressin analog) aggravates the EAE in NIL and HYPOX rats. Besides, the AIA severity is low in NIL and no signs of AIA occur in HYPOX animals, while AL induces severe arthritis. Here we compare the splenic expression (RT‐PCR) of mRNA of ILs 1, 2, 6, 10, 12 and INF‐¦Ã between sham operated (SHAM), AL, NIL, and HYPOX groups immunized for EAE (females) or AIA (males). Intact control groups were included for basal IL levels. Rats were operated 3 weeks before immunizations and sacrificed at day 15. Results showed over expression of IL‐2 in AL‐EAE group. Similar augments of IL‐1 and 2 in SHAM, NIL and HYPOX‐EAE groups, and low expression in ILs 6, 10 and INF‐¦Ã in NIL, AL and HYPOX groups. IL‐12 was higher in HYPOX‐EAE rats with no changes in the remaining groups. In AL‐AIA group, significant increases in ILs 1, 2, 6 and INF‐¦Ã occurred, while in NIL‐AIA group ILs 2, 12 and INF‐¦Ã were low respect the remaining groups. HYPOX group did not developed clinical signs of AIA and ILs 2, 6, 10, 12 and INF‐¦Ã levels were similar to the SHAM group. We conclude that neurohypophysial hormones are important in the control of autoimmune diseases and that the ILs involved in the immune responses are different for EAE and AIA.CONACYT. M̈¦xico

The effect of hypophysectomy on pancreatic islet hormone and insulin-like growth factor I content and mRNA expression in rat

The growth arrest after hypophysectomy in rats is mainly due to growth hormone (GH) deficiency because replacement of GH or insulin-like growth factor (IGF) I, the mediator of GH action, leads to resumption of growth despite the lack of other pituitary hormones. Hypophysectomized (hypox) rats have, therefore, often been used to study metabolic consequences of GH deficiency and its effects on tissues concerned with growth. The present study was undertaken to assess the effects of hypophysectomy on the serum and pancreatic levels of the three major islet hormones insulin, glucagon, and somatostatin, as well as on IGF-I. Immunohistochemistry (IHC), in situ hybridization (ISH), radioimmunoassays (RIA), and Northern blot analysis were used to localize and quantify the hormones in the pancreas at the peptide and mRNA levels. IHC showed slightly decreased insulin levels in the beta cells of hypox compared with normal, age-matched rats whereas glucagon in alpha cells and somatostatin in delta cells showed increase. IGF-I, which localized to alpha cells, showed decrease. ISH detected a slightly higher expression of insulin mRNA and markedly stronger signals for glucagon and somatostatin mRNA in the islets of hypox rats. Serum glucose concentrations did not differ between the two groups although serum insulin and C-peptide were lower and serum glucagon was higher in the hypox animals. These changes were accompanied by a more than tenfold drop in serum IGF-I. The pancreatic insulin content per gram of tissue was not significantly different in hypox and normal rats. Pancreatic glucagon and somatostatin per gram of tissue were higher in the hypox animals. The pancreatic IGF-I content of hypox rats was significantly reduced. Northern blot analysis gave a 2.6-, 4.5-, and 2.2-fold increase in pancreatic insulin, glucagon, and somatostatin mRNA levels, respectively, in hypox rats, and a 2.3-fold decrease in IGF-I mRNA levels. Our results show that the fall of serum IGF-I after hypophysectomy is accompanied by a decrease in pancreatic IGF-I peptide and mRNA but by partly discordant changes in the serum concentrations of insulin and glucagon and the islet peptide and/or mRNA content of the three major islet hormones. It appears that GH deficiency resulting in a "low IGF-I state" affects translational efficiency of these hormones as well as their secretory responses. The maintenance of normoglycemia in the presence of reduced insulin and elevated glucagon serum levels, both of which would be expected to raise blood glucose, may result mainly from the enhanced insulin sensitivity, possibly due to GH deficiency and the subsequent decrease in IGF-I production.

Neurointermediate pituitary lobectomy decreases the incidence and severity of experimental autoimmune encephalomyelitis in Lewis rats

Acute experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system, mediated by T lymphocytes. Immunization of Lewis rats with myelin antigens suspended in complete Freund's adjuvant induces EAE. In a previous study on rats we have found that neurointermediate pituitary lobectomy (NIL) decreased both the humoral and cell-mediated immune responses. Here we investigated the effect of NIL on the incidence and severity of EAE and on the function of the hypothalamic-pituitary-adrenal axis in Lewis rats. NIL, hypophysectomized (Hypox) and sham-operated (Sham) rats were immunized s.c. with guinea-pig brain extract suspended in complete Freund's adjuvant. Untreated rats were used as controls. Water intake, body weight gain, clinical and histopathologic incidence and severity of EAE were evaluated in the operated groups. On killing, plasma adrenocorticotropin and corticosterone levels were measured and adrenals, thymuses and spleens were weighed. Histopathologic lesions were counted in the brain and spinal cord. Water intake and body weight gain were significantly decreased in Sham and Hypox animals with EAE whereas higher intakes persisted in the NIL group. Plasma levels of adrenocorticotropin were within the normal range whereas corticosterone levels increased in Sham and occasionally in NIL animals. Thymus weights were decreased in NIL and Hypox groups. The clinical and histopathologic incidence and severity of EAE were significantly decreased in NIL animals as compared with Sham and Hypox rats. We concluded that NIL affects the cell-mediated immune response and plays a role in the development and progression of EAE in the Lewis rat.

The role of galanin and its receptor in the feedback regulation of growth hormone secretion.

GH controls its own secretion through a mechanism involving short-loop feedback regulation of the synthesis and release of GH-releasing hormone (GHRH). GHRH neurons coexpress the peptide galanin, but the functional significance of this coexpression is unknown. In this study, we tested the hypotheses that 1) galanin gene expression in GHRH neurons is regulated by GH and 2) somatostatin (SS) or GHRH neurons are a target for the action of galanin in the hypothalamus. First, we compared levels of galanin messenger RNA (mRNA) in GHRH neurons between normal male rats and Lewis dwarf rats, which have markedly reduced blood levels of GH. The brains of normal and dwarf animals were processed for detection of galanin mRNA and GHRH mRNA by double-label in situ hybridization. We observed that Lewis dwarf rats had significantly reduced levels of galanin mRNA in their GHRH neurons (P < 0.05). Next, we tested the hypothesis that GH regulates galanin gene expression in GHRH neurons by experimentally altering circulating levels of GH. Three groups of adult male rats were used: 1) intact rats (n = 7); 2) hypophysectomized (hypox) rats (n = 7); and 3) hypox rats treated with 1.5 mg of rat GH (rGH) over a 3-day period (n = 6). At the end of the treatment period, the animals were killed, and their brains were collected and processed for double-label in situ hybridization for GHRH mRNA and galanin mRNA. The signal level of galanin mRNA in GHRH neurons was reduced in hypox animals to less than 10% of that in intact controls (P < 0.0001); whereas, the levels of galanin mRNA signal in GHRH neurons did not differ significantly between the groups of intact and rGH-treated hypox rats. Finally, to determine whether SS or GHRH neurons are targets for galanin, we used double-label in situ hybridization to determine whether either of these populations of neurons express galanin receptor mRNA. A subset of SS neurons in the PeN appeared to express the galanin receptor mRNA, whereas few, if any, GHRH neurons appeared to do so. We conclude that galanin, like its cotransmitter GHRH, is a target for GH action, and we infer that galanin may play a role in the feedback control of GH secretion by exerting a direct effect on SS neurons.

The role of galanin and its receptor in the feedback regulation of growth hormone secretion

GH controls its own secretion through a mechanism involving short-loop feedback regulation of the synthesis and release of GH-releasing hormone (GHRH). GHRH neurons coexpress the peptide galanin, but the functional significance of this coexpression is unknown. In this study, we tested the hypotheses that 1) galanin gene expression in GHRH neurons is regulated by GH and 2) somatostatin (SS) or GHRH neurons are a target for the action of galanin in the hypothalamus. First, we compared levels of galanin messenger RNA (mRNA) in GHRH neurons between normal male rats and Lewis dwarf rats, which have markedly reduced blood levels of GH. The brains of normal and dwarf animals were processed for detection of galanin mRNA and GHRH mRNA by double-label in situ hybridization. We observed that Lewis dwarf rats had significantly reduced levels of galanin mRNA in their GHRH neurons (P < 0.05). Next, we tested the hypothesis that GH regulates galanin gene expression in GHRH neurons by experimentally altering circulating levels of GH. Three groups of adult male rats were used: 1) intact rats (n = 7); 2) hypophysectomized (hypox) rats (n = 7); and 3) hypox rats treated with 1.5 mg of rat GH (rGH) over a 3-day period (n = 6). At the end of the treatment period, the animals were killed, and their brains were collected and processed for double-label in situ hybridization for GHRH mRNA and galanin mRNA. The signal level of galanin mRNA in GHRH neurons was reduced in hypox animals to less than 10% of that in intact controls (P < 0.0001); whereas, the levels of galanin mRNA signal in GHRH neurons did not differ significantly between the groups of intact and rGH-treated hypox rats. Finally, to determine whether SS or GHRH neurons are targets for galanin, we used double-label in situ hybridization to determine whether either of these populations of neurons express galanin receptor mRNA. A subset of SS neurons in the PeN appeared to express the galanin receptor mRNA, whereas few, if any, GHRH neurons appeared to do so. We conclude that galanin, like its cotransmitter GHRH, is a target for GH action, and we infer that galanin may play a role in the feedback control of GH secretion by exerting a direct effect on SS neurons.

In vivo modulation of proliferating cell nuclear antigen in growth plate chondrocytes from normal, hypophysectomized, growth hormone-treated hypophysectomized rats: a comparative immunohistochemical study with image analysis.

Growth hormone (GH) regulates the proliferation and maturation of chondrocytes in the epiphyseal growth plate, in which a temporal dimension is superimposed on the septal organization of the tissue. In this study we investigated the in vivo effects of hypophysectomy (Hypox) and injection of GH into Hypox animals (Hypox + GH) on the proliferative activity of the growth plate chondrocytes. We assessed the immunohistochemical expression of proliferating cell nuclear antigen (PCNA) in paraffin-embedded tissues, using monoclonal antibody PC 10 against PCNA combined with immunogold-silver staining. We subjected the immunostained sections to computer-based image analysis by ACAS 570 interactive laser cytometry employing a conventional microscopic light source. Hypox was carried out on 20 rats at 8 weeks of age, half of which received a hypodermic injection of human GH at a dose of 1 IU/kg twice a day for 1 week after the operation. Another group of five rats of the same age were used as normal controls. In normal rats, a distinct PCNA immunoreaction was observed in the proliferative zone, whereas a remarkable diminution of the number of immunoreactive cells in this zone was apparent in Hypox animals. On the other hand, marked hyperplasia of PCNA-positive cells was seen in the proliferative zone of the Hypox + GH rat growth plate. The immunoreactive cells of this zone in Hypox + GH animals exhibited increased nuclear size and staining intensity of PCNA compared with those in normal and Hypox rats. These findings were further confirmed by computer-based image analysis of the specimens in terms of cell integrated value, area, perimeter, and shape factor. These different patterns of PCNA expression may imply that GH specifically promotes the proliferation phase of the chondrocytes in the proliferative zone. The data also suggest that GH influences not only cell replication activity but also cell kinetics of chondrocytes in the growth plate during their lifespan.

Multihormonal regulation of dehydroepiandrosterone sulfotransferase messenger ribonucleic acid levels in adult rat liver

Hydroxysteroid sulfotransferases (DHEA ST) represent a family of enzymes that catalyze the conversion of dehydroepiandrosterone and other 3 beta-hydroxysteroids into more hydrophilic water-soluble sulfate conjugates. The present study was designed to investigate the regulation of hepatic DHEA ST expression by sex steroids and pituitary hormones, namely GH and PRL, in both male and female rats. DHEA ST mRNA levels were measured by dot blot hybridization using a 332-basepair fragment of rat DHEA ST (ST-20) cDNA as a probe. Hepatic DHEA ST mRNA levels were 2.8-fold higher in females than in males. A 15-day gonadectomy did not affect DHEA ST mRNA levels in the male liver; in females, a 35% decrease in DHEA ST mRNA levels compared with those in intact controls was observed. Administration of 17 beta-estradiol (E2; 1 microgram/kg, twice daily) alone had no effect on the accumulation of DHEA ST mRNA, but the same treatment completely reversed the marked inhibitory effect of dihydrotestosterone (DHT; 400 micrograms/kg, twice daily) on this parameter in both gonadectomized males and females. In female rats, 24-day hypophysectomy (HYPOX) decreased DHEA ST mRNA levels by 62%, whereas no change was detected in males. In HYPOX animals, treatment with E2 or DHT for 9 days starting 15 days after surgery had no effect on hepatic DHEA ST mRNA levels in rats of both sexes. In intact males, the presence of pituitary implants under the kidney capsule increased DHEA ST mRNA levels by 190% (above the control), which reached levels similar to those in intact females, whereas pituitary implants exerted no effect on this parameter in intact females. However, treatment of HYPOX rats with ovine PRL (oPRL; 4 mg/kg, twice daily) had no significant effect on the accumulation of DHEA ST mRNA in male and female animals. In HYPOX females, administration of rat GH (80 micrograms/kg, twice daily) had no effect on DHEA ST mRNA levels, whereas continuous infusion of rat GH (3.6 micrograms/h), using osmotic minipumps to mimic the female GH secretory pattern, increased this parameter by 290% (above the control value), thus completely reversing the effect of hypophysectomy. In HYPOX males, both modes of GH administration increased DHEA ST mRNA levels by 70-90% (above the control). The present study demonstrates that in both gonadectomized male and female rats, administration of E2 completely blocks the marked inhibitory effect of DHT on hepatic DHEA ST mRNA levels.

Multihormonal regulation of dehydroepiandrosterone sulfotransferase messenger ribonucleic acid levels in adult rat liver.

Hydroxysteroid sulfotransferases (DHEA ST) represent a family of enzymes that catalyze the conversion of dehydroepiandrosterone and other 3 beta-hydroxysteroids into more hydrophilic water-soluble sulfate conjugates. The present study was designed to investigate the regulation of hepatic DHEA ST expression by sex steroids and pituitary hormones, namely GH and PRL, in both male and female rats. DHEA ST mRNA levels were measured by dot blot hybridization using a 332-basepair fragment of rat DHEA ST (ST-20) cDNA as a probe. Hepatic DHEA ST mRNA levels were 2.8-fold higher in females than in males. A 15-day gonadectomy did not affect DHEA ST mRNA levels in the male liver; in females, a 35% decrease in DHEA ST mRNA levels compared with those in intact controls was observed. Administration of 17 beta-estradiol (E2; 1 microgram/kg, twice daily) alone had no effect on the accumulation of DHEA ST mRNA, but the same treatment completely reversed the marked inhibitory effect of dihydrotestosterone (DHT; 400 micrograms/kg, twice daily) on this parameter in both gonadectomized males and females. In female rats, 24-day hypophysectomy (HYPOX) decreased DHEA ST mRNA levels by 62%, whereas no change was detected in males. In HYPOX animals, treatment with E2 or DHT for 9 days starting 15 days after surgery had no effect on hepatic DHEA ST mRNA levels in rats of both sexes. In intact males, the presence of pituitary implants under the kidney capsule increased DHEA ST mRNA levels by 190% (above the control), which reached levels similar to those in intact females, whereas pituitary implants exerted no effect on this parameter in intact females. However, treatment of HYPOX rats with ovine PRL (oPRL; 4 mg/kg, twice daily) had no significant effect on the accumulation of DHEA ST mRNA in male and female animals. In HYPOX females, administration of rat GH (80 micrograms/kg, twice daily) had no effect on DHEA ST mRNA levels, whereas continuous infusion of rat GH (3.6 micrograms/h), using osmotic minipumps to mimic the female GH secretory pattern, increased this parameter by 290% (above the control value), thus completely reversing the effect of hypophysectomy. In HYPOX males, both modes of GH administration increased DHEA ST mRNA levels by 70-90% (above the control). The present study demonstrates that in both gonadectomized male and female rats, administration of E2 completely blocks the marked inhibitory effect of DHT on hepatic DHEA ST mRNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)

The inhibition of liver ornithine decarboxylase expression in neonatal rats by maternal separation or CNS β-endorphin is independent of the pituitary

Previously we have shown, in rat pups, that either short-term maternal separation (MS) or central (but not peripheral) administration of β-endorphin (BE) markedly decrease basal levels of ornithine decarboxylase (ODC) activity throughout the body and suppresses liver ODC reponsiveness to injected growth hormone (GH). In this study, hypophysectomized (hypox) pups were used to determine whether the pituitary mediates these effects. Hypophysectomy clearly did not prevent the inhibitory actions of MS or intracisternal (i.c.) BE on liver ODC gene expression. The inability of GH to stimulate ODC activity in hypox animals exposed to MS or given BE i.c. is not due to nutritional deprivation, as glucose supplementation did not reverse the response. The results from these studies demonstrate that the pituitary is not the conduit by which either MS or centrally-administered BE regulates liver ODC activity. Also, they support the hypothesis that BE or an analogous opioid neuropeptide is a prime organizer within the CNS of the adaptive physiological response of neonatal rats to short-term MS. As we have previously shown that autonomic neuronal pathways are not involved in the effects of MS on peripheral tissues, the data obtained suggest that increased activity of this CNS opioid system during MS triggers the release of “neurochemicals” into the bloodstream capable of suppressing growth in the mammalian neonate.

Pituitary factors in blood plasma are necessary for smooth muscle cell proliferation in response to injury in vivo.

Intimal thickening in response to vascular injury is inhibited in animals previously subjected to hypophysectomy. We have investigated the nature and cell kinetics of this effect in a balloon catheter model of injury to the rat carotid artery. The ability of injury to stimulate [3H]thymidine labeling 48 hours after injury was almost completely eliminated in hypophysectomized (hypox) compared with control animals (0.1% versus 32.1%). Total DNA content of the developing neointima 14 days after injury was only 30% of the values found in ballooned carotid arteries of normal rats. If hypox rats were treated with recombinant human growth hormone, the proliferative response was not restored. There are two possible general explanations for the reduction of proliferative response in hypox animals: 1) that smooth muscle cells in the hypox animals have lost the ability to respond to the stimulus of injury or 2) that the ability of the smooth muscle cells to respond has not been reduced by prior hypophysectomy, but that the response itself requires the presence of pituitary-dependent factors. Transplantation experiments were performed in vivo to distinguish between these possibilities. Carotid arteries in inbred Lewis rats were excised 1 hour after balloon injury to give platelets the opportunity to adhere. These vessels were then transplanted from hypox into control animals and vice versa. At 48 hours, proliferation of smooth muscle cells in "control-to-hypox" transplants was 0.3% compared with 14.3% in "control-to-control" transplants, whereas vessels from hypox rats increased their indices to 4.8% if transplanted into control animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypophysectomized Rats Depend on Residual Prolactin for Survival*

Hypophysectomized (Hypox) female Fischer 344 rats had 10-20% lactogenic activity in their serum when compared to controls by the Nb2 lymphoma proliferation assay. If such animals were treated daily with a rabbit antirat PRL serum, their serum lactogenic activity diminished further; severe anemia and immunological anergy developed; and death occurred within 8 weeks. In contrast, untreated Hypox animals increased gradually their serum lactogenic activity, starting on the 7th week after pituitary removal, which rose up to 50% of control levels by week 9. Hypox animals showed normochromic normocytic anemia, a grossly reduced immunocompetence, decreased body, thymus, spleen, adrenal, and ovary weights, and decreased DNA and RNA synthesis in the thymus, spleen, and bone marrow. However, the condition of Hypox animals did not deteriorate further over the 9-week experimental period. All the hematological deficiencies and decrease in organ weights observed in Hypox rats were normalized after grafting with syngeneic pituitaries (SPG). These effects of SPG could be inhibited by additional treatment with antirat PRL serum. Treatment of Hypox animals with ovine PRL had a restoring effect similar to SPG, which was not inhibited by additional antirat PRL serum treatment. Rat and ovine PRL and GH and human placental lactogen all stimulated the incorporation of 3H-thymidine by rat bone marrow cells in vitro. These results indicate that PRL has a multiple trophic effect and is capable of maintaining vital bodily functions for long periods of time.

Prolactin Inhibition of Pregnant Mare's Serum Stimulated Follicle Development in the Rat Ovary

Prolactin (PRL) effects on the female reproductive system have been presumed to occur primarily at the hypothalamic-pituitary level. The following studies were designed to evaluate whether PRL can directly alter gonadotropin actions at the ovarian level. In the first experiment, 10 groups of 7 cycling adult female rats were given a daily dose of pregnant mare's serum (PMS: 25 IU) and either saline (SAL) or PRL (0.25, 0.8, 2.5, 8, or 25 micrograms) twice daily for 4 days. In the second experiment, PMS (6 doses: 0-75 IU/d; 16 animals/dose) was administered to all animals while half the animals at each PMS dose received PRL (25 micrograms twice daily) and half received an equal volume of diluent. Finally, hypophysectomized (hypox) adult rats (n = 5-6/group) received 25 IU PMS/d and PRL (0-75 micrograms) twice daily. An additional group received 0 PRL and 0 PMS. Ovarian weight and histology were evaluated at the completion of each study. In the first experiment, PRL inhibited PMS-stimulated ovarian weight gain in a dose-dependent manner (p less than 0.01). Numbers of preantral (p less than 0.005) and antral (p less than 0.05) follicles were decreased in animals receiving an inhibitory dose of PRL (25 micrograms BID) compared to controls. In the second experiment PRL (25 micrograms BID) again inhibited PMS-stimulated ovarian weight (p less than 0.01) at all doses of PMS. Finally, in hypox animals, PRL inhibited PMS-stimulated ovarian weight gain (25 and 75 micrograms PRL: p = 0.001), and mean number (p less than 0.001) and diameter (p less than 0.001) of antral ovarian follicles (8-75 micrograms PRL) compared to controls. In summary, administration of PRL inhibited PMS-stimulated ovarian weight gain, and antral follicle diameter (in hypox animals only) and number in adult female rats suggesting that in states of hyperprolactinemia, PRL alters gonadotropin-mediated activities (i.e., folliculogenesis) directly at the ovarian level in addition to its hypothalamic and pituitary actions.

Pituitary-dependent effects of estradiol-17-beta on catecholamine turnover rates, gamma-aminobutyric acid and glutamate concentrations in various hypothalamic and limbic brain structures.

Estrogens exert many effects in a variety of hypothalamic and mesolimbic structures. Whether the actions of the estrogens are direct or indirect, possibly involving anterior pituitary hormones, is largely unknown. We therefore studied catecholamine turnover rates, gamma-aminobutyric acid and glutamate concentrations in various hypothalamic and mesolimbic structures in estrogen-treated hypophysectomized (hypox) rats and compared the measured parameters with those in sham hypophysectomized (sham) animals. Results clearly demonstrate that many of the effects of estrogen require an intact pituitary. Dopamine turnover rates in hypox rats were significantly reduced in the hypothalamus and the striatum, whereas they increased in the medial septum and medial preoptic area in comparison with sham rats. Norepinephrine turnover rates were reduced in the anterior mediobasal hypothalamus, somatosensory cortex and the nucleus accumbens of hypox animals in comparison with the sham-operated animals. In contrast, norepinephrine turnover was accelerated in the mediocortical amygdala and posterior medial basal hypothalamus of hypox rats. A significant reduction of epinephrine turnover was evident in the nucleus accumbens of hypox rats. Gamma-aminobutyric acid concentrations increased in the medial septum, anterior and posterior part of the mediobasal hypothalamus, but decreased in the mediocortical amygdala of hypox animals. Concentrations of glutamate were also decreased in the mediocortical amygdala in hypox animals. These results indicate that many direct effects of estrogens in the brain are accompanied by indirect effects which require an intact pituitary.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-Like Growth Factor-Binding Proteins in Hypophysectomized Rat Liver: Characterization and Subcellular Localization*

We have characterized binding proteins for insulin-like growth factors (IGFs) in hepatic subcellular fractions and in the washed supernatants of these fractions in normal and hypophysectomized (hypox) rats. In the course of assessing IGF-II-binding sites on rat liver microsomes, we observed that [125I] IGF-II binding to the microsomal membranes of hypox rats was much lower than that in normal rats. Paradoxically, binding increased in hypox animals at low concentrations (0.5-5 ng/ml) of unlabeled IGF-II. After resuspension and centrifugation (washing) of the microsomes, no difference was found in [125I]IGF-II binding to hypox vs. normal microsomes. However, the binding of [125I]IGF-II to the washing supernatant (SN) from hypox rat microsomes was greater than binding to that from normal animals. Binding to SN was inhibited by unlabeled IGF-II in a dose-dependent manner. Scatchard analyses indicated that the affinity constant for binding by hypox SN was higher than that of normal SN and the microsomal fractions of both hypox and normal rats. After further subfractionation of the liver, no binding activity was found in SN from plasmalemma, whereas it was about 20% of input counts per min of [125I]IGF-II in SN from combined Golgi-endosome fractions of hypox rat liver. We next compared IGF-binding moieties in microsomal SN with those in plasma using cross-linking of [125I]IGF-II followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In normal rat plasma, we observed the presence of 42K, 39K, 31K, and 27K binding complexes. In hypox rat plasma only a 42-39K doublet was found. In the SN of normal rat microsomes, the predominant complex migrated at 39K and was distinguishable only after acidification. In the SN of hypox rat microsomes, the 42K complex was predominant, with a minor 34K complex. These studies have identified IGF-binding moieties in hepatic tissues, particularly in hepatic vesicular elements, which interfere in the binding of IGF-II to membrane receptors. Their presence in these receptor-rich elements may influence IGF binding to intracellular receptors and, hence, the biological activity of the peptide.

Effect of growth hormone on carbohydrate and lipid metabolism.

Introduction APPROXIMATELY 60 yr ago, Houssay and his colleagues (1) described hypersensitivity to insulin in hypophysectomized (hypox) animals. Further work documented a primary effect of GH in this phenomenon (1). Over the ensuing years, numerous investigators have sought to define the role of GH in carbohydrate and lipid metabolism. Unfortunately, no unified picture has emerged. Both insulin-like and antiinsulin-like effects have been documented, although the physiological significance of the former is uncertain. Major controversies surround the relationship between structure and function of the GH molecule. Indeed, some believe that most of the reported effects of GH are due to contaminants in the hormone preparation. With two exceptions (2, 3), reviews in this area (4–10) have focused mainly on the author's own work in a circumscribed field; e.g. in vivo effects after GH administration (6, 7, 9), tissue effects in vitro (5, 7), or effects on lipolysis (4, 8,10). Furthermore, these reviews were publis...

Insulin-like growth factor (IGF) binding in hypophysectomized rat liver microsomes: alteration by a soluble binding moiety.

In this study, we compared the binding of IGF-I and IGF-II to liver microsomes of normal and hypophysectomized (Hypox) rats. The binding of [125I]-IGF-II, measured by centrifugation of membrane-bound ligand, was lower in hypox than in normal rats (15 +/- 2 vs 26 +/- 1%, p less than 0.001) but binding was increased (46 +/- 1.5 vs 31 +/- 1%, p less than 0.001) when bound and free hormones were separated using polyethyleneglycol (PEG) precipitation. This was due to the presence of soluble binding activity which dissociated from membranes to compete for IGF binding. When soluble binding activity was first removed from microsomal membranes by a washing procedure no difference was found in [125I]-IGF-II binding to microsomes of Hypox and normal animals (33 +/- 1 s 32 +/- 1%). However, in the microsomal washing supernatant from Hypox (containing soluble binding activity) IGF-II binding was much higher than in that from normals (17 +/- 2 vs 6 +/- 0.5%, p less than 0.001). The binding of [125I]-IGF-I was lower than that of [125I]-IGF-II but was comparably changed. By contrast, [125I]-insulin binding was similar in Hypox and normal rats and was not influenced by PEG precipitation or prewashing of the membranes. Inhibition dose-response curves showed a paradoxical increase in [125I]-IGF-II binding to unwashed microsomes of Hypox rats in the range of 0.5-5 ng/ml cold IGF-II. In normal animals [125I]-IGF-II binding to microscomes was progressively inhibited by IGF-II in a range of 0.5-500 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of High-Density Lipoproteins to Luteal Membranes. The Role of Prolactin, Luteinizing Hormone, and Circulating Lipoproteins

Ovarian and adrenal membranes from immature gonadotropin-primed rats, treated with 4-amino-pyrazolopyrimidine (4APP) to reduce endogenous lipoprotein levels, displayed higher binding of porcine high-density lipoprotein (HDL) when compared to control rats. Immature, hypophysectomized (HYPOX) rats bearing corpora lutea (CL) on Day 5 after ovulation had lower levels of serum progesterone and reduced capacity for HDL and human chorionic gonadotropin (hCG) binding to ovarian membranes when compared with intact animals. Hypophysectomy also reduced the number of HDL binding sites in adrenal membranes. Treatment of HYPOX animals with luteinizing hormone (LH) and prolactin (Prl) alone or in combination increased the HDL binding sites in the ovary relative to HYPOX-untreated rats. Neither hormone affected binding to adrenals, where only adrenocorticotropic hormone (ACTH) enhanced HDL binding. LH treatment reduced the serum progesterone levels and hCG binding to the ovaries, whereas Prl administration increased progesterone levels with no effect on hCG binding. We conclude from this study that HDL binding in the luteinized ovary is regulated by Prl and LH and circulating lipoproteins, whereas in adrenals it is regulated by ACTH and circulating levels of lipoproteins.