Activation of the prolactin receptor but not the growth hormone receptor is important for induction of mammary tumors in transgenic mice.

Transgenic mice overexpressing the human growth hormone gene develop mammary carcinomas. Since human growth hormone gene can activate both the growth hormone receptor (GHR) and the prolactin (PRL) receptor (PRLR), it is not clear which receptor system is responsible for the malignant transformation. To clarify the receptor specificity, we created transgenic mice with two different genes: (a) transgenic mice overexpressing the bovine growth hormone (bGH) gene having high levels of bGH only activating the GHR and also high serum levels of IGF-I; and (b) transgenic mice overexpressing the rat PRL (rPRL) gene that have elevated levels of PRL (one line 150 ng/ml and one line 13 ng/ml) only binding to the PRLR and with normal IGF-I levels. When analyzed histologically, all of the PRL transgenic female mice developed mammary carcinomas at 11-15 mo of age. Only normal mammary tissue was observed among the bGH transgenic animals and the controls. Cell lines established from a tumor produced rPRL and expressed PRLR. In organ culture experiments, an auto/paracrine effect of rPRL was demonstrated. In conclusion, activation of the PRLR is sufficient for induction of mammary carcinomas in mice, while activation of the GHR is not sufficient for mammary tumor formation.

The role of prolactin and growth hormone in breast cancer.

This review will focus on the role for prolactin (PRL) and growth hormone (GH) in mammary tumor formation. Much attention has previously been focused on circulating levels of GH/PRL in relation to mammary tumor formation. We will review data demonstrating that these ligands also could be produced locally in different organs, including the mammary gland and mammary tumors, and suggest that this local production may be of importance for pathological conditions. We will also discuss mechanisms for crosstalk between steroids and GH/PRL. A crosstalk between GH- and PRL response is possible at multiple levels. In the human, GH can activate both the prolactin receptor (PRLR) and the growth hormone receptor (GHR). We have demonstrated that activation of the PRLR, but not the GHR, is inducing mammary tumors in transgenic mice. Furthermore, the elevated levels of insulin-like growth factor 1 (IGF-I) seen in the GHR activating transgenic mice is not sufficient for tumor induction. The induced tumors express functionally active prolactin that could be of importance for the tumor formation. Paracrine/aurocrine stimulation by PRL may be more important than PRL transported via the circulation. In women, the role for stimulation of the PRLR and/or the GHR in mammary tumor formation has not been proven, although experiments from primates suggest that the PRLR could be of importance.

Expression of Epstein-Barr nuclear antigen 2 in kidney tubule cells induce tumors in transgenic mice.

The effect of EBNA2 in normal cells in vivo has not as yet been explored. The experiments described here were initiated to follow the consequences of the expression of EBNA2 in different tissues in transgenic mice. EBNA2 transgenic strains were generated using a vector containing EBNA2 encoding sequences under the control of the simian virus 40 (SV 40) early enhancer/promoter fused to the endogenous EBNA2 Wp promoter. Control mice carrying a transgene with the same sequence but lacking the EBV DNA part remained healthy during observation periods of up to 15 months. The SV-EBNA2 transgenic animals, however, over time developed abdominal masses that on necropsy showed to be due to kidney tumors. Histological examination revealed the presence of tumors with the morphology of kidney adenocarcinoma with a solid growth pattern. At the age of 20 weeks the kidneys of all animals investigated showed disseminated islands of tubular hyperplasia but no true malignant neoplasms. At about 50 weeks of age multiple foci of microscopic tubular adenocarcinomas were found in both kidneys. Eventually, tumors could be diagnosed in about 90% of the SV-EBNA2 transgenic mice. EBNA2-encoding RNA was expressed in both non-malignant kidney tissue and in tumors as shown by cDNA/PCR analysis. Immunoprecipitation and immunoblot analysis showed that the tumor cells contained a polypeptide of the same size as EBNA2 in B95-8 cells that reacted with a monoclonal anti-EBNA2 antibody. Immunohistochemistry demonstrated nuclear expression of EBNA2 in hyperplastic tubules and in tumor tissue.

Liver-derived IGF-I is of importance for normal carbohydrate and lipid metabolism.

IGF-I is important for postnatal body growth and exhibits insulin-like effects on carbohydrate metabolism. The function of liver-derived IGF-I is still not established, although we previously demonstrated that liver-derived IGF-I is not required for postnatal body growth. Mice whose IGF-I gene in the liver was inactivated at 24 days of age were used to investigate the long-term role of liver-derived IGF-I for carbohydrate and lipid metabolism. Serum levels of leptin in these mice were increased by >100% at 3 months of age, whereas the fat mass of the mice was decreased by 25% at 13 months of age. The mice became markedly hyperinsulinemic and yet normoglycemic, indicating an adequately compensated insulin resistance. Furthermore, they had increased serum levels of cholesterol. We conclude that liver-derived IGF-I is of importance for carbohydrate and lipid metabolism.

Selective cerebral overexpression of growth hormone alters cardiac function, morphology, energy metabolism and catecholamines in transgenic mice.

BACKGROUND: Growth hormone (GH) has important regulatory effects on cardiac morphology and function both during normal development as well as in pathophysiological settings such as myocardial infarction (MI) and congestive heart failure (CHF). In order to investigate in more detail the interaction between GH and sympathetic nervous system (SNS) system we studied the effects of selective cerebral GH overexpression on myocardial content of catecholamines, myocardial and brain energy metabolism as well as on cardiac function during resting and stress conditions in a transgenic mouse model. METHODS: Transgenic mice with selective bovine GH overexpression under control of glial fibrillary acidic protein promoter in the brain (GFAP-bGH, n=15) were created and compared to genetically matched non-transgenic mates (Control, n=15). Cardiac morphology and function were evaluated in vivo using transthoracic echocardiography during resting and stress conditions induced pharmacologically by dopamine (D) and isoprotenolol (ISO). Myocardial and brain energy metabolism were evaluated non-invasively using in vivo volume-selective phosphorus magnetic resonance spectroscopy ((31)P MRS). Myocardial content of catecholamines was analyzed by means of HPLC. RESULTS: Compared to the C animals, the GFAP-bGH mice have showed several differences in the cardiac phenotype. Systolic (fractional shortening) and diastolic function (E/A wave ratio of mitral flow) was disturbed in the GFAP-bGH mice (both p<0.05). During the dopamine stress, there was chronotropic insufficiency in the GFAP-bGH group (p<0.01) while no difference was observed in response to isoprotenolol. Left ventricular dimensions were increased in GFAP-bGH mice (p<0.05). There was a tendency for higher body weight in GFAP-bGH compared to the control group (p=0.06) while no difference was observed in heart weight and brain weight when normalized for body weight. Myocardial content of noradrenaline was lower in the GFAP-bGH group (p<0.05). PCr/ATP ratio was higher (p<0.05) in the brain and lower in the heart (p<0.05) in the GFAP-bGH mice. CONCLUSIONS: Selective cerebral overexpression of GH results in alterations of cardiac function, morphology and metabolism in transgenic mice. Decreased myocardial content of catecholamines in the GFAP-bGH mice suggests central interaction between GH and sympathetic nervous system.

Atrial natriuretic peptide inhibits spontaneous rat oocyte maturation.

We report results of experiments demonstrating a dose-dependent inhibition of spontaneous maturation (resumption of meiosis) in rat oocyte-cumulus complexes by atrial natriuretic peptide (ANP). The inhibition was persistent over the time period studied. The ANP analog Tyr8-ANP, which mediates smooth muscle relaxation in other organs without elevating cGMP levels, did not inhibit the spontaneous maturation. ANP, but not Tyr8-ANP, dose-dependently stimulated cGMP accumulation in oocyte-cumulus complexes. Furthermore, sodium nitroprusside (SNP), that stimulates a soluble form of guanylate cyclase, inhibited spontaneous maturation in oocyte-cumulus complexes and stimulated cGMP accumulation in oocyte-cumulus complexes. Neither ANP nor SNP stimulated cAMP accumulation. In oocytes where the surrounding cumulus cells had been removed neither ANP nor SNP inhibited the spontaneous maturation. These results demonstrate that cumulus cells, but not the oocyte itself, have ANP receptors and guanylate cyclases. Furthermore, ANP, via cGMP, can influence oocyte meiosis, suggesting a possible involvement of ANP and cGMP in the control of the meiotic process in rat oocytes.

Transgenic mice overexpressing the prolactin gene develop dramatic enlargement of the prostate gland.

An altered endocrine status of elderly men has been hypothesized to be important for development of prostate hyperplasia. The present study addresses the question whether increased PRL expression is of importance for development of prostate hyperplasia in mice. Three lines of PRL transgenic mice were generated having serum levels of PRL of approximately 15 ng/ml, 100 ng/ml, and 250 ng/ml, respectively. These mice developed dramatic enlargement of the prostate gland, approximately 20 times the normal prostate weight and they had a 4- to 5-fold increased DNA content. Histologically, the prostate glands in the transgenic mice were distended from secretion, and the amount of interstitial tissue was increased. The levels oftestosterone and IGF-I were increased in the PRL transgenic animals. In mice overexpressing the bovine GH gene, displaying elevated IGF-I levels, the prostate gland was slightly larger compared with normal mice, indicating that the effect of PRL was not primarily mediated through elevated plasma IGF-I levels. The present study suggests that PRL is an important factor in the development of prostate hyperplasia acting directly on the prostate gland or via increased plasma levels of testosterone.

Disproportional bone growth and reduced weight gain in gonadectomized male bovine growth hormone transgenic and normal mice.

Sex steroids have been shown to influence longitudinal bone growth during sexual maturation, partially by increased GH secretion. Mice transgenic for metallothionein promoter bovine GH were developed by pronuclear injection as a model with sex steroid-independent GH secretion. Prepubertal normal and transgenic, male and female mice were either gonadectomized or sham operated. The growth was divided into two segments: peripubertal growth from 30-60 days of age and adult growth from 60-90 days of age. Orchidectomy resulted in a decreased growth rate of the lumbar spine and a decreased weight gain during the peripubertal growth, whereas tibia growth was unaffected. The alteration in proportions between the lumbar spine and the tibia was apparent for both normal and bovine GH transgenic mice, suggesting that the effect was not mediated via decreased GH secretion. Orchidectomy resulted in increased adult tibial growth, whereas weight gain and lumbar growth were unaffected. In female mice, gonadectomy did not influence these parameters during either time period studied. In summary, we present data indicating that the male gonads in a GH secretion-independent manner stimulate pubertal growth of the spine and inhibit the tibial growth of adult animals.

Bovine growth hormone transgenic mice display alterations in locomotor activity and brain monoamine neurochemistry.

Recent clinical and experimental data indicate a role for GH in mechanisms related to anhedonia/hedonia, psychic energy, and reward. In the present study we have investigated whether bovine GH (bGH) transgenic mice and nontransgenic controls differ in spontaneous locomotor activity, a behavioral response related to brain dopamine (DA) and reward mechanisms, as well as in locomotor activity response to drugs of abuse known to interfere with brain DA systems. The animals were tested for locomotor activity once a week for 4 weeks. When first exposed to the test apparatus, bGH transgenic animals displayed significantly more locomotor activity than controls during the entire registration period (1 h). One week later, after acute pretreatment with saline, the two groups did not differ in locomotor activity, whereas at the third test occasion, bGH mice were significantly more stimulated by d-amphetamine (1 mg/kg, ip) than controls. At the fourth test, a tendency for a larger locomotor stimulatory effect of ethanol (2.5 g/kg, ip) was observed in bGH transgenic mice. bGH mice displayed increased tissue levels of serotonin and 5-hydroxyindoleacetic acid in several brain regions, decreased DA levels in the brain stem, and decreased levels of the DA metabolite 3,4-dihydroxyphenylacetic acid in the mesencephalon and diencephalon, compared with controls. In conclusion, bGH mice display more spontaneous locomotor activity than nontransgenic controls in a novel environment and possibly also a disturbed habituation process. The finding that bGH mice were also more sensitive to d-amphetamine-induced locomotor activity may suggest that the behavioral differences observed are related to differences in brain DA systems, indicating a hyperresponsiveness of these systems in bGH transgenic mice. These findings may constitute a neurochemical basis for the reported psychic effects of GH in humans.

Elevated levels of growth hormone increase bone mineral content in normal young mice, but not in ovariectomized mice.

Both estrogens and GH are necessary for normal bone remodeling. This study investigates the effect of elevated GH levels on the amount and density of bone in young female mice and its dependence on intact ovarian function. Metallothionein promoter-GH-transgenic mice were either sham operated or ovariectomized at 25-29 days of age, and the bone measurements were made at about 90 days of age. A 6-mm high cylinder containing only cortical bone was cut from the right tibia, and lumbar vertebrae 6 was measured as a bone with predominantly cancellous bone. The amounts of tibial and vertebral bone, measured by dry weight, mineral weight, organic weight, bone mineral content (measured by dual energy x-ray analysis), and volume, were increased in GH-transgenic animals compared to those in normal littermates. This stimulatory effect of elevated GH levels was not seen in ovariectomized mice. The real density of the tibial bone were slightly decreased in GH-transgenic animals compared to normal littermates. In conclusion, elevated levels of GH increase the amounts of vertebral (predominantly cancellous) bone and tibial (cortical) bone in young mice. Intact ovaries are a prerequisite for the stimulatory effect of elevated levels of GH. The fact that ovariectomy decreases the stimulatory effect of elevated GH levels suggests that the effect of elevated GH levels in bone is dependent upon the presence of basic sex steroid secretion.

Expression of insulin-like growth factor-I (IGF-I) in the rat fallopian tube: possible autocrine and paracrine action of fallopian tube-derived IGF-I on the fallopian tube and on the preimplantation embryo.

Recent studies have indicated that growth factors such as insulin-like growth factors (IGFs) increase the growth rate of cultured preimplantation embryos. We therefore hypothesized that the fallopian tube may produce IGFs which in turn participate in the regulation of preimplantation embryo development in vivo. In the present study we examined the expression of IGF-I in the fallopian tube. We demonstrated that IGF-I transcripts (7.0, 1.7, and 1.2-0.8 kilobases) were abundant in the fallopian tube. Immunoreactive IGF-I was most abundant in the epithelial cells in the fallopian tube, and IGF-I messenger RNA (mRNA) was detected in the luminal region of the fallopian tube. A solution hybridization assay was used to examine the regulation of IGF-I mRNA. The abundance of IGF-I transcripts changed markedly during the 4-day estrous cycle with the highest levels on the day of proestrus. The increase in IGF-I mRNA between the day of diestrus II and the day of proestrus was 4-fold (P < 0.01). The pattern of IGF-I mRNA expression in the fallopian tube resembled the pattern of ovarian estrogen production during the estrous cycle. The level of IGF-I mRNA decreased after hypophysectomy. The expression of IGF-I mRNA in the fallopian tube was dose-dependently regulated by estradiol, and a single sc injection of estradiol [5 micrograms/100 g body wt (BW)] increased the IGF-I mRNA in a time-dependent manner with a significant increase after 3 h (P < 0.01). The lowest estradiol dose tested (0.1 microgram/100 g BW) increased the expression after 6 h, whereas progesterone (5 micrograms/100 g BW) was ineffective. The presence of embryos in the fallopian tube did not statistically significantly influence the abundance of IGF-I transcripts as measured with a solution hybridization assay on RNA extracted from whole fallopian tubes. In order to determine possible targets for fallopian tube-derived IGF-I we examined the expression of IGF-I receptor mRNA. Northern blot analysis revealed that an 11-kilobase IGF-I receptor transcript was expressed in the fallopian tube. Using a reverse transcriptase-polymerase chain reaction, IGF-I receptor mRNA was also detected in the eight-cell but not two-cell preimplantation embryo. The present study demonstrates that IGF-I is produced in the fallopian tube and its expression is regulated by estradiol. Both the fallopian tube and the eight-cell preimplantation embryo express IGF-I receptors and are therefore potential target tissues.

Embryonic stem cells express growth hormone receptors: regulation by retinoic acid.

Embryonic and fetal growth are generally considered to be independent of pituitary GH. However, it has been demonstrated recently that 18-day-old rat embryos and rat fetuses express GH receptors, suggesting that GH could play a role in early development. The aim of the present investigation was to determine whether preimplantation embryos also express GH receptors. Germ line competent mouse embryonic stem (ES) cells and cultured mouse preimplantation embryos were examined with Northern blot analysis, RNAse-protection solution-hybridization assays, reverse transcription/polymerase chain reaction assays and immunohistochemistry for the detection of GH receptors. Northern blot analysis of ES cells using a probe corresponding to the extracellular domain of the GH receptor demonstrated the presence of two transcripts (1.2 and 4.5 kilobases). The RNAse-protection solution-hybridization assay revealed that ES cells express approximately one sixth of the GH-receptor messenger RNA (mRNA) levels expressed in liver from pregnant mice. Treatment of cultured ES cells with retinoic acid (100 nM) for 6 days increased GH-receptor mRNA levels (P < 0.01). GH-receptor mRNA was further identified in ES cells, preimplantation embryos, muscle, liver, and placenta by a reverse transcription/polymerase chain reaction assay. In humans it has previously been shown that exon 3 of the GH-receptor is deleted in the placenta. However, none of the studied mouse tissues had a deletion of the GH-receptor mRNA corresponding to exon 3 of the human GH receptor. GH-receptor immunoreactivity was identified on the cultured ES-cells by immunohistochemistry. In conclusion, we have in the present study shown that germline competent ES cells and preimplantation mouse embryos express the GH receptor transcript and that this transcription is increased by retinoic acid in ES cells. Furthermore, the presence of GH-receptor immunoreactivity on the ES cells indicates that the GH-receptor transcript is translated.

Vascular function and blood pressure in GH transgenic mice.

Acromegaly is associated with cardiovascular disease. We studied vascular function and mean arterial blood pressure in transgenic mice overexpressing bovine GH. Mean arterial blood pressure was measured in conscious, unrestrained male and female bovine GH and littermate control mice during normal as well as high salt intake using telemetric devices. Structure in artificially perfused maximally dilated hindquarter vascular beds and vascular reactivity and endothelial function in small mesenteric vessels were studied in female bovine GH and control mice. Mean arterial blood pressure was increased in female bovine GH transgenic (126 +/- 3 mm Hg) and male bovine GH transgenic (129 +/- 4 mm Hg) compared with female (109 +/- 3 mm Hg, P < 0.05) and male (111 +/- 3 mm Hg, P < 0.05) controls respectively. Increased salt intake had no effect on mean arterial blood pressure. Perfusion studies showed a significant decrease in the average diameter of the female bovine GH transgenic hindquarter vascular bed (P < 0.05). The responses of isolated resistance arteries to nor-epinephrine, potassium-induced depolarization, acetylcholine, or sodium-nitroprusside did not significantly differ between bovine GH transgenic and control mice. We conclude that the phenotype of the bovine GH transgenic mice includes a salt-resistant form of hypertension. Furthermore, the increase in mean arterial blood pressure is accompanied by a significant structural narrowing of the resistance vasculature without changes in vascular reactivity or endothelial function. The results imply that hypertension in bovine GH transgenic mice is maintained mainly by a structurally based increase in peripheral vascular resistance.

Enhanced spontaneous locomotor activity in bovine GH transgenic mice involves peripheral mechanisms.

Clinical and experimental studies indicate a role for GH in mechanisms related to anhedonia/hedonia, psychic energy, and reward. Recently we showed that transgenic mice with general overexpression of bovine GH display increased spontaneous locomotor activity. In the present study, we investigated whether this behavioral change is owing to a direct action of GH in the central nervous system or to peripheral GH actions. A transgenic construct, containing the glial fibrillary acidic protein promoter directing specific expression of bovine GH to the central nervous system, was designed. The central nervous system-specific expression of bovine GH in the glial fibrillary acidic protein-bovine GH transgenic mice was confirmed, but no effect on spontaneous locomotor activity was observed. Serum bovine GH levels were increased in glial fibrillary acidic protein-bovine GH transgenic mice but clearly lower than in transgenic mice with general overexpression of bovine GH. In contrast to the transgenic mice with general overexpression of bovine GH, glial fibrillary acidic protein-bovine GH mice did not display any difference in serum IGF-I levels. The levels of free T(3) and the conversion of the free T(4) to free T(3) were only increased in transgenic mice with general overexpression of bovine GH, but serum corticosterone levels were similarly increased in both transgenic models. These results suggest that free T(3) and/or IGF-I, affecting dopamine and serotonin systems in the central nervous system, may mediate the enhanced locomotor activity observed in transgenic mice with general overexpression of bovine GH.

Impairment of cardiac function and bioenergetics in adult transgenic mice overexpressing the bovine growth hormone gene.

Cardiovascular abnormalities represent the major cause of death in patients with acromegaly. We evaluated cardiac structure, function, and energy status in adult transgenic mice overexpressing bovine GH (bGH) gene. Female transgenic mice expressing bGH gene (n = 11) 8 months old and aged matched controls (n = 11) were used. They were studied with two-dimensional guided M-mode and Doppler echocardiography. The animals (n = 6) for each group were examined with 31P magnetic resonance spectroscopy to determine the cardiac energy status. Transgenic mice had a significantly higher body weight (BW), 53.2+/-2.4 vs. 34.6+/-3.7 g (P < 0.0001) and hypertrophy of left ventricle (LV) compared with normal controls: LV mass/BW 5.6+/-1.6 vs. 2.7+/-0.2 mg/g, P < 0.01. Several indexes of systolic function were depressed in transgenic animals compared with controls mice such as shortening fraction 25+/-3.0% vs. 39.9+/-3.1%; ejection fraction, 57+/-9 vs. 77+/-5; mean velocity of circumferential shortening, 4.5+/-0.8 vs. 7.0+/-1.1 circ/sec, p < 0.01. Creatine phosphate-to-ATP ratio was significantly lower in bGH overexpressing mice (1.3+/-0.08 vs. 2.1+/-0.23 in controls, P < 0.05). Ultrastructural examination of the hearts from transgenic mice revealed substantial changes of mitochondria. This study provides new insight into possible mechanisms behind the deteriorating effects of long exposure to high level of GH on heart function.

Investigation of the role of prolactin in the development and function of the lacrimal and harderian glands using genetically modified mice.

PURPOSE: To determine whether prolactin receptor is essential for normal development and function of the lacrimal gland and whether hyperprolactinemia can alter lacrimal development. METHODS: Lacrimal gland morphology and function were examined in two genetic mouse models of prolactin action: a prolactin receptor knockout model that is devoid of prolactin action and a transgenic model of hyperprolactinemia. RESULTS: Image analysis of lacrimal and Harderian gland sections was used to quantify glandular morphology. In females, lacrimal acinar area decreased by 30% and acinar cell density increased by 25% over control subjects in prolactin transgenic animals, but prolactin receptor knockout mice showed no changes. In males, transgenic animals showed no changes, but prolactin receptor knockout mice showed a 5% reduction in acinar area and an 11% increase in acinar cell density, which was lost after castration. The morphology of the Harderian glands underwent parallel changes but to a lesser degree. A complete loss of porphyrin accretions was seen in the Harderian glands of male and female knockout animals. No differences in tear protein levels were seen in knockout animals by two-dimensional gels. Enzyme-linked immunosorbent assay (ELISA) and Western blot analysis showed that the level of secretory component and IgA in knockout mouse tears remained unchanged. There was no change in the predisposition of the 129 mouse strain to conjunctivitis in the knockout animals. CONCLUSIONS: Prolactin plays a small role in establishing the sexual dimorphism of male lacrimal glands. In females, hyperprolactinemia causes a hyperfemale morphology, suggesting a role in dry eye syndromes. Prolactin is required for porphyrin secretion by the Harderian gland but plays no essential role in the secretory immune function of the lacrimal gland.

GH substitution reverses the growth phenotype but not the defective ossification in thyroid hormone receptor alpha 1-/-beta-/- mice.

Thyroid hormone receptor alpha 1, beta 1 and beta 2-deficient mice (TR alpha 1-/-beta-/- mice) demonstrate growth retardation and defective ossification in the epiphyses associated with an inhibition of the GH/IGF-I axis. There are differences between TR alpha 1-/-beta-/- mice (receptor deficient) and the hypothyroid animal model (ligand deficient). Such differences include possible repressive actions exerted by unliganded receptors in the ligand-deficient (hypothyroid) model but not in the receptor-deficient model. In the present study we have investigated whether or not GH substitution rescues the skeletal phenotype of TR alpha 1-/-beta-/- mice. TR alpha 1-/-beta-/- and wild-type (WT) mice were treated with GH from day 18 until 10 weeks of age. GH substitution of mutant mice resulted in a significant and sustained stimulatory effect on the body weight that was not seen in WT mice. GH-treated mutant mice but not GH-treated WT mice demonstrated increased length and periosteal circumference of the femur. However, GH substitution did not reverse the defective ossification seen in TR alpha 1-/-beta-/- mice. TR alpha 1-/-beta-/- mice displayed increased width of the proximal tibial growth plate, which was caused by increased width of the proliferative but not the hypertrophic layer. GH substitution did not restore the disturbed morphology of the growth plate in TR alpha 1-/-beta-/- mice. In summary, GH substitution reverses the growth phenotype but not the defective ossification in TR alpha 1-/-beta-/- mice. Our data suggest that TRs are of importance both for the regulation of the GH/IGF-I axis and for direct effects on cartilage.

Effects of chronically elevated growth hormone levels on polyamine metabolism in elderly transgenic mice.

The polyamines are ubiquitous, multifunctional aliphatic amines with roles in cell growth, proliferation, differentiation, and malignant development. After growth stimulation, rapid and transient changes occur in polyamine regulatory enzymes. In this respect, acute effects of growth hormone (GH) injection on polyamine metabolic enzymes have earlier been shown. The present investigation comprises studies of the effects on polyamine metabolism of constitutively elevated levels of circulating GH in elderly transgenic (tg+) mice, overexpressing bovine GH. Polyamine levels were found to be constitutively altered in the liver and kidney of tg+ mice. Less changes were found in the spleen and none in the brain. The cellular uptake of polyamines in the liver from tg+ mice showed an increase and considerable changes were observed in the activity of ornithine decarboxylase (ODC) in the liver and kidney and S-adenosylmethionine decarboxylase (AdoMetDC) in the liver. A conspicuous finding was the distribution pattern of ODC protein in the liver and both tg- and tg+ animals. The results show that the effects of chronically elevated GH levels are organ-dependent and complex, and differ from acute GH effects. Despite high ODC activity and polyamine levels in liver, these mice did not display any malignant transformation even at an advanced age, indicating that high ODC activity is not sufficient to induce tumorigenesis in vivo.

High frequency of mammary adenocarcinomas in metallothionein promoter-human growth hormone transgenic mice created from two different strains of mice.

Transgenic mice were developed by injecting a mouse metallothionein promoter-human growth hormone (Mt-hGH) gene fragment into the pronucleus of C57Bl x DBA/2J-f2 or C57Bl x CBA-f2 one cell embryos. Six founder animals with the C57Bl x DBA genetic background grew 1.3-2.2 times larger than littermate controls and had higher levels of hGH in plasma (4.6-279 mU/l). Three of the four female transgenic founders developed malignant papillar adenocarcinomas of mammary origin at 27-43 weeks of age. One male transgenic founder was successfully mated and two of three female transgenic offsprings developed mammary tumors. To examine if the tumor induction was dependent on the strain of mice used the experiments were repeated using animals with different genetic background. Fourteen female hGH transgenic mice from five founder animals were generated using C57Bl x CBA-f2 mice. Thirteen of the animals had elevated levels of hGH in plasma (7-1960 mU/l) and grew larger than control animals. Nine of the animals developed mammary adenocarcinomas. Four of the hGH expressing animals did not demonstrate macroscopic tumor formation but have not yet been analyzed histologically. The present study suggests that markedly elevated endogenous levels of GH cause mammary carcinoma in hGH transgenic mice. The present animal model might prove useful for studying molecular mechanisms involved in the development of hormonally induced mammary tumors.

Oocyte maturation as regulated by follicular factors.

Follicular fluid (FFl), obtained from 24 women treated with clomiphene/hCG in an in vitro fertilization program, was characterized with respect to steroid hormone levels and oocyte maturation inhibitor (OMI) activity. Three FFl samples apparently were derived from cystic follicles and contained low steroid levels and no OMI activity in an in vitro rat oocyte assay. The remaining 21 follicles contained normal preovulatory steroid levels and mature and generally fertilizable oocytes. In 7 of these follicles the FFl (at 50% concentration) significantly inhibited rat oocyte meiosis, while 14 exerted no OMI activity. The results confirm earlier work on porcine and human FFl, suggesting that the putative OMI activity declines with follicular maturation.