Dramatic pituitary hyperplasia in transgenic mice expressing a human growth hormone-releasing factor gene

A transgenic animal model system was used to analyze the mitogenic effects of GRF on its target cell, the pituitary somatotroph. We have previously established a strain of mice that express a mouse metallothionein-I/human GRF (hGRF) fusion gene, and that grow to be abnormally large due to GH hypersecretion. We show here that chronic GRF production in these mice leads to the development of enormous pituitary glands. The increase in pituitary size appears to be largely the result of a selective proliferation (hyperplasia) of somatotrophs, the GH-producing cells. This observation provides direct evidence that a neuropeptide may act as a specific trophic factor for its target cell. In addition to this effect on pituitary development, we find that the pituitary is a major site of expression of mouse metallothionein-I/hGRF mRNA, and of hGRF peptide. This tissue specificity was unexpected in that neither component of the fusion gene is highly expressed in the normal pituitary. It suggests that pituitary somatotrophs might produce and respond to GRF in an essentially autocrine fashion in these transgenic animals.     

Fertility of transgenic female mice expressing bovine growth hormone or human growth hormone variant genes

Although growth hormone (GH) exerts various direct and indirect stimulatory effects on gonadal development and function, excessive levels of GH in acromegalic patients and in transgenic animals are often associated with reproductive disorders. We have examined reproductive performance of transgenic female mice expressing the following hybrid genes: mouse metallothionein-1 (MT)/human placental GH variant (hGH.V), MT/bovine GH(bGH), and phosphoenolpyruvate carboxykinase (PEPCK)/bGH. This allowed us to evaluate the effects of chronic GH excess in three animal models and to obtain some information on the significance of the lactogenic activity of the foreign GH (hGH.V vs. bGH) and on the developmental stage of transgene expression (MT vs. PEPCK). Transgenic animals from each line had elevated plasma insulin-like growth factor-I levels and greatly increased adult body weight. Plasma bGH levels were significantly higher in PEPCK/bGH than in MT/bGH transgenic mice. Approximately 20% of transgenic MT/hGH.V and MT/bGH females and over 60% of transgenic PEPCK/bGH females were infertile. Transgenic females that did reproduce ovulated either a normal or increased number of eggs but exhibited a variety of reproductive disorders including increased interval between pairing with a male and conception, increased interval between litters, reduced number of litters, reduced fetal growth, increased pre- and postnatal mortality, and alterations in sex ratio. Among adult offspring of these females, the proportion of transgenic animals was significantly less than the expected 50%. While some characteristics (e.g., fetal crown-rump length and weight on Day 14 of pregnancy) were affected to a comparable extent in transgenic females from all three lines, MT/hGH.V and PEPCK/bGH females were, in general, more severely affected than the MT/bGH animals. Sterility of PEPCK/bGH females appeared to be due to luteal failure since treatment with progesterone led to pregnancy. Greatly increased intervals between successive litters appeared to be due to failure to mate during postpartum estrus and to sterile matings during this period. Reduced fetal size and weight may have been due to chronic glucocorticoid excess because comparable changes could be induced in normal females by injections of dexamethasone during pregnancy, and plasma corticosterone levels were previously shown to be elevated in transgenic mice from each of these lines. Comparison of these results with data obtained from matings of normal female mice to transgenic males from the same lines suggests that reduced fetal growth is due primarily to maternal genotype, while reduced "transmission" of the hybrid genes is not, and presumably reflects increased mortality of transgenic progeny at various stages of development.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of every-other-day feeding on prolactin regulatory mechanism in transgenic human growth hormone mice

Transgenic mice overexpressing human growth hormone (hGH) exhibit accelerated aging with functional hyperprolactinemia and greatly depressed endogenous prolactin. Calorie restriction (CR) is widely recognized as the most effective experimental intervention to delay aging. The aim of the present work was to analyze the effects of lifelong overexpression of hGH on prolactin-gene expression as well as the dopamine production at the pituitary level and discern whether this mechanism changes as a function of feeding patterns. Ten-month-old mice fed every other day (EOD) were killed after one day of fasting. The results confirmed typical phenotypic features of these transgenic mice: an increase in body weight, very high hGH plasma concentrations, and hyperinsulinemia. There was a marked inhibition of the expression of the prolactin gene, together with an increased tyrosine hydroxylase (TH) and the long isoform of dopamine receptor type 2 (D2LR) gene expression at the pituitary level. These parameters were not affected by the EOD feeding pattern. These data may suggest an autocrine or paracrine effect of dopamine at the hypophyseal level on prolactin secretion that is independent of the feeding pattern.     

Pharmacokinetics of radioiodinated human and ovine growth hormones in transgenic mice expressing bovine growth hormone

Pharmacokinetics of radioiodinated human growth hormone (hGH) and ovine growth hormone (oGH) were studied in normal mice and in transgenic mice carrying the bovine growth hormone (bGH) gene fused to phosphoenolpyruvate carboxykinase promoter/regulator (PEPCK-bGH). Multiexponential plasma decay curves were obtained in both normal and transgenic mice after a¹²⁵I-oGH injection and pharmacokinetic parameters were estimated by fitting blood concentration data to a three compartment model. The half-life for the rapid compartment was shorter in transgenic than in normal mice (t_1/2:1.2±0.3 vs. 2.2±0.5 min). The slow compartment had a t_1/2 of 160±23 min for transgenic and 70±8 min for normal mice while the middle compartment had a t_1/2 of approximately 10 min for both groups of mice. The mean residence times were 167±24 and 55±5 min for transgenic and normal mice, respectively. Specific liver uptake of radioactivity after injection of¹²⁵I-oGH or¹²⁵I-hGH was found in both groups of animals. Specificity studies indicated that, similarly to normal mice, livers of transgenic mice possess a mixed population of somatotropic and lactogenic receptors. Uptake of labelled hGH by the liver was dose-dependent and the doses that prevented 50% of liver uptake (ED_50%) were 8 and 165 g per 50 g body weight for normal and transgenic mice, respectively. Thesein vivo results confirm and extend previousin vitro findings that a life-long excess of bGH increases hepatic somatotropic and lactogenic receptors. Since elevation in growth hormone (GH) receptors was reported to be associated with an increase in GH binding protein (GHBP), we suspect that both the increase in the mean residence time and the reduction in specific uptake of GH in the livers of transgenic mice may be the result of an increase in GHBP levels.     

Identification of somatogenic binding sites in liver microsomes from normal mice and transgenic mice expressing human growth hormone gene.

Somatogenic binding sites were detected and characterized in microsomal preparations from livers of normal mice and mice expressing metallothionein-I/hGH (mMT/hGH) hybrid gene, using 125I-labelled bovine or human GH, or a photoreactive derivative of hGH (125I-AP-hGH1). Specific binding of 125I-bGH was detected in liver microsomes from both normal and transgenic mice with an apparent Kd of 2 nM. 125I-hGH was partially displaced by bGH. 125I-AP-hGH1 was covalently bound to the microsomal preparations, and bGH prevented the formation of the 130 kDa species with no appreciable effect on 63 kDa and 70 kDa lactogenic complexes.     

Culture of human pituitary prolactin and growth hormone cells

Summary Fragments of pituitary tissue obtained from a total of 37 patients with either breast cancer, diabetic retinopathy, galactorrhea, or acromegaly were dissociated into single cell suspensions prior to cell culture. Release of human growth hormone (hGH) and human prolactin (hPRL) into the culture medium was measured by radioimmunoassay. During a 3-week culture period, prolactin cells released 9–13 times the intracellular levels of hPRL at the time of seeding, whereas hGH release from growth hormone cells was only 1–2 times that of their initial intracellular level during this same time. Both growth hormone and prolactin cells retained distinctive ultrastructural features during culture. The prolactin cells responded to TRH stimulation by elevated release of PRL into the medium. No evidence for mitotic division of prolactin cells in vitro was found.This work was supported by NCI Contract NO 1-CB-23863     

Somatotropic and lactotropic receptors in transgenic mice expressing human or bovine growth hormone genes

The somatotropic and lactotropic receptors were studied in liver microsomal preparations from transgenic mice carrying the human growth hormone (hGH) or bovine growth hormone (bGH) gene fused to mouse metallothionein-I (MT) or phosphoenolpyruvate carboxykinase promoter/regulator (PEPCK). Specificity studies indicated that, similarly to normal mice, liver microsomes from the transgenic animals possess a mixed population of somatotropic and lactotropic binding sites. In transgenic animals of both sexes, the binding capacity of somatotropic receptors was significantly increased without corresponding changes in affinity. Expression of the MT-hGH hybrid gene was associated with the induction of somatotropic receptors which was approximately twice as great as that measured in animals expressing the MT-bGH hybrid gene. The binding capacity of lactotropic receptors in liver microsomes (quantitated, by the use, of labelled ovine prolactin) was increased 2–3 fold in transgenic females and approximately 10-fold in transgenic males as compared to the respective normal controls. We conclude that lifelong excess of GH up-regulates hepatic GH and prolactin receptors, and that lactogenic activity of GH is not essential for induction of prolactin receptors in the liver of transgenic mice.     

Cryptic human growth hormone gene sequences direct gonadotroph-specific expression in transgenic mice

Our laboratory reported previously that chimeric genes encoding either rat somatostatin (SS) or human GH (hGH), but containing the identical mouse metallothionein-I (MT) promoter/enhancer sequences and hGH 3'-flanking sequences, were selectively expressed in the gonadotrophs of transgenic mice. The experiments reported here were designed to identify the DNA sequences responsible for this unexpected cell-specific expression within the anterior pituitary. We produced new transgenic mice expressing fusion genes that tested separately the requirement of the MT or 3'-hGH sequences for gonadotroph expression. A fusion gene that retained the original MT and SS sequences, with a simian virus 40 polyadenylation signal exchanged for the 3'-hGH sequences, no longer directed strong pituitary expression, but was active in the liver. In contrast, a cytomegalovirus promoter/enhancer-SS-hGH fusion gene was expressed at the same high level in the anterior pituitaries of transgenic mice as the originally studied MT-SS-hGH gene. Immunohistochemical analysis indicated that pituitary expression of the cytomegalovirus promoter/enhancer-SS-hGH fusion gene was also restricted to gonadotroph cells in adult mice. These studies indicate that sequences within the 3'-flanking region of the hGH gene can direct expression of chimeric genes to pituitary cells that do not normally produce growth hormone.     

Stress-induced secretion of human growth hormone in transgenic mice

The effect of stress on human growth hormone (hGH) secretion was studied in transgenic mice. Experiments were conducted on fourth, fifth, and sixth generation male mice carrying a fusion gene, consisting of the promoter sequence of the mouse metallothionein I gene ligated to the hGH structural gene (mMT-I/hGH). In animals adapted to a controlled photoperiod, basal (unstimulated) levels of plasma hGH exhibited a diurnal cycling, with peak values occurring during the later half of the light period (15.5 +/- 1.0 vs 10.7 +/- 0.9 ng/ml, mean +/- SE, light versus dark, respectively). Food deprivation (5 days) led to elevated levels of plasma hGH (11.0 +/- 0.7 vs 32.0 +/- 4.2 ng/ml, preversus post-fast, respectively) accompanied by weight loss (49.5 +/- 0.8 vs 34.3 +/- 0.7 g), and hypoglycemia (7.8 +/- 0.2 vs 5.0 +/- 0.3 mM); glucose administration (5% drinking solution ad libitum) blocked the changes in levels of plasma hGH (12.2 +/- 1.1 vs 13.8 +/- 0.8 ng/ml) and plasma glucose (7.4 +/- 0.3 vs 7.9 +/- 0.5 mM), although the animals still sustained significant weight loss (44.9 +/- 1.6 vs 35.2 +/- 1.1 g). Vigorous exercise (swimming, 4 hr) produced a small but significant increase in plasma hGH, 12.1 +/- 1.1 ng/ml (1 hr pre-swim) vs 16.7 +/- 0.6 ng/ml (immediately post-swim). These findings indicate that the mMT-I/hGH transgene is responsive to the physiologic status of the host animal. Taken together with information regarding the heterologous components of the fusion gene, these data are consistent with the view that the hGH (structural) sequence may play a role in the response to stress.     

Down regulation of masked and unmasked insulin receptors in the liver of transgenic mice expressing bovine growth hormone gene.

The interaction of insulin with its receptor was studied in microsomes from livers of transgenic mice expressing the bovine growth hormone gene with mouse metallothionein-1 promoter (MT/bGH) and in their normal (non-transgenic) littermates. Specific binding of 125I-insulin was detected in hepatic microsomes from normal and transgenic mice with an apparent Kd of 8 and 200 nM, for high and low affinity sites, respectively. The transgenic MT/bGH mice had a marked hyperinsulinism without significant elevation of plasma glucose levels. Under identical conditions of preparation and incubation, microsomes from the transgenic male and female mice bound 39% and 34% less insulin than those from their litter mates. Scatchard's analysis indicates that this decrease in binding is due to a decrease in the number of receptor sites. In contrast to the marked decrease in insulin binding to unmasked receptors, the levels of masked (also called cryptic) insulin receptors were similar (or slightly increased) in transgenic mice microsomes as compared to those of their normal litter mates.     

Immunocytochemical localisation of prolactin and growth hormone in the ovine pituitary

Summary Using the peroxidase-antiperoxidase immunocytochemical staining technique, prolactin and growth hormone cells have been identified and described in the ovine pituitary. The image analysing computer, Quantimet 720, was used to assess accurately the size range of the secretory granules in these cell types. The area size distributions of the prolactin and growth hormone granules are similar. An increased proportion of larger granules was observed in the prolactin cells post-partum. Serial sections stained alternately for prolactin or growth hormone confirmed that the cells contain either prolactin or growth hormone but not both.     

Tissue-specific and expression of porcine growth hormone gene in BAC transgenic mice

One of the primary goals of traditional livestock breeding is to improve growth rate and optimise body size. Growth rate can be significantly increased by integrating a growth hormone (GH) transgene under the control of a ubiquitous promoter, but while such animals do demonstrate increased growth there are also serious deleterious side-effects to the animals health. Here we report the generation and initial characterization of transgenic mice that carried a porcine BAC encoding the porcine GH gene. We show that GH expression is restricted specifically to the pituitary, is associated with elevated IGF-1 levels, and results in growth enhancement. No negative effects to the health of the transgenic animals were detected. This initial characterisation supports the use of BAC pGH transgene in livestock studies.     

Abnormalities of the human growth hormone gene and protooncogenes in some human pituitary adenomas

Hypersecretion of human GH (hGH) or PRL by human pituitary adenomas is not under normal homeostatic control despite normal receptor function mediating the regulatory effects of hypothalamic peptides for these trophic hormones. This implies that the defects underlying hormonal hypersecretion may not reside at the plasma membrane of the adenoma cell; instead, dysregulation may reside at the hormone gene level. To investigate this, genomic DNA derived from a prolactinoma and a hGH-secreting adenoma were digested with the restriction endonuclease EcoRI and the methylation sensitive restriction endonuclease HpaII and hybridized with the 32P-labeled genomic hGH (2.6 kilobase) probe. Our data revealed hypomethylation of genes of the hGH family (hGH and chorionic somatomammotropin) in the absence of gross abnormalities such as gene translocation. In a similar analysis using a 32P-labeled probe consisting of the EcoRI-BamHI (500 base pair) fragment in the 5'-flanking region upstream of the first exon of the hGH gene, hypomethylation of this specific site of the hGH genes was observed. These results are consistent with the concept that hypomethylation of genes is involved in gene expression. At the same time, protooncogene abnormalities in these adenomas were investigated to delineate any genetic basis for their neoplastic growth. Genomic DNA of adenomas were subjected to Southern blotting analysis using a panel of protooncogene probes. Amplification of the v-fos gene was observed in one prolactinoma. The significance of this observation is discussed.     

Ultrastructural immunocytochemical localization of prolactin and growth hormone in the porcine pituitary

Summary The immunocytochemical peroxidase-antiperoxidase technique was used to identify prolactin- and growth hormone-producing cells in the porcine pituitary at the ultrastructural level. The growth hormone-producing cells contain round secretory granules (300 nm to 500 nm in diameter). The prolactin-producing cells can be identified by their distinct round and ovoid secretory granules which vary in size. Most of these cells contain large granules (450 nm to 750 nm in diameter), but some prolactin-producing cells display smaller secretory granules (250 nm to 500 nm). The two hormones were localized exclusively in the secretory granules. Staining for prolactin was observed in round and ovoid granules, as well as in small and polymorphic granules within the Golgi complex. This study confirmed (i) that the two hormones are located in different cells, and (ii) that under normal physiological conditions no one cell can synthesize and store both hormones simultaneously.     

Production of transgenic rabbits and mice carrying the gene for bovine growth hormone

The results of experiments on the transfer of bovine gene for growth hormone into mice and rabbits are presented. The gene was transferred by the technique of microinjection into the zygote. In all cases transgene in rabbits occurred to be changed. In two transgenic mice the bovine growth hormone gene represented some tandem arranged copies. One of the mice had accelerated growth. This phenotypic changes is found to be inheritable.     

Effects of overexpression of growth hormone on T cell activity in transgenic mice

Growth hormone plays a key role in the maturation and maintenance of the immune response, however, the effects of chronic high circulating concentrations of the hormone on the immune system is poorly understood. Transgenic mice overexpressing bovine growth hormone (b-GH) gene, fused to the rat phosphoenolpyruvate carboxykinase promoter (PEPCK), with very high plasma concentration of heterologous b-GH and their littermate normal siblings were used. Spleen cellularity, percentages of total T lymphocytes, CD4+ and CD8+ cells, ratio of T cell subpopulations, mitogen-induced lymphocyte proliferation and natural killer (NK) cell activity were examined in male transgenic mice and normal littermate mice at 2 and 6 months of age. The number of splenic lymphocytes was greater in transgenic mice than in matched normal littermates at both ages. The NK cell activity was lower in transgenic mice than in the matched normal littermates at both ages, with the lowest values found in older mice. The b-GH transgenic mice had lower percentages of T cells at both ages, however, in young transgenic mice, the percentage of CD4+ cells was reduced while percentage of CD8+ cells was increased in comparison to normal controls. Both basal and mitogen-induced proliferation capacity of splenocytes were reduced in PEPCK-b-GH-25 mice as compared to normal littermates of both ages. Proliferative indexes in response to concanavalin A and phytohemagglutinin were markedly decreased in 6 month old PEPCK-b-GH-25 mice as compared to littermate controls or younger mice. These results indicate that overexpression of b-GH in mice is associated with decreased T cell function and that these abnormalities are age-dependent.     

Immunocytochemical localisation of prolactin and growth hormone in the perinatal sheep pituitary

Summary The peroxidase anti-peroxidase immunocytochemical staining technique has been used to identify prolactin and growth hormone cells in pituitaries from fetal and neonatal sheep. The size of the secretory granules in these cell types has been measured using the image analysing computer Quantimet 720. The area size distributions of the fetal prolactin and growth hormone granules were compared with those in the neonate and the adult. It appears that the gestational age of the fetus may influence the size range of prolactin secretory granules.     

Developmental expression of growth hormone and prolactin genes in the bovine pituitary

Cell-free translation was used to initially characterize the major mRNA species present in the bovine anterior pituitary as a function of development. The only detectable change in translation products, which occurred during the transition from fetus to adult, was a reversal in the relative ratio of pituitary growth hormone and prolactin. Subsequent hybridization analysis with cloned growth hormone and prolactin cDNA probes indicated that growth hormone mRNA comprised over 40% of the total fetal mRNA and was 50- to 100-fold higher than prolactin mRNA. The steady state levels of growth hormone mRNA remained relatively constant throughout gestation. In contrast, prolactin mRNA levels, which were low early in gestation, increased during development to become the principal mRNA in the adult pituitary. Since growth hormone and prolactin are synthesized and secreted by specialized cells (somatotrophs and mammotrophs, respectively) immunochemical staining was used to determine whether the changes in the mRNA levels for these two hormones were a reflection of specific cell proliferation. For growth hormone, there was a close correlation between the number of somatotrophs and the relative levels of growth hormone mRNA. In contrast, the increase in prolactin mRNA exceeded the increase in the number of mammotrophs. Thus, the cellular concentration of growth hormone mRNA remains relatively constant during development, while the cellular concentration of prolactin mRNA increases by more than an order of magnitude.     

Expression and regulation of the pituitary- and placenta-specific human glycoprotein hormone alpha-subunit gene is restricted to the pituitary in transgenic mice.

Expression of the glycoprotein hormone alpha subunit occurs in both the pituitary and placenta in humans. However, this study found that expression of this subunit is restricted to the pituitary in mice. An interspecies analysis of human alpha-subunit gene regulation was undertaken, using the transgenic-mouse approach. In mice transgenic for a genomic clone containing the complete human alpha-subunit gene and several kilobases of 5'- and 3'-flanking sequences, cell-type-specific expression and hormonal regulation of the human alpha-subunit transgene occurred in the mouse pituitary, whereas no expression of the transgene was detectable in the mouse placenta. These findings provide strong evidence that a common trans-acting factor(s) regulates glycoprotein hormone alpha-subunit gene expression in the human and mouse pituitaries; however, this factor(s) or a unique factor(s), though functional in the human placenta, is either nonfunctional or absent in the mouse placenta.