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.     

Regulation of growth hormone-releasing hormone gene expression and biosynthesis

Growth hormone-releasing hormone (GRH) was initially isolated, characterized, sequenced, and cloned from human tumors and subsequently from the hypothalamus of humans and other animal species. Extensive structure-function studies have indicated the amino terminus to be most important for its biologic action, and the primary mechanism of its bioinactivation occurs by cleavage of an amino terminal dipeptide. The GRH gene is expressed primarily in the hypothalamic arcuate nucleus but also in the placenta. Expression of the GRH gene is regulated by growth hormone in a classical feedback manner, with hypophysectomy leading to increased expression that is reversed by growth hormone treatment. GRH gene overexpression in transgenic mice leads to a syndrome similar to that of ectopic GRH secretion with massive pituitary hyperplasia and markedly enhanced growth. The transgenic mouse has been used for studies of GRH biosynthesis and provides a suitable model for the study of precursor processing to the mature hormone.     

Regulation of expression of a sheep metallothionein 1a-sheep growth hormone fusion gene in transgenic mice.

Transgenic mice containing a sheep metallothionein 1a-sheep growth hormone fusion gene exhibited low, tissue-specific basal levels of transgene mRNA expression, resulting in slightly elevated levels of circulating growth hormone that did not lead to a detectable increase in growth. After zinc stimulation, high levels of transgene mRNA expression were induced in a number of tissues; these levels correlated with increased levels of circulating growth hormone, resulting in growth increases of up to 1.5 times the levels of controls and unstimulated transgenic mice. After removal of the zinc stimulus, transgene expression and circulating growth hormone concentrations returned to basal levels. Additional evidence from the pattern of developmental expression of the transgene suggests that zinc is the main regulator of this promoter in mice. The demonstrated regulation and low basal level of expression of the sheep metallothionein 1a promoter make it a candidate for use in other mouse transgenic studies and for use in transgenic livestock, in which regulation of expression is essential.     

Interactions of human growth hormone and prolactin on pituitary and Leydig cell function in adult transgenic mice expressing the human growth hormone gene

Adult male transgenic mice expressing the human growth hormone (hGH) gene are hypoprolactinemic. To evaluate the effects of exogenous prolactin (PRL) and endogenously secreted hGH on pituitary and Leydig cell function, adult male transgenic and nontransgenic mice (10-16 wk of age) were treated s.c. with either saline-polyvinylpyrrolidone (PVP) or oPRL (100 micrograms/mouse) in saline-PVP. Animals were treated twice daily; a total of 7 injections were given. One hour after the last injection, each group of mice was treated i.p. either with saline or oLH (0.3 microgram/g BW); 2 h later, blood was obtained via heart puncture. Plasma FSH, LH, PRL, androstenedione (A-dione), and testosterone (T) levels were measured by validated RIAs. Basal PRL levels were significantly lower (p less than 0.001) and basal LH concentrations were significantly higher (p less than 0.01) in transgenic than in nontransgenic mice. Administration of PRL significantly decreased (p less than 0.01) plasma LH levels in transgenic mice, whereas similar treatment of nontransgenic mice increased (p less than 0.01) circulating LH concentrations. Plasma FSH levels were unaffected in transgenic and nontransgenic mice treated with saline or PRL. Basal plasma A-dione and T levels were similar in both groups of animals and were significantly increased after treatment with LH. Administration of PRL increased T levels in transgenic and nontransgenic mice, but the T response to LH treatment was greater in PRL-treated transgenic mice, indicating the synergistic effect of hGH in the biosynthesis of T.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

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.     

Adenoviral vector mediates high expression levels of human growth hormone in the milk of mice and goats

The production of large quantities of complex proteins with biopharmaceutical purposes is the main drawback for their more extensive use. Here we demonstrated that a direct instillation of a recombinant adenoviral vector containing an expression cassette for the human growth hormone gene into the mammary gland of mice and goats allowed for the efficient secretion of human growth hormone in the milk. Through this approach we were able to express human growth hormone at maximal levels of 2.8 mg/ml in the milk of mice and up to 0.3 mg/ml in goat milk. We found that the expression levels were closely dependent on both the degree of differentiation of the secretory epithelium and on the adenoviral dose used. Here we demonstrated that the direct transduction of mammary epithelial cells by means of a recombinant adenovirus could be a suitable alternative to transgenic technology for the production of recombinant proteins of biopharmaceutical interest.     

Seminal vesicle production and secretion of growth hormone into seminal fluid.

Production of foreign proteins in the tissues of transgenic animals represents an efficient and economical method of producing therapeutic and pharmaceutical proteins. In this study, we demonstrate that the mouse P12 gene promoter specific to the male accessory sex gland can be used to generate transgenic mice that express human growth hormone (hGH) in their seminal vesicle epithelium. The hGH is secreted into the ejaculated seminal fluids with the seminal vesicle lumen contents containing concentrations of up to 0.5 mg/ml. As semen is a body fluid that can be collected easily on a continuous basis, the production of transgenic animals expressing pharmaceutical proteins into their seminal fluid could prove to be a viable alternative to use of the mammary gland as a bioreactor.     

Increased milk yield in transgenic mice expressing insulin-like growth factor 1

Insulin-like growth factor 1 (IGF-1) mediates many of the actions of growth hormone. Overexpression of IGF-1 was reported to have endocrine and paracrine/autocrine effects on somatic growth in transgenic mice. To study the paracrine/autocrine effects of IGF-1 in mammary gland, transgenic mice were produced by pronuclear microinjection of a construct containing a bovine alpha-lactalbumin (alpha-LA) promoter linked to an ovine IGF-1 cNDA. This alpha-LA promoter has previously been shown to direct expression of a human factor VIII gene specifically to the mammary gland of transgenic mice. Three transgenic mouse lines were established as a result of microinjection of 398 embryos. Transgene expression was found in mammary gland at day 1 of lactation from these three lines. Progeny test were carried out by mating two transgenic males/one transgenic female to two nontransgenic females/one nontransgenic male. Mice from one line (line 1225) were all nonexpressors and the other (line 1372) failed to produce offspring. Milk yield was analyzed in the line 1137 that produced 10 mice, of which three were transgenic females and three nontransgenic females. All of the three transgenic females showed integration of the transgene and expressed transgene IGF-1 mRNA in the mammary gland. Milk yields from days 5, 10, and 15 of lactation were significant greater in transgenic expressors than in their nontransgenic littermates. Specifically, there is 17.9% increase in total milk yield from these three days for transgenics compared with nontransgenics. These results demonstrate that local overexpression of IGF-1 in transgenic mice is capable to stimulating milk yield during the first lactation.     

Tissue-specific expression and dietary regulation of a chimeric phosphoenolpyruvate carboxykinase/bovine growth hormone gene in transgenic mice

A series of transgenic mice was produced by microinjection of a segment of DNA, containing 460 base pairs of the phosphoenolpyruvate (P-enolpyruvate) carboxykinase promoter-regulatory region ligated to the bovine growth hormone structural gene, into the male pronucleus of fertilized mouse eggs. Founder animals which contained the gene were selected for further analysis and for breeding. The concentration of bovine growth hormone in the serum of animals which were shown to contain the gene ranged from a low of 5 ng/ml serum to approximately 2300 ng/ml serum. Mice with high levels of bovine growth hormone had growth rates double that of their litter mates which did not contain the transgene. The transgene was expressed only in the liver and kidney of the animals studied, and the level of specific mRNA for bovine growth hormone in these tissues could be regulated by diet in a manner similar to the endogenous P-enolpyruvate carboxykinase gene. Feeding the animals a diet high in carbohydrate for 1 week caused a 90% decrease in the concentration of bovine growth hormone in the blood, suggesting that the expression of the chimeric P-enolpyruvate carboxykinase/bovine growth hormone gene is sensitive to insulin. When the same animals were then refed a diet high in protein, but devoid of carbohydrate, the concentration of bovine growth hormone in their blood was induced 30-fold within a week. The administration of dibutyryl cyclic AMP to the transgenic mice caused a 2-fold induction in the level of bovine growth hormone in the serum within 90 min. Thus the region between -460/+73 in the P-enolpyruvate carboxykinase promoter-regulatory region contains sequences which can direct the tissue-specific expression, as well as hormonal and dietary responsiveness, of a linked structural gene.     

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.     

Ovarian follicle apoptosis in bovine growth hormone transgenic mice

Growth hormone directly or via insulin like-growth factor-I has been shown to inhibit preovulatory follicle apoptosis, which is the underlying mechanism of follicular atresia. We studied the levels of apoptosis in the ovaries of transgenic mice expressing bovine growth hormone. Female bovine growth hormone transgenic mice (n = 10) and nontransgenic litter mates (n = 8) were killed at early proestrus. Ovaries were collected, sectioned, and processed using a nonradioactive in situ method for apoptosis detection. Follicles were classified and counted on the basis of size and level of apoptosis. Our results demonstrate that the percentage of ovarian follicles containing apoptotic cells was lower in transgenic versus normal mice (30% vs. 46%; P < 0.05). The percentage of follicles undergoing heavy apoptosis was lower (P < 0.05) in transgenic versus control animals in preovulatory and early antral follicles, but it was not different in preantral follicles. The percentage of healthy preovulatory follicles was also higher in transgenic versus normal mice (7.4% vs. 4.3%; P < 0.05). These results indicate that growth hormone overexpression in transgenic mice significantly decreases follicle apoptosis, and thus atresia in the mouse ovary, therefore leading to increased propensity for ovulation in these animals.     

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.     

Rabbit whey acidic protein gene upstream region controls high-level expression of bovine growth hormone in the mammary gland of transgenic mice

Transgenic mice were produced which secreted high levels of bGH into milk. The 6.3-kb upstream region of the rabbit whey acidic protein (rWAP) gene was linked to the structural part of the bovine growth hormone (bGH) gene, and the chimeric gene was introduced into mouse oocytes. bGH was detected by radioimmunoassay in the milk of all resulting transgenic mice. bGH concentrations in milk varied from line to line, from 1.0–16 mg/ml. This expression was not correlated to the number of transgene copies. In all lines studied, the mammary gland was the major organ expressing bGH mRNA during lactation. bGH mRNA concentrations were barely detectable in the mammary gland of cyclic females; they increased during pregnancy. These results show that the upstream region of the rWAP gene harbors powerful regulatory elements which target high levels of bGH transgene expression to the mammary gland of lactating transgenic mice. © 1995 wiley-Liss, Inc.