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We have used immunohistochemistry and non-radioactive in situ hybridisation to localise the GH receptor and its transcript in the bovine mammary gland during mammogenesis, lactation and involution. We found a characteristic pattern of immunoreactive GH (irGH) receptor distribution in the epithelial and stromal compartments during the different stages of mammary gland development: The ductular epithelium showed a distinct staining for irGH receptor during most stages, whereas the alveolar epithelium contained a modest amount of GH receptor during pregnancy which increased during lactation and galactopoiesis. In dry cows, the immunostaining for GH receptors in the alveolar epithelium was very weak or negative. Curiously, the amount of GH receptor mRNA appeared relatively constant during mammogenesis and lactation. The epithelial cells of the alveoli and ducts as well as the endothelial cells showed a distinct signal in our in situ hy! bridisation studies. The predominant localisation of GH receptors in the epithelium of ducts and alveoli is supportive of a role for GH in epithelial differentiation and maintenance. Furthermore, the increased intensity of immunostaining in bovine mammary tissue post partum suggests a direct role for GH receptor in mediating the effect of GH in milk production and secretion.

The production of peptide hormones by skeletal muscle tissue is a promising area of gene therapy. Skeletal muscle myogenesis can be induced in vitro, resulting in the fusion of mononucleate myoblasts to form multinucleate myotubes, and delivery vectors are first tested in vitro. C2C12 myoblasts transfected with pcDNA3-GH, which used the human cytomegalovirus (CMV) promoter, secreted immunoreactive GH with comparable biological activity to pituitary GH. Mouse myeloid leukaemia cells, which express the mouse GH receptor were used for the bioassay, and activation of these cells by GH was measured by a colorimetric microculture tetrazolium assay. Cells were incubated with a tetrazolium salt (MTS) and an intermediate electron acceptor (phenazine methosulphate, PMS), and formazan production was measured as optical density (O.D.) at 490 nm. The efficiencies of several plasmid expression vectors were compared in differentiated and non-differentiated muscle cells, as a function of bioactive GH secreted by the transfected cells. Ten-day differentiated C2C12 myotubes transfected with pcDNA3E-GH, which used the CMV promoter and a rat myosin light chain enhancer element, secreted significantly more biologically active GH than myotubes transfected with pcDNA3-GH (0.82 O.D. units+/-0.06 vs 0.57+/-0.05 respectively, P

Based on localization studies of the GH receptor/binding protein (BP) in the gastrointestinal tract, we have recently demonstrated growth hormone regulation of gastric intrinsic factor. In order to define the role of GH in the submandibular gland (SMG) we have investigated the effect of GH on SMG structure and function with particular reference to haptocorrin. Bovine GH (65 micrograms/100 g body weight) was administered twice daily to adult male dwarf rats for 6 days (DW+) while control animals received vehicle (DW-). Administration of GH produced a significant increase in body weight (P < 0.001) and allometric increase in SMG weight (P = 0). There was no change in RNA or protein content per g SMG and GH administration produced a small decrease in DNA content normalized to SMG weight. Morphometric analysis of the SMG revealed a significant increase in the percentage area of the gland occupied by tubular (GH receptor/BP expressing) structures and a significant increase in the diameter of both the intralobular striated and granular convoluted tubules. The effect of GH on cellular proliferation in the ductular and acinar components was determined by the immunohistochemical detection of nuclear 5'-bromo-2'-deoxyuridine (BrdU) incorporated during a 2-h pulse of BrdU. GH treatment induced a 5.5-fold increase in the labelling index of tubular cells whereas the acinar cell labelling index increased only 3.3-fold. Soluble extracts of SMG were prepared for estimation of 57Co-cyanocobalamin (vitamin B12) binding. GH administration resulted in an increase in total 57Co-cyanocobalamin (CBL) binding per mg SMG protein. To determine the contribution of haptocorrin (R-protein) the amount of cobinamide dicyanide (CD) displaceable binding was calculated. GH administration produced a 70% increase in CD displaceable CBL binding per mg SMG indicating GH regulation of haptocorrin. A comparison of total SMG CBL binding and CD displaceable CBL binding between male and female rats detected no sex difference. Therefore sex-specific GH secretory profiles are unlikely to be of importance in the regulation of haptocorrin. In conclusion we have demonstrated that GH stimulates hypertrophy and hyperplasia of components of the SMG in the dwarf rat. The observed upregulation of haptocorrin may synergize with the GH-stimulated increase in intrinsic factor to facilitate absorption of CBL during the anabolic state.

There is evidence that prolactin (PRL) influences gastrointestinal function. However, the sites at which prolactin exerts these effects are not known. A monoclonal antibody was therefore generated against the rabbit mammary gland prolactin receptor (MAb 218) and used to study the distribution of the prolactin receptor in the rabbit gastrointestinal tract (GIT) by immunohistochemistry. MAb 218 is an IgG 1 κprecipitating antibody which precipitates major affinity cross-linked mammary gland prolactin receptor subunits of molecular masses 45 and 80 kDa. It has an affinity of 0·8 × 109 mol/l for the prolactin receptor and does not react with GH or insulin receptors in precipitation assays. MAb 218 immunoreactivity was observed in classical prolactin target cells such as mammary gland epithelium, and this immunoreactivity was abolished by preincubation of MAb 218 with purified prolactin receptor but not by preincubation with purified GH receptor. In the GIT, the most intense immunoreactivity was associated with the oesophageal epithelium, chief (zymogenic) cells of the fundic mucosa, pancreatic islets of Langerhans and surface epithelial cells of the duodenum and jejunum. Other specific elements of the GIT were immunoreactive at lower levels or were immunonegative. No immunoreactivity was observed in these locations with a control monoclonal antibody (MAb 50·8) of identical isotype to 218. To support the immunohistochemical findings, rabbit gastric mucosal membranes were used to show the presence of lactogen-specific binding. Scatchard analysis of 125I-labelled human GH binding to the gastric mucosal membranes with rat prolactin as displacing ligand yielded an affinity constant (Ka) of 1·0 ± 0·2 × 109 mol/l with a capacity of 3·5 ± 0·4 fmol/mg protein. Affinity cross-linking and sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the gastric receptor revealed lactogenic hormone-binding subunits of molecular masses 43, 68 and 83 kDa. The 68 kDa subunit was not seen in rabbit mammary gland or ovarian tissue, and may be unique to gastric mucosa. In conclusion, we have demonstrated the presence of a high affinity lactogenic receptor in specific epithelial cell subpopulations of the GIT. This localization of the prolactin receptor in the GIT will assist in further functional assignment of prolactin to gastrointestinal physiology. Journal of Endocrinology (1993) 139, 371–382

Thyrotrophin (4-256 μU/ml) promoted an increase in the rate of release of radioiodine from the organic iodine pool of cultured porcine thyroid cells in follicular formations. This action of TSH was antagonized by low concentrations of epidermal growth factor (EGF; 0·1–5 nmol/l). The maximal effect of EGF was reached by 0·5 nmol/l. EGF (0·5–5 nmol/l) also inhibited the stimulatory effect of 8-chloro cyclic AMP (0·06–1·0 nmol/l) on radioiodine turnover. Exposure of thyroid cultures to media with a calcium concentration of 17·7 μmol/l (1% of normal) resulted in a very marked increase in the rate of release of radioiodine. The effect of TSH in low-calcium media was to inhibit the increased release of radioiodine, and EGF (0·5 nmol/l) antagonized this inhibitory effect of TSH. The calcium ionophore, A23187, stimulated radioiodine release in a dose-dependent fashion, and EGF (1·7 nmol/l) inhibited this response. Fluid transport in thyroid monolayers was stimulated by prostaglandin E2 (PGE2; 1 μmol/l). EGF (5 nmol/l) also stimulated fluid transport, but antagonized the effect of PGE2 added subsequently. It was concluded that EGF exerted acute antagonistic effects on thyroid cell responses in vitro to cyclic AMP and agents promoting accumulation of cyclic AMP in time-frames too short for these inhibitory effects to be attributable to the dedifferentiative effect of the growth factor. Journal of Endocrinology (1991) 128, 213–218

Acromegaly is characterized by coarsening of facial features, acanthosis nigricans, hypertrichosis and oily skin. To determine the site through which GH exerts these effects, we have used immunohistochemistry to localize the GH receptor/binding protein (BP) in rat, rabbit and human skin. Three monoclonal antibodies (MAb 1, 43, 263) were immunoreactive in identical locations, whereas no immunoreactivity was evident when control monoclonal antibodies (MAb 50.8 and MAb 7 (rat)) were used. Skin from neonatal and adult animals was used to determine whether GH receptor/BP expression was developmentally regulated. Immunoreactivity of the GH receptor/BP in the three species was consistently localized in the stratum basale and stratum spinosum. Intermittent staining was observed in the stratum granulosum. Scattered basal epidermal cells often displayed more intense immunoreactivity. This distribution was observed at all maturational stages examined. Intense GH receptor/BP immunoreactivity was observed in all histological layers of the lower one-third of hair follicles and in hair matrix cells of the dermal papillae. Immunoreactivity was also detected in the outer epithelial root sheath of the upper two-thirds of hair follicles, in sebaceous glands and in fibroblasts of the connective tissue sheath surrounding the follicle. GH receptor/BP immunoreactivity was also present in the secretory duct and myoepithelial cells of human eccrine sweat glands. Fibroblasts, Schwann cells of peripheral nerve fascicles, skeletal muscle cells and adipocytes of the dermis were also immunoreactive as were medial smooth muscle and endothelial cells of arteries. These results provide evidence that GH acts locally on the epidermis and epidermal appendages concordant with our recent localization of GH receptor/BP to epithelial cell types of the gastrointestinal and reproductive systems. Journal of Endocrinology (1990) 126, 467–472

Hyperglycaemia was produced in chronically catheterized fetal lambs and pregnant ewes by the infusion of glucose into the fetus. Plasma concentrations of placental lactogen did not change significantly in either fetal or maternal circulations. Fetal and maternal hypoglycaemia was induced by administration of insulin to the fetus and ewe separately. Plasma concentrations of placental lactogen in the fetus did not change significantly but maternal plasma concentrations fell slightly after hypoglycaemia in either fetus or ewe. Plasma concentrations of placental lactogen rose in both the ewe and fetus during prolonged fasting of the ewe. These results neither confirm nor refute a role for placental lactogen in intermediary metabolism of the pregnant ewe and fetus but glucose concentration alone is unlikely to be a significant factor in the control of secretion of this hormone.

A panel of monoclonal antibodies (MAbs) reactive with distinct epitopes on the rabbit liver GH receptor and rabbit serum GH-binding protein (GHBP) were tested for cross-reactivity with the GHBP from human serum. Four of seven MAbs reacted with the human serum GHBP. Immunoprecipitation of the human binding protein enabled hormonal specificity identical to that previously reported for human GH receptors to be demonstrated. Scatchard analyses of 125I-labelled human GH binding to the serum GHBP were carried out with correction made for endogenous human GH which was measured by radioimmunoassay of each serum sample. This approach yielded the first reliable estimates of the affinity and capacity of the human GHBP. The binding capacity (mean ± s.e.m.) of female sera (804±126 pmol/l; n= 6) was greater than that of male sera (505 ± 36 pmol/l; n=9; P < 0·02). The affinity of the GHBP was 0·91 ±0·10 litres/nmol (n= 15). The presence of multiple epitopes common to the human serum GHBP and the rabbit liver GH receptor is consistent with identity between the extracellular domains of the human GHBP and the human GH receptor, as is the case for the rabbit GHBP and GH receptor. Journal of Endocrinology (1989) 123, 327–332