SUMMARY

A method is described for the quantitative bioassay of melanocytestimulating hormone (MSH) activity using the skin of the lizard Anolis carolinensis in vitro. The colour changes were registered photoelectrically as changes in reflexion of green monochromatic light. Synthetic α-MSH (CIBA-Geigy Ltd) was used as standard. The method can detect MSH activity at a concentration down to 0·01–0·05 ng α-MSH/ml, and allows the detection of MSH activity in normal rat plasma. The mean precision of the assay λ is about 0·13. This assay thus combines high sensitivity with good precision, and is also comparatively rapid, easy to handle and inexpensive.

It is well known that high levels of glucocorticoids cause osteoporosis and that physiologic levels of growth hormone (GH) are required for normal bone remodeling. It has been suggested that glucocorticoids regulate GH-responses via the regulation of GH-receptor expression. The aim of the present study was to investigate whether cortisol plays a role in the regulation of GH-receptor expression in cultured human osteoblasts. The effect of serum starvation and cortisol on GH-receptor expression was tested in human osteoblast (hOB)-like cells. Serum starvation for 24 h resulted in an increase in GH-receptor mRNA levels (90 +/- 1% over control culture). Cortisol increased GH-receptor mRNA levels in a dose-dependent manner with a maximal effect at 10(-6)M. The stimulating effect of cortisol on GH-receptor mRNA levels was time-dependent, reaching a peak 12 h after the addition of cortisol (126 +/- 29% over control culture) and remaining up to 12 h later. The increase in GH-receptor mRNA levels was accompanied by an increase in 125I-GH binding which reached a maximum at 24 h (196 +/- 87% over control culture). In conclusion, glucocorticoids increase GH-receptor expression in hOB-like cells. Further studies are needed to clarify whether glucocorticoid-induced regulation of the GH-receptor is important in human bone physiology.

ABSTRACT

The influence of various culture conditions was studied on the effect of GH and insulin-like growth factor-I (IGF-I) on DNA and matrix synthesis in epiphyseal rat chondrocytes in monolayer culture.

Chondrocytes from enzymatically digested rat tibia epiphyseal growth plates were seeded in 48-well culture plates and precultured for 10 days in Ham's F-12 medium supplemented with 1% (v/v) newborn calf serum and 1% (v/v) of a serum substitute. After preculture, the medium was changed to Ham's F-12 medium supplemented with 1% serum from hypophysectomized rats, and the effect of GH and IGF-I on DNA synthesis ([³H]thymidine incorporation) and matrix production ([³⁵S]sulphate uptake) was studied during an additional 96-h culture period. Isotopes were present during the last 24 h of culture.

Both hGH and IGF-I stimulated DNA synthesis in a dose-dependent manner. A maximal effect of GH was seen at a concentration of 25 μg/l (60 ± 11% stimulation over control) and for IGF-I at 10 μg/l (162 ± 12%). The stimulatory effects of the same concentrations of human GH (hGH) and IGF-I on [³⁵S]sulphate uptake were 135 ± 25 and 320 ± 42% respectively. In-vitro pulse labelling revealed that GH did not produce a response during the first 3 days of culture (after addition of GH) but was effective during days 4 and 5 of culture. In contrast, IGF-I was effective throughout the culture period. Pretreatment of cells with GH or IGF-I for 2·5 days showed that GH but not IGF-I produced a sustained effect on [³H]thymidine uptake.

In order to study the influence of cell density on the effect of GH and IGF-I on DNA synthesis, the effect of added peptides was evaluated after different preculture periods (5–15 days). A maximal stimulatory effect of hGH was seen at a cell density of 150 000–300 000 cells/cm². GH had no significant effect at a low (< 100 000 cells/cm²) or a high (>400 000 cells/cm²) cell density. The magnitude of the stimulatory effect of IGF-I was the same at densities between 10 000 and 250 000 cells/cm², but was reduced at higher cell densities (over 250 000 cells/cm²).

Chondrogenic properties of cells that had been cultured for 15 days were verified in vitro by positive alcian blue staining and identification of type II collagen, and in vivo by development of cartilage nodules in nude mice.

The results from the present study clearly show that GH and IGF-I both stimulate DNA synthesis and matrix production in epiphyseal chondrocytes in monolayer culture. The results also demonstrate that expression of the effect of GH is highly dependent upon the culture conditions.

Journal of Endocrinology (1992) 133, 291–300

ABSTRACT

In the present study a double-staining technique was developed to investigate simultaneous GH and insulin-like growth factor-I (IGF-I) binding to chondrocytes in a monolayer cell culture.

Rat tibial epiphyseal chondrocytes were isolated by enzymatic digestion and cultured in monolayer. GH and IGF-I were labelled with biotin. The affinity of the biotin-labelled ligands was compared with unlabelled ligands in a radioreceptor assay.

To study the distribution of GH and IGF-I binding in the monolayer, chondrocytes were incubated with biotinylated ligands with or without an excess of unlabelled ligands, followed by incubation with Vectastain ABC complex, which was then reacted with diaminobenzidine (DAB). Double staining was accomplished by carrying out the first reaction with DAB in the presence of nickel ammonium sulphate to give a black precipitate, followed by incubation with the second ligand, then ABC complex and finally DAB in the absence of nickel ammonium sulphate to give a brown stain. The presence of type-II collagen was demonstrated by immunohistochemistry and used as a marker for differentiated chondrocytes.

Biotin-labelled GH and biotin-labelled IGF-I exhibited dose-dependent displacements of ¹²⁵I-labelled GH and ¹²⁵I-labelled IGF-I respectively from the chondrocytes in a radioreceptor assay. The displacement curves were identical to those of unlabelled ligands indicating that the affinity was unaltered. Binding of biotinylated GH to cells was seen throughout the culture in regions where there was little or no type-II collagen staining. IGF-I binding was predominantly localized to cells at high density; areas which also showed a high degree of staining for type-II collagen.

The different locations of binding suggest that epiphyseal chondrocytes in monolayer culture comprise a heterogeneous cell population and that IGF-I and GH have different target cells.

Journal of Endocrinology (1993) 137, 361–367

ABSTRACT

The effect of human GH (hGH) and insulin-like growth factor-I (IGF-I) on colony formation of rabbit epiphyseal tibial chondrocytes at different stages of maturation was studied in suspension culture. The epiphyseal growth plate from the proximal tibia of 8-week-old male rabbits was dissected and divided into three different (proximal, intermediate and distal) zones, each zone representing an enrichment of chondrocytes from the germinative, proliferative and hypertrophic cell layers respectively. Chondrocytes from these three zones were isolated by collagenase digestion and cultured in the presence of 10% (v/v) newborn calf serum in suspension stabilized with 0·5% (w/v) agarose for 14 days.

The colonies were classified according to diameter and the number of colonies was estimated as a function of colony size (distribution of cloning efficiency). Cell clusters with a diameter of 56 μm or more were classified as colonies. The cloning efficiency (number of colonies formed per 1000 seeded cells) was 10·1 ± 0·7 and 6·0 ± 0·8 for chondrocytes isolated from the proximal and intermediate zones respectively.

Insulin-like growth factor-I (25–200 ng/ml) increased the number of colonies in chondrocytes isolated from the proximal zone (122 ± 9·0–156 ± 8·4%; control value, 100%) and from the intermediate zone (136 ± 14·0–191 ± 29%). Low concentrations of hGH (10–40 ng/ml) stimulated colony formation in chondrocytes isolated from the proximal zone (125 ± 6·4– 137 ± 7·9%) whereas a high concentration of hGH (160 ng/ml) was ineffective. Chondrocytes isolated from the intermediate zone showed no response to low concentrations of hGH (10–20 ng/ml). High concentrations of hGH (40–160 ng/ml) reduced colony formation of chondrocytes from this zone (81 ± 9·1–53 ± 4·2% of control value). Chondrocytes isolated from the distal zone formed either none or an insignificant number of colonies in the absence or presence of hGH or IGF-I.

Both IGF-I and hGH caused a significant change in distribution of cloning efficiency in chondrocytes isolated from the proximal zone. IGF-I potentiated the formation of small colonies (colony diameter 64–176 μm) while hGH increased the number of large colonies (colony diameter 112–320 μm). In fact, very large colonies (colony diameter > 272 μm) were only seen in cultures containing hGH. Among chondrocytes isolated from the intermediate zone, IGF-I stimulated colonies of all sizes and no change was seen in the distribution of cloning efficiency. Human GH at 160 ng/ml significantly altered the distribution of cloning efficiency in chondrocytes from the intermediate zone due to an inhibitory effect on the formation of colonies of small size.

These results show that the cloning efficiency of epiphyseal chondrocytes in suspension culture is dependent upon their previous spatial location in the intact growth plate.

The finding that GH stimulated the formation of colonies of large size cells in from the proximal part of the growth plate indicates that GH interacts with a limited number of cells which exhibit a high proliferative capacity, e.g. with stem cell chondrocytes or early proliferative chondrocytes.

J. Endocr. (1987) 115, 263–271

ABSTRACT

Growth hormone has been reported to exert direct effects on rat and rabbit epiphyseal chondrocytes in vitro, indicating that GH interacts with specific receptors on these cells. To investigate this possibility, binding of GH to cultured rat epiphyseal chondrocytes was studied under various experimental conditions. Chondrocytes were isolated enzymatically from epiphyseal growth plates of the proximal tibia of 20-day-old male rats and were cultured in monolayer in Ham's F-12 medium supplemented with 10% calf serum and 1% of a serum substitute. The cells were seeded at various densities (25 000–200 000 cells/well) and cultured for 5–16 days. Twenty-four hours before binding experiments, the medium containing calf serum was changed for one containing serum obtained from hypophysectomized rats, in order to avoid binding of GH present in the calf serum. Binding was studied by incubating ¹²⁵I-labelled human GH (hGH) with the cells in the presence or absence of various concentrations of unlabelled hGH, bovine GH (bGH), rat GH (rGH) and ovine prolactin (oPRL).

Specific binding could be demonstrated in cells cultured for 5–16 days. Binding was dependent upon time and temperature, and maximal binding was obtained by incubating the labelled hormone for 4–6 h at 24 °C. An increase in binding was noted between 7 and 12 days in culture. In cells cultured for 12 days, addition of unlabelled hGH, bGH or rGH caused a dose-dependent displacement of ¹²⁵I-labelled hGH, whereas oPRL was ineffective. Scatchard analysis resulted in a linear plot, and the number of binding sites/cell was approximately 5700, with a dissociation constant of 0·46 nmol/l. The increase in binding between days 7 and 12 was independent of the density of seeded cells, but total binding was higher if the cells were seeded at a low density. By using a monoclonal antibody to the rabbit GH receptor, specific staining could be demonstrated immunohistochemically in the cultured cells. The results show the presence of GH receptors in cultured rat epiphyseal chondrocytes and also show that the culture conditions influence the expression of GH receptors.

Journal of Endocrinology (1989) 122, 69–77

ABSTRACT

The effects of tri-iodothyronine (T₃) and insulin-like growth factor-I (IGF-I) on [³H]thymidine incorporation, alkaline phosphatase (ALP) activity and IGF-I receptor mRNA levels were studied in rat epiphyseal chondrocytes cultured in monolayer.

Chondrocytes from enzymatically digested rat tibia epiphyseal growth plates were seeded in monolayer culture and precultured for 7–14 days in Ham's F-12 medium supplemented with 10% (v/v) newborn calf serum and 1% (v/v) of a serum substitute. After preculture the medium was changed to Ham's F-12 medium containing 1% (v/v) serum from hypophysectomized rats, and the effects of T₃ and/or IGF-I on DNA synthesis ([³H]thymidine incorporation), ALP activity (a late marker of differentiated epiphyseal chondrocytes) and IGF-I receptor mRNA levels were studied.

ALP activity was increased by T₃ in a dose-dependent manner with a maximal response at 10 μg T₃/1 (678 ±86% compared with control culture). The increase in ALP activity was accompanied by a concomitant decrease in [³H]thymidine incorporation (52 ±14% compared with control culture). Human GH (hGH; 50 μg/l) and IGF-I (25 μg/l) had no stimulatory effect on ALP activity. However IGF-I (10 μg/l) exerted an inhibition on the T₃ (10 μg/l)-induced increase in ALP activity (64 ± 9% compared with T₃-treated culture). T₃ (3 μg/l) inhibited the increase in [³H]thymidine incorporation caused by 25 μg IGF-I/l(51 ± 13% compared with IGF-I-treated culture). Furthermore, IGF-I receptor mRNA levels were increased by 10 μg T₃/l (137 ±4·2% compared with control culture) while no effect of hGH (50 μg/l) or IGF-I (25 μg/l) was demonstrated.

Both T₃ and IGF-I were shown to interact with epiphyseal chondrocytes and both substances seemed to affect cell proliferation and maturation and therefore longitudinal bone growth. Furthermore, the results indicated that IGF-I is important for proliferation of the cells while T₃ initiates the terminal differentiation of epiphyseal chondrocytes.

Journal of Endocrinology (1992) 135, 115–123

ABSTRACT

Levels of mRNA for the insulin-like growth factor-I (IGF-I) in rat heart and skeletal muscle and its dependence on GH were investigated using a solution hybridization assay. Levels of IGF-I mRNA decreased following hypophysectomy, and replacement therapy with human GH (hGH) normalized heart and skeletal muscle levels. The stimulatory effect of hGH was dose-dependent, the lowest effective dose being 100 μg. A significant increase of IGF-I mRNA was observed 60 min after s.c. administration of 100 μg hGH and the maximum increase was apparent 6–12 h after hGH injection. Administration of 200 μg IGF-I or 11 μg insulin did not significantly change levels of IGF-I mRNA. The results show that GH regulates the level of IGF-I mRNA in rat heart and skeletal muscle and give further support to the hypothesis that locally produced IGF-I might be a local mediator for the direct stimulatory effect of GH on the growth and development of heart and skeletal muscle.

Journal of Endocrinology (1989) 120, 107–112

ABSTRACT

Expression of insulin-like growth factor I (IGF-I) mRNA and its dependence on GH was studied in rat epiphyseal growth plate by in-situ hybridization. Methodological aspects of the technique were first evaluated on fixed sagittal cryosections from 28-day-old rat epiphyseal growth plates to ascertain specific hybridization. In-situ hybridization was compared on sections from 10- and 28-day-old rats and the GH-dependence was studied in 35-day-old hypophysectomized rats. Expression of IGF-I mRNA was apparent in chondrocytes of the proliferative, hypertrophic and early degenerative zones of the growth plate of 28-day-old rats. The epiphyseal growth plate from 10-day-old rats showed a weak hybridization signal compared with 28-day-old rats. In contrast, the mesenchymal cells of the periosteum of 10-day-old rats showed a rather strong hybridization signal. Hypophysectomy resulted in a reduction in hybridization signal and cell number of the growth plate compared with 35-day-old age-matched normal rats. GH-Replacement therapy (200 μg human GH s.c. every 4 h for 24 h) resulted in partially restored expression of IGF-I mRNA.

The present study has shown that the IGF-I gene is expressed in the rat epiphyseal growth plate chondrocytes and that the expression is dependent on GH. The results support a paracrine/autocrine role of IGF-I for the expression of the stimulatory effect of GH on longitudinal bone growth.

Journal of Endocrinology (1990) 125, 67–74

Events occurring early in life or prenatally are able to play important roles in the pathogenesis of diseases in adult life. Different sorts of stress or hormonal influences, during particular periods of pregnancy, may result in persisting or transient changes in physiology. Glucocorticoids are used for the treatment of a variety of diseases, to promote organ maturation and to prevent preterm delivery. Glucocorticoids are also known to affect skeletal growth and adult bone metabolism. The aim of the present study was to investigate whether exposure to dexamethasone (Dex) during fetal life has any effect on skeletal growth and/or bone mineral density in adult rat offspring. Pregnant rats were given injections of either Dex (100 micro g/kg) or vehicle on days 9, 11 and 13 of gestation. Dex-exposed male but not female rat offspring showed transient increases in crown-rump length and tibia and femur lengths at 3-6 weeks of age. In contrast, the cortical bone dimensions were altered in 12-week-old female but not male Dex-exposed offspring. The areal bone mineral densities of the long bones and the spine, as determined by dual X-ray absorptiometry, and trabecular as well as cortical volumetric bone mineral density, as measured using peripheral quantitative computerized tomography, were unchanged in both male and female Dex-exposed offspring. In conclusion, prenatal Dex exposure affects skeletal growth in a gender-specific manner, while the mineralization of bones is unaffected in both male and female offspring.