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Insulin-like growth factor I (IGF-I) is required for the maintenance of differentiated functions of bovine adrenal fasciculata cells in culture. We have investigated, by immunocytochemistry, the presence of IGF-I in cells cultured in the absence or presence of corticotropin (ACTH) and angiotensin II (A-II), as well as the secretion of IFG-I and its binding proteins (IGF-BP). In control cultures, very few cells were specifically stained with the anti-IGF-I serum. Following 2 days treatment with A-II (10(-6)M) or ACTH (10(-8)M) the number of stained cells increased 5 and 14 fold, respectively. In all cases the staining was specific since it was abolished when non-immune rabbit serum replaced the anti-IGF serum or when the anti-IGF-I serum was preincubated with saturating concentrations of the peptide. Under the same experimental conditions the secretion of IGF-I in the medium, evaluated by a specific radioimmunoassay, was increased 2- and 7-fold by A-II and ACTH, respectively. Using the method of western ligand blot, we found that the major form of IGF-BP secreted by control adrenal cells is a 38-42 kDa doublet protein. Two minor forms with apparent mol wt of 28-31 kDa and 24 kDa have also been identified. Following acid-ethanol extraction of the conditioned medium all the IGF-BP were recovered in the pellet, whereas most of the IGF-I was in the supernatant. ACTH and, to a lesser extent.(ABSTRACT TRUNCATED AT 250 WORDS)

In this paper the effects of growth factors on the differentiated function of pig Leydig cells and other steroidogenic cells are reviewed. Two types of action have been observed, i.e. positive or negative acute effects on testosterone secretion, and long-term trophic effects of hCG receptor and responsiveness to hCG. Among the growth factors, insulin-like growth factor I (IGF-I) and transforming growth factor beta (TGF beta-1) are of particular interest. IGF-I is required for the maintenance and probably the expression of differentiated functions of several steroidogenic cells, including the Leydig cells. TGF beta-1 has effects opposite to IGF-I on Leydig cell functions. When considering effects of growth factors on Leydig cells, caution should be taken in extrapolating results obtained in one species to another.

The production of insulin-like growth factor I (IGF-I) by pig Leydig cells and pig Sertoli cells cultured alone or together was investigated. Human chorionic gonadotropin (hCG) and basic fibroblast growth factor (FGF) stimulate in a dose-dependent manner IGF-I production by Leydig cells. At maximal concentrations the effects of both factors were almost additive. Insulin at micromolar concentrations enhanced IGF-I production and potentiated the effects of hCG and FGF. The secretion of IGF-I by Sertoli cells was stimulated by FSH and FGF. Under basal conditions, the production of IGF-I by the coculture was similar to the addition of the production by each cell cultured alone. In contrast, in the presence of hCG, FSH or FGF, the production of IGF-I by the coculture largely exceeded that expected from the monocultures. Moreover, stimulation of the coculture with both hCG and FSH resulted in a further increase in IGF-I production. These results indicate that Leydig as well as Sertoli cells secrete IGF-I and that the secretion of both cell types is stimulated by the corresponding gonadotropin. In addition, they indicate that Leydig-Sertoli interactions play a role in the control of IGF-I production, supporting the contention that this growth factor plays an important role in the paracrine and autocrine control of testicular functions.

Most of the somatomedin in serum exists as a high molecular weight complex (approximately equal to 140,000) composed of binding protein subunits and small molecular weight somatomedin. These binding proteins may interfere with measurements of the somatomedins by various radioligand assays. In a companion paper, we reported that when serum is incubated at neutral pH the detectability of the somatomedin-C (Sm-C) is increased. Using gel chromatography, however, it was not possible to demonstrate release of free Sm-C from the macromolecular complex. In the studies reported here, incubation of heparinized plasma at pH 7.4 followed by gel chromatography in a heparin-containing buffer caused 70-80% of the immunoreactive Sm-C to shift from the gamma-globulin region to a molecular weight which approximates that of free Sm-C. This conversion is a time-dependent process which is inhibited by the proteolytic enzyme inhibitor antipain. Similar changes in the elution profile of Sm-C were observed when heparinized plasma was acidified and chromatographed in heparin. These findings suggest that 1) at neutral pH, serum proteolytic enzymes reduce the affinity between small molecular weight Sm-C and its binding proteins. 2) At acid pH, a similar effect is observed. 3) In the presence of heparin, reassociation of these components does not occur. These results suggest a possible mechanism whereby the somatomedin macromolecular complex could be disrupted so that small molecular weight somatomedin is made available to tissues.

The effects of insulin, somatomedin-C (Sm-C), epidermal growth factor (EGF), fibroblast growth factor (FGF), vitamin E, and retinoic acid on growth and function of immature cultured pig Sertoli cells were investigated. All these factors, except vitamin E, stimulated Sertoli cell DNA synthesis and proliferation. The mitogenic effects of insulin observed only at micromolar concentrations were similar to those induced by nanomolar concentrations of Sm-C or EGF, but significantly less than those induced by FGF. The effects of EGF and Sm-C were almost additive, whereas those of Sm-C and FGF were synergistic. After a 6-day treatment, FGF and retinoic acid induced a significant increase in the number of follicle-stimulating hormone (FSH) receptors per cell, and in FSH-induced cyclic adenosine 3',5'-monophosphate (cAMP) production. Sm-C, which alone had no effect on these two parameters, potentiated FGF action. Basal plasminogen activator activity was enhanced after the 6-day treatment with EGF plus insulin and, particularly, with FGF plus insulin. Similarly, the response of the latter group to FSH was significantly higher than in any other group of cells. FGF was also able to stimulate cell multiplication and enhanced the FSH receptor number of Sertoli cells isolated from 15- and 26-day-old rats. Thus, FGF is the most potent known mitogenic factor for cultured Sertoli cells, and it stimulates the phenotypic expression of these cells.

The potential regulatory action of Sertoli cells on Leydig cell functions has been investigated by using a coculture system of Leydig and Sertoli cells obtained from immature pig or by culturing Leydig cells with Sertoli cell-conditioned medium (SCCM). Coculture of Leydig and Sertoli cells for 48 h in the absence of insulin or somatomedin-C (Sm-C), produced a small but significant increase in both hCG receptors and hCG-induced testosterone production. Addition to the medium of pFSH (100 ng/ml), insulin (5 micrograms/ml) or somatomedin-C (50 ng/ml) produced a marked increase in these two parameters of Leydig cell function. A further significant increase was observed when pFSH was associated with insulin or Sm-C. In contrast, coculture of Leydig cells with aortic endothelial cells decreased both the hCG receptor number and the hCG responsiveness. SCCM stimulated Leydig cell testosterone production following a 4 h incubation. The stimulation depended upon the amount of SCCM used and the conditions in which Sertoli cells were cultured. The most active was the medium from cells cultured in the presence of pFSH and insulin at high concentrations. Since pig Sertoli cells have specific somatomedin-C receptors, but not insulin receptors, it is likely that the effect of micromolar concentrations of insulin are exerted through Sm-C receptors. In addition, SCCM produced a long-term effect after 48 h incubation. SCCM from cells cultured in the absence of insulin and pFSH inhibited both the hCG receptor number and hCG responsiveness. A similar inhibition was observed with SCCM medium from cells cultured without insulin but with pFSH.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of human somatomedin C/insulin-like growth factor I(SM-C/IGF-1) and human growth hormone (hGH) on colony formation by erythroid precursor cells (CFU-E and BFU-E) from children's bone marrow or blood was studied by methylcellulose cloning assay. We found that physiological concentrations of IGF-1, but not of hGH, stimulated erythropoiesis in vitro in the presence of erythropoietin, as demonstrated by the increased activity of a cytosolic enzyme of the heme pathway (uroporphyrinogen I synthase). The results suggest that IGF-I could be involved in the regulation of erythroid differentiation.

These data describe the effects of purified preparations of several growth factors on thymidine incorporation into phyto-haemagglutinin-activated (PHA) human lymphocytes. The somatomedins selected for this study included human somatomedins A and C, insulin-like growth factors (IFG1 and IGF2) and multiplication stimulating activity (MSA). Assays were carried out with and without serum. Complementary assays were performed with a low-molecular serum ultrafiltrate added to somatomedin C and to MSA. We found that all the peptides tested, except MSA, stimulated thymidine incorporation into PHA-activated lymphocytes in a dose-dependent manner, even though different doses were required to obtain a response. The data reported point out the multiplicity and the interrelationships of the serum factors involved in the stimulation of human cells growth.

We have characterized somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) receptors in cultured pig Leydig cells by binding and cross-linking affinity experiments. At equilibrium the dissociation constant and the number of binding sites per cell were 1.8 +/- 0.2 X 10(-9) M and 12,200 +/- 3200 respectively. Under reducing conditions, disuccinimidyl suberate cross-linked 125I-Sm-C to one receptor complex with apparent Mr = 120,000. Continuous treatment of Leydig cells with increasing concentrations of human chorionic gonadotropin (hCG) for 48 h resulted in a dose-dependent (ED50 0.05 nM) increment in IGF type I receptors (2.5-3-fold increase). Conversely, treatment of Leydig cells for 48 h with increasing concentrations of Sm-C/IGF-I produced a 3-4-fold increase in hCG receptors. This effect was dose-dependent (ED50 = 7 ng/ml). Sm-C/IGF-I treatment also enhanced Leydig cell responsiveness to hCG for both cAMP (6-fold) and testosterone (8-fold). Moreover the stimulatory effects of Sm-C/IGF-I on cells cultured either in the absence or presence of 5% human serum from an hypopituitary patient were inhibited by anti-(Sm-C/IGF-I) antibodies. Taken together these results not only show that Leydig cells must be considered as targets for Sm-C/IGF-I, but also lend support to the possibility that Sm-C/IGF-I plays a role in the regulation of Leydig cell function. Moreover, they suggest that Sm-C/IGF-I might be responsible for the delayed puberty observed in some patients with isolated growth hormone deficiency.

Somatomedin-C/insulin-like growth factor I was purified from human plasma using a monoclonal antibody affinity column. Combining immunoaffinity chromatography with standard protein purification methods resulted in an overall recovery of 18%. The 35 micrograms of somatomedin-C/insulin-like growth factor I purified from 500 ml of plasma appeared as a single band when analyzed by polyacrylamide gel electrophoresis and could be used in radioimmunoassay and receptor binding studies.

Conditioned medium from pig Sertoli cells cultured in a chemically defined medium containing 3H-leucine contains a peptide with properties similar to that of human purified plasma and recombinant DNA somatomedin-C/insulin-like growth factor 1 (Sm-C/IGF-1). Purification of this peptide was achieved by affinity chromatography using mouse monoclonal anti-Sm-C/IGF-1 antibodies and reverse-phase high pressure liquid chromatography on a Bondapak C18 column. Polyacrylamide gel electrophoresis of the purified material gives a single spot after staining or in the autoradiogram, with identical molecular weight to that of pure human Sm-C/IGF-1. The purified peptide behaves like both the pure and recombinant DNA Sm-C/IGF-1 in the specific RIA and/or radioreceptor assays. Under basal conditions the amount of Sm-C/IGF-1 secreted by Sertoli cells was about 4 ng/10(6) cells/24 h, but it decreased during culture. Neither growth hormone nor follitropin were able to stimulate Sm-C/IGF-1 secretion, but both fibroblast growth factor and epidermal growth factor enhanced two- to three-fold its secretion. In addition, cells pretreated for 24 h with these growth factors became sensitive to the stimulatory effect of FSH. Since previous in vitro studies have shown that Sm-C/IGF-1 is a mitogenic and differentiating factor for both Sertoli and Leydig cells, it is possible that Sm-C/IGF-1 secreted by Sertoli cells might play a paracrine and/or autocrine role in the regulation of testicular function.

The present study examined the effects of both insulin and insulin-like growth factor-I (IGF-I) on cell division and specific functions of cultured adrenocortical cells from 100- to 122-day-old ovine fetuses. When culture was performed in a serum-free medium containing transferrin and ascorbic acid, the number of cells increased only slightly (1.2-fold) over a 4-day period. Addition of insulin or IGF-I in the culture medium enhanced the number of cells counted on Day 5. The effect of both peptides was dose-dependent, but 10 ng/ml IGF-I was as potent as 10 micrograms/ml insulin. The acute cyclic adenosine 3',5'-monophosphate (cAMP) and steroidogenic responses to adrenocorticotropin (ACTH1-24) decreased in fetal cells cultured in the absence of insulin or ACTH. Insulin at micromolar concentrations not only prevented this decrease but enhanced the acute ACTH1-24-induced cAMP output on Day 5 over that observed on Day 2. Treatment of fetal cells for 4 days with increasing concentrations of insulin or IGF-I enhanced the acute cAMP and steroidogenic responses (3- to 4-fold) to ACTH1-24 over that of control cells. The ED50 of IGF-I was about 3 ng/ml (congruent to 0.4 nM) whereas that of insulin was about 10 ng/ml (1.7 nM). However, a second plateau was apparent at concentrations of insulin above 1 microgram/ml. The acute cholera toxin stimulation of cAMP production of cells cultured in the absence of insulin or ACTH.(ABSTRACT TRUNCATED AT 250 WORDS)

Using primary culture of steroidogenic cells in vitro, in serum-free chemically defined medium, strong evidence has been provided for actions of IGF-I on the differentiation of immature Leydig cells and on the maintenance of a differentiated function of mature adrenocortical cells. These data point to the steroidogenic cells as a target for IGF-I action. They also add further evidence for a role of IGF-I in the differentiation process. These actions can be of importance in normal physiological situations as well as in abnormal conditions where IGF-I is decreased, such as hypopituitarism and malnutrition.

Maximum 125I-IGF-I/Sm-C total binding to chick embryo fibroblasts was 3% at +37 degrees C and decreased to less than 1% in presence of 2.8 X 10(-9) M unlabelled IGF-I/Sm-C. Insulin did not compete with IGF-I/Sm-C for the binding to cells. Biological action of IGF-I/Sm-C was evaluated on 2-deoxyglucose and alpha-aminoisobutyrate uptake. Results are compared with those obtained with insulin. Maximal peptide effects on the two transport processes were obtained at a 0.65 X 10(-7) M concentration and for a 120 minute association time, whereas cells were markedly less sensitive to insulin and time response curves were different. These results suggest that insulin action on nutrient uptake in chick embryo fibroblasts is not mediated by the binding of the hormone to IGF-I/Sm-C receptors.

Porcine cultured Leydig cells (LC) lose hCG receptors and hCG responsiveness (cAMP and testosterone) when they are cultured for three days in a defined medium without insulin or somatomedin C (Sm-C) (Insulin-like growth factor I). In the presence of insulin (50 ng/ml) or of Sm-C (10 ng/ml) the loss of the hCG receptor number and the decreased cAMP response to hCG were prevented, but the steroidogenic response to hCG was only partially prevented. This parameter became normal when cells were pretreated with either Sm-C (10 ng/ml) plus insulin (50 ng/ml) or with insulin alone at high concentrations (5 micrograms/ml). These results indicate that both Sm-C and insulin acting through their own receptors increase Leydig cell steroidogenic capacity by increasing hCG receptor number and improving some step beyond cAMP formation.

The proportion of immunoreactive somatomedin-C (IR-Sm-C) in blood that is available for measurement in the RIA is influenced by whether the sample is processed as serum or plasma, by how promptly the sample is chilled and frozen, by whether the reaction is carried out in glass or polystyrene tubes and by whether the incubation mixture contains protamine or heparin. Although protamine buffers and polystyrene tubes increase the availability of purified somatomedin-C (Sm-C), they decrease the detectability of Sm-C in serum. By incubating serum at neutral or acid pH, this IR-Sm-C can be made available for measurement, suggesting that incubation alters the nature of the linkage between Sm-C and its binding proteins or causes a conformational change in the binding protein, resulting in greater exposure of IR-Sm-C. The increment in measurable IR-Sm-C that occurs at neutral pH appears to be due to the action of proteolytic enzymes since it is time, temperature, and pH dependent and is inhibited by a variety of protease inhibitors and chelating agents. The increment which occurs at acid pH is not inhibited by chelating agents or elevated temperature and must be due in part to acid hydrolysis of Sm-C and its binding proteins. However, since the acid-induced increment is optimal within a narrow pH range and falls off sharply below pH 3.5, acid proteases may also be involved. These observations, which provide insight into the nature of the serum proteins with which Sm-C is associated, bear on the interpretation of results of serum somatomedin measurements carried out with different methods. They also may aid in delineating the mechanisms by which the somatomedin contained in the macromolecular complex in serum is made available to tissues.

Using primary culture in a chemically defined medium of somatic testicular cells from immature pig, we were able to demonstrate synthesis and secretion of Somatomedin-C/Insulin-Like Growth Factor 1 (Sm-C/IGF-1) by Sertoli cells, a process that is stimulated by Fibroblast Growth Factor (FGF). The presence of IGF type 1 receptor was demonstrated on Sertoli cells. Sm-C/IGF-1 stimulates and potentiates the effects of FGF on both cell multiplication and secretion of plasminogen activator. The presence of both IGF type 1 and insulin receptors was also documented on immature Leydig cells. Pre-incubation of immature Leydig cells for 48 hours with Sm-C/IGF-1 resulted in a dramatic dose-dependent increment of both the LH receptor number and steroidogenic response to LH as well as a clear stimulation of DNA synthesis. These data provide new important insights on the role played by Sm-C, a growth and differentiating factor, on the maturation of the male gonad endocrine function.

The effects of human GH and insulin-like growth factor I on the proliferation and differentiation of erythroid progenitor cells from the bone marrow and peripheral blood of children were studied in a hormone-depleted culture system. Growth of erythroid progenitors was quantified by directly scoring colonies and by biochemical determination of the activity of a cytosolic enzyme of the heme pathway, uroporphyrinogen I synthase. In the presence of erythropoietin, high concentrations (50-100 ng/mL) of human GH induced an increase in the number of erythroid colonies (and their uroporphyrinogen I synthase activity) formed by bone marrow or peripheral blood erythroid precursors. In the same conditions, physiological concentrations of insulin-like growth factor I (0.5-1 ng/mL) stimulated erythroid cell growth and differentiation (P less than 0.03) from bone marrow or peripheral blood.

Using an in vitro system of pig Leydig cells (LC) and Sertoli cells (SC) we have demonstrated that: 1) LC contained specific receptors for both somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) and insulin, whereas SC contained only Sm-C/IGF-I receptors; 2) pretreatment of LC with insulin or Sm-C/IGF-I increased hCG receptor number and the cAMP and testosterone responses to this hormone. The enhanced steroidogenic capacity was related to an increased activity of several enzymes of the steroidogenic pathway. At physiological concentrations Sm-C/IGF-I was more potent than insulin, but the effects of the latter peptide at micromolar concentrations were similar to those produced by nanomolar concentrations of Sm-C/IGF-I. However, at maximal concentrations of both peptides, there was no additive effect; 3) the specificity of the effect of Sm-C/IGF-I was proven by the fact that all the effects induced by this peptide, but not by insulin, were blunted by an anti-Sm-C/IGF-I antibody; 4) pretreatment of SC with Sm-C/IGF-I at nM concentrations or with insulin (but only at microM concentrations) enhanced the stimulatory effect of FSH on cAMP production and the secretion of plasminogen activator; 5) in both LH and SC, Sm-C/IGF-I had small mitogenic effects but potentiated the mitogenic action of fibroblast growth factor (FGF). The effect of insulin was observed only at microM concentrations; 6) SC secreted a factor which had physico-chemical and biological properties similar to that of Sm-C/IGF-I. The secretion of this factor was stimulated by FGF and EGF.

The role of insulin-like growth factor I (IGF-I) on the specific function of several steroidogenic cells has been recently reported. Since IGF-I is produced by several tissues, we have investigated whether bovine adrenal cells secrete this peptide. Purification of conditioned medium from adrenal cells incubated with [35S]methionine through affinity chromatography (monoclonal anti-IGF-I antibody), high pressure liquid chromatography, and polyacrylamide gel electrophoresis revealed a single band of similar Mr as pure recombinant IGF-I. Moreover, the purified adrenal-secreted IGF-I displaced bound 125I-IGF-I to its adrenal receptors, and pretreatment of adrenal cells with the purified peptide enhanced the acute corticotropin (ACTH)-induced cAMP production as recombinant IGF-I. The basal secretion of IGF-I (6 +/- 1 ng/48 h/10(6) cells) was stimulated 3-, 4.5-, and 9.5-fold by fibroblast growth factor, angiotensin II (A-II), and ACTH, respectively, but not by growth hormone. The stimulatory effects of A-II and ACTH were dose-dependent (ED50 congruent to 2.5 x 10(-8) and 1.5 x 10(-10) M, respectively), and the effects of both hormones were additive. Glucocorticoids were not the mediators of the effect of the two hormones on IGF-I secretion, since inhibition of their steroidogenic action by aminoglutethimide did not significantly modify IGF-I secretion. An immunoreactive IGF-I material was also secreted by mouse adrenal tumor cell line Y-1, but the stimulatory effect of ACTH was only 2-fold, and there was no effect of A-II. Since bovine adrenal cells contain specific IGF-I receptors and this peptide is required for the maintenance of some adrenal cell-specific function, the present data suggest that IGF-I may act in an autocrine fashion to stimulate adrenal cell differentiation stimulated by ACTH and A-II.

This review focuses on the action of somatomedins IGF-1 and IGF-2, and in particular the role of IGF-1 in cell multiplication and differentiation. A brief overview is given of the factors controlling IGF-1 flow rate expression in the tissues. Factors influencing IGF-1 plasma levels, i.e. age, and insufficiency or excess of growth hormone, are discussed.

We have investigated the effects of insulin and somatomedin-C/insulinlike growth factor I(Sm-C) in purified porcine Leydig cells in vitro on gonadotrophins (hCG) receptor number, hCG responsiveness (cAMP and testosterone production), and thymidine incorporation into DNA. Leydig cells cultured in a serum-free medium containing transferrin, vitamin E, and insulin (5 micrograms/ml) maintained fairly constant both hCG receptors and hCG responsiveness. When they were cultured for 3 days in the same medium without insulin, there was a dramatic decline (more than 80%) in both hCG receptor number and hCG responsiveness. However the cAMP but not the testosterone response to forskolin was normal. Both insulin and Sm-C at nanomolar concentrations prevent the decline of both hCG receptors and hCG-induced cAMP production. This effect of both peptides was dose dependent with an ED50 of about 1 ng/ml and 5 ng/ml for SM-C and insulin, respectively. Insulin and Sm-C had no additive effect on these parameters. At nanomolar concentrations, Sm-C and insulin enhanced hCG-induced testosterone production but the effect of Sm-C was significantly higher than that of insulin. However, the effect of insulin at higher concentrations (5 micrograms/ml) was significantly higher than that of Sm-C at 50 ng/ml. In contrast, at nanomolar concentrations only Sm-C stimulated [3H]-thymidine incorporation into DNA and cell multiplication, the stimulatory effect of insulin on these parameters, was seen only at micromolar concentrations. These results indicate that both Sm-C and insulin acting through their own receptors increase Leydig cell steroidogenic responsiveness to hCG by increasing hCG receptor number and improving some step beyond cAMP formation. In contrast, the mitogenic effects of insulin are mediated only through Sm-C receptors.