Steroidogenesis of cultured purified pig Leydig cells: secretion and effects of estrogens

Using a primary culture of purified immature pig Leydig cells we have demonstrated: (1) that during the first 3 days of culture there is a 'spontaneous' maturation of the steroidogenic response to hCG, as expressed by a 50-fold increase of the steroidogenic capacity, an increased secretion of both dehydroepiandrosterone sulfate (DHAS) and testosterone (T), a shift of the DHAS/T ratio (5 on day 0 vs. 0.5 on day 3) without significant changes in the number of hCG-binding sites; (2) that purified cells, on day 3 following hCG stimulation, secrete large amounts of T and small amounts of E2 (T/E2 congruent to 150) and detectable amounts of estrone, while crude pig interstitial cells under the same conditions secrete less T but 40-50 times more estrogens (T/estrogens congruent to 1.5); (3) that the steroidogenic responsiveness of purified Leydig cells is not impaired by E2 treatment, in spite of the fact that Leydig cells contain specific estradiol receptors (approximately 10000 sites/cell). These data suggest that in this model the main source of testicular estrogens are not Leydig cell but some other testicular cell types, and that the lack of effect of estrogens on pig Leydig cell steroidogenesis is not due to absence of estrogen receptors.     

Secretion of chorionic gonadotropin by cultured human pituitary cells

To investigate the possibility that the human pituitary gland secretes CG, we used a highly specific, two-site, double monoclonal immunoradiometric assay to measure CG in the medium in which the dispersed cells of pituitary glands from human fetuses of 20-24 weeks gestation were cultured. The cross-reactivity of immunopurified human LH in the CG assay was less than 0.03%. LH was also measured by a double monoclonal immunoradiometric assay. Secretion of CG by the cultured fetal pituitary cells was readily detectable, although in gradually decreasing amounts, for the 11 days of culture. LH secretion paralleled CG secretion and was much greater in magnitude. Pituitary cells from female fetuses generally secreted more CG as well as more LH than those from male fetuses. Dilutions of the medium showed that the secreted CG exhibited parallelism with the CG standard. Chromatofocusing of the medium across a pH 6-9 gradient yielded several peaks of LH immunoreactivity between pH 6.5 and 8.5, but no peaks of CG. Chromatofocusing of the medium across a pH 3-7 gradient yielded peaks of CG, but not LH, between pH 4.0-5.5. These data indicate that CG immunoreactivity, distinct and separable from LH immunoreactivity, is secreted by the dispersed cells of fetal human pituitary glands.     

Isolation of human Leydig cells which are highly responsive to human chorionic gonadotropin

Leydig cells were purified on discontinuous Percoll gradients after collagenase digestion of human or rat testes. Leydig cells from both species were found in three bands. As determined by positive staining for 3 beta-hydroxysteroid dehydrogenase, band 1 (lowest density cells) from both species contained only 12-28% Leydig cells. However, while band 3 was the most Leydig cell-enriched fraction in rat cell preparations (70-90% Leydig cells), human band 2 (48-70% Leydig cells) was consistently more Leydig cell enriched than was band 3 (30-56% Leydig cells). Despite their slightly different fractionation pattern, Leydig cells prepared from five men with prostatic carcinoma were similar to those from the rat, both in terms of the amount of testosterone produced basally per 10(6) Leydig cells (80-234 ng/20 h) and in terms of the magnitude of their response to hCG (764-1342 ng/10(6) Leydig cells X 20 h; 5- to 17-fold stimulation above basal). Cells prepared from five other men with prostatic carcinoma produced much lower amounts of testosterone, but still had up to a 6-fold response to hCG. Plasma LH, FSH, and testosterone concentrations in the latter group could not be distinguished from those in the group whose Leydig cells produced large amounts of testosterone in vitro. Morphologically, the testes from the latter group appeared to contain more darkly staining than lightly staining Leydig cells than did the former group. Rat Leydig cells responded in a dose-dependent fashion to hCG over the range 0.03-0.5 mU/mL, whereas human Leydig cells were 10- to 100-fold less sensitive, responding to hCG in the range 0.4-100 mU/mL. The number and affinity of Leydig cell LH (hCG) receptors were assessed from Scatchard analysis of [125I]hCG binding. Compared with rat cells, human Leydig cells contained approximately 20% of the number of LH receptors, while the affinity of the receptors (Kd, approximately 10(-10) M) was similar to that in rats. In conclusion, a method for the isolation of highly responsive human Leydig cells has been developed. The results so far suggest that the function of human Leydig cells may be more similar to that of the rat than thought previously.     

The effects of chronic human chorionic gonadotropin treatment on Leydig cell function

The purpose of this study was to examine the effect of chronic daily hCG treatment on interstitial cell function in the rat, as judged by plasma testosterone levels, the testicular binding of labeled hCG, and the capacity of the testis to respond to gonadotropin stimulation by the production of testosterone in vitro. TWenty-four hours after the first injection of 100 IU hCG there was a significant decline in hCG binding to testis homogenates and an inability to respond to hCG stimulation in vitro, After 7 days of daily injections of 10 IU or 100 IU hCG, the loss of hCG binding was maintained. However, despite the marked decline in hCG binding, there was an enhanced testosterone response to hCG stimulation in vitro, and plasma testosterone levels were significantly elevated. With continued injections of hCG for 14 or 21 days, the testes remained hyperresponsive to hCG stimulation in vitro, but hCG binding returned to control levels, and plasma testosterone concentrations declined and were not statistically different from controls. The latter changes probably result from the formation of specific hCG antibodies (Kd at 4 C, 7.8 +/- 4.5 X 10(-10) M) that were detected in plasma from rats treated for 14 or mopre days with hCG. The formation and levels of the hCG antibodies in these animals were sufficient to neutralize the effects of the exogenous hCG, thereby returning plasma testosterone levels to normal and restoring the complement of hCG receptors.     

Inhibition of apoptosis in cultured immature rat Leydig cells by human chorionic gonadotropin associated with Bcl-2 mRNA expression

Bcl-2 is the first identified negative regulator of apoptotic cell death. When the level of Bcl-2 mRNA in rat whole testes was examined in the present study, it gradually decreased in rats from 2.5 to 9 weeks old. We also examined the effect of human chorionic gonadotropin (hCG) on apoptosis and the level of Bcl-2 mRNA expression in immature Leydig cells in vitro. When the cells were cultured with serum free media (SFM), Bcl-2 mRNA levels gradually decreased. On the other hand, the level of Bcl-2 mRNA in cells treated with 50 ng/ml of hCG decreased at 6 h, but increased after 12 h. At 24 h, the level of Bcl-2 mRNA in the treated cells was almost the same as that of control cells (time = 0). At 12 h after the addition of various concentrations (from 0.1-1000 ng/ml) of hCG, Bcl-2 mRNA levels increased in a dose-dependent manner. An analysis of DNA fragmentation showed that treatment with hCG prevents the apoptosis of immature Leydig cells. Our findings suggest that Bcl-2 mRNA may be related to the programmed cell death of immature rat Leydig cells in vitro, which are inhibited by hCG.     

Acute stimulation of aromatization in Leydig cells by human chorionic gonadotropin in vitro.

Aromatization of testosterone in Leydig cells purified from mature rat testes was assessed. Leydig cells incubated for 4 hr with increasing concentrations of [3H]testosterone. At saturating concentrations of testosterone, human chorionic gonadotropin (hCG) acutely stimulated aromatization. This stimulation was first observed at 1 hr, an 8-fold increase being found during a 4-hr incubation. The maximal amount of estradiol produced at saturating concentrations of testosterone and hCG was 1.8 ng per 10(6) cells. These results demonstrate that purified Leydig cells have a high capacity for aromatization and that hCG can acutely stimulate aromatization independently of stimulating testosterone synthesis in vitro.     

Effect of a second injection of human chorionic gonadotropin on the desensitized Leydig cells

Experimental evidence has demonstrated that multiple doses of LH will increase the steroidogenic capacity of Leydig cells. This work was undertaken with the aim of defining the effect of a second hCG administration on the desensitized state, measuring binding of the gonadotropin and the steroidogenic capacity of rat testes. A single injection of 200 IU hCG induced a sharp increase of plasma testosterone which was still evident 24 h later. A second peak was observed at 72 h. The in vivo refractoriness of Leydig cells between 24 and 72 h after the single injection was proved by the fact that a second administration of hCG, 2 h before sacrifice, did not induce any increase in plasma testosterone. A second administration of 200 IU hCG, 48 h after the first injection, showed a similar pattern but on the 5th day there was an increased stimulation of testosterone production with respect to that obtained after a single dose of hCG. The in vitro studies on testicular binding capacity and steroidogenic responsiveness showed that the second administration of hCG, 48 h after the first injection, maintained the testicular binding capacity at the lowest level and the 'adenylate cyclase desensitization' but restored the steroidogenic capacity to even supramaximal values, compared to normal rats, 3 days after this second hCG administration. These results would support a dissociation between receptor loss and maximal testosterone synthesis as well as possibly indicating an alternative pathway different from the classical.     

Regulation of human chorionic gonadotropin synthesis in cultured human placental cells by glucocorticoids

The synthesis of hCG and its subunits in temperature-sensitive, simian virus 40 tsA mutant-transformed human first trimester and term placental cells and in choriocarcinoma cells was studied in the presence and absence of glucocorticoid hormones. The tsA-transformed placental cells were studied at 33 C (the temperature at which the cells exhibit the transformed phenotype) and at 40 C (the temperature at which the cells regain their differentiated nontransformed phenotype). The glucocorticoids cortisol and dexamethasone did not affect the synthesis of hCG or hCG alpha in either transformed term placental cells or choriocarcinoma cells. In the tsA-transformed first trimester placental cells, however, glucocorticoids greatly inhibited hCG synthesis but induced hCG alpha synthesis. The inhibition on hCG synthesis was immediate, stronger at 40 C than at 33 C, and independent of the growth stage of the cells. The glucocorticoid effects were specific; neither estradiol nor progesterone affected the synthesis of hCG or hCG alpha. Furthermore, progesterone did not block the action of the glucocorticoid. These selective effects in cultured first trimester placental cells suggest that glucocorticoids may play an important role in the regulation of hCG production during early pregnancy.     

Synthesis and processing of human chorionic gonadotropin subunits in cultured choriocarcinoma cells.

Pulse and pulse-chase experiments have identified the presence of partially glycosylated precursors of the alpha and beta subunits of human chorionic gonadotropin (hCG) in cultured JAR choriocarcinoma cells. The alpha subunit precursor has an apparent molecular weight (by sodium dodecyl sulfate/polyacrylamide gel electrophoresis) of 18,000 (compared to 22,000 for fully processed alpha subunit); the beta subunit precursor has an apparent molecular weight of 24,000 (fully processed, 34,000). Both of these precursors appear to have an intracellular half-life of at least 1 hr and to contain the mannose core but not the terminal carbohydrate sequences. Fully processed alpha and beta subunits do not accumulate intracellularly, indicating that further processing of the precursors is followed by rapid secretion.     

Leydig cell responsiveness to single and repeated human chorionic gonadotropin administration.

A single im injection of 1500 IU hCG significantly increased plasma testosterone levels for at least 96--120 h in normal men (n = 7), patients with isolated gonadotropin deficiency (n = 6), and boys with delayed puberty (n = 7); the maximum values [1315 +/- 309, 370 +/- 177, and 963 +/- 249 ng/100 ml (mean +/- SD), respectively] were achieved after 72 h in each group. Repeated daily injections of 1500 IU hCG for 3 days increased plasma testosterone levels in the same subjects at 72 h after the start to levels (1342 +/- 412, 407 +/- 199, and 1052 +/- 449 ng/100 ml, respectively) similar to those found in the single dose experiment. The levels achieved at 24 and 48 h also did not differ significantly in the two experiments. The data indicate the lack of additional leydig cell stimulation by repeated hCG injections given within 48 h after a single dose.     

Epidermal growth factor stimulates secretion of human chorionic gonadotropin by cultured human choriocarcinoma cells.

Epidermal growth factor (EGF) binds to JEG-3 cells, a tissue culture line of human choriocarcinoma. EGF also stimulates secretion of human chorionic gonadotropin (hCG) and to a lesser extent the secretion of free hCG-alpha.     

On the fates of receptor-bound ovine luteinizing hormone and human chorionic gonadotropin in cultured Leydig tumor cells. Demonstration of similar rates of internalization

Using a clonal strain of cultured Leydig tumor cells (designated MA-10), we have compared the fate of the receptor-bound ovine LH (oLH) and human CG (hCG) in cells incubated in the presence or absence of extracellular Na+. We have previously shown that Na+ does not affect the number of LH/CG receptors or the binding affinity of hCG, but it decreases the binding affinity of oLH. Thus, it was possible to compare the fate of these two hormones under conditions where their binding affinities differ by a factor of 8 (i.e. in the presence of Na+) or by a factor of less than 2 (i.e. in the absence of Na+). Moreover, since only the affinity of oLH is affected by Na+, we were able to distinguish between those effects mediated by a change in binding affinity from those effects that are more general in nature by comparing the behavior of hCG in cells incubated in the presence or absence of Na+. The results presented herein show that the rates of internalization of oLH and hCG are very similar regardless of the presence or absence of Na+; and the absence of Na+ leads to a 2- to 3-fold decrease in the rate of degradation of the internalized oLH and hCG. We have found, however, that the binding affinities of oLH and hCG have significant effects on the pathway of receptor-mediated endocytosis under conditions where there is no free hormone present in the medium. The results presented show that in the absence of free hormone in the medium, the rate of hormone internalization can be approximated from the rate of disappearance of the surface-bound hormone only if the binding affinity of the hormone is high enough so that there is little or no dissociation of the hormone from the receptor during the course of the experiment (i.e. hCG in the presence or absence of Na+, but oLH only in the absence of Na+). If the binding affinity of the hormone is low (i.e. oLH in the presence of Na+), then the rate of disappearance of the surface-bound hormone represents the sum of the rates of internalization and dissociation of the hormone and thus cannot be used to approximate the rate of hormone internalization.     

Human chorionic gonadotropin up-regulates insulin-like growth factor-I receptor gene expression of Leydig cells.

The effects of hCG, 8-bromo-cAMP, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, and forskolin on insulin-like growth factor-I (IGF-I) receptor gene expression of Leydig cells were studied. The treatment of purified Leydig cells with hCG caused a dose-dependent increase in [125I]IGF-I binding to Leydig cells without changes in binding affinity, indicating that the increased binding was due to increased receptor numbers and not to increased affinity. The minimal time required for hCG to induce IGF-I binding was 6 h, and it had reached a plateau at 16 h. 8-Bromo-cAMP (1 mM) increased IGF-I binding about 2-fold, and forskolin (10 microM) increased binding about 51%. Using the ribonuclease protection assay, we found that hCG and 8-bromo-cAMP could increase IGF-I receptor mRNA expression as early as 2 h before the increase in IGF-I binding. The induction by hCG was over 3.5-fold at 4 h and decreased to about 2-fold at 6 h. 4 beta-Phorbol 12 beta-myristate 13 alpha-acetate had a very small effect on IGF-I receptor mRNA levels (1.5-fold increase at 2 h and no changes at 4 and 6 h). In conclusion, IGF-I receptors can be up-regulated by hCG, 8-bromo-cAMP, and forskolin. The up-regulation of IGF-I receptor number is associated with transient increases in IGF-I receptor mRNA levels. This could be a mechanism by which hCG and IGF-I interact to enhance Leydig cell steroidogenesis.     

Heterogeneity of the human chorionic gonadotropin alpha-subunit secreted by cultured choriocarcinoma (JEG) cells.

The cultured human choriocarcinoma cell line, JEG-clone 3, secretes substantial quantities of both biologically active hCG and an immunoreactive alpha-subunit (JEG-alpha). This study is concerned with a comparative characterization, using RIA, of the chromatographic properties (via gel exclusion and isoelectric focusing) of JEG-alpha and standard urinary hCG-alpha. Most of the molecules comprising the JEG-alpha fraction have an apparent molecular weight greater than that of hCG-alpha. Both hCG-alpha and JEG-alpha exhibit heterogeneity on electrofocusing. However, JEG-alpha contains a major component with an isoelectric pH (pI) of 4.8; this is a minor component, if present at all, in hCG-alpha. The JEG-alpha pI 4.8 component chromatographs with an apparent molecular weight greater than hCG-alpha, while a minor JEG-alpha pI 7.0 component chromatographs with an apparent molecular weight similar to that of the standard. Heterogeneity is expected in the carbohydrate moieties of the glycoprotein hormone subunits. Results of studies on the incorporation of 14C- and 3H labeled amino acids into JEG-alpha suggest that heterogeneity also exists in the protein moiety of JEG-alpha. An interesting possibility is that the form(s) of JEG-alpha with larger apparent molecular weight represents a precursor of the alpha-subunit used to form hCG.     

Luteinizing and human chorionic gonadotropin hormones increase intercellular communication and gap junctions in cultured mouse leydig cells

The effect of luteinizing (LH) and human chorionic gonadotropin (hCG) hormones on gap junctions (Gjs) and intercellular communication (ic) was evaluated in Leydig (interstitial) cells from mouse testes. Cell cultures enriched in Leydig cells were studied under control conditions and when maintained in the presence of 100 ng/mL LH, 10 ng/mL hCG, or 1 mM dibutiryl-cAMP (db-cAMP), for 8, 24, and 36 h. To monitor the extent of ic, Lucifer yellow (LY) was injected through a patch pipet into one cell of-small cell aggregates (6–10), and its transfer was evaluated using fluorescent microscopy. The expression of GJs was monitored using immunofluorescent (IF) labeling of connexin 43 (Cx43) with a specific antibody. Testosterone secretion was determined by radioimmunoassay. At all culture times, testosterone levels in the medium were higher in treated than in control cell cultures. In cell cultures of 8 h, LY transferred to most of the neighboring cells (93%) and cell membrane appositions showed abundant Cx43; no difference was found between control and treated cells. In contrast, in control cell cultures of 24 and 36 h, LY transferred to a reduced fraction of neighboring cells (46 and 21%, respectively) and Cx43 labeling was markedly decreased. Addition of LH, hCG, or db-cAMP, to cell cultures for 24 and 36 h completely prevented the decrease in ic and Cx43 expression. Immunoblot studies, from total protein homogenates of cell cultures of 36 h, showed that relative levels of 40- and 43-kDa bands, characteristic of Cx43, were higher in treated than in control cells. These results demonstrate that the expression of Cx43 and ic in Leydig cells is modulated by LH and hCG, and suggest that their effect is mediated by the second messenger of these hormones, cAMP.     

Characterization of several clonal lines of cultured Leydig tumor cells: gonadotropin receptors and steroidogenic responses

Several clonal lines of cultured Leydig tumor cells have been established and characterized in terms of gonadotropin receptors and steroid production. Although freshly isolated cells derived from the M5480P tumor have functional hCG receptors, only two of the five clonal lines established were shown to bind significant quantities of hCG. In these clones, steroid production can be stimulated to the same extent by hCG, cholera toxin, and 8-Br-cAMP. The other three clones bind a small amount of hCG and respond to the hormone with a marginal increase in steroidogenesis. Steroid production, however, is significantly stimulated by cholera toxin or 8-Br-cAMP. A comparison of the steroids produced by freshly isolated cells and two of the clones revealed some changes in the steroidogenic pathway. The most obvious change is an increase in the ability of the cultured cells to synthesize 20 alpha-dihydroprogesterone (20 alpha-hydroxypregn-4-en-3-one). These clonal lines may provide a suitable model system for the study of gonadotropin actions and regulation of the expression of differentiated functions of Leydig cells.