Internalization and degradation of receptor-bound human choriogonadotropin in Leydig tumor cells. Fate of the hormone subunits

The studies presented herein were aimed at characterizing the pathway involved in the internalization and degradation of human choriogonadotropin by cultured Leydig tumor cells. A quick biochemical method that differentiates between the surface-bound and internalized hormone was developed. Using this method and two hormone derivatives labeled exclusively (with 125I) in the alpha or beta subunits, it was possible to follow the fate of each hormone subunit during hormone binding, internalization, and degradation. The results show that the hormone is internalized in the intact form and that it reaches its place of degradation (presumably the lysosomes) in the intact form. The pathway for degradation of the internalized hormone is complex, and it appears to involve processing of one or both subunits of the intact hormone, followed by subunit dissociation and further degradation of the individual subunits. The alpha subunit is quickly degraded by the cells. The only detectable degradation products are extracellular amino acids. The beta subunit is degraded slower, and several intracellular degradation products are detectable before amino acids appear in the medium.     

Inhibition of the degradation of receptor-bound human choriogonadotropin by leupeptin.

The ability of leupeptin to block the degradation of receptor-bound human choriogonadotropin has been studied. It was found that this compound inhibited hormone degradation and intracellular cathepsin B activity in a parallel fasion, without affecting hormone-stimulated steroidogenesis.     

Removal of the surface-bound human choriogonadotropin results in the cessation of hormonal responses in cultured Leydig tumor cells

The relationship between the binding and internalization of human choriogonadotropin and the stimulation of cAMP and steroid production was studied in cultured Leydig tumor cells. It was found that removal of the surface-bound hormone results in a rapid cessation of both cAMP and steroid production. We propose that the surface-bound hormone is responsible for the activation of steroidogenesis and that hormone internalization is involved in the deactivation of this process.     

Lutropin is processed much more rapidly than human choriogonadotropin by porcine Leydig cells in primary culture

Lutropin (LH) and human choriogonadotropin (hCG) share the same receptor and stimulate testosterone production in porcine Leydig cells in primary culture. Cells were pulsed with [125I]LH or [125I]hCG. During the chase, more than 80% of cell-bound LH consisted in internalized material which was degraded and excreted (half-time : 25 min) NH4Cl largely inhibited this degradation. On the contrary, hCG remained essentially bound to the cell surface and was not degraded by the cells with or without NH4Cl up to 160 min.     

Lutropin is processed much more rapidly than human choriogonadotropin by porcine Leydig cells in primary culture

Lutropin (LH) and human choriogonadotropin (hCG) share the same receptor and stimulate testosterone production in porcine Leydig cells in primary culture. Cells were pulsed with [125I]LH or [125I]hCG. During the chase, more than 80% of cell-bound LH consisted in internalized material which was degraded and excreted (half-time: 25 min) NH4Cl largely inhibited this degradation. On the contrary, hCG remained essentially bound to the cell surface and was not degraded by the cells with or without NH4Cl up to 160 min.     

Immunoprecipitation of the lutropin/choriogonadotropin receptor from biosynthetically labeled Leydig tumor cells. A 92-kDa Glycoprotein

The structure of the lutropin/choriogonadotropin (LH/CG) receptor has been studied by immunoprecipitating the receptor from biosynthetically labeled cultured Leydig tumor cells (designated MA-10). This was performed by binding human choriogonadotropin (hCG) to the labeled cells, solubilizing the hormone-receptor complex, partially purifying the complex by lectin chromatography, and immunoprecipitating the complex with an antibody that recognizes receptor-bound hCG. The conditions used for the release of the radiolabeled receptor from the immunoprecipitate and the subsequent analysis of this material on sodium dodecyl sulfate gels allowed us to determine directly the structure of the free (not hormone-occupied) LH/CG receptor. From experiments using cells labeled with [35S]methionine and [35S]cysteine, we show that the LH/CG receptor is composed of a single polypeptide chain that migrates as a 92-kDa protein on sodium dodecyl sulfate gels whether analyzed in the absence or presence of reducing agents. Other studies presented demonstrate that the LH/CG receptor is a glycoprotein.     

Internalization and recycling of lutropin receptors upon stimulation by porcine lutropin and human choriogonadotropin in porcine Leydig cells

Porcine lutropin (pLH) and human choriogonadotropin (hCG) recognize the same hormonal receptor and elicit the same steroidogenic response in porcine Leydig cells in primary culture. We compared the variation in the number of occupied and free receptors present on the cell surface under short-term stimulation by the two hormones at 35 degrees C. Both hormones produced a rapid dose-dependent decrease in the total number of the gonadotropin receptors present on the cell surface with a half-life of 8-10 min. This decrease was reversible upon hormone removal and receptors were recovered on the cell surface with the same half-life of 8-10 min. With pLH, the receptors were recycled in a free state, but in the presence of hCG the receptors were recycled in an occupied state. This difference could be related to the higher affinity of hCG for the receptor in 150 mM NaCl buffer (Ka = 1.6 X 10(9) for hCG and 1.5 X 10(7) for pLH) and higher stability to acid pH of the hCG-receptor complex (dissociation pK = 3.7 for hCG and 4.5 for pLH).     

Characterization of the expression products of recombinant human choriogonadotropin and subunits

Human choriogonadotropin (hCG) is a placental glycoprotein hormone composed of a 92-amino acid alpha subunit noncovalently linked to a 145-amino acid beta subunit. We report here the expression of biologically active hCG in mouse C127 cells transfected with expression vectors containing the DNA coding for both subunits. In addition, the same cell line was used to express the alpha subunit alone. The expression products were purified by affinity chromatography using specific monoclonal antibodies to hCG or its subunits. The system secreting biologically active hCG also produced a 10-fold or greater molar excess of free beta subunit. The dimeric hormone, as well as the excess beta subunit, resembles the standard urinary hCG and beta subunit by chemical and biological criteria. In contrast, when the vector encoding for the alpha subunit was expressed alone, the alpha subunit had a higher molecular weight than both standard alpha and the alpha found in the expressed dimeric hormone. The molecular weight difference between expressed alpha subunit and standard alpha was found to reside in the alpha peptide consisting of residues 52-91 which contained all of the carbohydrate of the alpha subunit. The N-asparagine-linked carbohydrate moieties in the recombinant alpha were found to be triantennary in contrast to biantennary in urinary alpha, and this hyperglycosylation was responsible for the higher molecular weight of the alpha subunit when it was expressed alone. We found no evidence of O-threonine glycosylation at position alpha 39 reported to be present in free forms of the alpha subunit; however, the companion paper (Corless, C.L., Bielinska, M., Ramabhadran, T. V., Daniels-McQueen, S. Otani, T., Reitz, B. A., Tiemeier, D. C., and Boime, I. (1987) J. Biol Chem. 262, 14197-14203) finds a small quantity of O-glycosylation. Since the excess beta subunit appears to be of normal size and contains the expected complement of sugars, only free alpha subunit seems to be a potential substrate for addition of extra sugar moieties. No large beta subunit forms have been found by others, while large alpha subunits have been described both clinically and in tissue culture systems. These observations imply that the conformation of the free alpha subunit, in the regions of the glycosylation recognition sites, allows easier access for glycosyltransferases than those same sites in the beta subunit. When alpha is combined with beta, the local structures around the alpha glycosylation sites are apparently altered so as to make the synthesis of triantennary chains less favorable.     

Inhibition of the degradation of receptor-bound human choriogonadotropin by lysosomotropic agents, protease inhibitors, and metabolic inhibitors.

A previous report from this laboratory showed that binding of iodine-labeled human choriogonadotropin to Leydig tumor cells is not a reversible process (Ascoli, M., and Puett, D. (1978) J. Biol. Chem. 253, 4892--4899). Most of the cell-bound hormone was found to be degraded to 3'-monoiodotyrosine before being released from the cells, and the degradation process could be inhibited by the lysosomotropic agents NH4Cl, chloroquine, and Triton WR-1339. It is reported herein that the degradation of receptor-bound human choriogonadotropin is an energy-dependent process, which can be inhibited by compounds that interfere with glycolysis or oxidative phosphorylation (e.g. NaF, NaN3, NaCN, and 2-deoxyglucose). Hormone degradation is also inhibited by some protease inhibitors such as the chloromethyl ketones of lysine and phenylalanine, but not by specific trypsin inhibitors (e.g. p-aminobenzamidine and p-tosyl-L-arginine methyl ester). With the exception of NH4Cl, it was found that the compounds which inhibit hormone degradation also inhibit hormone-stimulated steroidogenesis. However, the present results involving dose dependency, and those given in the following paper (Ascoli, M. (1978) J. Biol. Chem. 253, 7839--7843), indicate that these two phenomena are not related.     

Processing of human choriogonadotropin and its receptors by cultured pig Leydig cells. Role of cyclic AMP and protein synthesis

We have examined the process by which human choriogonadotropin/luteinizing hormone (hCG/LH) receptors are regulated in cultured porcine Leydig cells. Treatment of Leydig cells with human choriogonadotropin, cholera toxin, forskolin and cyclic 8-bromoAMP (8-BrcAMP) produced a loss of surface receptors without modification of the binding affinity. This negative regulation of the number of receptors mediated by maximal concentrations of hCG was higher than that induced by the other agents. The extent of receptor loss in cells treated with increasing concentrations of hCG was highly correlated with their capacity to stimulate cAMP production. However, there was little correlation between down-regulation and cAMP production of these cells treated by hCG plus forskolin or cholera toxin plus forskolin, where a synergistic cAMP production was obtained. Following exposure of Leydig cells to both hCG and 8-BrcAMP, the surface receptor disappearance began after an initial lag period of about 6-8 h. Thereafter a 50% loss of surface receptor was observed in the next 8-h incubation. Monensin with hCG shortens this lag period before initiation of receptor loss. Kinetic studies with 125I-hCG, in the presence or absence of monensin, showed that the half-life of the receptor-bound hormone complexes at the cell surface was 10.5 h and 8 h respectively. Therefore, the steady state of the surface receptor during the lag phase of 8 h is probably related to recycling of internalized receptors and/or translocation of performed receptors. Cycloheximide and actinomycin D inhibit hCG-mediated and 8-BrcAMP-mediated down-regulation. Cycloheximide lengthens ligand-receptor complexes at the surface by slowing down the rate of internalization (half-life of 20 h), but this mechanism is not enough per se to explain the effect of cycloheximide. Pulses of hCG or 8-BrcAMP for 4 h and 8 h sufficed to induce nearly maximal down-regulation. However, it was possible to attenuate this triggering effect by adding cycloheximide after pulse of the cells. Thus, even after removal of the triggering agent (hCG or 8-BrcAMP), the loss of surface receptor could be triggered by a protein-sensitive signal. Taken as a whole these results indicate that a coordinated interaction is involved in the cell-surface hCG/LH receptor regulation. The apparent steady state of the number of receptors during the first hours of stimulation passed through a reuptake of internalized receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

The inositol phosphate/diacylglycerol pathway in MA-10 Leydig tumor cells. Activation by arginine vasopressin and lack of effect of epidermal growth factor and human choriogonadotropin

It is now well established that mouse epidermal growth factor (mEGF) modulates the hormonal responsiveness of MA-10 Leydig tumor cells but does not affect cell multiplication. The studies presented herein are the first in a series of experiments designed to characterize the intracellular signaling systems activated by mEGF and their possible roles in mediating the diverse biological actions of this growth factor in MA-10 cells. We show that (i) MA-10 cells express a hormone-sensitive inositol phosphate/diacylglycerol pathway that can be stimulated with arginine vasopressin (AVP), (ii) mEGF does not activate this pathway, and (iii) activation of this pathway with arginine vasopressin does not mimic the biological actions of mEGF. Other data presented show that lutropin/choriogonadotropin, the principal endocrine regulators of Leydig cell function, also do not stimulate the inositol phosphate/diacylglycerol pathway in MA-10 cells.     

Demonstration of a direct effect of inhibitors of the degradation of receptor-bound human choriogonadotropin on the steroidogenic pathway.

Results presented in the previous paper (Ascoli, M., and Puett, D. (1978) J. Biol. Chem. 253, 7832--7838) show that the degradation of receptor-bound 125I-labeled human choriogonadotropin can be inhibited by chloroquine, protease inhibitors, and metabolic inhibitors. These compounds were also shown to inhibit gonadotropin-stimulated steroidogenesis. It is reported herein that these inhibitors also block the stimulation of steroidogenesis by both cholera toxin and 8-Br-adenosine 3':5'-monophosphate, thus showing that they are not specific for the hormonal stimuli. These results, taken together with previous observations that show that NH2Cl can block hormone degradation without inhibiting hormone-stimulated steroidogenesis, strongly suggest that the degradation of choriogonadotropin is not required for its stimulatory action on progesterone production.     

Epidermal growth factor activates steroid biosynthesis in cultured Leydig tumor cells without affecting the levels of cAMP and potentiates the activation of steroid biosynthesis by choriogonadotropin and cAMP

The studies presented herein were designed to investigate the effects of mouse epidermal growth factor (mEGF) on steroid biosynthesis in a clonal strain of cultured murine Leydig tumor cells (designated MA-10). We show that in short-term incubations (up to 8 h), mEGF activates steroid biosynthesis without affecting cAMP levels. The maximal activation of steroid biosynthesis by mEGF (about 10-fold) is, however, much lower than the maximal activation detected with human choriogonadotropin (hCG) or cAMP analogues (about 1000-fold). We also show that mEGF has two (opposing) effects on the activation of steroidogenesis by hCG. Initially, it transiently attenuates the increase in intracellular cAMP and steroid biosynthesis provoked by submaximal concentrations of hCG. At later times, however, it potentiates the stimulatory effects of submaximal concentrations of hCG on steroid biosynthesis in a synergistic fashion. Last, we show that mEGF and submaximal concentrations of cAMP analogues also activate steroidogenesis in a synergistic fashion and that the degree of synergism attained with cAMP analogues plus mEGF is much higher than that attained with hCG plus mEGF. Taken together, our results show that mEGF (i) activates steroidogenesis without affecting cAMP levels and (ii) modulates the activation of steroidogenesis by the cAMP second messenger system.     

Characterization of the desensitized state of Leydig tumor cells

A perifusion system has been used to study the in vitro desensitization of isolated Leydig tumor cells. It was observed that the cells become refractory, as measured by decreased rates of steroidogenesis, during continuous perifusions with saturating concentrations of either human choriogonadotropin (CG), cholera toxin, or 8-bromo-cyclic AMP. Furthermore, an initial perifusion of the cells with either human CG, cholera toxin, or 8-bromo-cyclic AMP causes subsequent desensitization towards all three stimuli. Thus, each of these stimuli is equally effective in inducing a state of desensitization in these cells that is manifested by a steroidogenic lesion(s) distal to cyclic AMP formation. It was found that the post-cyclic AMP lesion(s) in the desensitized state occurs prior to the formation of pregnenolone. However, the decreased rates of steroidogenesis do not seem to arise from a depletion of intracellular cholesterol.     

Dual internalization pathway for lutropin and choriogonadotropin in porcine Leydig cells in primary culture

The role of the high affinity receptor in the internalization of porcine lutropin (pLH) and human choriogonadotropin (hCG) by porcine Leydig cells in primary culture during short-term stimulation by the two hormones was investigated. The fate of the hormones was followed either by electron microscopy (with colloidal gold-labeled hormones) or by measurement of the cellular distribution of [125I]pLH and [125I]hCG. With both techniques, the internalization of pLH was found to be one order of magnitude greater than hCG, though the recycling rate of the high affinity receptors was the same with both hormones. However, when the cell surface was progressively depleted of its high affinity receptors by preincubation with increasing doses of hCG or pLH, the internalization of [125I]pLH remained high and largely independent of the number of high affinity receptors still available on the cell surface, while that of [125I]hCG was found to be proportional to this number. The endocytosis of [125I]pLH could only be inhibited by the simultaneous presence of micromolar concentrations of unlabeled pLH, hCG or alpha or beta subunits of ovine LH (oLH). The intact alpha-hCG subunit and the deglycosylated alpha-oLH subunit were less potent, while beta-hCG and deglycosylated beta-oLH had no significant effect. These results could be explained by the existence of a "carrier" or "scavenger" receptor for LH, but with a low affinity (congruent to 3.10(6) M-1) and present in excess on the cell surface as compared to the high affinity receptor. The possible physiological significance of this receptor is discussed.     

Mouse Leydig cells express multiple P2X receptor subunits

ATP acts on cellular membranes by interacting with P2X (ionotropic) and P2Y (metabotropic) receptors. Seven homomeric P2X receptors (P2X₁–P2X₇) and seven heteromeric receptors (P2X_1/2, P2X_1/4, P2X_1/5, P2X_2/3, P2X_2/6, P2X_4/6, P2X_4/7) have been described. ATP treatment of Leydig cells leads to an increase in [Ca²⁺]_i and testosterone secretion, supporting the hypothesis that Ca²⁺ signaling through purinergic receptors contributes to the process of testosterone secretion in these cells. Mouse Leydig cells have P2X receptors with a pharmacological and biophysical profile resembling P2X₂. In this work, we describe the presence of several P2X receptor subunits in mouse Leydig cells. Western blot experiments showed the presence of P2X₂, P2X₄, P2X₆, and P2X₇ subunits. These results were confirmed by immunofluorescence. Functional results support the hypothesis that heteromeric receptors are present in these cells since 0.5 μM ivermectin induced an increase (131.2 ± 5.9%) and 3 μM ivermectin a decrease (64.2 ± 4.8%) in the whole-cell currents evoked by ATP. These results indicate the presence of functional P2X₄ subunits. P2X₇ receptors were also present, but they were non-functional under the present conditions because dye uptake experiments with Lucifer yellow and ethidium bromide were negative. We conclude that a heteromeric channel, possibly P2X_2/4/6, is present in Leydig cells, but with an electrophysiological and pharmacological phenotype characteristic of the P2X₂ subunit.     

Histogenesis of human extraparenchymal Leydig cells

From 64 consecutive autopsies of patients with neither testicular nor hormonal pathology, 26 showed extraparenchymal Leydig cells, located mainly in the epididymis and in the spermatic cord. The ultrastructural study of these specimens plus those obtained from 2 patients affected with functional testicular tumors leads to the following conclusions: (1) The origin of ectopic Leydig cells is not interstitial Leydig cells having infiltrated the testicular nerves and migrated along them towards ectopic locations. (2) The ectopic Leydig cells are considered to develop from undifferentiated precursor cells, located extraparenchymally, mainly inside and beside the testicular nerves. These precursor cells are similar to those observed in the testicular interstitium and have an ovoid shape and some cytoplasmic projections. The cytoplasm contains vesicles of smooth endoplasmic reticulum, lysosomes, lipid droplets and abundant microfilament bundles. The transformation from these cells into mature Leydig cells implies a progressive differentiation of the cytoplasmic components involved in steroid biosynthesis.     

Mitosis in adult human Leydig cells

Summary The ultrastructural study of testicular biopsies from 87 adult men revealed mitosis in two mature Leydig cells, each from a different man. The men showed normal hormone levels and had received no previous chemotherapy or hormone treatment, nor had they been exposed to known toxic agents. The presence of mitotic Leydig cells suggests that differentiated Leydig cells may divide and contribute either to the increase in the number of Leydig cells or to the formation of multinucleate Leydig cells.This work was partially supported by a grant from the Comisión Asesora de Investigación Científica y Técnica, Madrid, Spain