Dynamic Remodeling of Membranes and Their Lipids during Acute Hormone-Induced Steroidogenesis in MA-10 Mouse Leydig Tumor Cells.

Lipids play essential roles in numerous cellular processes, including membrane remodeling, signal transduction, the modulation of hormone activity, and steroidogenesis. We chose steroidogenic MA-10 mouse tumor Leydig cells to investigate subcellular lipid localization during steroidogenesis. Electron microscopy showed that cAMP stimulation increased associations between the plasma membrane (PM) and the endoplasmic reticulum (ER) and between the ER and mitochondria. cAMP stimulation also increased the movement of cholesterol from the PM compared to untreated cells, which was partially inhibited when ATPase family AAA-domain containing protein 3 A (ATAD3A), which functions in ER and mitochondria interactions, was knocked down. Mitochondria, ER, cytoplasm, PM, PM-associated membranes (PAMs), and mitochondria-associated membranes (MAMs) were isolated from control and hormone-stimulated cells. Lipidomic analyses revealed that each isolated compartment had a unique lipid composition, and the induction of steroidogenesis caused the significant remodeling of its lipidome. cAMP-induced changes in lipid composition included an increase in phosphatidylserine and cardiolipin levels in PAM and PM compartments, respectively; an increase in phosphatidylinositol in the ER, mitochondria, and MAMs; and a reorganization of phosphatidic acid, cholesterol ester, ceramide, and phosphatidylethanolamine. Abundant lipids, such as phosphatidylcholine, were not affected by hormone treatment. Our data suggested that PM-ER-mitochondria tethering may be involved in lipid trafficking between organelles and indicated that hormone-induced acute steroid production involves extensive organelle remodeling.

Interstitial Leydig Cell Tumorigenesis-Leptin and Adiponectin Signaling in Relation to Aromatase Expression in the Human Testis.

Although epidemiological studies from the last years report an increase in the incidences of Leydig cell tumors (previously thought to be a rare disease), the biochemical characteristics of that tumor important for understanding its etiology, diagnosis, and therapy still remains not completely characterized. Our prior studies reported G-protein coupled estrogen receptor signaling and estrogen level disturbances in Leydig cell tumors. In addition, we found that expressions of multi-level-acting lipid balance- and steroidogenesis-controlling proteins including peroxisome proliferator-activated receptor are altered in this tumor. In order to get deeper into the other molecular mechanisms that regulate lipid homeostasis in the Leydig cell tumor, here we investigate the presence and expression of newly-described hormones responsible for lipid homeostasis balancing (leptin and adiponectin), together with expression of estrogen synthase (aromatase). Samples of Leydig cell tumors (n = 20) were obtained from patients (31-45 years old) and used for light and transmission electron microscopic, western blotting, and immunohistochemical analyses. In addition, body mass index (BMI) was calculated. In tumor mass, abundant lipid accumulation in Leydig cells and various alterations of Leydig cell shape, as well as the presence of adipocyte-like cells, were observed. Marked lipid content and various lipid droplet size, especially in obese patients, may indicate alterations in lipid homeostasis, lipid processing, and steroidogenic organelle function in response to interstitial tissue pathological changes. We revealed significantly increased expression of leptin, adiponectin and their receptors, as well as aromatase in Leydig cell tumors in comparison to control. The majority of patients (n = 13) were overweight as indicated by their BMI. Moreover, a significant increase in expression of phospholipase C (PLC), and kinases Raf, ERK which are part of adipokine transductional pathways, was demonstrated. These data expand our previous findings suggesting that in human Leydig cell tumors, estrogen level and signaling, together with lipid status, are related to each other. Increased BMI may contribute to certain biochemical characteristics and function of the Leydig cell in infertile patients with a tumor. In addition, altered adipokine-estrogen microenvironment can have an effect on proliferation, growth, and metastasis of tumor cells. We report here various targets (receptors, enzymes, hormones) controlling lipid balance and estrogen action in Leydig cell tumors indicating their possible usefulness for diagnostics and therapy.

Preneoplastic and neoplastic changes in the Leydig cells population in mice exposed to low doses of cadmium.

The morphological consequences of chronic exposition to low doses of cadmium (Cd) in the Leydig cells population were investigated in 40 sexually mature male mice at morphological and ultrastructural levels. Animals were orally exposed to cadmium (0.015 g/L of CdCl(2) in drinking water) for 3, 6, 12 and 18 months and then sacrificed, samples were collected for toxicological, light and electron microscope studies. Vascular lesions were evident from 6 months of Cd exposure, the severity of the morphological changes observed in the testicular vases were highly and clearly correlated to the time of exposure to Cd. The severity of the Leydig cells morphological changes were increasing along the time of exposure. Presence of cytoplasm vacuolization and degenerative images of the cells were frequent after 12 months of Cd exposure. Also two Leydig cells tumours after 12 and 18 months Cd exposure were presented. These results indicate that prolonged exposures to low doses of Cd are able to induce morphological damage on the Leydig cells.

Leydig cell tumor and hyperplasia: a review.

We describe the role of Leydig cells in normal, hyperplastic and neoplastic testis. Recent acquisitions on etiology and pathobiology of Leydig cell proliferations, unusual microscopic presentations and clinical and morphologic features predictive of malignancy are reported.

Urethral stromal tumor with pacemaker cell phenotype.

Penile malignancies are rare in developed countries. The authors present a case of a penile urethral mesenchymal tumor occurring in a 51-year-old Caucasian male and displaying light microscopic, immunohistochemical, and ultrastructural features suggestive of a pacemaker cell type, combined with a lack of diagnostic features of any other established tumor category. The immunohistochemical profile was intensely positive for vimentin, PKC theta, and NSE and weakly positive to nonreactive for CD34 and smooth muscle actin, and entirely negative for CD117 (c-kit), S-100, and other markers. C-kit and PDGFRA gene analysis showed no mutations. Electron microscopy revealed tumor cells with plentiful cytoplasm and cytoplasmic processes/filopodia, both filled with intermediate filaments and occasional solitary focal densities. There were also prominent smooth endoplasmic reticulum cisternae, caveolae, neurosecretory granules, particularly concentrated in cytoplasmic processes, and synaptic-type structures. Poorly formed basal lamina, gap junctions, and intercellular collagen aggregates, consistent with skeinoid-type fibers, were also noted. Interstitial cells with potential pacemaker function have been recently described in the lower urinary tract, including the urethra, and this tumor may be related to this cellular phenotype.

Acute action of choriogonadotropin on Leydig tumor cells: qualitative and quantitative transformation of lipid moieties.

BACKGROUND: Testicular Leydig cells use either exogenous or de novo synthesized cholesterol as the substrate for the production of testosterone with hormone stimulation. Although the long-term effect of trophic hormones on Leydig cell cholesterol uptake, storage, and deesterification has been well documented, the early effects of the human choriogonadotropin (hCG) on cell cholesterol/lipid distribution are not yet known. METHODS: Sections of cells treated with hCG for 15 sec to 30 min were examined by electron microscopy (EM) for the surface density of lipid moieties in the cytoplasm. In addition, the time-dependent distribution of lipids within the cytoplasmic inclusions and the ultimate destination of this substrate were evaluated by EM. The results were analyzed with standard morphometric methods. RESULTS AND CONCLUSION: The surface density of cytoplasmic lipid pools increased significantly within the 15 sec following the exposure of cells to hCG, and it tapered off to the control level in the subsequent 30 min. Such a fluctuation in the amount of cytoplasmic lipids may be due to (1) the quantity of released substrate from the reticular compartment or (2) the rate of its transport across the outer mitochondrial membrane to the inner mitochondrial cytochrome P450 side-chain cleavage, where steroidogenesis begins with the conversion of cholesterol to pregnenolone. These two processes were not quantitatively coordinated in the stimulated cell during the initial 30 min, resulting in a surplus of cytoplasmic lipid pools. To compensate for such uneven metabolic balance, the cell apparently disposed of the excess substrate by a mechanism of molecular regrouping from a micellar configuration to a bilayer structure followed by exocytosis.

ATP and a mitochondrial electrochemical gradient are required for functional activity of the steroidogenic acute regulatory (StAR) protein in isolated mitochondria.

The Steroidogenic Acute Regulatory (StAR) protein has been put forth as the rapidly synthesized, cycloheximide-sensitive protein that is required for the transport of cholesterol to the inner mitochondrial membrane and the P450scc enzyme and thereby acutely regulates steroidogenesis in steroidogenic tissues. In this study, several of the factors that may be required for StAR activity were examined using an in vitro system. Lysates from StAR-transfected COS-1 cells were added to mitochondria isolated from MA-10 Leydig tumor cells. Results obtained demonstrated that StAR-containing cell lysate increased steroidogenesis in isolated mitochondria, but failed to do so in the presence of m-CCCP, apyrase, or AMP-PNP, suggesting that StAR function requires ATP hydrolysis as well as an electrochemical gradient for maximal steroidogenic activity.

Leydig cell tumor of the testis: analysis of testosterone production and secretion by three-dimensional histoculture.

We treated an 11-year-old boy with a testicular Leydig cell tumor. We analyzed the testosterone production of this tumor by immunolocalization of steroidogenic enzymes and in vitro three-dimensional histoculture. Spermatic venous blood from the tumor bearing testis had noticeably high concentrations of testosterone and androstenedione. The tumor had the characteristic ultrastructural features of steroid producing cells and was immunoreactive for P450scc (side chain cleavage), 3 beta HSD (hydroxysteroid dehydrogenase) and P450c17 (17 alpha-hydroxylase). Three-dimensional collagengel-supported histoculture demonstrated that the tumor tissue in the culture maintained its histologic architecture, expression of steroidogenic enzymes, and secretion of testosterone into the medium for up to 7 days in culture. Histoculture preserved in vitro testosterone production in this case of testicular Leydig cell tumor.

Steroid production after in vitro transcription, translation, and mitochondrial processing of protein products of complementary deoxyribonucleic acid for steroidogenic acute regulatory protein.

We have previously demonstrated that steroidogenic acute regulatory protein (StAR) is essential for the rate-limiting step in the acute regulation of steroidogenesis, which is the transport of cholesterol from the outer to the inner mitochondrial membrane. We have hypothesized that this transport occurs as the 37-kilodalton (kDa) precursor form of StAR is imported into the mitochondria and processed to its 30-kDa mature forms. Using an in vitro transcription and translation system in the presence of mitochondria isolated from unstimulated mouse MA-10 Leydig tumor cells, we now directly show that the 37-kDa form is indeed the cytosolic precursor of StAR and can be processed by mitochondria to all four 30-kDa mature forms. To determine the subcellular location of StAR in steroidogenic cells, ultrastructural immunocytochemistry was performed in adrenal zona fasciculata cells using the protein A-gold technique. We show that StAR is associated exclusively with the mitochondria. There, StAR is primarily localized in the intermembrane space and the intermembrane space side of the cristae membrane. StAR was shown to induce steroid production in isolated mitochondria. StAR protein was expressed in COS1 cells and the cell lysate, which was shown to contain abundant levels of StAR by Western blot analysis, was incubated with mitochondria isolated from unstimulated MA-10 cells. In these experiments, StAR increased steroid production by at least 4-fold over control mock-transfected lysate, and this increase was time and dose dependent. Furthermore, the increase in steroid production induced by StAR-containing lysate was not observed when COS1 lysate containing high levels of another mitochondrially imported protein, adrenodoxin, was used. We conclude from these results that in response to tropic hormone stimulation of steroidogenic cells, StAR is synthesized as a 37-kDa precursor, imported into the mitochondria, processed to its 30-kDa mature forms, and localized to the intermembrane space. During import and processing in vitro, StAR induces steroid production in isolated mitochondria in a specific manner.

Topography of the Leydig cell mitochondrial peripheral-type benzodiazepine receptor.

Native MA-10 mouse Leydig tumor cell mitochondrial preparations were examined by transmission electron (TEM) and atomic force (AFM) microscopic procedures in order to investigate the topography and organization of the peripheral-type benzodiazepine receptor (PBR). Mitochondria were immunolabeled with an anti-PBR antiserum coupled to gold-labeled secondary antibodies. Results obtained indicate that the 18,000 MW PBR protein is organized in clusters of 4-6 molecules. Moreover, on many occasions, the interrelationship among the PBR molecules was found to favor the formation of a single pore. Taking into account recent observations that the 18,000 MW PBR protein is functionally associated with the pore forming 34,000 MW voltage-dependent anion channel (VDAC) these results suggest that (i) the mitochondrial PBR complex could function as a pore, thus allowing the translocation of cholesterol and other molecules to the inner mitochondrial membrane, and (ii) the native receptor is a multimeric complex of an approximate 140,000 MW composed on an average of five 18,000 PBR subunits, one 34,000 VDAC subunit, and associated lipids.

Intranuclear Reinke's crystals in a testicular Leydig cell tumor diagnosed by aspiration cytology. A case report.

Fine needle aspiration cytologic findings in a Leydig cell tumor of the testis are described. Besides the rarity of case reports on fine needle aspiration cytologic diagnosis of this tumor, the present case was of interest because of the finding of numerous intranuclear and intracytoplasmic Reinke's crystals as well as some lying free between the cells. In a few cells the intranuclear crystals were seen to orient themselves in a row, with a nipplelike protrusion of the nuclear membrane as if being pushed by the crystals. Besides the well-formed crystals, many nuclei showed irregular, thin, groovelike spaces that may have been earlier stages of crystal formation. These findings suggested the intranuclear formation of Reinke's crystals. Extranuclear crystals were seen to fuse in pairs and hence to appear thicker than the intranuclear crystals. The crystals lying free between the cells also showed a linear arrangement in places. The crystals were more numerous with May-Grünwald-Giemsa (MGG) staining. Intranuclear crystals were seen only in MGG-stained smears.

A cholesteryl ester hydrolase inhibitor blocks cholesterol translocation into the mitochondria of MA-10 Leydig tumor cells.

The present studies describe an unexpected action of a cholesteryl ester hydrolase inhibitor on MA-10 Leydig tumor cells. These studies were initially intended to use the inhibitor, diethylumbelliferyl phosphate, to block cholesteryl ester hydrolysis and, thus, determine the contributions of this form of cholesterol to steroidogenesis and reveal any direct hormone effects on cholesterol esterification. Although this compound acted as an effective inhibitor of the cholesteryl ester hydrolase in intact MA-10 cells, it inhibited steroidogenesis at lower concentrations and to a greater extent than could be explained by simple inhibition of the ester hydrolase enzyme. This compound proved not to be generally toxic, but blocked some process occurring between cAMP formation and cholesterol side-chain cleavage. The diethylumbelliferyl phosphate block of steroidogenesis was readily bypassed by 22-hydroxycholesterol. These data indicated that the compound inhibited cholesterol transport. The lesion in cholesterol transport was not general, but very specific; cholesterol translocation to the mitochondrial site of cholesterol side-chain cleavage was blocked by this organophosphate compound.

Ultrastructure and immunohistochemistry of a fetal-type Leydig cell tumor.

A symptomless scrotal mass was removed from a 34-year-old man. The lesion was 7 cm in diameter and it was grossly a hemorrhagic cyst with indurated walls. By light microscopy tumor cell clusters and cords were seen infiltrating the testicle, tunica albuginea, and paratesticular tissue. In the immunohistochemical analysis the tumor cells were immunoreactive with anti-S-100 protein and anticarcinoembryonic antigen, but they did not express cytokeratin or alpha-fetoprotein as tested with paraffin sections. Tumor cell clusters were enveloped by a laminin-positive basement membrane. Electron microscopy revealed abundant smooth endoplasmic reticulum, lipid droplets, and membranous whorls in the cytoplasm. Lamellar whorled bodies were also seen in mitochondria, which contained tubulovesicular cristae. The presence of a well-developed, often multilayered basement membrane was confirmed at ultrastructural level. The activity of 3-beta-hydroxysteroid dehydrogenase suggested that the tumor cells were capable of androgen synthesis. The morphological features are reminiscent of fetal-type Leydig cells and are distinctly different from the Leydig cell tumors described so far.

Benign testicular tumors in children with congenital adrenal hyperplasia.

The association between testicular tumors/nodules and congenital adrenal hyperplasia (CAH) has been previously reported. From 1960 to 1989, three patients (13 to 18 years old) with long-standing CAH developed testicular masses. Two patients with 21-hydroxylase deficiency were diagnosed in the neonatal period while one other with 11-hydroxylase deficiency was diagnosed at 3 years of age when he presented with sexual precocity. In all three patients, medical compliance was poor. The testicular masses were bilateral in two patients and unilateral in one, measured 1 to 2 cm, and occupied only the upper half of the testicle. Testicular biopsy specimens were obtained after at least 6 months of evidence of compliance with the adrenocorticotrophic hormone (ACTH) suppressive medication and failure of the nodules to regress. On gross examination the masses appeared to be firm yellow brown nodules. Light microscopy showed interlacing strands, cords, and rests of cells resembling interstitial (Leydig) cells but with no Reinke crystalloids. Electronmicroscopy in all patients showed variable amounts of both smooth and rough endoplasmic reticulum, the later with occasional dilated cisternae. Follow-up ranged from 6 months to 6 years. No further surgical treatment has been necessary. There has been no evidence of recurrence, distant metastases, or secondary malignancies during the time of follow-up. These findings suggest that testicular tumors may develop from chronic excessive ACTH stimulation of a putative pluripotential testicular cell, a Leydig cell, or an adrenal cortical rest. Unlike other testicular tumors these do not require orchiectomy as the initial form of therapy.

Leydig cell tumor of the testis: a case diagnosed by fine-needle sampling without aspiration with histologic, immunohistologic, and electron microscopic analysis.

Fine-needle sampling without aspiration was performed in a patient with a testicular mass. The cytologic diagnosis was consistent with Leydig cell tumor. Cytologic features included abundant grey-blue cytoplasms with spherical or oval nuclei in May-Grünwald-Giemsa-stained smears. Intranuclear inclusions were observed but no Reinke's crystals were detected. Histologic findings confirmed the diagnosis and tumor cells were positive for vimentin. Electron microscopic analysis of the tumor showed abundant smooth endoplasmic reticulum and mitochondria with tubulovesicular cristae but no Reinke's crystals.

Cholesterol movement between the plasma membrane and the cholesteryl ester droplets of cultured Leydig tumour cells.

The present studies characterize the turnover of plasma membrane cholesterol in MA-10 Leydig tumour cells. Plasma membrane cholesterol of MA-10 cells was slowly internalized and converted into cholesteryl ester. Low-density lipoprotein (LDL) stimulated, in a dose- and time-dependent fashion, plasma membrane cholesterol conversion into intracellular esters. Stimulation of membrane internalization was not simply the consequence of accelerated uptake of membrane with LDL, since binding and internalization of epidermal growth factor and transferrin had no effect on turnover of plasma membrane cholesterol. The protein of LDL is unimportant as well, since delipidated LDL had no effect on membrane turnover. The action of LDL on cholesterol turnover was explained entirely by its contribution to cholesteryl ester stores. The degree of plasma membrane cholesterol internalization and esterification was directly proportional to the size of cellular ester stores.

Malignant Sertoli and Leydig cell tumour in a boar.

A rare case of Sertoli and Leydig cell tumour was investigated in a 6-month-old boar. The neoplasm was found in the right testis and had metastasized to the liver, spleen, kidneys and diaphragmatic peritoneum. Metastatic nodules were also present in the tissues near the right testis and some neoplastic cells were present in the superficial inguinal lymph node. The neoplastic cells exhibiting severe pleomorphism were divided into Sertoli and Leydig cell types, although in some sites, it was not possible to classify tumour cells clearly as Sertoli or Leydig in type. In the metastatic lesions there were anaplastic Sertoli cells with abundant collagen fibres. Some neoplastic Sertoli cells and a few neoplastic Leydig cells revealed cytoplasmic reactivity for testosterone. Ultrastructurally, the neoplastic cells were characterized by mitochondria with tubulovesicular cristae, smooth endoplasmic reticulum, membranous structures, lysosomes, lipofuscin granules or lipid droplets.

Steroid biosynthesis in the Sertoli-Leydig cell tumor: effects of insulin and luteinizing hormone.

In vitro steroid production by a virilizing Sertoli-Leydig cell tumor of the ovary was studied. For comparison, stromal tissue from the opposite normal ovary was also incubated under similar conditions. The tumor fragments secreted significantly more testosterone (527 +/- 168 versus 48 +/- 29 pg/mg tissue, p less than 0.001), androstenedione (1188 +/- 400 versus 40 +/- 10 pg/mg tissue, p less than 0.001), and dehydroepiandrosterone (419 +/- 132 versus 73 +/- 25 pg/mg tissue, p less than 0.004) than that of normal ovarian stroma. Measurement of steroids in the ovarian venous serum draining the tumor indicated a peripheral ovarian gradient for both delta 4 and delta 5 steroids. Incubation of tumor fragments with luteinizing hormone alone resulted in a significant increase in the secretion of androstenedione and dehydroepiandrosterone (p less than 0.05). Addition of insulin to luteinizing hormone resulted in significantly greater release of androstenedione than that of treatment with luteinizing hormone alone (p less than 0.04). Addition of insulin had no effect on the release of dehydroepiandrosterone. Luteinizing hormone and insulin, either alone or in combination, failed to produce any change in the secretion of testosterone. We conclude that (1) increased testosterone secretion by Sertoli-Leydig cell tumor resulted from increased availability of precursors from both delta 4 and delta 5 pathways; (2) the tumor was responsive to luteinizing hormone with an increase in the secretion of androstenedione and dehydroepiandrosterone; (3) insulin acts synergistically with luteinizing hormone to increase secretion of androstenedione; (4) the tumor has specific binding sites for insulin; and (5) the increased levels of insulin and luteinizing hormone in polycystic ovarian disease may play a role in the pathogenesis of Sertoli-Leydig cell tumor.

Plasma membrane cholesterol: removal and insertion into the membrane and utilization as substrate for steroidogenesis.

The plasma membrane cholesterol content of MA-10 Leydig tumor cells is depleted by trophic hormone stimulation and repleted by incubating the cells with low density lipoprotein. The present studies used subcellular fractionation to investigate the membranes involved in steroid hormone synthesis. The results showed that the plasma membrane was the major source of cholesterol substrate and that the cholesterol content changed independently of any mass changes in membrane protein or phospholipid. Membrane phospholipid composition also did not change as membrane cholesterol content decreased or increased, a finding inconsistent with the proposal that phospholipid composition dictates the amount of cholesterol contained in a membrane. The mitochondria of the MA-10 cells were cholesterol rich, containing more cholesterol per unit protein or phospholipid than the plasma membrane. This cholesterol was presumably in the outer mitochondrial membrane, since virtually all of the cholesterol of intact mitochondria was accessible to cholesterol oxidase. Although there was a high concentration of mitochondrial cholesterol, this cholesterol was largely inert as a substrate for steroidogenesis, and plasma membrane cholesterol was incorporated into steroid hormones without ever equilibrating with the mitochondrial cholesterol pool.

Ultrastructure and light microscopical study of a Leydig cell tumor of the testis associated with bilateral gynaecomastia.

Light and electronmicroscopic study of a Leydig cell testicular tumor in an 18-year-old male is presented. Bilateral gynaecomastia and normal hormonal blood levels were found. Emphasis on the diagnostic value of electronmicroscopy is remarked upon, based on the following ultrastructural characteristics of the cells; 1) Ovoid shaped nuclei with ondulating contours and dispersed and homogeneous chromatin, 2) Rich agranular endoplasmic reticulum with frequent special modifications, such as membranous whorls with a central cytoplasmic mass or lipid droplets, 3) Numerous mitochondria with occasional tubular cristae, 4) Numerous lipid vacuoles. Other structures also identified in this tumor are Reinke crystalloids, cytoplasmic microbodies, myelin figures, gap-type junctional complexes and paracrystalline inclusions of Payer type E, which are less common.