The effects of ethylene dimethane sulphonate (EDS) on rat Leydig cells: evidence to support a connective tissue origin of Leydig cells

Ethylene dimethane sulphonate (DS) administered to adult male rats in a single dose of 75 mg/kg body weight results in a rapid destruction of Leydig cells which, in turn, is associated with a marked decline in levels of serum testosterone. For 24-72 h after treatment with EDS (post-EDS) the Leydig cells undergo degenerative changes consisting of chromatin condensation and cytoplasmic vacuolation, and testicular macrophages progressively remove Leydig cells from the intertubular tissue by phagocytosis. This results in the total absence of Leydig cells on Days 7-14 and the absence of any detectable specific 125I-hCG binding to testis homogenates. Associated with the low levels of serum testosterone, levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in serum rise, LH to levels found in castrate rats. Morphometric and 125I-hCG binding studies indicate that a new generation of Leydig cells develop from Day 21 and reach control levels by Day 49. Morphologic observations suggest that the Leydig cells arise by differentiation from a pool of connective tissue cells that includes fibroblasts, lymphatic endothelial cells and pericytes. The new Leydig cells, which appear around Day 21 post-EDS, have the features of fetal Leydig cells. The latter appear to transform into Leydig cells typical of normal adult rats between 35-49 days post-EDS. The differentiation of new Leydig cells is associated with a reestablishment of normal levels of testosterone 21 days post-EDS. Serum LH and FSH return to normal at 28 days and 49 days respectively.     

11beta-hydroxysteroid dehydrogenase type 2 expression in the newly formed Leydig cells after ethane dimethanesulphonate treatment of adult rats

The enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD) catalyzes the reversible conversion of physiologically active corticosterone to the biologically inert 11beta-dehydrocorticosterone in rat testis and protect the Leydig cells (LCs) against the suppressive effect of glucocorticoids. The developmental pathway of the adult LCs population is accompanied with an increase in the 11beta-HDS activity. Thus, 11beta-HDS together with its role in controlling the toxicological effect of glucocorticoids on LCs can be used as a marker for their functional maturity. Ethane 1,2-dimethanesulphonate (EDS) treatment of adult rats become unique appropriate model, which enable to answer many questions related to the differentiation of adult LCs in the prepubertal rat testis. The aim of the present study was to investigate the specific changes in the 11beta-HDS type 2 immunoreactivity in tandem with the expression of androgen receptor (AR) during renewal of LCs population after EDS treatment. In the present study, we observed the first appearance of immunostaining for 11beta-HSD2 in new LCs population on day 14 after EDS administration when the progenitor LCs were detected. Our immunohistochemical analysis revealed progressive increases in the 11beta-HSD2 reaction intensity on 21 days after EDS treatment and reached a maximum on day 35. AR immunoexpression was found in new LCs on day 14 and 21 after EDS injection with an increasing curve of intensity. The most prominent AR immunostaining in new population LCs was evident by 35 days after EDS and that coincided with the increased number of LCs and restoration of adult LCs population. Our results demonstrated similar pattern of immunoreactivity for 11beta-HSD2 and AR in new LCs population after EDS treatment and suggested that the changes in 11beta-HSD2 expression can be used for evaluation of adult LCs differentiation in rat testis.     

Morphometric changes in the adrenal medulla of intact and ethane dimethanesulphonate (EDS)-injected rats subjected to chronic treatment with isoproterenol or propranolol

Morphometric characteristics of adrenal medulla were analysed stereologically in adult male rats injected with a single dose of ethane dimethanesulphonate (EDS), an agent that causes atrophy of the inner adrenocortical zone, or vehicle, and subjected to isoproterenol (ISO) or propranolol (PROP) treatment over the following 15 days. Compared with dimethylsulphoxide (DMSO) vehicle-injected controls, in EDS-administered rats the volume of chromaffin cell nuclei was decreased. ISO treatment in these rats increased the volumes of chromaffin cells and their nuclei. Furthermore, in both EDS- and vehicle-injected rats ISO significantly enlarged the total volume of medullary blood vessels, suggesting a vasodilatatory effect of β-adrenoceptor stimulation. However, unexpectedly, in EDS-injected rats PROP treatment also caused an increase in the volumes of chromaffin cells and their nuclei. This finding, most likely, may be related to a non-β-adrenoceptor-related action of PROP. Collectively, the present results suggest that the response of adrenomedullary chromaffin cells to chronic ISO-induced β-adrenoceptor stimulation is dependent on the functional status of adrenal cortex, so that the stereologically detectable changes were found only in rats previously exposed to the destructive action of EDS on the adrenal gland cortical cells. Moreover, they indicate that ISO treatment exerts a reversing effect on the morphometrical changes of chromaffine cells induced by EDS administration.     

Ethylene dimethane sulfonate (EDS) ablation of Leydig cells in adult rat depletes testosterone resulting in epididymal sperm granuloma: Testosterone replacement prevents granuloma formation

Sperm granuloma may develop in the epididymis following vasectomy or chemical insults. Inflammation due to sperm granuloma causes abdominal and scrotal pain. Prolonged and persistent inflammation in the epididymis due to sperm granuloma may lead to infertility. Extravasation of germ cells into the interstitium of epididymis following damage of the epididymal epithelium is one of the primary reasons for sperm granuloma-associated pathology. Since testosterone is vital for the maintenance of epididymal epithelium, we investigated the pathology of sperm granuloma and its relationship with testosterone. Adult rats were treated with a Leydig cell-specific toxicant ethylene dimethane sulfonate (EDS) to eliminate testosterone. At 7 days post-EDS, disrupted epididymal epithelium and sperm granuloma were observed in the caput epididymis. Sperm granuloma and caput were collagen-filled indicating fibrosis. Numerous round apoptotic cells were localized inside the caput lumen and dispersed through the sperm granuloma. Tnp1 (round spermatid marker) was significantly higher in the epididymis of the EDS-treated group compared to controls suggesting the apoptotic cells were round spermatids. Increases in CD68⁺ macrophages and T cells (CD4 and CD8) support an inflammatory immune infiltration in post-EDS epididymis. However, testosterone replacement following EDS prevented the sperm granuloma-associated pathology. We suggest that the immune response in the sperm granuloma may be due to the increased numbers of apoptotic round spermatids or other testicular tissue components that may be released, in addition to the regression of epididymal epithelium due to testosterone loss. Thus, testosterone replacement prevents EDS-induced sperm granuloma and ameliorates sperm granuloma-associated pathology.     

Functional multiplicity of atrial natriuretic peptide receptors on cultured rat Leydig tumor cells

Native rat atrial natriuretic peptide (NANP) was shown to bind with high affinity and to increase intracellular levels of cGMP in cultured rat Leydig tumor cells. A linear analog of NANP which lacks the disulfide-linked bridge structure also bound with high affinity but did not increase levels of intracellular cGMP or antagonize the increase of this cyclic nucleotide by NANP. These data are consistent with the existence of two functional subpopulations of ANP receptors on cultured rat Leydig tumor cells; one which is capable of activating guanylate cyclase and one which is not linked to this enzyme.     

Functional states of the luteinizing hormone/choriogonadotropin-receptor complex in rat Leydig cells

Three classes of gonadotropins with different ratios of stimulating to binding activities (S/B ratio) in rat Leydig cells have been identified. An S/B ratio of 1 was observed for rat luteinizing hormone (LH), porcine LH, and equine choriogonadotropin (CG) (class I), whereas ovine and equine LH exhibited and S/B ratio of 10-20 (class II) and human CG (hCG) (class III) an S/B ratio of 60. We coined the term "superactivity" to designate this particular behavior. This phenomenon was further studied by comparing the competitive activities of porcine LH (pLH) and hCG in radioreceptor assays using rat Leydig cell membranes and either radiolabeled oLH or hCG as the tracer, in the presence or absence of 150 mM NaCl. At equilibrium, both native hormones were equipotent in competing with 125I-oLH binding, but hCG was 4-fold more potent than pLH when 125I-hCG was used. Moreover, the binding rates of both hormones were considerably diminished in the presence of NaCl, but hCG binding at equilibrium was not affected, whereas that of oLH was almost completely abolished. From these results and previous data on the binding and internalization of these hormones, we suggest the existence of two interconvertible functional states of the hormone-receptor complex: (formula; see text). The equilibrium constant k3/k4 would be extremely high for hCG and lower and lower for the hormones in class II and class I, respectively. The equilibrium constant k1/k2 would be the one affected by the presence of NaCl and seems to be similar for all the hormones tested. The normal activity or superactivity of gonadotropins would thus be primarily dependent on the equilibrium between HR1 and HR2.     

Effects of prolonged cyclosporine-A treatment on the Leydig cells of the rat testis

The effects of a prolonged (30-day) treatment with daily therapeutical doses of cyclosporin A (CAS) (20 mg/kg) on testicular Leydig cells were studied in adult rats. CSA administration provoked a significant decrease in both basal and human chorionic gonadotropin (hCG)-stimulated testosterone concentration in the peripheral blood without affecting the volume of the testes or the interstitial space. However, there was conspicuous atrophy of the Leydig cells, due mainly to a decrease in mitochondria and smooth endoplasmic reticulum, the organelles containing the enzymes of testosterone synthesis. Lipid droplets, in which cholesterol is stored, were notably increased. The nuclear volume and the surface area per cell of rough endoplasmic reticulum fell significantly in Leydig cells of CAS-treated animals. In light of these findings, it is concluded that CSA inhibits the growth and steroidogenic capacity of rat Leydig cells, probably by depressing their protein synthesis. Whether the mechanism underlying the action of CSA on Leydig cells is only indirect, by blockade of hypophyseal gonadotropin release, or also direct is unsettled and requires further investigation.     

Effect of desialylated human chorionic gonadotropin (hCG) on the bioactivity of rat Leydig cells

Human chorionic gonadotropin is a glycoprotein hormone that, like LH, stimulates steroidogenesis in gonadal cells. Using a desialylation process, 95 per cent of the sialic acid residues from an intact standard hCG molecule were eliminated and then the electrophoretic properties and the bioactivity of the desialylated hCG were determined. Using rat Leydig cells as a biological model, the binding affinity to LH receptors of Leydig cell membranes, steroidogenic activity and second messenger production were studied. The results indicate that the loss of sialic acid from the hCG molecule slightly increases the binding activity to LH receptors and results in steroidogenic activity with an increased ED₅₀. Cyclic AMP production was significantly reduced however and arachidonic acid release was not observed. Several possible mechanisms that could explain these results are discussed. © 1998 John Wiley & Sons, Ltd.     

Sertoli cell-secreted protein(s) stimulates DNA synthesis in purified rat Leydig cells in vitro

We have examined the effects of Sertoli cell-secreted proteins (SCSP) on [3H]thymidine incorporation by purified preparations (greater than 96%) of rat Leydig cells to determine whether Sertoli cells influence DNA synthesis in these cells in vitro. Incubation of Leydig cells isolated from testes of rats of ages 16 to 90 days with SCSP (Mr greater than 10,000) induced significant dose-, time- and age-related increases in [3H]thymidine incorporation by the cells. A dose-response curve to SCSP showed that as little as 0.2 micrograms SCSP/ml consistently induced a small but significant increase (31% and 10% above control; P less than 0.001) in [3H]thymidine incorporation by Leydig cells isolated from immature (26 days) and mature (70 days) rats, respectively. The maximum response (230% and 48% above control) was obtained with a concentration of 18 micrograms SCSP/ml in cells isolated from immature and mature rats, respectively. Hydroxyurea, a specific inhibitor of replicative DNA synthesis, significantly (P less than 0.001) inhibited both basal and SCSP-induced [3H]thymidine incorporation in Leydig cells from immature and adult rats without affecting the viability of the cells. Incubation of immature rat Leydig cells in SCSP for 48 h also stimulated a 3-fold increase in cell number. The component of the crude SCSP which stimulated Leydig cell [3H]thymidine incorporation is trypsin-sensitive, heat-stable, and adsorbs to a heparin-agarose affinity column but not to concanavalin A-Sepharose. The secretion of this factor(s) by Sertoli cells is stimulated independently by FSH and testosterone. These results demonstrate for the first time that cultured Sertoli cells secrete a protein(s) which, in vitro, stimulates rat Leydig cell replicative DNA synthesis.     

Growth properties of RSV-transformed rat sarcoma (XC) cells

RSV-transformed rat sarcoma (XC) cells were characterized as to their ability to anchorage-dependent and anchorage-independent growth in various culture conditions. The proliferation of XC cells was dependent on serum concentration and initial cell density. The relationship between seeding density and the growth rate of XC cells indicated that the investigated cells produce factors with growth stimulating and growth inhibiting activity. The anchorage-independent growth inhibiting activity was found in ultrafiltrate (500 Mr 10,000) of XC cells conditioned medium. The obtained results suggests that anchorage-independent growth of XC cells may be regulated by two types of autocrine factors, which are antagonistic in their biological affects.     

Stem cell factor functions as a survival factor for mature Leydig cells and a growth factor for precursor Leydig cells after ethylene dimethane sulfonate treatment: implication of a role of the stem cell factor/c-Kit system in Leydig cell development

The significance of the interaction between Sertoli cell-produced stem cell factor (SCF) and its receptor, c-kit, on Leydig cells (LCs) during LC development and differentiation is unknown. In the present study, we investigated the potential role of the SCF/c-kit system in LC apoptosis and precursor LC proliferation after ethylene dimethane sulfonate (EDS) treatment in rats. A function-blocking anti-c-kit antibody, ACK-2, was used to block SCF/c-kit interaction at four time points, corresponding to the peak of LC apoptosis and three waves of proliferation of precursor LCs. Blockade of SCF/c-kit interaction by ACK-2 accelerated LC apoptosis and inhibited proliferation of precursor LCs during the first two waves of precursor LC proliferation around days 3-4 and day 10, but not the third wave of precursor LC proliferation around day 20 after EDS treatment. The data suggest that the soluble SCF might act as a survival factor for mature LCs and a growth factor for precursor LCs after EDS-induced LC depletion. This is also supported by a close correlation between the oscillating levels of soluble SCF mRNA and the profiles of LC apoptosis and regeneration. Since regeneration of the LC population after EDS treatment resembles the development of adult-type LCs during prepubertal life, the present findings imply that soluble SCF might participate in regulation of the formation of the LC population during testicular development. Our data also support a model in which delicate and reciprocal regulation exists between soluble SCF production by Sertoli cells, testosterone production by LCs, and pituitary gonadotropins.     

Diversity of methyl acceptor proteins in rat pheochromocytoma (PC12) cells revealed after treatment with adenosine dialdehyde

Protein N-methylation is a widespread modification whose functions are poorly understood. To overcome the inherent technical difficulties in identification of N-methylated proteins, we cultured PC12 cells with a methylation inhibitor, in expectation that proteins would accumulate in a hypomethylated state. Cell extracts were then incubated with [methyl-3H]S-adenosyl-L-methionine to label methyl-accepting sites via endogenous methyltransferases. Using two-dimensional gel electrophoresis we detected over 50 methyl acceptors, ranging from 18 to 120 kDa. Most had isoelectric points greater than 7.0. NG,NG-Dimethylarginine and NG-monomethylarginine accounted for about 90% of the methyl-3H-amino acids recovered after acid hydrolysis and thin-layer chromatography. The production of hypomethylated proteins should prove useful, not only in the identification of new methyl acceptors, but also in the isolation and characterization of new methyltransferases.     

D-Aspartate stimulation of testosterone synthesis in rat Leydig cells

D-Aspartate increases human chorionic gonadotropin-induced testosterone production in purified rat Leydig cells. L-Aspartate, D-,L-glutamate or D-,L-asparagine could not substitute for D-aspartate and this effect was independent of glutamate receptor activation. Testosterone production was enhanced only in cells cultured with D-aspartate for more than 3 h. The increased production of testosterone was well correlated with the amounts of D-aspartate incorporated into the Leydig cells, and L-cysteine sulfinic acid, an inhibitor of D-aspartate uptake, suppressed both testosterone production and intracellular D-aspartate levels. D-Aspartate therefore is presumably taken up into cells to increase steroidogenesis. Intracellular D-aspartate probably acts on cholesterol translocation into the inner mitochondrial membrane, the rate-limiting process in steroidogenesis.     

Antisteroidogenic actions of hydrogen peroxide on rat Leydig cells

It has been well known that reactive oxygen species (ROS) are produced in the steroidogenic pathway and spermatozoa. H2O2, one of ROS produced by spermatozoa, appears to be a primary toxic agent. In the present study, we examined the effects of H2O2 on the basal and evoked-testosterone release from primary Leydig cells, the protein expressions of cytochrome P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory (StAR) protein were also investigated. Our preparation was found to contain approximately 87% Leydig cells and very few macrophages. The results demonstrated that H2O2 (>1 x 10(-4) M) significantly inhibited the basal and hCG-stimulated testosterone release. H2O2 abolished forskolin- or 8-Br-cAMP-evoked testosterone release. In the presence of pregnenolone, progesterone, or androstenedione, the inhibitory effect of H2O2 on testosterone release was prevented. H2O2 also inhibited pregnenolone production in the presence of trilostane (an inhibitor of 3beta-hydroxysteroid dehydrogenase), therefore diminished the activity of P450scc in Leydig cells. In addition to the inhibition of hormone secretion, H2O2 also regulated steroidogenesis by diminishing protein expression of StAR. These results suggest that H2O2 acts directly on rat Leydig cells to diminish testosterone production by inhibiting P450scc activity and StAR protein expression.     

Stimulatory effect of follicle-stimulating hormone on rat Leydig cells

Summary The effects of FSH on the testicular interstitial tissue of immature hypophysectomized rats were studied by comparing morphological changes in Leydig cells with quantitative changes in interstitial tissue histology using morphometric analysis. Three groups of rats received subcutaneous injections of 0.5 ml saline vehicle or 10 g rFSH or 20 ng oLH (equivalent to the amount of LH known to contaminate the FSH), twice daily for 7 days. Administration of FSH significantly increased testis weight and stimulated more advanced spermatogenesis compared to saline or LH. Morphometric analysis of testes of LH-treated rats showed a small but significant increase in total interstitial cell volume compared to saline treatment. FSH caused much greater increases in the total volume of interstitial tissue and interstitial cells than either saline or LH and significantly increased the total volume of interstitial fluid by comparison with the other groups. FSH but not saline or LH treatment resulted in a striking hypertrophy of Leydig cells, to produce cells ultrastructurally identical to Leydig cells from adults. Since the target tissue of FSH is the seminiferous epithelium, the observed effects on Leydig cells by FSH treatment suggest that the secretion of factors by the seminiferous tubules may mediate the maturation of Leydig cells.     

Percoll-gradient separation of Leydig cells from postnatal rat testes

The characteristics of the fetal and adult populations of Leydig cells from postnatal rat testes were compared by Percoll gradient centrifugation. A single peak of hCG binding, due to the presence of fetal Leydig cells, was obtained after purification of intertubular cells from 8-day-old animals. Two peaks of specific hCG binding were obtained after purification of intertubular cells from 15-day-old rats: it was confirmed by autoradiographic techniques that the hCG was bound by adult-like Leydig cells in one peak and fetal Leydig cells in the other. Similarly, intertubular cell preparations from 21- and 25-day-old rats resolved into two peaks of hCG binding; adult-like Leydig cells were observed in the first peak, but fetal Leydig cells were rarely observed in the second of these peaks. These results demonstrate the separation of two Leydig cell populations from intertubular cells obtained from animals aged up to 15 days. Thereafter the pattern of the hCG binding profile is similar but is not due to the presence of the same cell types. Therefore these results emphasize the necessity for morphological identification of cell types to permit the correct interpretation of the corresponding biochemical data.     

The industrial chemical bisphenol A (BPA) interferes with proliferative activity and development of steroidogenic capacity in rat Leydig cells

The presence of bisphenol A (BPA) in consumer products has raised concerns about potential adverse effects on reproductive health. Testicular Leydig cells are the predominant source of the male sex steroid hormone testosterone, which supports the male phenotype. The present report describes the effects of developmental exposure of male rats to BPA by gavage of pregnant and lactating Long-Evans dams at 2.5 and 25 μg/kg body weight from Gestational Day 12 to Day 21 postpartum. This exposure paradigm stimulated Leydig cell division in the prepubertal period and increased Leydig cell numbers in the testes of adult male rats at 90 days. Observations from in vitro experiments confirmed that BPA acts directly as a mitogen in Leydig cells. However, BPA-induced proliferative activity in vivo is possibly mediated by several factors, such as 1) protein kinases (e.g., mitogen-activated protein kinases or MAPK), 2) growth factor receptors (e.g., insulin-like growth factor 1 receptor-beta and epidermal growth factor receptors), and 3) the Sertoli cell-secreted anti-Mullerian hormone (also called Mullerian inhibiting substance). On the other hand, BPA suppressed protein expression of the luteinizing hormone receptor (LHCGR) and the 17beta-hydroxysteroid dehydrogenase enzyme (HSD17B3), thereby decreasing androgen secretion by Leydig cells. We interpret these findings to mean that the likely impact of deficits in androgen secretion on serum androgen levels following developmental exposure to BPA is alleviated by increased Leydig cell numbers. Nevertheless, the present results reinforce the view that BPA causes biological effects at environmentally relevant exposure levels and its presence in consumer products potentially has implication for public health.