Functional properties of developing rat Leydig cells after treatment with ethylene dimethanesulphonate (EDS)

The development of a new population of Leydig cells after specific elimination of existing Leydig cells in mature rats by ethylene dimethanesulphonate (EDS) was characterized by investigating the testicular activities of 5 alpha-reductase and non-specific esterase, the serum concentrations of 3 alpha-androstanediol and testosterone and the Leydig cell morphology. Plasma concentrations of both androgens were strongly reduced up to 15 days after administration of EDS. Thereafter, in contrast to the gradual and continuous increase of serum testosterone values, the changes in serum 3 alpha-androstanediol were transient, with the highest level on Day 35. The temporal pattern of testicular 5 alpha-reductase activity was almost similar to that of serum 3 alpha-androstanediol. The testicular esterase activity increased gradually from Day 25 until Day 76. The temporal changes in steroid concentrations and enzyme activities after EDS administration indicate that the development of the Leydig cells in EDS-treated rats occurs in a fashion similar to that in pubertal rats. However, the numerous lipid droplets and large nuclei in these Leydig cells indicate that these cells may also be classified as fetal cells. It is concluded that, after treatment with EDS, fetal and pubertal characteristics are present in Leydig cells. It is, however, unknown whether both characteristics are present in one or in two distinct cell populations.     

Differential effects of the destruction of Leydig cells by administration of ethane dimethane sulphonate to postnatal rats

Ethane dimethane sulphonate (EDS) is a cytotoxic drug that selectively destroys Leydig cells in adult testes. This study has examined the effect of a single injection of EDS on the Leydig cell populations present in the testes of rats aged 5, 10, or 20 days. Microscopic examination of the tissue demonstrated that the fetal Leydig cell population was destroyed at all ages, but that subsequent development of the adult population of Leydig cells was not affected. Whilst the destruction of the fetal Leydig cells in this acute phase of EDS on 5-day-old rats was accompanied by a decline in serum testosterone levels, there was no apparent effect on this hormone when EDS administered at 10 or 20 days of age, despite the destruction of fetal Leydig cells in these rats. The long-term effects of EDS on Day 5 of age resulted in proliferation of the intertubular tissue in which more Leydig cells were observed, but serum testosterone and testosterone levels in response to human chorionic gonadotropin stimulation in vitro were normal despite moderate or severe disruption of the seminiferous epithelium. These data show that the fetal Leydig cells of immature testes are sensitive to the cytotoxic effects of EDS in the adult, but the response of the testes differs depending on the age at which the drug is administered.     

Effects of thyroid hormone on Leydig cell regeneration in the adult rat following ethane dimethane sulphonate treatment

We tested the effects of thyroid hormone on Leydig cell (LC) regeneration in the adult rat testis after ethane dimethyl sulphonate (EDS) treatment. Ninety-day-old, thyroid-intact (n = 96) and thyroidectomized (n = 5) male Sprague-Dawley rats were injected intraperitoneally (single injection) with EDS (75 mg/kg) to destroy LC. Thyroid-intact, EDS-treated rats were equally divided into three groups (n = 32 per group) and treated as follows: control (saline-injected), hypothyroid (provided 0.1% propyl thiouracil in drinking water), and hyperthyroid (received daily subcutaneous injections of tri-iodothyronine, 100 microg/kg). Testing was done at Days 2, 7, 14, and 21 for thyroid-intact rats and at Day 21 for thyroidectomized rats after the EDS treatment. Leydig cells were absent in control and hyperthyroid rats at Days 2, 7, and 14; in hypothyroid rats at all ages; and in thyroidectomized rats at Day 21. The LC number per testis in hyperthyroid rats was twice as those of controls at Day 21. 3beta-Hydroxysteroid dehydrogenase (LC marker) immunocytochemistry results agreed with these findings. Mesenchymal cell number per testis was similar in the three treatment groups of thyroid-intact rats on Days 2 and 7, but it was different on Days 14 and 21. The highest number was in the hypothyroid rats, and the lowest was in the hyperthyroid rats. Serum testosterone levels could be measured in control rats only on Day 21, were undetectable in hypothyroid rats at all stages, and were detected in hyperthyroid rats on Days 14 and 21. These levels in hyperthyroid rats were twofold greater than those of controls on Day 21. Serum androstenedione levels could be measured only in the hyperthyroid rats on Day 21. Testosterone and androstenedione levels in the incubation media showed similar patterns to those in serum, but with larger values. These findings indicate that hypothyroidism inhibits LC regeneration and hyperthyroidism results in accelerated differentiation of more mesenchymal cells into LC following the EDS treatment. The observations of the EDS-treated, thyroidectomized rats confirmed that the findings in hypothyroid rats were, indeed, due to the deficiency of thyroid hormone.     

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.     

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.     

Direct inhibitory effects of estrogens on rat Leydig cells in vitro.

In dispersed rat interstitial cells in vitro both natural and synthetic estrogens inhibited the action of pituitary luteinizing hormone (LH), as assessed by testosterone production. The estrogens also inhibited dibutyryl cyclic AMP induced steroidogenesis, suggesting that one point of inhibition could be distal to the formation of cyclic AMP in the cells. Diethyl stilbestrol and its clinically used sodium phosphate derivative (Honvol), also affected hormone-receptor interaction when tested with rat testicular homogenates. Among estradiol, estradiol benzoate, Honvol and diethyl stilbestrol only the latter at high concentration had toxic effects on Leydig cells as noted from loss of thier viability.     

The Rutilus alburnoides complex (Cyprinidae): evidence for a hybrid origin

A genetic survey of 18 presumptive enzyme loci was conducted on members of the diploid-triploid R. alburnoides complex in order to test the hypothesis of hybrid origin. Most specimens examined were heterozygous at a high proportion of loci, with seven loci showing virtually fixed heterozygosity in some or all populations. This observation strongly supports the hypothesized hybrid ancestry. Involvement in the origin of the R. alburnoides complex was examined by comparison of allels with those observed in the other diploid cyprinids that inhabit the same Portuguese drainages. Allelic composition at the homozygous loci IDDH* and LDH-B* seems to rule out the genus Chondrostoma and other members of the genus Rutilus as one of the ancestors, and supports Leuciscus sp. as one parental species involved in the putative hybrid origin of the R. alburnoides complex. Moreover, with few exceptions, all specimens exhibited at every locus one or two alleles also present in extant populations of Leuciscus. The indentity of the other parental taxon remains unclear. Five diploid males exhibited multilocus genotypes that fit to the hypothetical genotype(s) of the ‘missing’ ancestor. However, the possibility exists that a hybrid female could produce or re-create a genotype of a parental species.     

The connective tissue of the rat lung: electron immunohistochemical studies

The ultrastructural distribution of specific connective-tissue components in the normal rat lung was studied by electron immunohistochemistry. Three of these components were localized: type I collagen, fibronectin and laminin. Type I collagen was present not only in major airways and vascular structures, but also in alveolar septa. Laminin was found in all basement membranes, and only in basement membranes, demonstrating once more that this glycoprotein is an intrinsic component of the basement membrane. Fibronectin was found free in the interstitium and on the surfaces of collagen fibers. The basement membranes of bronchial, glandular and endothelial cells of large vessels lacked fibronectin; however, capillary endothelial and occasionally epithelial alveolar basement membranes contained some fibronectin in an irregular, spotty distribution. This localization suggests that in the lung, as in other tissues, fibronectin is not an intrinsic component of the basement membrane, but rather a stromal and plasma protein. Only basement membranes in the alveolar parenchyma contained "trapped" plasma fibronectin.     

The effects of reduced O2 and antioxidants on steroidogenic capacity of cultured rat Leydig cells

We have examined the effects of reduced O2 tension and the antioxidant dimethylsulfoxide (DMSO) to determine if O2-derived free radicals are the cause of decreased steroidogenic capacity (testosterone and progesterone production) of cultured rat Leydig cells. Rat Leydig cells were initially cultured under standard conditions of 5% CO2, 95% air (19% O2) with or without DMSO. Addition of DMSO resulted in increased basal testosterone production on days 2, 3 and 4 of culture. hCG (10 mIU)-stimulated testosterone secretion was 2-3 times greater on days 2 and 3 in the presence of DMSO. Lowering the O2 concentration to 5% in the presence of DMSO resulted in even greater hCG-stimulated testosterone production on days 1 to 3. However, the effect of DMSO or low O2 and DMSO were not seen after 5 days. The reduced O2 concentration resulted in an increase in hCG (10 mIU)-stimulated progesterone synthesis throughout the culture, particularly on days 4 to 8. Also, when total steroid (progesterone and testosterone) was determined, cells cultured under reduced O2 conditions responded with increased steroid production on days 1 to 8 in comparison to controls (19% O2). These results demonstrate that lowered O2 concentration and DMSO provide a protective effect resulting in the maintenance of testosterone production and an increase in progesterone synthesis. These findings suggest that free radical-mediated damage of enzymes may result in decreased steroidogenic capacity of cultured Leydig cells.     

Effects of multiple injections of ethane 1,2-dimethane sulphonate (EDS) on the frog, Rana esculenta, testicular activity

Ethane 1,2-dimethane sulphonate (EDS) is an alkylating agent, which has a selective cytotoxic effect on Leydig cells in some mammalian species. Similarly, in the frog, Rana esculenta, Leydig cells are destroyed after a single EDS injection and regenerate after 28 days. Regeneration of Leydig cells in frogs appears to be independent of the pituitary. The present experiments in R. esculenta were carried out: a) to investigate Leydig cell responsiveness to gonadotropin stimulation during 58 days after a single EDS injection; and b) to assess whether four consecutive EDS injections induce additional effects on the testicular cell population. Our results show that androgen stimulation after gonadotropin injections is restored after 44 days from a single EDS injection. Since the interstitial compartment appears to be normal at least 28 days after EDS treatment, it is likely that new Leydig cells lack gonadotropin receptors. With respect to multiple-EDS injections, Leydig cells completely disappear in several areas and the adjacent germinal compartment is disorganised. In some cases damaged germinal compartment is still surrounded by intact Leydig cells. Surprisingly, testicular and plasma androgens strongly increase in EDS-treated animals. Therefore, Sertoli cells may produce substances inhibiting androgen production in Leydig cells. J. Exp. Zool. 287:384-393, 2000.     

The use of rat Leydig tumor (R2C) and human hepatoma (HEPG2) cells to evaluate potential inhibitors of rat and human steroid aromatase

The efficacies of 10-propargylestr-4-ene-3,17-dione (PED), 4-hydroxyandrostenedione (4-OHA) and the imidazole broad spectrum antimycotic drugs, econazole, imazalil, miconazole and ketoconazole, to inhibit the steroid aromatase activities of rat Leydig tumor (R2C) cells and human hepatoma (HEPG2) cells have been determined. The analysis of inhibition of steroid aromatase activity of intact cells provided further insight into the potential use of such drugs to block cellular estrogen synthesis. The IC50 values for the inhibition of aromatase activity of R2C cells by econazole, imazalil, miconazole, ketoconazole, 4-OHA and PED were 4, 9, 40, 1100, 11 and 10 nM, respectively. These drugs also inhibited the steroid aromatase activity of HEPG2 cells with corresponding IC50 values of 13, 27, 20, 15000, 2 and 2 nM, respectively; these findings were suggestive that the steroid aromatase of rat has many similarities to the human enzyme in its interaction with putative inhibitory compounds. Importantly, however, ketoconazole inhibited the rat aromatase more effectively than it did the human enzyme, while PED and 4-OHA were less effective inhibitors of the rat enzyme compared to that of human. These findings indicate differences in the potencies of various drugs to inhibit estrogen biosynthesis in human and rat cells. These may relate to differences in the two aromatase systems and/or differences in the stability of the drugs in the human hepatoma and rat Leydig tumor cells.     

The effects of interstitial cell-stimulating hormone, its subunits, and recombinants on isolated rat Leydig cells.

The stimulation of cyclic AMP accumulation and testosterone synthesis in isolated rat Leydig cells by ovine and bovine ICSH, their subunits prepared by a new, mild procedure (dissociation of subunits at pH 3 and salt fractionation) and the recombined hormones have been studied. Whereas the isolated subunits exhibit less than 0.2% of the potency of the native hormones, recombination of the subunits results in full restoration of the biological activity. In contrast to this, recombination of the subunits prepared by a countercurrent distribution method resulted in only partial regeneration of the biological activity. The ovine hormone was found to be twice as active as the bovine ICSH. Both heterologous hybrids of the subunits of the ovine and bovine hormones were significantly more active than the bovine hormone. The utility of the isolated rat Leydig cell system as a rapid, sensitive bioassay for ICSH is also discussed.     

Stimulation of cholesterol side-chain cleavage by a luteinizing-hormone-releasing hormone (luliberin) agonist (ICI 118630) in rat Leydig cells.

The action of a luliberin (luteinizing-hormone-releasing hormone) agonist (ICI 118630) and lutropin (luteinizing hormone) on the activity of the cytochrome P-450 cholesterol side-chain cleavage enzyme in rat Leydig cells has been investigated. This has been carried out by studying the metabolism of exogenous (22R)-22- and 25-hydroxycholesterol to testosterone. It was found that both hydroxycholesterols increased testosterone production to higher levels than achieved by lutropin alone. Addition of luliberin agonist but not lutropin was found to increase further the metabolism of the hydroxycholesterol to testosterone; this occurred in the presence of saturating and subsaturating levels of the hydroxycholesterols. This effect of luliberin agonist was potentiated in the presence of lutropin. The protein synthesis inhibitor, cycloheximide, inhibited the luliberin agonist-induced stimulation of the hydroxycholesterol metabolism. At low calcium levels (1.1 microM), testosterone production was increased by addition of (22R)-22-hydroxycholesterol but the luliberin agonist effect was negated. The calmodulin inhibitor trifluoperazine inhibited (22R)-22-hydroxycholesterol-stimulated steroidogenesis and negated the luliberin agonist effect. These results indicate that luliberin agonist specifically increases the synthesis of the cytochrome P-450 cholesterol side-chain cleavage enzyme in rat testis Leydig cells.     

The mast cell as an effector of connective tissue degradation: a study of matrix susceptibility to human mast cells

The susceptibility of connective tissue elements to degradation by human mast cells was explored using purified mast cell tryptase and sonicated mast cell preparations. The R-22 strain of smooth muscle cells from rat heart was used for preparation in vitro of a labelled anchored matrix. Digestion of 11.9 +/- 1.2% (n = 5) of this matrix was observed after overnight incubation with the mast cell sonicates. Pretreatment of the sonicate with a tryptase inhibitor TLCK reduced the digestion by 42%. Digestion of 12 +/- 1% (n = 4) of the matrix was observed with purified tryptase. The susceptible substrate within this anchored insoluble matrix resided in the glycoprotein compartment as defined by enzymatic characterization of the residual matrix. Mast cells may play a role in mediating connective tissue degradation through the release of proteases specifically synthesized by this cell.