Loss and recovery of androgen receptor protein expression in the adult rat testis following androgen withdrawal by ethane dimethanesulfonate

Androgens are especially important for the maintenance of spermatogenesis in adulthood and the experimental withdrawal of testosterone (T) production by ethane dimenthanesulfonate (EDS) is a valuable tool for studying androgen-dependent events of spermatogenesis. The aim of the present study was to investigate the specific changes in immunoexpression of androgen receptor (AR) in the testis in relation to degeneration and regeneration of Leydig cell (LC) population and seminiferous epithelium. Immunohistochemistry for AR and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) as well as TUNEL assay for apoptosis were performed on testicular sections of control and EDS-treated rats. Serum LH and T levels were measured by RIA. Our results revealed a total loss of AR immunoexpression from the nuclei of Sertoli (SCs), LCs and peritubular cells during the first week after EDS administration and that coincided with severe drop in T levels. Two weeks after EDS administration, the AR expression was recovered in these cells but normal stage-specificity in SCs was replaced by uniform intensity of AR immunostaining at all the stages of the spermatogenic cycle. The stage-specific pattern of androgen expression in SCs with a maximum at stages VII-VIII appeared 5 weeks after treatment. LC immunoreactivity for 3beta-HSD at different time points after EDS administration correlated with values of T concentration. The maximal germ cell apoptosis on day 7 was followed by total loss of elongated spermatids 2 weeks after EDS treatment. Regeneration of seminiferous epithelium 3 weeks after EDS administration and onwards occurred in tandem with the development of new LC population indicated by the appearance of 3beta-HSD-positive cells and gradual increase in T production. The specific changes in AR after EDS including their loss and recovery in Sertoli cells paralleled with degenerative and regenerative events in Leydig and germ cell populations, confirming close functional relationship between Sertoli, Leydig and germ cells.     

Stimulation of interstitial cell growth after selective destruction of foetal Leydig cells in the testis of postnatal rats

Summary Five-day-old male rats received a single treatment of ethane dimethanesulphonate (EDS), and the response of the testis on days 6–10 and 21 was examined by light microscopy and morphometry, supplemented by measurement of peripheral testosterone levels. One day after treatment, foetal Leydig cells degenerated, showing fragmentation, condensation and nuclear pyknosis. Macrophages phagocytosed the foetal Leydig cells resulting in their disappearance by day 7. Destruction of foetal Leydig cells was followed by an arrest of testicular growth in comparison to testes of intact age-matched control rats. In testes of EDS-treated rats, gonocytes and spermatogonia also degenerated, forming pyknotic bodies within the seminiferous cords. In contrast, interstitial fibroblasts and mesenchymal cells showed proliferative activity, which on days 4 and 5 after treatment resulted in peritubular hyperplasia surrounding each seminiferous cord. Thereafter, on day 21 after EDS administration, the previously depressed serum testosterone levels became markedly elevated coincident with the development of many immature-type Leydig cells, of which the total volume per testis was similar to that of Leydig cells in control testes, despite a four- to five-fold difference in testicular volumes. The results indicate that, although EDS destroys the foetal Leydig cells and impairs spermatogenesis, the interstitial tissue exhibits increased cell growth. The latter probably occurs in response to altered gonadotrophic stimulation and/or disturbances in the interaction between the seminiferous cords and the interstitial tissue.     

Androgen regulation of stage-dependent cyclin D2 expression in Sertoli cells suggests a role in modulating androgen action on spermatogenesis

Regulation of spermatogenesis involves stage-dependent androgen action on Sertoli cells, but the pathways involved are unclear. We assessed if cyclin D2 could play a role. In rats, Sertoli cell nuclear, stage-dependent immunoexpression of cyclin D2 switched on after Day 10 and persisted through Day 35, but disappeared by adulthood. However, ethane dimethane sulfonate (EDS)-induced testosterone withdrawal in adult rats for 6 days induced stage-dependent cyclin D2 immunoexpression in Sertoli cells, with highest expression at stages IX-XII and nondetectable at stages VI-VIII (opposite that for androgen receptor [AR] immunoexpression). In EDS-treated rats, a single injection of testosterone but not of estrogen reversed this change in 4 h, and testosterone administration from the time of EDS treatment prevented expression of cyclin D2 in Sertoli cells. The EDS-induced changes in cyclin D2 immunoexpression were matched by changes in expression of Ccnd2 (cyclin D2) mRNA in isolated stage-dissected tubules. Treatment of adult rats with flutamide induced stage-dependent cyclin D2 immunoexpression in Sertoli cells within 18 h, and confocal microscopy revealed that immunoexpression of AR and cyclin D2 were mutually exclusive within individual seminiferous tubules in these animals. Sertoli cell-selective ablation of the AR in mice using Cre/loxP technology also resulted in stage-dependent Sertoli cell cyclin D2 immunoexpression. Downstream from cyclin D2 action is retinoblastoma 1 (RB1), a tumor suppressor protein, immunoexpression of which paralleled stage-dependent AR expression in Sertoli cells; RB1 stage specificity disappeared after EDS treatment. These results point to a non-cell cycle role for cyclin D2 and RB1 in mature Sertoli cells in the stage-dependent mechanisms regulated by AR expression and androgen action.     

Testicular blood flow and microcirculation in rats after treatment with ethane dimethyl sulfonate

The effects of ethane dimethyl sulfonate (EDS) on total testicular blood flow, microcirculation, and the testicular interstitial fluid volume (IFV) in rats were studied. In agreement with previous studies, treatment of control rats with human chorionic gonadotropin (hCG) induced an increase in IFV and total testicular blood flow as measured with radioactive microspheres. These effects of hCG were completely abolished in rats pretreated with EDS; in EDS-treated rats not receiving any hCG, there were decreases in IFV when compared with untreated control rats. Furthermore, the pulsatile pattern of testicular microcirculation registered with laser-Doppler flowmetry was abolished after EDS treatment, and this effect was not influenced by hCG treatment. The hCG-induced increase in IFV is associated with an increased accumulation of polymorphonuclear leukocytes locally in the testis, but this accumulation of leukocytes was not observed in rats pretreated with EDS. It was concluded from the present study that hCG-induced changes in total testicular blood flow and testicular microcirculation require functionally intact Leydig cells.     

Identification of markers for precursor and leydig cell differentiation in the adult rat testis following ethane dimethyl sulphonate administration.

Administration of ethane dimethane sulphonate (EDS) to adult rats results in the destruction of all Leydig cells, followed by a complete regeneration. We investigated this regeneration process in more detail, using different markers for precursor and developing Leydig cells: the LH receptor, 3beta-hydroxysteroid dehydrogenase (3beta-HSD), transforming growth factor alpha (TGFalpha), and a new marker for Leydig cell maturation, relaxin-like factor (RLF). LH receptor immunoreactivity was found in Leydig cell-depleted testes at 3 and 8 days after EDS administration. The positive (precursor) cells had a mesenchymal-like morphology. The number of LH receptor-positive cells 8 days after EDS administration was 15 +/- 4 per 500 Sertoli cell nuclei. Fifteen days after EDS administration, the first new Leydig cells could be observed. These cells stained positively with both the antibodies against the LH receptor and 3beta-HSD, while some cells also stained positively for TGFalpha. After EDS administration, RLF mRNA disappeared from the testis and reappeared again at the time of the appearance of the first Leydig cells. Concomitant with the increase in the number of Leydig cells, the number of RLF-expressing cells increased. The observations of the present study give further support to the hypothesis that Leydig cell development in the prepubertal testis, and in the adult testis following EDS administration, takes place along the same cell lineage and suggest, therefore, that the adult EDS-treated rat can serve as a model for studying the adult-type Leydig cell development that normally occurs in the prepubertal rat testis.     

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.     

Abnormal development of the testis after administration of the Leydig cell cytotoxic ethylene-1,2-dimethanesulphonate to the immature rat

Male rats were injected with 50 mg ethylene-1,2-dimethanesulphonate/kg from Day 5 to Day 16 after birth and control rats received injections of the same volume of vehicle. Testes were studied at various times from Day 6 to Day 108 using histochemistry, light and electron microscopy. Fine structural degenerative changes were observed in the Leydig cells and seminiferous tubules of EDS-treated animals as early as Day 6. By Day 11 no Leydig cells could be detected and the interstitia of EDS-treated testes contained large numbers of fibroblast-like cells which formed peritubular collars 3-5 cells thick; the tubules contained Sertoli cells with heterogeneous inclusions and large numbers of lipid droplets. A small number of Leydig cells was found at Day 14 and their numbers increased so that, in animals of 28 days and older, large clusters of Leydig cells were present between severely atrophic tubules. These tubules contained Sertoli cells with few organelles; germinal cells were not observed after 28 days in EDS-treated animals. These results show that EDS destroys the fetal population of Leydig cells postnatally and this mimics the well documented effect of EDS on adult Leydig cells. The seminiferous tubules were permanently damaged by EDS in the present experiments. Tubular damage could have been due to a direct cytotoxic effect of multiple injections of EDS on the tubule before the blood-testis barrier develops or due to withdrawal of androgen support secondary to Leydig cell destruction.     

Androgen secretion and characteristics of testicular HCG binding in cryptorchid rats

Response of the cryptorchid testis to gonadotrophic stimulation was assessed by comparison of the androgen production capability in vivo and in vitro with that of the normal scrotal testis. Serum androgen concentrations in cryptorchid rats were similar to those in normal rats, and the incremental increase 60 min after 50 i.u. hCG (i.v.) was about 7-fold for both groups. Basal and hCG-stimulated androgen production in vitro was higher for abdominal testes (557 and 3286 ng/pair) than for scrotal tests (157 and 504 ng/pair). Specific binding of hCG by testicular homogenates was slightly higher (P < 0.05) for cryptorchid testes when expressed per unit weight, but Scatchard analysis indicated that although hCG binding affinities did not differ (Ka = 2 x 10(10) M-1), hCG binding capacity of cryptorchid testes was only 75 ng, compared to 219 ng for scrotal testes. These data indicate that a discrepancy exists between androgen production in vivo and in vitro by cryptorchid testes and that normal serum androgen concentrations are maintained in the presence of decreased numbers of testicular LH/hCG receptors.     

Expression and regulation of lipocalin-type prostaglandin d synthase in rat testis and epididymis

Lipocalin-type prostaglandin D synthase (L-PGDS), a bifunctional protein, is expressed in the male reproductive organs of many species. However, the expression and regulation of L-PGDS in rat are still uncertain. The present study investigated the regionalization and regulation of L-PGDS expression in rat testis and epididymis by in situ hybridization and immunohistochemistry under the conditions of sexual maturation, castration, and ethylene dimethane sulfonate (EDS) treatments. In sexually mature rats, L-PGDS mRNA was weakly expressed only in the testicular peritubular cells, whereas L-PGDS immunostaining was highly detected in the Leydig cells by Day 70 postpartum. During sexual maturation, L-PGDS mRNA expression was highly detected in the caput, corpus, and cauda of the epididymis 70 days after birth. Compared with normal L-PGDS expression in adult epididymis, both L-PGDS mRNA expression and protein immunostaining were significantly reduced in the caput, corpus, and cauda epididymis after castration. Testosterone propionate treatment induced a significant increase of L-PGDS expression in the epididymis of castrated rats. Compared with adult rat epididymis, L-PGDS mRNA and protein expression was down-regulated after EDS treatment. Testosterone propionate treatment could induce an increase of L-PGDS mRNA and protein expression in the epididymis of EDS-treated rats. In conclusion, both castration and EDS treatments caused a significant decrease of L-PGDS expression in the epididymis, whereas testosterone propionate treatment could induce an increase of L-PGDS expression in the epididymis of both castrated and EDS-treated rats, indicating that L-PGDS expression in the rat epididymis can be up-regulated by testosterone.     

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.     

Gene expression profiling of androgen receptor antagonists in the rat fetal testis reveals few common gene targets

The androgen receptor (AR) is expressed in the fetal testis; however, the role of AR in fetal testicular development is poorly understood. Disrupted AR activity and subsequent gene expression alterations may disturb developmental programming of the fetal testis and result in testicular abnormalities later in life. The present study was performed to examine global gene expression patterns in rat fetal testis following in utero exposure to various AR antagonists. Pregnant Sprague-Dawley rats were treated with flutamide (50 mg/kg/day), linuron (50 mg/kg/day), vinclozolin (200 mg/kg/day), p,p'-DDE (100 mg/kg/day) or corn oil vehicle by gavage daily from gestation day (GD) 12-19. Testes were isolated on GD 19, and AR immunostaining, histology, and global changes in gene expression were determined. There were no alterations in the pattern or expression level of AR and no apparent histological changes in the fetal testes in any treatment group. Microarray analysis using Dunnett's test with multiple testing correction revealed no significant gene expression alterations following exposure to flutamide, linuron, vinclozolin, and p,p'-DDE. A less stringent analysis yielded some chemical specific effects on gene expression, and these effects were further evaluated by real-time RT-PCR. Vinclozolin treatment reduced the expression of several genes involved in cholesterol biosynthesis, though the testosterone levels were unchanged in the fetal testes in any treatment group. In flutamide, linuron, and p,p'-DDE treatment groups, the expression of hemoglobin Y, beta-like embryonic chain (Hbb-y) was reduced. Myomesin 2 (Myom2) expression was increased following linuron treatment. Given the lack of a common set of genes and the absence of overt histopathology, we conclude that the fetal testis is not a major target for AR activity at this stage of development although some cell-type specific gene expression changes cannot be ruled out.     

Morphofunctional alterations in testicular cells of deslorelin-treated boars: an immunohistochemical study

In this study we thoroughly scrutinized testes morphology and investigated whether treatment of recipient boars with gonadotropin-releasing hormone (GnRH)-agonist deslorelin could alter the expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), luteinizing hormone receptors (LHRs), and androgen receptors (ARs) in testicular cells. An implant containing 4.7 mg of the GnRH-agonist deslorelin was subcutaneously inserted into crossbred male pigs at 91 and 147 days of age. Testicular traits, morphology of the testes, the proteins' expression, and testosterone concentration in blood plasma were analyzed in all boars after slaughter at 175 days of age. Histological analysis revealed significant alterations in both the interstitial tissue and seminiferous tubules of experimental animals after 28 and 84 days of deslorelin treatment. The intensity of the AR immunostaining within the testis appeared as a function of the severity of testicular dysgenesis. Time-dependent action of deslorelin on the expression of LHR and 3beta-HSD in Leydig cells was also detected. Staining for LHR and 3beta-HSD was very weak or the Leydig cells were immunonegative. Concomitantly, a significant decrease in plasma testosterone level was found in both groups of deslorelin-treated boars when compared with the control group. This is the first report showing the cellular distribution of AR, LHR, and 3beta-HSD in testicular cells of deslorelin-treated boars. It is concluded that morphological and immunohistochemical studies are important for the evaluation of testicular histoarchitecture and steroidogenic function. Subsequently, the endocrine control of reproduction in the GnRH-agonist deslorelin-treated males will be better understood.     

Effects of androgen on androgen receptor expression in rat testicular and epididymal cells: a quantitative immunohistochemical study

Androgen is essential for maintenance of spermatogenesis in the testis and for maturation of spermatozoa in the epididymis. The effects of androgen are mediated through its receptor (AR), the levels of which are, in turn, regulated by androgen. Previous studies have shown that AR concentrations in Leydig and Sertoli cells are differentially regulated during development. The aim of the present study was to determine if cell-type-specific regulation of AR by androgen occurs in testicular and epididymal cells during adulthood. Adult male rats were treated with the LHRH-antagonist Azaline B (100 g/day) by osmotic pump for 1, 2, 3, 4, or 8 wk to suppress endogenous androgen, with identical numbers of intact control animals at each time period. An androgen replacement group was simultaneously treated with the antagonist and a synthetic androgen, 7 alpha-methyl-19-nortestosterone (MENT), during the final 4 wk of the experiment. Levels of nuclear AR protein in specific cell types were quantified by immunohistochemistry in conjunction with computer-assisted image analysis. Levels of AR in testicular cells declined sharply after treatment with the LHRH antagonist. In Sertoli cells, nuclear AR levels decreased to 8% of control (P < 0. 01) after 4 wk treatment; and to 12% and 17% of control (P < 0.01) in Leydig and myoid cells, respectively. Androgen replacement resulted in complete recovery of nuclear AR levels in Sertoli cells (93%, P > 0.05) but in only partial recovery in myoid (69%, P < 0. 01) and Leydig cells (56%, P < 0.01). In the epididymis, tubular epithelial cells and stromal cells differed in their responses to the LHRH antagonist. After 1 wk, nuclear AR levels in caput stromal cells decreased dramatically to 34% of control (P < 0.01) and in cauda stromal cells to 43% (P < 0.01). In contrast, the decline of AR levels in epididymal epithelial cells was not as dramatic as that in stromal cells. After 1 wk, the decline in the caput and cauda was to 87% and 76% of control, respectively. After 8 wk, nuclear AR levels in stromal cells further declined to 1.1% in caput and 1.4% in cauda, whereas in the epithelial cells, a smaller decline in nuclear AR was noted (to 30% in the caput and 45% in the cauda). After androgen replacement with MENT, nuclear AR levels recovered to more than 90% of control in both epididymal cell types. These results indicate that AR levels in the nuclei of adult Sertoli cells depend mainly on the level of androgen, whereas in the adult Leydig and myoid cells, the androgen dependency is more limited. The results also indicate that in the epididymis, stromal cells are more sensitive than epithelial cells to the regulation of AR levels by androgen.     

Role of aromatase and androgen receptor expression in gonadal sex differentiation of ZW/ZZ-type frogs,Rana rugosa

To elucidate the mechanisms of amphibian gonadal sex differentiation, we examined the expression of aromatase and androgen receptor (AR) mRNAs for days 17-31 after fertilization. The effects of inhibitors and sex steroid hormones were also examined. In ZZ males, expression of AR decreased after day 19, while aromatase expression was low throughout the sampling period. Males treated with 17beta-estradiol (E2) showed increasing aromatase expression after day 21, and formed ovaries. AR antagonist treatment also induced high-level aromatase expression and ovarian differentiation. In males co-treated with an aromatase inhibitor and E2, the undifferentiated gonads developed into testes despite high-level aromatase expression. Males treated with androgen and E2 before and during an estrogen sensitive period, respectively, also formed testes. In ZW females, AR expression persisted at a low-level, while aromatase expression increased after day 18. Short-term treatment with an aromatase inhibitor was ineffective in preventing ovarian differentiation, whereas long-term treatment resulted in testes developing from ovarian structure. Compared with the ZZ males and ZW females, WW females did not exhibit detectable expression of AR, suggesting that the active AR gene(s) itself, or a putative gene regulating AR gene expression, is located on Z chromosomes. From the time lag of aromatase expression between ZW females and ZZ males treated with E2 and the effect of AR antagonist, it was found that in males elevated AR expression suppresses aromatase expression directly or indirectly. Consequently, endogenous androgens, accumulated by blocking estrogen biosynthesis, induced testicular differentiation. The gonadogenesis of males is dependent on sex hormone, whereas that of females has evolved to hormone-independence.     

Evidence for an androgen receptor in the seminiferous tubules of the human testis

Androgen receptors (AR) were studied in seminiferous tubule cytosol and testicular nuclear extracts prepared from testes of previously untreated elderly men undergoing orchiectomy as therapy for prostatic carcinoma. Cytosol exhibited high affinity (Kd = 0.8 nM), saturable binding of [3H]methyltrienolone; however, the synthetic progestin, promegestone was a stronger competitor for MT binding sites than were 5 alpha-dihydrotestosterone (DHT) or testosterone (T), suggesting the presence of progesterone-like binding sites. Addition of triamcinolone acetonide (TA) produced the usual relative steroid specificity for AR binding and reduced the measured AR binding capacity by 19 +/- 8% (Mean +/- SD, n = 3). The umber of MT binding sites was 30-40 fmol/mg protein, or an average of 65 fmol/g testis, and the equilibrium dissociation constant at 0 degrees C was 0.6-1.4 nM. In the presence of sodium molybdate, binding was stable for 40 h at 0 degrees C and the half-time of dissociation of the MT-AR complex was 12-16 h. The binding of salt extractable (600 mM KCl) nuclear sites to MT was saturable and was specific for androgens. The number of binding sites in nuclear extracts was 170 fmol/g testis and the apparent equilibrium dissociation constant was 4.2 nM. Thus, the binding of MT to human seminiferous tubule cytosol and testicular nuclear extract exhibits properties which are nearly identical to those of the prostate AR. Further study of this androphilic protein may provide insight into the role of androgen in normal and abnormal spermatogenesis in man.     

Prostatic androgen receptor and plasma testosterone levels in streptozotocin-induced diabetic rats.

Diabetes was induced in male Sprague-Dawley (S-D) rats by streptozotocin (STZ) administration. Following STZ injection, plasma glucose levels in the treated rats were significantly elevated from values of untreated controls. Over the experimental period (140 days) plasma testosterone (T) levels, prostatic nuclear androgen receptor (AR) contents and prostatic weights declined with increasing age in the rats. The declines in both STZ-treated and untreated rats were similar in manner and no notable differences were discerned in the data obtained from the two groups. On the contrary, prostatic cytosolic AR contents in untreated rats remained unchanged with advancing age, but was reduced to 50% of normal control values in diabetic rats following STZ treatment. Correlation analyses revealed that prostatic nuclear AR contents correlated positively with plasma T levels while prostatic cytosolic AR contents correlated negatively with plasma glucose levels. These data support former claims that prostatic nuclear AR content is dependent on circulating T level and suggest a possible link between prostatic cytosolic AR content and plasma glucose concentrations.