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.     

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.     

Effects of thyroid and luteinizing hormones on the onset of precursor cell differentiation into leydig progenitor cells in the prepubertal rat testis

Leydig cells in the adult rat testis differentiate during the neonatal-prepubertal period. However, the stimulus for the initiation of their differentiation is still not clear. In the present study our objectives were to test the effects of thyroid hormone and LH on the initiation of precursor cell differentiation into Leydig cells in the prepubertal rat testis. Four groups of Sprague-Dawley rats were used. All treatments began at postnatal Day 1. Rats in groups I, II, and III received daily s.c. injections of saline (200 microl, controls), triiodothyronine (T(3), 50 microg/kg body weight, hyperthyroid), and LH (ovine LH 10 microg/rat/day), respectively. Rats in group IV were made hypothyroid from postnatal Day 1 by adding 0.1% propylthiouracil (PTU) to their mother's drinking water. Testes of rats were collected at 7, 8, 9, 10, 11, 12, 16, and 21 days of age, fixed in Bouin's solution, and embedded in paraffin for immunocytochemical studies. Immunoexpression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and LH receptors (LHR) in testicular interstitial cells (other than the fetal Leydig cells) was observed using the avidin-biotin method. In control rats, out of all spindle-shaped cell types in the testis interstitium, only the peritubular mesenchymal cells showed positive immunolabeling for 3beta-HSD, beginning from the postnatal Day 11. However, positive immunolabeling for LHR was first detected in these cells at Day 12, i.e., after acquiring the steroidogenic enzyme activity. In T(3)-treated rats 3beta-HSD positive spindle-shaped cells were first observed at Day 9 (i.e., 2 days earlier than controls), and LHR-positive cells were first observed on Day 11 (2 days later than obtaining 3beta-HSD immunoactivity); they were exclusively the peritubular mesenchymal cells. The 3beta-HSD- and LHR-positive spindle-shaped cells were absent in the testis interstitium of LH-injected rats from Days 7 through 12 but were present at postnatal Day 16. In addition, more fetal Leydig cell clusters and fetal Leydig cells in mitosis were present in LH-treated rats compared to rats in all other treatment groups. Following their first detection, the number of positive cells for each protein continued to increase at each subsequent age in controls, T(3)-, and LH-injected groups. In PTU rats, 3beta-HSD and LHR-positive spindle-shaped cells were absent throughout the experimental period. From these observations, it is possible to suggest the following regarding the developing rat testis interstitium. 1) The precursor cells for the adult generation of Leydig cells in the postnatal rat testis are the peritubular mesenchymal cells. 2) Luteinizing hormone does not initiate the onset of mesenchymal cell differentiation into Leydig cells, instead it delays this process. However, daily LH treatment causes mitosis in fetal Leydig cells and increase in fetal Leydig cell clusters. 3) Thyroid hormone is critical to initiate the onset of mesenchymal cell differentiation into adult Leydig cells.     

Gestational exposure to ethane dimethanesulfonate permanently alters reproductive competence in the CD-1 mouse

Although the adult mouse Leydig cell (LC) has been considered refractory to cytotoxic destruction by ethane dimethanesulfonate (EDS), the potential consequences of exposure during reproductive development in this species are unknown. Herein pregnant CD-1 mice were treated with 160 mg/kg on Gestation Days 11-17, and reproductive development in male offspring was evaluated. Prenatal administration of EDS compromised fetal testosterone (T) levels, compared with controls. EDS-exposed pups recovered their steroidogenic capacities after birth because T production by hCG-stimulated testis parenchyma from prepubertal male offspring was unchanged. However, prepubertal testes from prenatally exposed males contained seminiferous tubules (STs) devoid of germ cells, indicating a delay in spermatogenesis. In adults, some STs in exposed males still contained incomplete germ cell associations corroborating observed reductions in epididymal sperm reserves, fertility ratios, and litter size. Morphometry revealed an EDS-induced increase in interstitial area and a concomitant decrease in ST area, but stereology revealed an unexpected decrease in the number and size of the LCs per testis in exposed males. Paradoxically, there was an increase in both serum LH and T production by adult testis parenchyma, indicating that the LCs were hyperstimulated. These data demonstrate permanent lesions in LC development and spermatogenesis caused by prenatal exposure in mice. Thus, although adult mouse LCs are insensitive to EDS, EDS appears to have direct action on fetal LCs, resulting in abnormal testis development.     

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.     

Detection of platelet-derived growth factor-alpha (PDGF-A) protein in cells of Leydig lineage in the postnatal rat testis

Platelet-derived growth factor-A (PDGF-A) is a locally produced growth factor in the rat testis secreted by both Sertoli cells and Leydig cells. It has been suggested that PDGF-A may be involved in modulation of testosterone production and may be essential to Leydig cell differentiation, however it is not known at what stage of differentiation PDGF-A begins to be expressed in the cells of Leydig lineage in the postnatal rat testis. Therefore, the objectives of this research were to determine at what postnatal age and in which cell type is PDGF-A first expressed in cells of the adult Leydig cell lineage, and does PDGF-A expression coincide with expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), an indicator of steroid hormone synthesis. Male Sprague Dawley rats of postnatal day 1, 7, 9-14, 21, 28, 40, 60, and 90 were used (n=6). Animals were euthanized and their testicles removed, fixed in Bouin's solution, embedded in paraffin, and 5 micrometers sections were prepared. Immunolocalization of PDGF-A and 3beta-HSD was carried out using a peroxidase-streptavidin-biotin method. PDGF-A was first detected in cells of the Leydig cell lineage at postnatal day 10 in progenitor cells, which were surrounding the seminiferous tubules (peritubular). These cells were confirmed to be the progenitor cells and not the mesenchymal or any other spindle-shaped cells in the testis interstitium by immunolocalization of 3beta-HSD and PDGF-A in the cells in adjacent sections of testis tissue from rats of postnatal days 10-14. After postnatal day 10, PDGF-A was continued to be expressed in subsequent cells of the Leydig lineage through day 90 (adult), however, was not present in peritubular mesenchymal precursor cells of the Leydig cell lineage or any other spindle-shaped cells in the testis interstitium at any tested age. These results revealed that PDGF-A first appears in Leydig progenitor cells in the postnatal rat testis at the onset of mesenchymal cell differentiation into progenitor cells at postnatal day 10 and suggest that a functional role(s) of PDGF-A in postnatally differentiated Leydig cells in the rat testis is established at the time of the onset of postnatal Leydig stem cell differentiation. It is suggested that the significance of the first expression of PDGF-A in the Leydig progenitor cells may be associated with inducing cell proliferation and migration of this cell away from the peritubular region during Leydig cell differentiation.     

High androgen receptor immunoexpression in human "Sertoli cell only" testis

Our purpose was to evaluate cellular androgen receptor (AR) distribution and intensity of immunostaining in the human azoospermic testis. Thirty six biopsy specimens from azoospermic men were immunostained, using a monoclonal antibody of human AR. The localization and the intensity of AR immunostaining was evaluated in Sertoli Cell Only (SCO) testis (G1, n = 21), in spermatogenesis arrest testis (G2, n = 11) and in histologically normal testis (G3, n = 4). We found an AR immunostaining in Sertoli, peritubular myoid and Leydig cells, but not in germ cells. The intensity of the immunostaining varied substantially between biopsy specimens of different patients. Sertoli and Leydig cells AR immunostaining (score and intensity) in SCO group was higher than in the other groups. For Sertoli cells, the score means of AR immunoreactivity were 20 +/- 2.36, 10.18 +/- 1.0 and 1 +/- 1, for G1, G2 and G3 groups, respectively. For Leydig cells, the score means were 10.24 +/- 1.37, 6 +/- 0.71 and 0, for G1, G2 and G3 groups, respectively. We found significant differences between G1 and G2 (p = 0.0008), between G1 and G3 (p = 1.54 10-7) and G2 and G3 (p = 0.00032). These results suggest that in the testis AR is located exclusively in somatic cells and its expression is higher in SCO syndrome than in normal and in arrest spermatogenesis testes.     

The regenerative capacity of testicular interstitial tissue

Summary A single intraperitoneal injection of ethane dimethanesulphonate (EDS) destroys all Leydig cells in the adult rat testis but 1–2 weeks later new foetal-type Leydig cells begin to regenerate within the interstitial tissue. A further EDS treatment at 4 weeks failed to kill the new population of foetal-type Leydig cells. Between 10–20 weeks, the new Leydig cells exhibited the characteristics of adult-type Leydig cells. These cells responded to another EDS treatment by exhibiting a second phase of complete degeneration followed by regeneration of a foetal-type and subsequently an adult-type cell population. The results indicate that the testis retains the ability to replenish its supply of Leydig cells despite successive phases of total degradation of Leydig cells.     

Localisation du récepteur des androgènes dans le testicule

Whether or not germ cells contain the androgen receptor remains a matter of controversy. In the present study we performed biotinstreptavidin immunoperoxidase using an affinity purified rabbit polyclonal antibody made to a 21 amino acid peptide of the amino terminus of the rat AR to determine androgen receptor (AR) distribution in the rat and mouse testes. Specificity of the antibody was confirmed as follows: 1) Western immunoblots rendered a specific band at approximately 110 kD; 2) preadsorption of the antibody with the 21 amino acid peptide eliminated specifice immunostaining; 3) the intensity of staining in all AR positive cells diminished as a function of antisera dilution; 4) tissues known to express abundant AR (e.g., epididymis, prostate, seminal vesicles) all rendered a robust, nuclear AR immunostaining pattern in the epithelial cells; 5) prostate cell lines known to express AR immunostained positive with the antibody; 6) AR negative COS-7 cells became AR immunopositive when transfected with a vector expressing the rat AR and intracellular AR distribution was a function of androgens. AR immunostaining results revealed the following: Within the interstitial compartment of adult rats, AR was detected in some Leydig cells and all smooth muscle cells forming the walls of blood vessels, but endothelial cells were negative. In the seminiferous tubules AR was observed in all peritubular myoid cell nuelei, but not in the distal layer of Iymphatic endothelial cells. In Sertoli cells, nuclear AR immunostaining was stage specific; moderate AR immunostaining became evident at late stage IV of the cycle, reached a robust peak at stages VII-VIII, and then disappeared completely. Specific AR immunostaining was also discerned in the nuclei of stage XI elongated spermatids, in which nuclear elongation is apparent but chromatin condensation has not yet begun. With onset of chromatin condensation, nuclear AR immunostaining in elongated spermatids was not discerned concomitant with its detection in the cytoplasm. In general, similar observations have now been confirmed in the adult mouse testis, except that an Leydig cells were strongly AR positive. Nucleic acid in situ hybridization studies for AR were performed in adult rat testis using a 236 bp antisense cRNA probe (rat AR cDNA was provided by Dr. C. Chang, U. Wisconsin, Madison, WI) to confirm the AR immunostaining. A prominent hybridization signal at the base of the seminiferous epithelium was observed, in the area occupied by Sertofi and spermatogonia. This led us to re-examine the immunostaining results to determine if spermatogonia were also AR positive. Preliminary results are consistent with the interpretation that AR is present in certain spermatogonial populations. Taken together, these results concur with prior observations suggesting that AR is present in the somatic cells of the testis; thus, it is these cell types that likely respond to circulating androgens to control spermatogenesis. However, they raise anew the controversy of whether germ cells respond directly to androgens.     

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.     

Differential modulation of 3T3-L1 adipogenesis mediated by 11beta-hydroxysteroid dehydrogenase-1 levels

The localized activation of circulating glucocorticoids in vivo by the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) plays a critical role in the development of the metabolic syndrome. However, the precise contribution of 11beta-HSD1 in the initiation of adipogenesis by inactive glucocorticoids is not fully understood. 3T3-L1 fibroblasts can be terminally differentiated to mature adipocytes in a glucocorticoid-dependent manner. Both inactive rodent dehydrocorticosterone and human cortisone were able to substitute for the synthetic glucocorticoid dexamethasone in 3T3-L1 adipogenesis, suggesting a potential role for 11beta-HSD1 in these effects. Differentiation of 3T3-L1 cells caused a strong increase in 11beta-HSD1 protein levels, which occurred late in the differentiation protocol. Reduction of 11beta-HSD1 activity in 3T3-L1 fibroblasts, achieved by pharmacological inhibition or adenovirally mediated delivery of short hairpin RNA constructs, specifically blocked the ability of inactive glucocorticoids to drive 3T3-L1 differentiation. However, even modest increases in exogenous 11beta-HSD1 expression in 3T3-L1 fibroblasts, to levels comparable with endogenous 11beta-HSD1 in differentiated 3T3-L1 adipocytes, were sufficient to block adipogenesis. Luciferase reporter assays indicated that overexpressed 11beta-HSD1 was catalyzing the inactivating dehydrogenase reaction, because the ability of both active and inactive glucocorticoids to activate the glucocorticoid receptor were largely suppressed. These results suggest that the temporal regulation of 11beta-HSD1 expression is tightly controlled in 3T3-L1 cells, so as to mediate the initiation of differentiation by inactive glucocorticoids and also to prevent the inhibitory activity of prematurely expressed 11beta-HSD1 during adipogenesis.     

Immunolocalization of albumin and transferrin in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis

The localization of albumin and transferrin was examined immunohistochemically in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis. These proteins appeared as early as the 13th day of gestation in migrating primordial germ cells before Sertoli cell differentiation. In the fetal testis, strong immunoreactivity was only detected in the gonocytes. In the prepubertal testis, spermatogonia, primary spermatocytes, and some Sertoli cells accumulate albumin and transferrin. At puberty, different patterns of immunostaining of the germ cells were observed at the various stages of the cycle of the seminiferous epithelium. Diplotene spermatocytes at stage XIII, spermatocytes in division at stage XIV, and round spermatids at stages IV–VIII showed maximal staining. Labeling was evident in the cytoplasm of adult Sertoli cells. Albumin and transferrin staining patterns paralleled each other during ontogenesis.     

Suppression of 11beta-hydroxysteroid dehydrogenase type 1 with RNA interference substantially attenuates 3T3-L1 adipogenesis.

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which regulates the local level of glucocorticoids, has been suggested to be involved in the development of obesity. A definitive functional role for 11beta-HSD1 in adipogenesis, however, remains to be established. We developed 3T3-L1 cell lines stably transfected with a small hairpin RNA (shRNA) targeting 11beta-HSD1. A shRNA containing two nucleotide substitutions was used as a control. Silencing of 11beta-HSD1 substantially attenuated the accumulation of lipid droplets and the expression of adipogenesis marker genes, which was induced by a mixture containing either corticosterone or dexamethasone. Silencing of 11beta-HSD1 increased the concentration of 11-dehydrocorticosterone in the culture supernatant but did not significantly affect the levels of corticosterone or dexamethasone. Translocation of glucocorticoid receptors to the nucleus in response to glucocorticoids was significantly attenuated by silencing 11beta-HSD1. The number of cells entering the S phase of the cell cycle following the induction of adipogenesis was significantly reduced by silencing 11beta-HSD1. 11beta-HSD1 shRNA delivered by lentiviral vectors after the induction of differentiation, however, did not affect the progression of adipogenesis. These results indicate that 11beta-HSD1 plays a significant functional role in the initiation of 3T3-L1 adipogenesis and provide new mechanistic insights into the role of 11beta-HSD1 in the development of obesity and related diseases.     

Differential regulation of the oxidative 11beta-hydroxysteroid dehydrogenase activity in testis and liver

11Beta-hydroxysteroid dehydrogenase (11beta-HSD) Type I enzyme is found in testis and liver. In Leydig cell cultures, 11beta-HSD activity is reported to be primarily oxidative while another report concluded that is primarily reductive. Hepatic 11beta-HSD preferentially catalyzes reduction and the reaction direction is unaffected by the external factors. Recent analysis of testicular 11beta-HSD revealed two kinetically distinct components. In the present study, various steroid hormones or glycyrrhizic acid (GCA), given for 1 week, or thyroxine given for 5 weeks to normal intact rats had different effects on the 11beta-HSD oxidative activity in testis and liver. Deoxycorticosterone, dexamethasone, progesterone, thyroxine, and clomiphene citrate increased testicular 11beta-HSD oxidative activity, but decreased hepatic enzyme activity except for deoxycorticosterone (unchanged). Corticosterone and testosterone decreased 11beta-HSD oxidative activity in testis but not that of liver (which was unchanged). Estradiol, GCA and adrenalectomy lowered oxidative activity of 11beta-HSD in testis and liver, but the degrees of reduction were different. The in vivo effects of glucocorticoids too were different, even in the same organ. Dexamethasone, a pure glucocorticoid, has greater affinity for glucocorticoid receptors (GR) than corticosterone. The direct effects of dexamethasone via GR in increasing testicular 11beta-HSD oxidative activity may override its indirect effects. Possibly, the reverse occurs with corticosterone treatment, as it has both glucocorticoid and mineralocorticoid effects. Because both organs have Type I isoenzyme, the difference in 11beta-HSD oxidative activities of these two organs could be attributable to the presence of an additional isozyme in testis or differences in tissue-specific regulatory mechanisms.     

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.     

11 Beta-hydroxysteroid dehydrogenase type 1 is induced in human monocytes upon differentiation to macrophages.

11beta-hydroxysteroid dehydrogenases (11beta-HSD) perform prereceptor metabolism of glucocorticoids through interconversion of the active glucocorticoid, cortisol, with inactive cortisone. Although the immunosuppressive and anti-inflammatory activities of glucocorticoids are well documented, the expression of 11beta-HSD enzymes in immune cells is not well understood. Here we demonstrate that 11beta-HSD1, which converts cortisone to cortisol, is expressed only upon differentiation of human monocytes to macrophages. 11beta-HSD1 expression is concomitant with the emergence of peroxisome proliferator activating receptor gamma, which was used as a surrogate marker of monocyte differentiation. The type 2 enzyme, 11beta-HSD2, which converts cortisol to cortisone, was not detectable in either monocytes or cultured macrophages. Incubation of monocytes with IL-4 or IL-13 induced 11beta-HSD1 activity by up to 10-fold. IFN-gamma, a known functional antagonist of IL-4 and IL-13, suppressed the induction of 11beta-HSD1 by these cytokines. THP-1 cells, a human macrophage-like cell line, expressed 11beta-HSD1 and low levels of 11beta-HSD2. The expression of 11beta-HSD1 in these cells is up-regulated 4-fold by LPS. In summary, we have shown strong expression of 11beta-HSD1 in cultured human macrophages and THP-1 cells. The presence of the enzyme in these cells suggests that it may play a role in regulating the immune function of these cells.     

Studies on the expression of 80-kDa human sperm antigen in rat testis and epididymis.

The 80-kDa human sperm antigen (HSA) has demonstrated to be a promising candidate for development of an antifertility vaccine because it is a sperm-specific, conserved, and immunogenic protein. The present study demonstrates the androgen-regulated expression of 80-kDa HSA in testis and epididymis of rat by immunohistochemistry (IHC), using its specific antibodies. Developmental expression of 80-kDa HSA was investigated on days 10, 20, 40, 60, and 90 of age in the testis and epididymis by IHC, and relative staining intensity was estimated by image analysis using BIOVIS software. On days 10 and 20, no significant staining was observed in the testis and epididymis, whereas it gradually increased from day 40 onwards. The highest staining was seen on day 90 in both testis and epididymis. Gradual increase in expression of 80-kDa HSA after day 40 suggests that it is possibly regulated by androgen. To study the androgen-regulated expression of 80-kDa, adult male rats were treated with 75 mg/kg body weight of ethylene dimethane sulfonate (EDS), which selectively destroys Leydig cells and thus induces complete androgen withdrawal. It was observed that the staining intensity decreased following EDS treatment in rat testis as well as epididymis, and it was regained after supplementation with dihydrotestosterone. Increased expression during sexual maturation at the time of testosterone surge and its regulation by antiandrogen/androgen treatment suggest androgen-dependent expression of 80-kDa HSA in rat testis and epididymis.     

Differentiation of human adipose derived stem cells into Leydig‐like cells with molecular compounds

Leydig cells (LCs) are the primary source of testosterone in the testis, and testosterone deficiency caused by LC functional degeneration can lead to male reproductive dysfunction. LC replacement transplantation is a very promising approach for this disease therapy. Here, we report that human adipose derived stem cells (ADSCs) can be differentiated into Leydig‐like cells using a novel differentiation method based on molecular compounds. The isolated human ADSCs expressed positive CD29, CD44, CD59 and CD105, negative CD34, CD45 and HLA‐DR using flow cytometry, and had the capacity of adipogenic and osteogenic differentiation. ADSCs derived Leydig‐like cells (ADSC‐LCs) acquired testosterone synthesis capabilities, and positively expressed LC lineage‐specific markers LHCGR, STAR, SCARB1, SF‐1, CYP11A1, CYP17A1, HSD3B1 and HSD17B3 as well as negatively expressed ADSC specific markers CD29, CD44, CD59 and CD105. When ADSC‐LCs labelled with lipophilic red dye (PKH26) were injected into rat testes which were selectively eliminated endogenous LCs using ethylene dimethanesulfonate (EDS, 75 mg/kg), the transplanted ADSC‐LCs could survive and function in the interstitium of testes, and accelerate the recovery of blood testosterone levels and testis weights. These results demonstrated that ADSCs could be differentiated into Leydig‐like cells by few defined molecular compounds, which might lay the foundation for further clinical application of ADSC‐LC transplantation therapy.     

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.