Effects of luteinizing hormone and androgen on the development of rat progenitor Leydig cells in vitro and in vivo.

Progenitor Leydig cells are derived from stem cells. The proliferation and differentiation of progenitor Leydig cells significantly contributes to Leydig cell number during puberty. However, the regulation of these processes remains unclear. The objective of the present study was to determine whether luteinizing hormone (LH) or androgen contributes to the proliferation and differentiation of progenitor Leydig cells. Fourteen-day-old male Sprague-Dawley rats were treated for 7 days with NalGlu, which is a gonadotropin-releasing hormone antagonist, to reduce the secretion of LH in the pituitary and thus, androgen in the testis. Rats were co-administered with LH or 7α-methyl-nortestosterone (MENT), which is an androgen resistant to metabolism by 5α-reductase 1 in progenitor Leydig cells, and the subsequent effects of LH or androgen were measured. (3)H-Thymidine was also intravenously injected into rats to study thymidine incorporation in progenitor Leydig cells. Progenitor Leydig cells were examined. NalGlu administration reduced progenitor Leydig cell proliferation by 83%. In addition, LH or MENT treatment restored Leydig cell proliferative capacity to 73% or 50% of control, respectively. The messenger RNA levels of proliferation-related genes were measured using real-time PCR. The expression levels of Igf1, Lifr, Pdgfra, Bcl2, Ccnd3 and Pcna were upregulated by MENT, and those of Pdgfra, Ccnd3 and Pcna were upregulated by LH. Both LH and MENT stimulated the differentiation of progenitor Leydig cells in vitro. We concluded that both LH and MENT were involved in regulating the development of progenitor Leydig cells.

The inhibition of human and rat 11ß-hydroxysteroid dehydrogenase 2 by perfluoroalkylated substances.

11ß-Hydroxysteroid dehydrogenase 2 (11ß-HSD2) regulates active glucocorticoid access to glucocorticoid and mineralocorticoid receptors by metabolizing it to an inactive form. Perfluoroalkylated substances (PFASs) are man-made polyfluorinated compounds that are widely used and persistent in the environment. We tested the inhibitory potencies of four PFASs including perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), perfluorohexanesulfonate (PFHxS) and perfluorobutane sulfonate (PFBS) on human and rat 11ß-HSD2. PFOS was a potent inhibitor of both human (IC(50)=48 nM) and rat (IC(50)=293 nM) 11ß-HSD2 activities. The potencies for the inhibition of human and rat 11ß-HSD2 activities were PFOS>PFOA>PFHxS>PFBS. PFASs showed competitive inhibition of both human and rat 11ß-HSD2 activities. This observation indicates that PFOS is a potent endocrine disruptor for glucocorticoid metabolism. Article from the Special issue on Targeted Inhibitors.

Deletion of the Igf1 gene: suppressive effects on adult Leydig cell development.

Deletion of the insulin-like growth factor 1 (Igf1) gene was shown in previous studies to result in reduced numbers of Leydig cells in the testes of 35-day-old mice, and in reduced circulating testosterone levels. In the current study, we asked whether deletion of the Igf1 gene affects the number, proliferation, and/or steroidogenic function of some or all of the precursor cell types in the developmental sequence that leads to the establishment of adult Leydig cells (ALCs). Decreased numbers of cells in the Leydig cell lineage (ie, 3ß-hydroxysteroid dehydrogenase-positive cells) were seen in testes of postnatal day (PND) 14-90 Igf1(-/-) mice compared with age-matched Igf1(+/+) controls. The development of ALCs proceeds from stem Leydig cells (SLCs) through progenitor Leydig cells (PLCs) and immature Leydig cells (ILCs). The bromodeoxyuridine labeling index of putative SLCs was similar in the Igf1(-/-) and Igf1(+/+) mice. In contrast, the labeling index of PLCs was reduced in the Igf1(-/-) mice on each day of PND 14 through PND 35, and that of more mature Leydig cells (referred to herein as LCs, a combination of ILCs plus ALCs) was reduced from PND 21 through PND 56. In Igf1(-/-) mice that received recombinant IGF-I, the labeling indices of PLCs and LCs were similar to those of age-matched Igf1(+/+) mice, indicating that the reductions in the labeling indices seen in the PLCs and LCs of the Igf1(-/-) mice were a consequence of reduced IGF-I. On each day of PND 21 through PND 90, testicular testosterone concentrations were significantly reduced in the Igf1(-/-) mice, as were the expressions of testis-specific mRNAs involved in steroidogenesis, including Star, Cyp11a1, and Cyp17a1. The increased expression of the gene for 5α-reductase (Srd5a1) in adult Igf1(-/-) testes suggests that the depletion of Igf1 might suppress or delay Leydig cell maturation. These observations, taken together, indicate that the reduced numbers of Leydig cells in the adult testes of Igf1(-/-) mice result at least in part from altered proliferation and differentiation of ALC precursor cells, but not of the stem cells that give rise to these cells.

Curcumin derivatives inhibit testicular 17beta-hydroxysteroid dehydrogenase 3.

Non-steroidal compounds that inhibit 17beta-hydroxysteroid dehydrogenase isoform 3 (17beta-HSD3), an enzyme catalyzing the final step in testosterone biosynthesis in Leydig cells, are under development for male contraceptive or treatment of androgen dependent diseases including prostate cancer. A series of curcumin analogues with more stable chemical structures were compared to curcumin as inhibitors of 17beta-HSD3 in rat intact Leydig cells as well as rat and human testis microsomes.

Identification and characterization of an androgen-responsive Kap promoter enhancer located in the intron II region of human angiotensinogen gene.

Transgenic expression of the human angiotensinogen (HAGT) gene directed by the mouse kidney androgen-regulated protein (Kap) gene promoter is proximal tubule cell-specific and androgen-regulated in vivo. The same Kap promoter fragment did not support similar regulation of other genes, but a transgene based on the original chimeric KAP-hAGT construct successfully directed NHE3 to kidney, suggesting that sequences within the HAGT gene fragment of the construct contributed to the regulation of its expression in vivo. In the present study, androgen-responsive regulatory sequences in the HAGT gene portions of the transgene were examined in transfected renal cells. A 1.4-kb enhancer between exons 2 and 3 was identified that increased the basal expression of Kap promoter 1.5- to 2-fold, its induction by dihydrotestosterone (DHT) 2- to 3-fold and its induction by dexamethasone (Dex) 4- to 5-fold. Sequence analysis revealed two potential hormone-responsive elements. Mutational assays and electrophoretic mobility shift assay showed one of these elements was androgen-specific. These findings may influence future strategies for the design of inducible, cell-specific transgenes.

Glucocorticoid suppresses steroidogenesis in rat progenitor Leydig cells.

Glucocorticoid (GC) inhibits testosterone production in adult Leydig cells by the glucocorticoid receptor (GR). However, whether GC affects the development of Leydig cells is unclear. The goal of the present study is to investigate the effects of GC on steroidogenesis of rat progenitor Leydig cells (PLCs) in vitro. Dexamethasone (DEX) inhibited androsterone (AO) production in PLCs. The GR antagonist RU38486 reversed the DEX-induced inhibition of AO, whereas the mineralocorticoid receptor antagonist RU28318 did not. RU38486 also reversed DEX-induced reductions in steady-state mRNA levels of steroidogenic acute regulatory protein (Star) and 3ß-hydroxysteroid dehydrogenase 1 (Hsd3b1). Steroidogenic acute regulatory protein (StAR) protein expression and 3ß-hydroxysteroid dehydrogenase (3ßHSD) enzyme activity were affected similarly. These results show that GCs inhibit steroidogenesis of PLCs by suppression of StAR and 3ßHSD via a GR-mediated mechanism.

Structure-dependent inhibition of human and rat 11beta-hydroxysteroid dehydrogenase 2 activities by phthalates.

Phthalates are diesters of phthalic acid and an alcohol moiety. Phthalates have been classified as endocrine disruptors and have a broad range of effects with unknown mechanisms. Some of the effects of phthalate are consistent with disruptions of normal glucocorticoid homeostasis, and in particular, with defective function of 11beta-hydroxysteroid dehydrogenase 2 (11beta-HSD2). In the present study, we tested 12 phthalate diesters and four monoesters for the inhibition of human and rat kidney 11beta-HSD2. We examined the modes of inhibition and looked for a relationship between the potency for inhibition and the chemical structures. Of the phthalate diesters we tested, dipropyl phthalate (DPrP) and di-n-butyl phthalate (DBP) significantly inhibited both human and rat 11beta-HSD2 activities. The IC(50)s were 85.59 microM for DPrP and 13.69 microM for DBP when calculated for rat 11beta-HSD2. As diesters, 8 of the phthalates did not affect 11beta-HSD2 enzyme activity. Compared to the diesters that were inhibitory, the 8 non-inhibitory phthalates, had either fewer carbons, that is 1 or 2 carbons in the alcohol moiety, or more carbons, 5-10, as a branched or unbranched chain in the alcohol moeity. However, phthalates could be inhibitors with six carbons in the alcohol moiety if the carbons were cyclized, as in dicyclohexyl phthalate (DCHP), which inhibited rat 11beta-HSD2 with an IC(50) of 32.64 microM. Thus, whether a phthalate is an inhibitor may reflect the size and shape of the compound. Although the diesters are the compounds used in manufacturing and present as environmental contaminants, it is the monoester metabolites that are detected in human serum and urine. We showed that mono (2-ethylhexyl) phthalate (MEHP) significantly inhibited human (IC50)=110.8+/-10.9) and rat (121.8+/-8.5 microM) 11beta-HSD2 activity even though its parent compound, di(2-ethylhexyl) phthalate (DEHP) did not. MEHP was a competitive inhibitor of 11beta-HSD2 enzymatic activity. We conclude that phthalates of a certain size act as competitive inhibitors.

Inhibition of 3beta- and 17beta-hydroxysteroid dehydrogenase activities in rat Leydig cells by perfluorooctane acid.

Perfluorooctane acid (PFOA) is classified as a persistent organic pollutant and as an endocrine disruptor. The mechanism by which PFOA causes reduced testosterone production in males is not known. We tested our hypothesis that PFOA interferes with Leydig cell steroidogenic enzymes by measuring its effect on 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17beta-hydroxysteroid dehydrogenase 3 (17beta-HSD3) activities in rat testis microsomes and Leydig cells. The IC(50)s of PFOA and mode of inhibition were assayed. PFOA inhibited microsomal 3beta-HSD with an IC(50) of 53.2+/-25.9 microM and 17beta-HSD3 with an IC(50) 17.7+/-6.8 microM. PFOA inhibited intact Leydig cell 3beta-HSD with an IC(50) of 146.1+/-0.9 microM and 17beta-HSD3 with an IC(50) of 194.8+/-1.0 microM. The inhibitions of 3beta-HSD and 17beta-HSD3 by PFOA were competitive for the substrates. In conclusion, PFOA inhibits 3beta-HSD and 17beta-HSD3 in rat Leydig cells.

Inhibition of LH-stimulated androgen production in rat immature Leydig cells: Effects on nuclear receptor steroidogenic factor 1 by FGF2.

Both fibroblast growth factor 2 (FGF2) and luteinizing hormone (LH) have been reported to regulate androgen production in Leydig cells in progenitor Leydig cells. The objective of the present study is to examine the regulation of androgen production in rat immature Leydig cells (ILCs). ILCs were isolated from 35-day-old rat testes and cultured in DMEM/F12 medium with LH (1 ng/ml) or FGF2 (10 ng/ml). 5alpha-Androstane-3alpha, 17beta-diol (3alpha-DIOL), the primary androgen in ILCs, and testosterone (T) were measured by Radioimmuno assay. The results showed the LH stimulated androgen production in ILCs, and FGF2 did not. However, FGF2 decreased the LH-stimulated androgen production. Real-time PCR and enzyme assay showed that FGF2 decreased levels of several steroidogenic enzymes, inhibited the expressions of steroidogenic acute regulatory (StAR) protein and steroidogenic factor 1 (Nr5a1) in LH-stimulated ILCs. FGF2-mediated inhibition of Nr5a1gene expression may be the mechanism through which FGF2 inhibits LH-stimulated androgen production.

Mono-(2-ethylhexyl) phthalate (MEHP) regulates glucocorticoid metabolism through 11beta-hydroxysteroid dehydrogenase 2 in murine gonadotrope cells.

Di-(2-ethylhexyl) phthalate (DEHP) and its metabolite mono-(2-ethylhexyl) phthalate (MEHP) have been classified as toxicants to the reproductive system at the testis level and DEHP may also impair reproductive axis function at the pituitary levels. However, MEHP is 10-fold more potent than DEHP in toxicity and little is known about the toxicological effect of MEHP on pituitary. In this study, we demonstrated that 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), not 11beta-HSD1, is strongly expressed in murine gonadotrope LbetaT2 cells. Interestingly, MEHP inhibited Hsd11b2 mRNA level and 11beta-HSD2 enzyme activity in LbetaT2 cells at as low as 10(-7)M. Corticosterone (CORT) at a concentration of 10(-6)M significantly inhibited LbetaT2 cell proliferation after 2-day culture, and 10(-6)M RU486, an antagonist of glucocorticoid receptor (GR), reversed this inhibition. However, in the presence of 10(-5) or 10(-4)M MEHP, the minimal concentration of CORT to inhibit the proliferation of LbetaT2 cells was lowered to 10(-7)M, and 10(-6)M RU486 was not able to completely reverse the CORT effect. In conclusion, along with the regulation of GR, 11beta-HSD2 may have a key role in glucocorticoid metabolism in LbetaT2 cells. MEHP may participate in the glucocorticoid metabolism in LbetaT2 cells through inhibition of 11beta-HSD2 enzyme activity. Such perturbation may be of pathological significance as MEHP may interfere with the reproductive system at pituitary level through regulation of glucocorticoid metabolism, especially in neonates with higher risk of phthalates exposure.

The (+)- and (-)-gossypols potently inhibit both 3beta-hydroxysteroid dehydrogenase and 17beta-hydroxysteroid dehydrogenase 3 in human and rat testes.

Androgen deprivation is commonly used in the treatment of metastatic prostate cancer. The (-)-gossypol enantiomer has been demonstrated as an effective inhibitor of Bcl-2 in the treatment of prostate cancer. However, the mechanism of gossypol as an inhibitor of androgen biosynthesis is not clear. The present study compared (+)- and (-)-gossypols in the inhibition of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17beta-HSD isoform 3 (17beta-HSD3) in human and rat testes. Gossypol enantiomers were more potent inhibitors of rat 3beta-HSD with IC(50)s of approximately 0.2microM compared to 3-5microM in human testes. However, human 17beta-HSD3 was more sensitive to inhibition by gossypol enantiomers, with IC(50)s of 0.36+/-0.09 and 1.13+/-0.12 for (-)- and (+)-gossypols, respectively, compared to 3.43+/-0.46 and 10.93+/-2.27 in rat testes. There were species- and enantiomer-specific differences in the sensitivity of the inhibition of 17beta-HSD3. Gossypol enantiomers competitively inhibited both 3beta-HSD and 17beta-HSD3 by competing for the cofactor binding sites of these enzymes. Gossypol enantiomers, fed orally to rats (20mg/kg), inhibited 3beta-HSD but not 17beta-HSD3. This finding was consistent with the in vitro data, in which rat 3beta-HSD was more sensitive to gossypol inhibition than rat 17beta-HSD3. As the reverse was true for the human enzymes, gossypol might be useful for treating metastatic prostate cancer.

Normal responses to restraint stress in mice lacking the gene for neuronal nitric oxide synthase.

The hormonal changes associated with immobilization stress (IMO) include a swift increase in corticosterone (CORT) concentration and a decrease in circulating testosterone (T) levels. There is evidence that the production of the short-lived neuromodulator nitric oxide (NO) is increased during stress in various tissues, including the brain. NO also suppresses the biosynthesis of T. Both the inducible and the neuronal isoforms of NO synthase (iNOS and nNOS, respectively) have been implicated in this suppression, but the evidence has not been conclusive. We used adult wild-type (WT) and nNOS knockout male mice (nNOS-/-) to assess the respective roles of CORT and nNOS-derived NO in stress mediated inhibition of T production. Animals were assigned to either basal control or 3-hour IMO groups. No difference in basal plasma and testicular T levels were observed between WT and nNOS-/-, although testicular weights of mutant mice were slightly lower compared to WT animals. The plasma contents of luteinizing hormone (LH) and CORT in unstressed mice of both genotypes were similar. Exposure to 3 hours of IMO increased plasma CORT and decreased T concentrations in mice of both genotypes. However, comparable levels of plasma LH and testicular nitrite and nitrate (NOx), NO stable metabolites, were detected in control and stressed WT and nNOS-/- mice. Adrenal concentrations of NOx declined after IMO, but the reduction was not statistically significant. These findings implicate CORT rather than NO generated by nNOS in the rapid stress-induced suppression of circulating T.

Phthalate-induced testicular dysgenesis syndrome: Leydig cell influence.

Phthalates, the most abundantly produced plasticizers, leach out from polyvinyl chloride plastics and disrupt androgen action. Male rats that are exposed to phthalates in utero develop symptoms characteristic of the human condition referred to as testicular dysgenesis syndrome (TDS). Environmental influences have been suspected to contribute to the increasing incidence of TDS in humans (i.e. cryptorchidism and hypospadias in newborn boys and testicular cancer and reduced sperm quality in adult males). In this review, we discuss the recent findings that prenatal exposure to phthalates affects Leydig cell function in the postnatal testis. This review also focuses on the recent progress in our understanding of how Leydig cell factors contribute to phthalate-mediated TDS.

In utero and lactational exposures to diethylhexyl-phthalate affect two populations of Leydig cells in male Long-Evans rats.

Diethylhexylphthalate (DEHP) has been classified as an antiandrogen. However, whether in utero and lactational exposures of DEHP affect Leydig cells has not been well established. In the present study, the effects of DEHP exposures on fetal Leydig cells (FLCs) and adult Leydig cells (ALCs) were assessed. Pregnant dams of Long-Evans rats were treated with 0, 10, and 750 mg/kg body weight DEHP from Gestational Day 12.5 to Postnatal Day (PND) 21.5. Fetal Leydig cell clustering and FLC-specific gene expression were examined. Anogenital distances (AGDs) of male pups were assessed at PND 2. Serum testosterone levels of male pups and mRNA levels of ALC-specific genes were measured at PNDs 21 and 49. The AGDs of male pups were significantly shorter in the group treated with 750 mg/kg DEHP (mean +/- SEM, 3.68 +/- 0.16 mm) compared with control (4.62 +/- 0.13 mm). The FLCs were aggregated after 10 and 750 mg/kg DEHP exposures. Several FLC-specific genes, including luteinizing hormone receptor (Lhcgr) and steroidogenic enzyme genes, were downregulated at both doses. Serum testosterone levels were significantly lower compared with control at PND 21 after treatment of 10 or 750 mg/kg DEHP, and continued to be lower even up to 49 days postpartum at the higher dose. The mRNA levels for Lhcgr and steroidogenic enzyme genes were significantly lower at both doses of DEHP at PND 21, whereas there were no significant differences for these genes at PND 49. In conclusion, in utero and continued lactational exposures to DEHP exert long-term disruption of steroidogenesis of ALCs.

The (+)- and (-)-gossypols potently inhibit human and rat 11beta-hydroxysteroid dehydrogenase type 2.

Gossypol has been proven to be a very effective male contraceptive. However, clinical trials showed that the major side effect of gossypol was hypokalemia. Gossypol occurs naturally as enantiomeric mixtures of (+)-gossypol and (-)-gossypol. The (-)-gossypol is found to be the active component of antifertility. 11beta-Hydroxysteroid dehydrogenase 2 (11betaHSD2) has been demonstrated to be a mineralocorticoid receptor (MR) protector by inactivating active glucocorticoids including corticosterone (CORT) in rats, and therefore mutation or suppression of 11betaHSD2 causes hypokalemia and hypertension. In the present study, the potency of gossypol enantiomers was tested for the inhibition of 11betaHSD1 and 2 in rat and human. Both (+) and (-)-gossypols showed a potent inhibition of 11betaHSD2 with the half maximal inhibitory concentration (IC(50)) of 0.61 and 1.33 microM for (+) and (-)-gossypols, respectively in rats and 1.05 and 1.90 microM for (+) and (-)-gossypols, respectively in human. The potency of gossypol to inhibit 11betaHSD1 was far less; the IC(50) was > or =100 microM for racemic gossypol. The gossypol-induced hypokalemia is likely associated with its potent inhibition of kidney 11betaHSD2.

Identification and characterization of the minimal androgen-regulated kidney-specific kidney androgen-regulated protein gene promoter.

The kidney androgen-regulated protein (Kap) gene is tissue specific and regulated by androgen in mouse kidney proximal tubule cells (PTCs). In the present study, we aimed to identify the minimal PTC-specific androgen-regulated Kap promoter and analyze its androgen response elements (AREs). A deletion series of the Kap1542 promoter/luciferase constructs were assayed in opossum kidney (OK) PTCs in the presence or absence of 15 nM dihydrotestosterone (DHT). Kap1542 and Kap637 had low activity and no androgen induction; Kap224 had a basal activity that was 4- to 5-fold higher than that of Kap1542, but was only slightly induced by DHT. Kap147 had a basal activity that was 2- to 3-fold higher than that of Kap1542 and was induced by DHT 4- to 6-fold. Kap77 abolished basal promoter activity but was still induced by DHT. Results showed that, in vitro, Kap147 was a minimal androgen-regulated promoter. Transient transfection in different cells demonstrated that Kap147 specifically initiated reporter gene expression in PTCs. Sequence analysis revealed two potential AREs located at positions -124 and -39 of Kap147. Mutational assays showed that only the ARE at -124 was involved in androgen response in OK cells. Electrophoretic mobility shift assay also verified -124 ARE bound specifically to androgen receptor. In conclusion, we defined the minimal Kap147 promoter that may be a good model for the study of kidney PTC-specific expression and molecular mechanisms that lead to an androgen-specific responsiveness in vivo.

Involvement of testicular growth factors in fetal Leydig cell aggregation after exposure to phthalate in utero.

Exposures to di-(2-ethylhexyl) phthalate (DEHP) have been shown to be associated with decreased adult testosterone (T) levels and increased Leydig cell numbers. As yet, little is known about DEHP effects in utero on fetal Leydig cells (FLC). The present study investigated effects of DEHP on FLC function. Pregnant Long-Evans female rats received vehicle (corn oil) or DEHP at 10, 100, or 750 mg/kg by oral gavage from gestational day (GD)2-20. At GD21, T production, FLC numbers and distribution, and testicular gene expression were examined. The percentage of FLC clusters containing 6-30 cells increased in all treatment groups, with 29 +/- 2% in control vs. 37 +/- 3, 35 +/- 3, and 56 +/- 4% in rats receiving 10, 100, and 750 mg/kg DEHP, respectively. In contrast, FLC numbers were 33% and 39% lower than control after exposures to 100 and 750 mg/kg DEHP, respectively. At these doses, mRNA levels of leukemia inhibitory factor (LIF) increased. LIF was found to induce cell aggregation in FLCs in vitro, consistent with the hypothesis that DEHP induced FLC aggregation. Testicular T levels were doubled by the 10 mg/kg dose and halved at 750 mg/kg. The mRNA levels of IGF-1 and c-Kit ligand (KITL) were induced by 10 mg/kg DEHP. These results, taken together, indicate that fetal exposures to DEHP have effects on FLC number, distribution, and most importantly, steroidogenic capacity and suggest that abnormal expressions of IGF1, KITL, and LIF genes may contribute to the reproductive toxicity of phthalates.

11{beta}-Hydroxysteroid dehydrogenase 2 in rat leydig cells: its role in blunting glucocorticoid action at physiological levels of substrate.

Corticosterone (CORT) suppresses Leydig cell steroidogenesis by inhibiting the expression of proteins involved in testosterone biosynthesis including steroidogenic acute regulatory protein and steroidogenic enzymes. In most cells, intracellular glucocorticoid levels are controlled by either or both of the two known isoforms of 11beta-hydroxysteroid dehydrogenase (11beta HSD): the nicotinamide adenine dinucleotide phosphate reduced-dependent low-affinity type I 11beta HSD (11beta HSD1) oxidoreductase and the nicotinamide adenine dinucleotide-dependent 11beta HSD2 high-affinity unidirectional oxidase. In Leydig cells, 11beta HSD1 alone may not be sufficient to prevent glucocorticoid-mediated suppression due to its low affinity for CORT at basal concentrations. The high-affinity unidirectional 11beta HSD2, if also present, may be critical for lowering intracellular CORT levels. In the present study, we showed that 11beta HSD2 is present in rat Leydig cells by PCR amplification, immunohistochemical staining, enzyme histochemistry, immunoprecipitation, and Western blotting. Real-time PCR showed a 6-fold enrichment of 11beta HSD2 mRNA in these cells, compared with whole testis and that the amount of 11beta HSD2 message was about 1000-fold lower, compared with 11beta HSD1. Diffuse immunofluorescent staining of 11beta HSD2 protein in the Leydig cell cytoplasm was consistent with its localization in the smooth endoplasm reticulum. 11beta HSD1 or 11beta HSD2 activities were selectively inhibited using antisense methodology: inhibition of 11beta HSD1 lowered reductase activity by 60% and oxidation by 25%, whereas inhibition of 11beta HSD2 alone suppressed oxidase activity by 50%. This shows that the high-affinity, low-capacity 11beta HSD2 isoform, present at only one thousandth the level of the low-affinity isoform may significantly affect the level of CORT. The inhibition of either 11beta HSD1 or 11beta HSD2 significantly lowered testosterone production in the presence of CORT. These data suggest that both types I and II 11beta HSD in Leydig cells play a protective role, opposing the adverse effects of excessive CORT on testosterone production.

Estrogen receptor-alpha gene deficiency enhances androgen biosynthesis in the mouse Leydig cell.

Leydig cells, which produce the primary male steroid hormone testosterone (T), express the two estrogen receptor (ER) subtypes, ERalpha and ERbeta, and have the capacity to convert testosterone to the natural estrogen 17beta-estradiol. Thus, Leydig cells are subject to estrogen action. The development of transgenic mice that are homozygous for targeted deletion of genes encoding the ER subtypes provides an opportunity to examine the role of estrogen in Leydig cell function. In this study androgen biosynthesis was analyzed in Leydig cells from mice that were homozygous for targeted deletion of the ERalpha gene (alphaERKO). T production by alphaERKO Leydig cells was 2-fold higher than that in wild-type (WT) cells. Serum T levels were accordingly higher in alphaERKO compared with WT mice (5.1 +/- 1.1 vs. 2.2 +/- 0.4 ng/ml; P

Hormone-specific regulation of the kidney androgen-regulated gene promoter in cultured mouse renal proximal-tubule cells.

The kidney androgen-regulated protein (KAP) is specifically expressed and differentially regulated by androgens and tri-iodothyronine (T(3)) in intact mouse early (PCT) and late (PR) proximal-tubule cells. Until now, detailed characterization of the molecular elements mediating androgen-responsive gene expression in the kidney has been hampered by the lack of appropriate cultured cell systems suitable for DNA transfection studies. In the present study we have analysed the hormone-dependent transactivation of the KAP gene promoter in immortalized differentiated PCT and PR proximal-tubule cells derived from L-PK/Tag1 transgenic mice. Transient transfection studies with different KAP promoter constructs indicated that a 224 bp-truncated fragment was sufficient to mediate cell-specific expression of the KAP promoter. Dihydrotestosterone (DHT) stimulated in an androgen-dependent manner the transactivation of KAP in PCT and PR cells, while mutation of a putative androgen-response element (ARE) sequence located at -39 bp from the transcription initiation site abolished the transactivation induced by DHT. Furthermore, insulin-like growth factor 1 (IGF-1), but not T(3), enhanced the androgen-dependent transactivation of KAP in cultured PCT cells. These results demonstrate that the short 224 bp fragment of the KAP promoter is sufficient to drive the proximal-tubule androgen-specific regulated expression of KAP and reveal synergistic interactions between IGF-1 and androgens for KAP regulation in PCT cells.