Dianne O. Hardy

The Kidney Androgen-regulated Protein Promoter Confers Renal Proximal Tubule Cell-specific and Highly Androgen-responsive Expression on the Human Angiotensinogen Gene in Transgenic Mice*

Transgenic mice were generated containing a 1542-base pair fragment of the kidney androgen-regulated protein (KAP) promoter fused to the human angiotensinogen (HAGT) gene with the goal of specifically targeting inducible expression of renin-angiotensin system components to the kidney. High level expression of both KAP-HAGT and endogenous KAP mRNA was evident in the kidney of male mice from two independent transgenic lines. Renal expression of the transgene in female mice was undetectable under basal conditions but could be strongly induced by administration of testosterone. Testosterone treatment did not cause a transcriptional induction in any other tissues examined. However, an analysis of six androgen target tissues in males revealed that the transgene was expressed in epididymis. No other extra-renal expression of the transgene was detected. In situ hybridization demonstrated that expression ofHAGT (and KAP) mRNA in males and testosterone-treated females was restricted to proximal tubule epithelial cells in the renal cortex. Although there was no detectable human angiotensinogen protein in plasma, it was evident in the urine, consistent with a pathway of synthesis in proximal tubule cells and release into the tubular lumen. These results demonstrate that 1542 base pairs of the KAP promoter is sufficient to drive expression of a heterologous reporter gene in a tissue-specific, cell-specific, and androgen-regulated fashion in transgenic mice.

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.

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.

Developmental Changes in Glucocorticoid Receptor and 11β-Hydroxysteroid Dehydrogenase Oxidative and Reductive Activities in Rat Leydig Cells1

Glucocorticoids directly regulate testosterone production in Leydig cells through a glucocorticoid receptor (GR)-mediated repression of the genes that encode testosterone biosynthetic enzymes. The extent of this action is determined by the numbers of GR within the Leydig cell, the intracellular concentration of glucocorticoid, and 11β-hydroxysteroid dehydrogenase (11βHSD) activities that interconvert corticosterone (in the rat) and its biologically inert derivative, 11-dehydrocorticosterone. As glucocorticoid levels remain stable during pubertal development, GR numbers and 11βHSD activities are the primary determinants of glucocorticoid action. Therefore, in the present study, levels of GR and 11βHSD messenger RNA (mRNA) and protein were measured in rat Leydig cells at three stages of pubertal differentiation: mesenchymal-like progenitors (PLC) on day 21, immature Leydig cells (ILC) that secrete 5α-reduced androgens on day 35, and adult Leydig cells (ALC) that are fully capable of testosterone biosynthesi...

In Utero and Lactational Exposures to Diethylhexyl-Phthalate Affect Two Populations of Leydig Cells in Male Long-Evans Rats

Abstract 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.

Identification of the Oxidative 3α-Hydroxysteroid Dehydrogenase Activity of Rat Leydig Cells as Type II Retinol Dehydrogenase1

Dihydrotestosterone (DHT) is the most potent naturally occurring androgen, and its production in the testis may have important consequences in developmental and reproductive processes. In the rat testis, three factors can contribute to intracellular DHT levels: 1) synthesis of DHT from T by 5α-reductase, 2) conversion of DHT to 5α-androstane-3α,17β-diol (3α-DIOL) by the reductive activity of 3α-hydroxysteroid dehydrogenase (3α-HSD), and 3) conversion of 3α-DIOL by an oxidative 3α-HSD activity. While the type I 3α-HSD enzyme (3α-HSD1 or AKR1C9) is an oxidoreductase in vitro and could theoretically be responsible for factors 2 and 3, we have shown previously that rat Leydig cells have two 3α-HSD activities: a cytosolic NADP(H)- dependent activity, characteristic of 3α-HSD1, and a microsomal NAD(H)-dependent activity. The two activities were separable by both developmental and biochemical criteria, but the identity of the second enzyme was unknown. To identify the microsomal NAD(H)-dependent 3α-HSD in rat Le...

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.

The (+)- and (−)-gossypols potently inhibit human and rat 11β-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.

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.