The effects of chronic oxytocin administration on body weight and food intake in DHT-induced PCOS model rats.

Polycystic ovary syndrome (PCOS) is commonly associated with metabolic disorders, which are exacerbated by obesity. Recent studies have revealed that oxytocin contributes to metabolic, appetite, and body weight regulation. In the present study, we evaluated the effects of chronic administration of oxytocin on body weight, food intake, and fat mass in a dihydrotestosterone-induced rat model of PCOS. Body weight, body weight change, and relative cumulative food intake were significantly lower in the oxytocin-treated PCOS rats than in the vehicle-treated control PCOS rats. Similarly, visceral adipocyte size was significantly smaller in the oxytocin-treated PCOS rats than in the vehicle-treated control PCOS rats. On the other hand, the numbers of cystic follicles in the ovary did not differ between the two groups. The chronic administration of oxytocin did not affect the rats' serum aspartate aminotransferase, alanine aminotransferase, or lactate dehydrogenase levels, indicating that it does not have adverse effects on hepatic function. These findings suggest that oxytocin could be a candidate drug for preventing the onset of obesity-related metabolic disorders in PCOS patients.

D-Chiro-Inositol Treatment Affects Oocyte and Embryo Quality and Improves Glucose Intolerance in Both Aged Mice and Mouse Models of Polycystic Ovarian Syndrome.

Polycystic ovarian syndrome (PCOS) is the main cause of female infertility. It is a multifactorial disorder with varying clinical manifestations including metabolic/endocrine abnormalities, hyperandrogenism, and ovarian cysts, among other conditions. D-Chiro-inositol (DCI) is the main treatment available for PCOS in humans. To address some of the mechanisms of this complex disorder and its treatment, this study examines the effect of DCI on reproduction during the development of different PCOS-associated phenotypes in aged females and two mouse models of PCOS. Aged females (8 months old) were treated or not (control) with DCI for 2 months. PCOS models were generated by treatment with dihydrotestosterone (DHT) on Days 16, 17, and 18 of gestation, or by testosterone propionate (TP) treatment on the first day of life. At two months of age, PCOS mice were treated with DCI for 2 months and their reproductive parameters analyzed. No effects of DCI treatment were produced on body weight or ovary/body weight ratio. However, treatment reduced the number of follicles with an atretic cyst-like appearance and improved embryo development in the PCOS models, and also increased implantation rates in both aged and PCOS mice. DCI modified the expression of genes related to oocyte quality, oxidative stress, and luteal sufficiency in cumulus-oocyte complexes (COCs) obtained from the aged and PCOS models. Further, the phosphorylation of AKT, a main metabolic sensor activated by insulin in the liver, was enhanced only in the DHT group, which was the only PCOS model showing glucose intolerance and AKT dephosphorylation. The effect of DCI in the TP model seemed mediated by its influence on oxidative stress and follicle insufficiency. Our results indicate that DCI works in preclinical models of PCOS and offer insight into its mechanism of action when used to treat this infertility-associated syndrome.

Hyperandrogenism and insulin resistance-induced fetal loss: evidence for placental mitochondrial abnormalities and elevated reactive oxygen species production in pregnant rats that mimic the clinical features of polycystic ovary syndrome.

KEY POINTS: Women with polycystic ovary syndrome (PCOS) commonly suffer from miscarriage, but the underlying mechanisms remain unknown. Herein, pregnant rats chronically treated with 5α-dihydrotestosterone (DHT) and insulin exhibited hyperandrogenism and insulin resistance, as well as increased fetal loss, and these features are strikingly similar to those observed in pregnant PCOS patients. Fetal loss in our DHT+insulin-treated pregnant rats was associated with mitochondrial dysfunction, disturbed superoxide dismutase 1 and Keap1/Nrf2 antioxidant responses, over-production of reactive oxygen species (ROS) and impaired formation of the placenta. Chronic treatment of pregnant rats with DHT or insulin alone indicated that DHT triggered many of the molecular pathways leading to placental abnormalities and fetal loss, whereas insulin often exerted distinct effects on placental gene expression compared to co-treatment with DHT and insulin. Treatment of DHT+insulin-treated pregnant rats with the antioxidant N-acetylcysteine improved fetal survival but was deleterious in normal pregnant rats. Our results provide insight into the fetal loss associated with hyperandrogenism and insulin resistance in women and suggest that physiological levels of ROS are required for normal placental formation and fetal survival during pregnancy. ABSTRACT: Women with polycystic ovary syndrome (PCOS) commonly suffer from miscarriage, but the underlying mechanism of PCOS-induced fetal loss during pregnancy remains obscure and specific therapies are lacking. We used pregnant rats treated with 5α-dihydrotestosterone (DHT) and insulin to investigate the impact of hyperandrogenism and insulin resistance on fetal survival and to determine the molecular link between PCOS conditions and placental dysfunction during pregnancy. Our study shows that pregnant rats chronically treated with a combination of DHT and insulin exhibited endocrine aberrations such as hyperandrogenism and insulin resistance that are strikingly similar to those in pregnant PCOS patients. Of pathophysiological significance, DHT+insulin-treated pregnant rats had greater fetal loss and subsequently decreased litter sizes compared to normal pregnant rats. This negative effect was accompanied by impaired trophoblast differentiation, increased glycogen accumulation, and decreased angiogenesis in the placenta. Mechanistically, we report that over-production of reactive oxygen species (ROS) in the placenta, mitochondrial dysfunction, and disturbed superoxide dismutase 1 (SOD1) and Keap1/Nrf2 antioxidant responses constitute important contributors to fetal loss in DHT+insulin-treated pregnant rats. Many of the molecular pathways leading to placental abnormalities and fetal loss in DHT+insulin treatment were also seen in pregnant rats treated with DHT alone, whereas pregnant rats treated with insulin alone often exerted distinct effects on placental gene expression compared to insulin treatment in combination with DHT. We also found that treatment with the antioxidant N-acetylcysteine (NAC) improved fetal survival in DHT+insulin-treated pregnant rats, an effect related to changes in Keap1/Nrf2 and nuclear factor-κB signalling. However, NAC administration resulted in fetal loss in normal pregnant rats, most likely due to PCOS-like endocrine abnormality induced by the treatment. Our results suggest that the deleterious effects of hyperandrogenism and insulin resistance on fetal survival are related to a constellation of mitochondria-ROS-SOD1/Nrf2 changes in the placenta. Our findings also suggest that physiological levels of ROS are required for normal placental formation and fetal survival during pregnancy.

Sex disparity in colonic adenomagenesis involves promotion by male hormones, not protection by female hormones.

It recently has been recognized that men develop colonic adenomas and carcinomas at an earlier age and at a higher rate than women. In the Apc(Pirc/+) (Pirc) rat model of early colonic cancer, this sex susceptibility was recapitulated, with male Pirc rats developing twice as many adenomas as females. Analysis of large datasets revealed that the Apc(Min/+) mouse also shows enhanced male susceptibility to adenomagenesis, but only in the colon. In addition, WT mice treated with injections of the carcinogen azoxymethane (AOM) showed increased numbers of colonic adenomas in males. The mechanism underlying these observations was investigated by manipulation of hormonal status. The preponderance of colonic adenomas in the Pirc rat model allowed a statistically significant investigation in vivo of the mechanism of sex hormone action on the development of colonic adenomas. Females depleted of endogenous hormones by ovariectomy did not exhibit a change in prevalence of adenomas, nor was any effect observed with replacement of one or a combination of female hormones. In contrast, depletion of male hormones by orchidectomy (castration) markedly protected the Pirc rat from adenoma development, whereas supplementation with testosterone reversed that effect. These observations were recapitulated in the AOM mouse model. Androgen receptor was undetectable in the colon or adenomas, making it likely that testosterone acts indirectly on the tumor lineage. Our findings suggest that indirect tumor-promoting effects of testosterone likely explain the disparity between the sexes in the development of colonic adenomas.

20-HETE and CYP4A2 ω-hydroxylase contribute to the elevated blood pressure in hyperandrogenemic female rats.

In male rats, androgen supplements increase 20-hydroxyeicosatetraenoic acid (20-HETE) via cytochrome P-450 (CYP)4A ω-hydroxylase and cause an increase in blood pressure (BP). In the present study, we determined the roles of 20-HETE and CYP4A2 on the elevated BP in hyperandrogenemic female rats. Chronic dihydrotestosterone (DHT) increased mean arterial pressure (MAP) in female Sprague-Dawley rats (96 ± 2 vs. 108 ± 2 mmHg, P < 0.05) and was associated with increased renal microvascular CYP4A2 mRNA expression (15-fold), endogenous renal 20-HETE (5-fold), and ω-hydroxylase activity (3-fold). Chronic DHT also increased MAP in low salt-fed Dahl salt-resistant female rats (81 ± 4 vs. 95 ± 1 mmHg, P < 0.05) but had no effect on MAP in Dahl salt-sensitive female rats (154 ± 3 vs. 153 ± 3 mmHg), which are known to be 20-HETE deficient. To test the role of CYP4A2, female CYP4A2(-/-) and SS.5(Bn) (wild type) rats were treated with DHT. DHT increased MAP in SS.5(Bn) female rats (104 ± 1 vs. 128 ± 1 mmHg, P < 0.05) but had no effect in CYP4A2(-/-) female rats (118 ± 1 vs. 120 ± 1 mmHg). Renal microvascular 20-HETE was reduced in control CYP4A2(-/-) female rats and was increased with DHT in SS.5(Bn) female rats (6-fold) but not CYP4A2(-/-) female rats. ω-Hydroxylase activity was 40% lower in control CYP4A2(-/-) female rats than in SS.5(Bn) female rats, and DHT decreased ω-hydroxylase activity in SS.5(Bn) female rats (by 50%) but significantly increased ω-hydroxylase activity in CYP4A2(-/-) female rats (3-fold). These data suggest that 20-HETE via CYP4A2 contributes to the elevation in BP in hyperandrogenemic female rats. The data also suggest that 20-HETE synthesis inhibition may be effective in treating the elevated BP in women with hyperandrogenemia, such as women with polycystic ovary syndrome.

Effects of trilostane and fipronil on the reproductive axis in an early life stage of the Japanese medaka (Oryzias latipes).

Given the critical role of the hypothalamic-pituitary-gonadal (HPG) axis, it is conceivable that perturbations at any point along this axis can potentially affect reproduction in fish and other vertebrates. We investigated the effects of a 3ß-hydroxysteroid dehydrogenase (3ß-HSD) inhibitor, trilostane (TRI), and a gamma-aminobutyric acid (GABA)-receptor antagonist, fipronil (FIP), on the HPG axis using an early life stage of the Japanese medaka. The newly hatched larvae were exposed to TRI (100, 300 and 1000 µg/L) and FIP (3, 10 and 30 µg/L), respectively, until 28 days post-hatching. Exposure to TRI decreased the body length in males, whereas FIP inhibited growth in both sexes. The induction of steroidogenesis-regulating genes (including 3ß-hsd) in males exposed to TRI, accompanied by increased vtg and er transcription, indicating a compensatory response to the presumed 3ß-HSD inhibition. These compensatory responses were not observed in TRI-treated females. Regarding FIP exposure, the GABA blocker resulted in the down-regulation of fshr and lhr. A compensatory up-regulation of steroidogenesis-regulating genes partially explained the elevated transcripts of vtg genes in both males and females after FIP exposure. These results suggest that both the inhibition of 3ß-HSD and the antagonism of GABA receptors are relevant modes of endocrine disruption that could impact the normal regulation of the HPG axis.

Hepatic protein expression networks associated with masculinization in the female fathead minnow (Pimephales promelas).

Endocrine disruptors that act via the androgen receptor (AR) are less well studied than environmental estrogens, and there is evidence that treatment with AR agonists can result in masculinization of female fish. In this study, female fathead minnows (FHM) were exposed to the model nonaromatizable androgen 5-alpha dihydrotestosterone (DHT) (100 µg/L), the ureic-based herbicide linuron (LIN) (100 µg/L), and a mixture of DHT and LIN (100 µg/L each) to better characterize androgen action in females. LIN was used because of reports that this chemical has an antiandrogenic mode of action in fish. After 21d, DHT and LIN treatments resulted in a significant depression of plasma vitellogenin (Vtg) and DHT and DHT+LIN increased the prevalence of nuptial tubercles in female FHMs indicating masculinization. Using iTRAQ and an LTQ Orbitrap Velos, ∼2000 proteins were identified in the FHM liver and the number of proteins quantified after exposures was >1200. Proteins that significantly and consistently changed in abundance across biological replicates included prostaglandin E synthase 3, programmed cell death 4a, glutathione S transferases, canopy, selenoprotein U, and ribosomal proteins. Subnetwork enrichment analysis identified that interferon and epidermal growth factor signaling were regulated by DHT and LIN, suggesting that these signaling pathways are correlated to depressed plasma vitellogenin. These data provide novel insight into hepatic protein networks that are associated with the process of masculinization in teleosts.

Establishing brown trout primary hepatocyte spheroids as a new alternative experimental model-Testing the effects of 5α-dihydrotestosterone on lipid pathways.

Three-dimensional (3D) fish liver cultures mimic the in vivo cellular microenvironment, which is ideal for ecotoxicological research. Despite that, the application of these cultures to evaluate toxic effects in fish is scarce. A 3D model of brown trout (Salmo trutta f. fario) primary hepatocyte spheroids was optimized in this study by using DMEM/F-12 with 15 mM of HEPES, 10 mL/L of an antibiotic and antimycotic solution and FBS 10% (v/v), at 18 °C with ∼100 rpm. The selection of optimal conditions was based on a multiparametric characterization of the spheroids, including biometry, viability, microanatomy and immunohistochemistry. Biometric and morphologic stabilization of spheroids was reached within 12-16 days of culture. To our knowledge, this study is the first to culture and characterize viable spheroids from brown trout primary hepatocytes for over 30 days. Further, the 3D model was tested to explore the androgenic influences on lipidic target genes after 96 h exposures to control, solvent control, 10 and 100 µM of 5α-dihydrotestosterone (DHT), a non-aromatizable androgen. Spheroids exposed to 100 µM of DHT had decreased sphericity. DHT at 100 µM also significantly down-regulated Acox1-3I, PPARγ and fatty acid synthesis targets (i.e., ACC), and significantly up-regulated Fabp1. Acsl1 was significantly up-regulated after exposure to both 10 and 100 µM of DHT. The results support that DHT modulates distinct lipidic pathways in brown trout and show that this 3D model is a new valuable tool for physiological and toxicological mechanistic studies.

Prenatal testosterone and dihydrotestosterone exposure disrupts ovine testicular development.

Androgens play important roles during the first trimester of intrauterine life, coinciding with genital tract differentiation, during virilization and maintenance of secondary male characteristics, and during initiation of spermatogenesis. Little is known about the impact of inappropriate exposure to excess androgens during fetal development on male sexual maturation and reproduction. The objectives of this study were to determine the effects of prenatal 5α-dihydrotestosterone (DHT) and testosterone treatment during ovine sexual differentiation on post-pubertal testicular formation and subsequent potential for fertility as assessed by epididymal sperm characteristics. Rams prenatally treated with testosterone exhibited increased testicular weight relative to age-matched controls and prenatal DHT-treated rams (P<0.05), as well as elevated total and free testosterone concentrations compared with DHT-treated rams (P=0.07 and P<0.05 respectively). The percentage of progressively motile sperm from the epididymis was significantly reduced in prenatal DHT-treated but not testosterone-treated rams compared with control rams (P<0.05). The testosterone-treated rams had a greater number of germ cell layers than DHT-treated rams, but comparable to the controls. Prenatal testosterone-treated rams had significantly larger seminiferous tubule diameter and lumen diameter compared with prenatal DHT-treated (P<0.05). Significantly, more prenatal DHT- and testosterone-treated rams (P<0.05) had occluded tubule lumen than control rams. Findings from this study demonstrate that exposure to excess testosterone/DHT during male fetal sexual differentiation have differential effects on post-pubertal testicular size, seminiferous tubule size and function, sperm motility, and testosterone concentrations.

Transcriptional regulatory dynamics of the hypothalamic-pituitary-gonadal axis and its peripheral pathways as impacted by the 3-beta HSD inhibitor trilostane in zebrafish (Danio rerio).

To study mechanisms underlying generalized effects of 3ß hydroxysteroid dehydrogenase (HSD3B) inhibition, reproductively mature zebrafish (Danio rerio) were exposed to trilostane at two dosages for 24, 48, or 96 h and their gonadal RNA samples profiled with Agilent zebrafish microarrays. Trilostane had substantial impact on the transcriptional dynamics of zebrafish, as reflected by a number of differentially expressed genes (DEGs) including transcription factors (TFs), altered TF networks, signaling pathways, and Gene Ontology (GO) biological processes. Changes in gene expression between a treatment and its control were mostly moderate, ranging from 1.3 to 2.0 fold. Expression of genes coding for HSD3B and many of its transcriptional regulators remained unchanged, suggesting transcriptional up-regulation is not a primary compensatory mechanism for HSD3B enzyme inhibition. While some trilostane-responsive TFs appear to share cellular functions linked to endocrine disruption, there are also many other DEGs not directly linked to steroidogenesis. Of the 65 significant TF networks, little similarity, and therefore little cross-talk, existed between them and the hypothalamic-pituitary-gonadal (HPG) axis. The most enriched GO biological processes are regulations of transcription, phosphorylation, and protein kinase activity. Most of the impacted TFs and TF networks are involved in cellular proliferation, differentiation, migration, and apoptosis. While these functions are fairly broad, their underlying TF networks may be useful to development of generalized toxicological screening methods. These findings suggest that trilostane-induced effects on fish endocrine functions are not confined to the HPG-axis alone. Its impact on corticosteroid synthesis could also have contributed to some system wide transcriptional changes in zebrafish observed in this study.

Assessing potential indicator of endocrine-disrupting property of chemicals using soil invertebrates.

Vitellogenin has been regarded as an acceptable indicator for evaluating the endocrine-disrupting property of chemicals using fish. However, the endocrine-disrupting property of chemicals has been rarely evaluated using soil species. This study aimed to find evidence that endocrine-disrupting chemicals (including the natural hormones estradiol and dihydrotestosterone) can affect the reproductive organs of earthworms. Earthworms were exposed to 17ß-estradiol, dihydrotestosterone, bisphenol A, and methylparaben for seven days. The four EDCs inhibited normal oogenesis and maturation of oocytes in earthworm ovary, and dihydrotestosterone and bisphenol A were observed to damage earthworm seminal vesicle tissues and inhibit normal spermatogenesis. The evidence showed that the tested EDCs have an adverse effect on female and male reproductive systems of soil invertebrates. The results suggest that the evaluations of oogenesis and spermatogenesis in the ovary and seminal vesicles of earthworms are useful indicators for investigating the endocrine-disrupting property of chemicals. Additionally, our results encourage further studies on developing novel indicators using soil invertebrates to evaluate the effects of the toxicity of endocrine-disrupting chemicals on the soil ecosystem.

Deciphering influences of testosterone and dihydrotestosterone on lipid metabolism genes using brown trout primary hepatocytes.

Despite of physiological and toxicological relevance, the potential of androgens to influence fish lipid metabolism remains poorly explored. Here, brown trout primary hepatocytes were exposed to six concentrations (1 nM to 100 µM) of dihydrotestosterone (DHT) and testosterone (T), to assess changes in the mRNA levels of genes covering diverse lipid metabolic pathways. Acsl1, essential for fatty acid activation, was up-regulated by T and DHT, whereas the lipogenic enzymes FAS and ACC were up-regulated by the highest (100 µM) concentration of T and DHT, respectively. ApoA1, the major component of high-density lipoprotein (HDL), was down-regulated by both androgens. PPARγ, linked to adipogenesis and peroxisomal ß-oxidation, was down-regulated by T and DHT, while Acox1-3I, rate-limiting in peroxisomal ß-oxidation, was down-regulated by T. Fabp1, StAR and LPL were not altered. Our findings suggest that androgens may impact on lipid transport, adipogenesis and fatty acid ß-oxidation and promote lipogenesis in fish liver.

Neonatal androgenization in rats affects oocyte maturation.

Androgens are relevant in order to achieve a normal growth and maturation of the follicle and oocyte, since both excess and absence of androgens may affect the correct ovarian function. The current study analyzes the impact of neonatal androgenization in the first ovulation and oocyte maturation in response to exogenous gonadotrophin stimulation. Neonatal rats were daily treated with testosterone, dihydrotestosterone, or vehicle during follicle assembly period (days 1 to 5). At juvenile period, rats were stimulated sequentially with PMSG and hCG. Ovulation, ovarian histology, hormonal milieu, morphological characteristics of meiotic spindle, and in vitro fertilization rate in oocytes were analyzed. Our data shows that oocytes from androgenized rats displayed a major proportion of aberrant spindles and altered meiotic advance that control animals. These alterations were accompanied with an increase in both fertilization rate and aberrant embryos after 48 h of culture. Our findings showed a direct impact of neonatal androgens on oocyte development; their effects may be recognized at adulthood, supporting the idea of a programming effect exerted by neonatal androgens. These results could be relevant to explain the low fertility rate seen in polycystic ovary syndrome patients after in vitro fertilization procedures.

Psoralea corylifolia L. extract ameliorates benign prostatic hyperplasia by regulating prostate cell proliferation and apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Psoralea corylifolia L. seed (PCL), commonly known as "Poguzhi" or "BuguZhi", has been widely used to treat kidney yang deficiency in traditional Chinese medicine (TCM) where tonifying the yang deficiency is a representative understanding for treatment of hormonal deficiency disorders such as enuresis, oliguria, and prostatic diseases. Although PCL has been commonly used to treat problems of the urinary system, its efficacy against benign prostatic hyperplasia (BPH) has not yet been reported. AIM OF THE STUDY: In the present study, we aimed to assess the in vitro and in vivo efficacy of PCL against BPH, a condition which negatively impacts quality of life in men. MATERIALS AND METHODS: Normal human prostate cell lines, RWPE-1 and WPMY-1 cells, were stimulated with 10 nM dihydrotestosterone (DHT) to establish an in vitro BPH model. Subsequently, cells were treated with 100 or 200 µg/ml PCL, which inhibited cell proliferation without cytotoxicity, to evaluate the anti-BPH effect of PCL. Eight-week-old male Wistar rats were castrated, except for those in the control group (Con), and BPH was induced by subcutaneous injection of 10 mg/kg testosterone propionate (TP). Concurrent with daily TP injections, 5 mg/kg of finasteride (Fina) and 50 or 100 mg/kg PCL were orally administrated daily for four weeks, excluding the weekends. RESULTS: In DHT-stimulated RWPE-1 and WPMY-1 cells, expression of androgen receptor (AR) androgen signaling-related markers such as 5α-reductase 2 (5AR2), AR, and prostate-specific antigen (PSA) was upregulated, whereas 100 or 200 µg/ml of PCL treatment downregulated these markers. Furthermore, PCL significantly reduced the mRNA expression of anti-apoptotic genes and increased the mRNA expression of pro-apoptotic gene. In vivo, administration of PCL reduced prostate size and weight in TP-induced BPH rats. Moreover, histological alterations in epithelium thickness were significantly restored by the administration of PCL. Immunohistochemical analysis revealed increased expression of AR and proliferating cell nuclear antigen (PCNA) in TP-induced BPH prostates; these changes were suppressed by administration of 50 or 100 mg/kg PCL. CONCLUSIONS: We demonstrated the effect of PCL against BPH, mediated by the regulation of prostate cell proliferation and apoptosis, in DHT-stimulated normal human prostate cell lines and TP-induced BPH rats. These findings suggest that PCL could be a potential therapeutic agent against BPH.

Transgenic Medaka Identify Embryonic Periods Sensitive to Disruption of Sex Determination.

Gonadal development in medaka (Oryzias latipes) is dependent on the synergy between estrogens and androgens. Disruption of steroid hormone levels can lead to ovo-testis. To determine the sensitive windows for hormonally induced sex reversal in medaka, we developed a novel 42sp50-GFP_ChgH-GFP transgenic medaka line, allowing the identification of female gonadal tissue by fluorescence present in developing oocytes. Germinal transgenesis resulted in a stable line exhibiting a strong green fluorescent protein signal constitutively in the ovaries and in the liver in response to estrogens. The sensitivity of this line to disruption of sex determination following 16-d chronic exposures was in the nanograms per liter range. To identify the developmental period sensitive to exogenous agents, fry were exposed to 24-h pulses of high concentrations of 17ß-estradiol (E2) or 5α-dihydrotestosterone (DHT) at various time points between days postfertilization (dpf) 0 and 12. Evaluation of phenotype followed by genotyping at 16 dpf revealed sensitivity to E2 between 1 and 8 dpf as well as 2 periods of susceptibility to DHT between 0 and 1 dpf and 4 and 8 dpf. No phenotypic sex reversal was detected after exposure to DHT or E2 on 11 or 12 dpf. The observed effects persisted to at least 24 dpf. The identified sensitive embryonic time periods for disruption of sex determination will aid future research on sex determination and the development of screening assays using early embryonic life stages. Environ Toxicol Chem 2020;39:842-851. © 2020 SETAC.

Disruption of classical estrogenic targets in brown trout primary hepatocytes by the model androgens testosterone and dihydrotestosterone.

Estrogenic effects triggered by androgens have been previously shown in a few studies. Aromatization and direct binding to estrogen receptors (ERs) are the most proposed mechanisms. For example, previously, a modulation of vitellogenin A (VtgA) by testosterone (T), an aromatizable androgen, was reported in brown trout primary hepatocytes. The effect was reversed by an ER antagonist. In this study, using the same model the disruption caused by T and by the non-aromatizable androgen - dihydrotestosterone (DHT), was assessed in selected estrogenic targets. Hepatocytes were exposed (96 h) to six concentrations of each androgen. The estrogenic targets were VtgA, ERα, ERß1 and two zona pellucida genes, ZP2.5 and ZP3a.2. The aromatase CYP19a1 gene and the androgen receptor (AR) were also included. Modulation of estrogenic targets was studied by quantitative real-time PCR and immunohistochemistry, using an HScore system. VtgA and ERα were up-regulated by DHT (1, 10, 100 µM) and T (10, 100 µM). In contrast, ERß1 was down-regulated by DHT (10, 100 µM), and T (100 µM). ZP2.5 mRNA levels were increased by DHT and T (1, 10, 100 µM), while ZP3a.2 was up-regulated by DHT (100 µM) and T (10, 100 µM). Positive correlations were found between VtgA and ERα mRNA levels and ZPs and ERα, after exposure to both androgens. The mRNA levels of CYP19a1 were not changed, while AR expression tended to increase after micromolar DHT exposures. HScores for Vtg and ZPs corroborated the molecular findings. Both androgens triggered estrogen signaling through direct binding to ERs, most probably ERα.

Baicalin alleviates benign prostate hyperplasia through androgen-dependent apoptosis.

BPH is a disease prevalent among elderly men that is characterized by abnormal proliferation of prostatic epithelial and stromal tissues. No effective treatment exists for BPH owing to lack of a clear understanding of its molecular etiology. Although several studies have reported therapeutic effects of baicalin against numerous diseases, including prostate cancer, its beneficial effects on BPH have not yet been explored. The present study investigated the therapeutic effects of baicalin on the development of BPH and its mechanism of action. We established a testosterone-treated BPH animal model and DHT-stimulated prostate cell lines, including RWPE-1 and WPMY-1. Administration of baicalin ameliorated the pathological prostate enlargement, suppressed the production of DHT, and inhibited the activity of 5α- reductase Type II in the animal model. BC exerted these effects via its anti-proliferative effects by restoring the Bax/Bcl-2 ratio, activating caspase-3 and caspase-8, and inducing the phosphorylation of AMPK. In vitro studies using DHT-stimulated prostate cells demonstrated an up-regulation of BPH-related and proliferation markers, whereas baicalin clearly reduced the overexpression of AR, PSA, PCNA, and Bcl-2. These results suggested that baicalin could suppress androgen-dependent development of BPH both in vivo and in vitro by inducing apoptosis.

Dose-dependent effects of dihydrotestosterone in the streptozotocin-induced diabetic rat kidney.

We recently reported that castration exacerbates albuminuria, glomerulosclerosis, and tubulointerstitial fibrosis associated with diabetic renal disease. The aim of the present study was to examine whether these effects of castration can be attenuated with dihydrotestosterone (DHT) supplementation. The study was performed in castrated male Sprague-Dawley, streptozotocin-induced diabetic rats treated with 0 mg/day DHT (DHT(0)), 0.75 mg/day DHT (DHT(0.75)), or 2.0 mg/day DHT (DHT(2.0)) for 14 wk. Treatment with 0.75 mg/day DHT attenuated castration-associated increases in urine albumin excretion (DHT(0), 81.2 +/- 18.1; DHT(0.75), 26.57 +/- 5.8 mg/day; P < 0.05), glomerulosclerosis (DHT(0), 1.1 +/- 0.79; DHT(0.75), 0.43 +/- 0.043 arbitrary units; P < 0.001), tubulointerstitial fibrosis (DHT(0), 1.3 +/- 0.12; DHT(0.75), 1.1 +/- 0.096 AU; P < 0.05), collagen type IV [DHT(0), 3.2 +/- 0.11; DHT(0.75), 2.1 +/- 0.070 relative optical density (ROD); P < 0.01], transforming growth factor-beta (DHT(0), 3.2 +/- 0.16; DHT(0.75), 2.1 +/- 0.060 ROD; P < 0.01), IL-6 (DHT(0), 0.37 +/- 0.011; DHT(0.75), 0.27 +/- 0.014 ROD; P < 0.05), and protein expression and reduced CD68-positive cell abundance (DHT(0), 17 +/- 0.86; DHT(0.75), 4.4 +/- 0.55 cells/mm(2); P < 0.001). In contrast, treatment with 2.0 mg/day DHT exacerbated all these parameters. These data suggest that the detrimental effects of castration in the diabetic kidney can be attenuated with low doses of DHT, whereas high doses augment the adverse effects of castration, and these effects appear to be influenced by estradiol. We conclude that the effects of DHT are dose dependent but caution should be taken when DHT supplementation is considered in the treatment of diabetic renal disease.

Proteomics of rat prostate lobes treated with 2-N-hydroxylamino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 5alpha-dihydrotestosterone, individually and in combination.

Epidemiological and preclinical studies suggest that environmental factors, hormonal responses and lifestyle, including diet and physical inactivity, are likely contributors to the initiation and progression of prostate cancer in humans. Although the effects of the food derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and/or testosterone (T) in the development of prostate cancer in the rat have been reported, the extent to which such compounds impact cancer related proteins is not clear. Knowledge of cancer-related proteins impacted by PhIP and/or T is prerequisite to developing novel strategies to early-detect prostate cancer. Male F344 rats were sacrificed, the prostate tissue isolated and separated into dorsolateral, ventral, and anterior lobes. The lobes were cultured and treated with 10(-3) M NHPhIP and/or 10(-7) M DT for 24 h. NHPhIP is the genotoxic form of PhIP and DT is the more proliferative form of T. We used 2D-DIGE and LC/MS/MS technologies to study the proteome of the prostate lobes to determine if the compounds will trigger detectable changes in expression of cancer-related proteins. Analysis of the signals from 2D-DIGE revealed that about 10% of proteins were differentially expressed in the NHPhIP and/or DT treatments compared to controls. Eight candidate protein spots detected by 2D-DIGE in at least two out of three lobes showed > or =2-fold difference between treated and control samples. Five out of the eight spots contained single proteins; including, phospholipase Calpha (PLP-Calpha), Rab7, SAR1a, ribosomal protein S7 (RPS7), and nucleoside diphosphate kinase (NDPK). A survey of the literature shows that NDPK expression is altered in human cancers, including prostate cancer. Thus, we validated the altered expression of NDPK by Western blot analysis. The concordance between 2D-DIGE and Western blot analysis was 80%. The results of this study demonstrate, for the first time, that the combination of 2D-DIGE and LC/MS/MS is a powerful tool for identification of proteins in the prostate tissue that are altered by environmental carcinogens and/or hormones.

6-sialyllactose ameliorates dihydrotestosterone-induced benign prostatic hyperplasia through suppressing VEGF-mediated angiogenesis.

Benign prostatic hyperplasia (BPH), a common disease in elderly males, is accompanied by non-malignant growth of prostate tissues, subsequently causing hypoxia and angiogenesis. Although VEGF-related angiogenesis is one of the therapeutic targets of prostate cancer, there is no previous study targeting angiogenesis for treatment of BPH. Dihydrotestosterone (DHT)- induced expressions of vascular endothelial growth factor (VEGF) in prostate epithelial RWPE-1 cells and human umbilical vascular endothelial cells (HUVECs). Conditioned media (CM) from DHT-treated RWPE-1 cells were transferred to HUVECs. Then, 6SL inhibited proliferation, VEGFR-2 activation, and tube formation of HUVECs transferred with CM from DHT-treated RWPE-1 cells. In the rat BPH model, 6SL reduced prostate weight, size, and thickness of the prostate tissue. Formation of vessels in prostatic tissues were also reduced with 6SL treatment. We found that 6SL has an ameliorative effect on in vitro and in vivo the BPH model via inhibition of VEGFR-2 activation and subsequent angiogenesis. These results suggest that 6SL might be a candidate for development of novel BPH drugs. [BMB Reports 2019; 52(9): 560-565].