Vascular endothelium expresses both the estrogen receptors (ERs) α and β, and ERα mediates development of early atherosclerosis in male mice. This process is thought to be testosterone-dependent. We hypothesized that male murine aortic endothelium produces robust levels of estradiol by aromatase conversion of testosterone, and that regulation of this process is mediated by the presence of ERs, primarily ERα. Aortic endothelium was isolated from ERα knockout (ERα -/-) and wild-type (ERα +/+) male mice and treated with testosterone or the <em>5α</em> reduction product dihydrotestosterone (<em>DHT</em>), with or without the P450 aromatase inhibitor anastrazole, or a non-specific estrogen receptor antagonist. Aromatase gene expression and estradiol production were assayed. Treatment with testosterone, but not <em>DHT</em>, caused increased aromatase expression and estradiol production in ERα +/+ endothelium that was attenuated by disruption of ERα in the ERα -/- group. Anastrazole inhibition of aromatase reduced testosterone-induced aromatase expression and estradiol levels in both ERα -/- and ERα +/+ endothelium. Antagonism of both ERs decreased testosterone-induced aromatase expression in both wild-type and knockout groups. The effects of the receptor antagonist on estradiol production differed between the two groups, however, with a reduction in estradiol release from the ERα +/+ cells and complete abolition of estradiol release from the ERα -/- cells. Thus, estradiol production in vascular endothelium from male mice is robust, depends on the aromatic conversion of testosterone and requires functional ERα to achieve maximal levels of estradiol generation. Local vascular production of aromatase-mediated estradiol in response to circulating testosterone may affect ERα-dependent mechanisms to increase susceptibility to early atheroma formation in male mice. This pathway may have important therapeutic relevance for reducing the risk of atherosclerotic cardiovascular disease in human males.

<AbstractText>Women with polycystic ovary syndrome (PCOS) commonly suffer from miscarriage, but the underlying mechanisms remain unknown. Herein, pregnant rats chronically treated with <em>5α</em>-dihydrotestosterone (<em>DHT</em>) 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 <em>DHT</em>+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 <em>DHT</em> or insulin alone indicated that <em>DHT</em> 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 <em>DHT</em> and insulin. Treatment of <em>DHT</em>+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.</AbstractText><AbstractText>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 <em>5α</em>-dihydrotestosterone (<em>DHT</em>) 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 <em>DHT</em> and insulin exhibited endocrine aberrations such as hyperandrogenism and insulin resistance that are strikingly similar to those in pregnant PCOS patients. Of pathophysiological significance, <em>DHT</em>+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 <em>DHT</em>+insulin-treated pregnant rats. Many of the molecular pathways leading to placental abnormalities and fetal loss in <em>DHT</em>+insulin treatment were also seen in pregnant rats treated with <em>DHT</em> alone, whereas pregnant rats treated with insulin alone often exerted distinct effects on placental gene expression compared to insulin treatment in combination with <em>DHT</em>. We also found that treatment with the antioxidant N-acetylcysteine (NAC) improved fetal survival in <em>DHT</em>+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.</AbstractText>

The development, growth, and function of the prostate gland depend on androgen stimulation. The primary androgen in prostate is <em>5α</em>-dihydrotestosterone (<em>DHT</em>) which is synthesized from circulating testosterone (T) through the action of <em>5α</em>-reductase (<em>5α</em>-R). Although <em>5α</em>-R occurs as five isozymes, only <em>5α</em>-R1 and <em>5α</em>-R2 are physiologically involved in steroidogenesis. The endocrine disruptor bisphenol A (BPA) alters sexual organs, including the prostate. Our previous findings indicated that BPA decreased the expression of <em>5α</em>-R1 and <em>5α</em>-R2 in rat prostate but also circulating T. Thus, it is unclear whether BPA exerts this effect on <em>5α</em>-R isozymes by reducing circulating T or by any other mechanism. In this study, we examine the effects of short-term exposure to BPA at doses below 25 μg/Kg/d and above 300 μg/Kg/d of the TDI on mRNA levels of <em>5α</em>-R1 and <em>5α</em>-R2 in prostate of adult castrated rats supplemented with T to achieve constant circulating T levels. mRNA levels were measured by absolute quantitative RT-PCR, T levels by RIA, and <em>DHT</em> levels by ELISA. Our results indicated that in castrated rats treated with T BPA at the two doses studied significantly decreased the mRNA levels of both <em>5α</em>-R isozymes in a dose-dependent manner without modifications in circulating T.

BACKGROUND

Through the actions of one or more isoforms of the enzyme <em>5α</em>-reductase in many male reproductive tissues, circulating testosterone (T) undergoes metabolic conversion into <em>5α</em>-dihydrotestosterone (<em>DHT</em>), which binds to and activates androgen receptors (AR) with greater potency than T. In birds, T is also subject to local inactivation into 5β-<em>DHT</em> by the enzyme 5β-reductase. Male golden-collared manakins perform an androgen-dependent and physically elaborate courtship display, and these birds express androgen receptors in skeletal muscles and spinal cord at levels far greater than those expressed in species with more limited courtship routines, including male zebra finches. To determine if local T metabolism facilitates or impedes activation of male manakin courtship, we examined expression of two isoforms of <em>5α</em>-reductase, as well as 5β-reductase, in forelimb muscles and spinal cords of males and females of the two aforementioned species.

RESULTS

We found that all enzymes were expressed in all tissues, with patterns that partially predict a functional role for <em>5α</em>-reductase in these birds, especially in both muscle and spinal cord of male manakins. Moreover, we found that 5β-reductase was markedly different between species, with far lower levels in golden-collared manakins, compared to zebra finches. Thus, modification to neuromuscular deactivation of T may also play a functional role in adaptive behavioral modulation.

CONCLUSIONS

Given that such a role for <em>5α</em>-reductase in androgen-sensitive mammalian skeletal muscle is in dispute, our data suggest that, in birds, local metabolism may play a key role in providing active androgenic substrates to peripheral neuromuscular systems. Similarly, we provide the first evidence that 5β-reductase is expressed broadly through an organism and may be an important factor that regulates androgenic modulation of neuromuscular functioning.

Androgens are vasoactive steroids that induce acute vasodilation in a number of isolated vascular beds from different species, but the effects of these hormones on systemic blood pressure (BP) have been studied little. Although it has been reported that androgens exert systemic hypotensive effects through peripheral vasodilation in normotensive rats, there have not been any reports of systemic hypotensive effects of androgens in animals with hypertension. This study was designed to evaluate the acute effects of testosterone (TES) and its 5-reduced metabolites on systemic BP in hypertensive rats and to test the hypothesis that hypotestosteronemia may be involved in the pathogenesis of hypertension. Chronic, indwelling catheters were implanted in carotid artery and jugular vein of 18-21-week-old male spontaneously hypertensive rats (SHR) and normotensive-control Wistar-Kyoto (WKY) rats, for BP recording and drug administration, respectively. Bolus injections of TES, <em>5α</em>- or 5β-dihydrotestosterone (<em>5α</em>- and 5β-<em>DHT</em>), were administrated cumulatively to conscious rats at doses of 0.1-100μmolkg-1min-1. 5β-<em>DHT</em> was also administrated during the pressor effect of Bay K 8644, an L-type voltage-operated Ca2+ channel (L-VOCC) agonist. In separate experiments, BP of orchidectomized normotensive male WKY and Wistar rats, with or without androgen-replacement therapy, was evaluated weekly for 10 weeks by tail-cuff plethysmography. TES and its metabolites reduced BP in a dose-dependent manner, while heart rate was reduced with some androgens at the highest doses. The hypotensive effects of androgens were markedly greater in SHR, with a rank order potency of: 5β-<em>DHT</em>)TES><em>5α</em>-<em>DHT</em>. 5β-<em>DHT</em>, the most potent antihypertensive androgen, abolished the pressor response to Bay K 8644 in SHR. TES deprivation by orchidectomy increased BP in normotensive WKY and Wistar rats, but this hypertension was prevented by TES replacement therapy. BP responses to androgens are androgen structure-dependent. These data indicate that: 1) androgens play a significant role in the control of BP and may contribute to the pathogenesis of hypertension; 2) blockade of L-VOCC is involved in the antihypertensive effects of androgens, which are non-genomically mediated; and 3) hypotestosteronemia may be a risk factor for hypertension.

The endocrine treatment of metastatic prostate cancer includes castration which reliably lowers the serum testosterone (T); however, the effect on intratumor levels of T and dihydrotestosterone (<em>DHT</em>) is less predictable. In vitro work demonstrated that the human prostate cancer cell line PC-3 had significant 5-a-reductase activity that could be inhibited with 17b-N,N-diethylcarbamoyl-4-aza-<em>5a</em>-androstan-3-one (4MA). In this study, we examined the effect of 5-a-reductase inhibition with 4MA and androgen suppression with dexamethasone on the growth characteristics and intratumor androgen levels in the PC-3 cell line in male athymic nude mice (Balb/c). The mice were randomized into six treatment groups: 1) noncastrate vehicle control, 2) 4MA, 0.25 mg/day, 3) 4MA, 1 mg/day, 4) dexamethasone, 25 micrograms/day, 5) 4MA, 1 mg/day, and dexamethasone, 25 micrograms/day, and 6) castrate control group. After 21 days of treatment the animals were sacrificed, serum collected, and tumors harvested. Each treatment produced intratumor <em>DHT</em> levels equivalent to the castrate group. Only the low dose 4MA caused a reduction in intratumor <em>DHT</em> without producing castrate levels of circulating T. The combination of dexamethasone and 4MA was less effective in lowering the intratumor <em>DHT</em>/T ratio than 4MA alone. No significant differences in tumor growth parameters were noted between intact control animals and any of the treatment arms. Serum T levels correlated poorly with intratumor androgen levels. Five-a-reductase inhibition produced castrate levels of intratumor <em>DHT</em> in the nonandrogen-dependent prostate cancer cell line PC-3. The combination of dexamethasone and 5-a-reductase inhibition with 4MA appears to be less effective in lowering intratumor androgen levels than either therapy alone.

Recent clinical studies have raised the clinically important question of the relationship between dihydrotestosterone (<em>DHT</em>) and prostate cancer (PCa) progression. The significance of <em>DHT</em> or <em>5α</em>-reductase inhibitors (5ARI) in PCa development and progression has not yet been fully characterized. The aim of this study was to determine whether the initiation of DNA replication was influenced by <em>DHT</em> in PCa. Three cell lines were used. LNCaP: a human PCa cell line that exhibits androgen-dependent proliferation, C4-2: a human PCa cell line that exhibits androgen-independent proliferation, and C4-2AT6: a castration resistant prostate cancer cell line. Two 5ARIs, finasteride and dutasteride, were used. We examined the mRNA expression of the components of pre-replication complex (Pre-RC), CDC6, CDT1, and MCM2-7. <em>DHT</em> induced cell proliferation of LNCaP accompanied by significantly increased CDC6, CDT1, and MCM2-7 expression. In contrast to LNCaP, <em>DHT</em> inhibited cell proliferation in C4-2AT6 cells accompanied by decreased expression of CDC6, CDT1, and MCM2-7. These reverse effects resemble the effects of 5ARIs in Pre-RC. Treatment with finasteride or dutasteride inhibited CDC6 expression in LNCaP, but both 5ARIs induced CDC6 expression in C4-2 and C4-2AT6 cells.These results indicate that <em>DHT</em> showed reversal effects on PCa cell proliferation among prostate cancer cells based on androgen-dependence, accompanied by regulation of the initiation of DNA replication. 5ARIs may modulate the DNA replication system in someaggressive PCa through up-regulation of CDC6 expression.

Androgens produce nongenomic effects in several cells by different mechanisms, including ion channel modulation. Adenohypophyseal cells express several K(+) channels, including voltage and Ca(2+)-dependent K(+) (BK) channels, which might be the target of androgens to modulate cellular action potentials and hormonal secretion. Androgen effects were studied in GH3 cells (from anterior pituitary rat tumor) by means of the patch-clamp technique. Cells were continuously perfused with saline solution, in the absence or presence of the androgens studied, while applying 40 mV pulses of 400 ms from a holding potential of -60 mV in whole-cell configuration with nystatin-perforated patches. Androgens reversibly blocked noninactivating K(+) currents in a concentration-dependent manner without a latency period and with an order of efficacy of: 5β-dihydrotestosterone (<em>DHT</em>>>testosterone><em>5α</em>-<em>DHT</em>. RT-PCR showed two isoforms of the α-pore forming subunits of BK channels. These channels are responsible for one third of the noninactivating current, according to the blockade of paxilline, a selective BK antagonist. Androgens seem to directly interact with BK channels since they were blocked in excised inside-out patches and independent of the whole-cell configuration and the NO-cGMP-dependent pathway. Testosterone, but not <em>5α</em>- or 5β-<em>DHT</em>, increased BK currents in HEK-293 cells overexpressing the short isoform, suggesting a cellular selectivity based on the α-subunits. The effect on noninactivating currents may be responsible for the decrease of spontaneous action potential frequency. Long-term cellular incubation with testosterone did not modify noninactivating currents density in GH3 cells. It is remarkable that 5β-<em>DHT</em>, a reductase metabolite with weak androgenic activity, was the most efficient blocker.

Benign prostatic hyperplasia (BPH) is characterized by uncontrolled proliferation of the prostate gland. Cynanchum wilfordii has been reported to improve sexual behavior in male rats. In this study, we investigated the protective effect of an aqueous extract of C. wilfordii (CWW) against BPH development in a testosterone-induced BPH rat model. The rats were divided into the following six groups: sham/vehicle; BPH/vehicle; BPH/finasteride; and three CWW doses (50, 100, and 200 mg/kg). After a 4-week treatment with CWW, the rats were euthanized at scheduled times, and their prostates were weighed, followed by a histopathological examination. Prostate growth inhibition rates in rats administered CWW 50, 100, and 200 mg/kg were 54.5%, 51.8%, and 50.1%, respectively. The BPH/CWW group showed decreased serum testosterone and dihydrotestosterone (<em>DHT</em>) levels compared to the BPH/vehicle group. Furthermore, the BPH/CWW group showed reduced prostate testosterone and <em>DHT</em> levels compared to the BPH/vehicle group. Mechanistically, the reverse transcription-polymerase chain reaction revealed downregulated mRNA expression levels of the androgen receptor, <em>5α</em>-reductase, and B-cell lymphoma-2 (Bcl-2) in the BPH/CWW200 group compared with those in the testosterone-induced groups. In conclusion, these findings show the effectiveness of CWW in slowing the progression of testosterone-induced BPH in rats.

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 <em>DHT</em>-stimulated prostate cell lines, including RWPE-1 and WPMY-1. Administration of baicalin ameliorated the pathological prostate enlargement, suppressed the production of <em>DHT</em>, and inhibited the activity of <em>5α</em>- 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. <i>In vitro</i> studies using <em>DHT</em>-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 <i>in vivo</i> and <i>in vitro</i> by inducing apoptosis.

In 1974, a lack of <em>5α</em>-dihydrotestosterone (<em>5α</em>-<em>DHT</em>), the most potent androgen across species except for fish, was shown to be the origin of a type of pseudohermaphrodism in which boys have female-like external genitalia. This human intersex condition is linked to a mutation in the steroid-<em>5α</em>-reductase type 2 (SRD<em>5α</em>2) gene, which usually produces an important enzyme capable of reducing the Δ⁴-ene of steroid C-19 and C-21 into a <em>5α</em>-stereoisomer. Seeing the potential of SRD<em>5α</em>2 as a target for androgen synthesis, pharmaceutical companies developed <em>5α</em>-reductase inhibitors (5ARIs), such as finasteride (FIN) and dutasteride (DUT) to target SRD<em>5α</em>2 in benign prostatic hyperplasia and androgenic alopecia. In addition to human treatment, the development of 5ARIs also enabled further research of SRD<em>5α</em> functions. Therefore, this review details the morphological, physiological, and molecular effects of the lack of SRD<em>5α</em> activity induced by both SRD<em>5α</em> mutations and inhibitor exposures across species. More specifically, data highlights 1) the role of <em>5α</em>-<em>DHT</em> in the development of male secondary sexual organs in vertebrates and sex determination in non-mammalian vertebrates, 2) the role of SRD<em>5α</em>1 in the synthesis of the neurosteroid allopregnanolone (ALLO) and <em>5α</em>-androstane-3α,17β-diol (3α-diol), which are involved in anxiety and sexual behavior, respectively, and 3) the role of SRD<em>5α</em>3 in N-glycosylation. This review also features the lesser known functions of SRD<em>5α</em>s in steroid degradation in the uterus during pregnancy and glucocorticoid clearance in the liver. Additionally, the review describes the regulation of SRD<em>5α</em>s by the receptors of androgens, progesterone, estrogen, and thyroid hormones, as well as their differential DNA methylation. Factors known to be involved in their differential methylation are age, inflammation, and mental stimulation. Overall, this review helps shed light on the various essential functions of SRD<em>5α</em>s across species.

BACKGROUND

Androgenic alopecia (AGA) is a major type of human scalp hair loss, which is caused by two androgens: testosterone (T) and <em>5α</em>-dihydrotestosterone (<em>5α</em>-<em>DHT</em>). Both androgens bind to the androgen receptor (AR) and induce androgen-sensitive genes within the human hair dermal papilla cells (HHDPCs), but <em>5α</em>-<em>DHT</em> exhibits much higher binding affinity and potency than T does in inducing the involved androgen-sensitive genes. Changes in the induction of androgen-sensitive genes during AGA are caused by the over-production of <em>5α</em>-<em>DHT</em> by the <em>5α</em>-reductase (<em>5α</em>-R) enzyme; therefore, one possible method to treat AGA is to inhibit this enzymatic reaction.

METHODS

RT-PCR was used to identify the presence of the <em>5α</em>-R and AR within HHDPCs. A newly developed AGA-relevant HHDPC-based assay combined with non-radioactive thin layer chromatography (TLC) detection was used for screening crude plant extracts for the identification of new <em>5α</em>-R inhibitors.

RESULTS

HHDPCs expressed both <em>5α</em>-R type 1 isoform of the enzyme (<em>5α</em>-R1) and AR in all of the passages used in this study. Among the thirty tested extracts, Avicennia marina (AM) displayed the highest inhibitory activity at the final concentration of 10 μg/ml, as the production of <em>5α</em>-<em>DHT</em> decreased by 52% (IC50 = 9.21 ± 0.38 μg/ml).

CONCLUSIONS

Avicennia marina (AM) was identified as a potential candidate for the treatment of AGA based on its <em>5α</em>-R1-inhibitory activity.

Sex steroid hormones are important players in the control of sex differentiation by regulating gonadal development in teleosts. Although estrogens are clearly associated with the ovarian differentiation in teleosts, the effects of androgens on early gonadal development are still a matter of debate. Traditionally, 11-ketotestosterone (11-KT) is considered the major androgen in fish; however, <em>5α</em>-dihydrotestosterone (<em>5α</em>-<em>DHT</em>), the most potent androgen in tetrapods, was recently found in fish testis and plasma, but its physiological role is still unknown. In this context, the expression of genes associated with steroidogenesis and spermatogenesis, body growth and sex differentiation were assessed in Odontesthes bonariensis larvae fed with food supplemented with two doses of <em>5α</em>-<em>DHT</em> (0.1 and 10μg/g of food) from hatching to 6weeks of age. At the lowest dose, <em>5α</em>-<em>DHT</em> treated larvae showed an estrogenic gene expression pattern, with low hsd11b2 and high cyp19a1a and er2 expression levels with no differences in sex ratio. At the highest dose, <em>5α</em>-<em>DHT</em> produced a male-shifted sex ratio and the larvae exhibited a gene expression profile characteristic of an advancement of spermatogenesis, with inhibition of amh and stimulation of ndrg3. No differences were observed in somatic growth. These results suggest that in this species, <em>5α</em>-<em>DHT</em> could have a role on sex differentiation and its effects can differ according to the dose.

Castration resistant prostate cancer (CRPC) remains androgen dependant despite castrate levels of circulating testosterone following androgen deprivation therapy, the first line of treatment for advanced metstatic prostate cancer. CRPC is characterized by alterations in the expression levels of steroidgenic enzymes that enable the tumour to derive potent androgens from circulating adrenal androgen precursors. Intratumoral androgen biosynthesis leads to the localized production of both canonical androgens such as <em>5α</em>-dihydrotestosterone (<em>DHT</em>) as well as less well characterized 11-oxygenated androgens, which until recently have been overlooked in the context of CRPC. In this review we discuss the contribution of both canonical and 11-oxygenated androgen precursors to the intratumoral androgen pool in CRPC. We present evidence that CRPC remains androgen dependent and discuss the alterations in steroidogenic enzyme expression and how these affect the various pathways to intratumoral androgen biosynthesis. Finally we summarize the current treatment strategies for targeting adrenal derived androgen biosynthesis.

Androgens are known to play a critical role in prostate cancer progression, but their effect on malignant phenotypes in salivary gland cancer is unclear. The androgen-androgen receptor (AR) axis may be involved in malignant phenotypes of salivary duct carcinoma (SDC) cells and therefore may be a new target for SDC treatment. To test this hypothesis, we investigated the effect of the androgen <em>5α</em>-dihydrotestosterone (<em>DHT</em>) on proliferation, migration, and invasiveness of SDC cells.

We used a wound-healing assay to measure cell migration and a Boyden chamber invasion assay to investigate SDC cell invasive capacity.

DHT treatment increased cell proliferation, migration, and invasion. However, treatment with flutamide, an AR inhibitor, blocked the effects of DHT.

These results suggest that the androgen-AR axis is involved in SDC malignancy and may be an effective therapeutic target for treatment of human SDC.

Anti-Müllerian hormone (AMH) and AMH type II receptor (AMHR2) are overexpressed in granulosa cells (GCs) from women with polycystic ovary syndrome (PCOS), the most common cause of female infertility.

The aim of the study was to compare the regulation of the AMH/AMHR2 system by <em>5α</em>-dihydrotestosterone (<em>5α</em>-<em>DHT</em>) and estradiol (E2) in GCs from control subjects and women with PCOS.

Experiments were performed on follicular fluids (FF) and GCs from women undergoing in vitro fertilization.

FF steroid levels were measured by mass spectrometry, and messenger RNA (mRNA) accumulation was quantified by reverse transcription real-time polymerase chain reaction.

Total testosterone (T), free T, and <em>5α</em>-<em>DHT</em> FF levels were significantly higher (P < 0.001) in women with PCOS than in controls. However, E2 and sex hormone-binding globulin concentrations were comparable between the two groups. In GCs from control women, the AMH and AMHR2 expression were not affected by <em>5α</em>-<em>DHT</em> treatment, whereas AMH mRNA levels were upregulated by <em>5α</em>-<em>DHT</em> in GCs from patients with PCOS (2.3-fold, P < 0.01) overexpressing the androgen receptor (1.4-fold, P < 0.05). E2 downregulated the AMH and AMHR2 expression in GCs from control women (1.4-fold, P < 0.001 and 1.8-fold, P < 0.01, respectively) but had no effect on these genes in GCs from women with PCOS. This differential effect of E2 was associated with a higher estrogen receptor 1 expression in GCs from women with PCOS (1.9-fold, P < 0.05).

In GCs from women with PCOS, the regulation of AMH and AMHR2 expression is altered in a way that promotes the overexpression of the AMH/AMHR2 system, and could contribute to the follicular arrest observed in these patients.

We sought to determine the role of ovarian vascularity and neo-angiogenesis in the development of mature follicles in polycystic ovary syndrome (PCOS) and to identify any changes induced by low-frequency electro-acupuncture (EA). Twenty-eight 21-day-old female Wistar rats were randomly divided into four groups-Control, Obesity, PCOS-like, and PCOS-like-EA (n = 7/group). Rats in the Obesity group were fed a high-fat diet throughout the experiment. Rats in the PCOS-like and PCOS-like-EA groups were implanted with a sustained-release tube containing <em>5α</em>-dihydrotestosterone (<em>DHT</em>) beneath the skin of the neck. Rats in the PCOS-like-EA group received low-frequency EA treatment starting at 70 days for 30 min five times a week for four weeks. At the end of the experiment, all rats were euthanized and perfused with hydrogel. The ovaries were collected for clarification and imaging, and ovarian vascularity and neo-angiogenesis were analyzed. Compared with Control and Obesity rats, the ovaries in <em>DHT</em>-induced PCOS-like rats were smaller in size and had fewer mature follicles and corpora lutea. EA increased angiogenesis in the antral follicles of PCOS-like rats, which in turn promoted follicle maturation, ovulation, and CL formation. Therefore, endogenous ovarian angiogenesis plays a very important role in follicular maturation and might be one of the peripheral and direct mechanisms of EA on PCOS.

<strong class="sub-title"> Introduction: </strong> Glioblastomas (GBM) are the most frequent and aggressive human brain tumors due to their high capacity to migrate, invade healthy brain tissue, and resist anticancer therapies. It has been reported that testosterone (T) levels are higher in patients with GBM than in healthy controls. It has also been dem{}onstrated that T induces proliferation, migration, and invasion of human GBM cell lines. T is mainly metabolized to <em>5α</em>-dihydrotestosterone (<em>DHT</em>) by the enzyme <em>5α</em>-reductase (<em>5α</em>R), but the role of this metabolite in GBM cells is unknown.

<strong class="sub-title"> Methods: </strong> The expression of <em>5α</em>R isoenzymes and <i>AR</i> in biopsies of GBMs was determined by the analysis of TCGA. U87 and U251 GBM cell lines were grown in supplemented DMEM. For evaluating the expression of <i>AR</i> in U251 and U87 cells, a RT-qPCR was performed. The cells were treated with T, <em>DHT</em>, finasteride (FIN), dutasteride (D), and the combined treatments, FIN+T and D+T or vehicle. After treatments, the viability was quantified by the trypan blue exclusion assay, the proliferation was evaluated by BrdU incorporation, and migration and invasion were analyzed by the scratch-wound and the transwell assays, respectively.

<strong class="sub-title"> Results: </strong> In a set of glioma biopsies from TCGA, we observed that <i>SRD5A2</i> (<em>5α</em>R2) expression was higher in GBM and in low-grade gliomas than in normal brain tissue. We observed that <em>DHT</em> and T increased proliferation, migration, and invasion of human GBM cell lines: U87 and U251. F and D, drugs that inhibit <em>5α</em>R activity, blocked the effects of T on GBM cells.

Discussion: These data suggest that T induces human GBM progression through its conversion into DHT. These results can be related to the chemical structure of DHT, which increases its affinity for AR and decreases five times the rate of dissociation compared to T. Also, it is possible that DHT mediates the effects of T on cell human GBM cells motility by changing the expression of genes involved in tumor infiltration.

<strong class="sub-title"> Keywords: </strong> <em>5α</em>-reductase; dihydrotestosterone; dutasteride; finasteride; glioblastoma.

In brainstem slices of young male rat, we investigated the influence of the neuroactive steroid testosterone (T) on the synaptic responses by analyzing the field potential evoked in the medial vestibular nucleus (MVN) by vestibular afferent stimulation. T induced three distinct and independent long-term synaptic changes: fast long-lasting potentiation (fLP), slow long-lasting potentiation (sLP) and long-lasting depression (LD). The fLP was mediated by 17β-estradiol (E(2)) since it was abolished by blocking the estrogen receptors (ERs) or the enzyme converting T to E(2). Conversely, sLP and LD were mediated by <em>5α</em>-dihydrotestosterone (<em>DHT</em>) since they were prevented by blocking the androgen receptors (ARs) or the enzyme converting T to <em>DHT</em>. Therefore, the synaptic effects of T were mediated by its androgenic or estrogenic metabolites. The pathways leading to estrogenic and androgenic conversion of T might be co-localized since, the occurrence of fLP under block of androgenic pathway, and that of sLP and LD under estrogenic block, were higher than those observed without blocks. In case of co-localization, the effect on synaptic transmission should depend on the prevailing enzymatic activity. We conclude that circulating and neuronal T can remarkably influence synaptic responses of the vestibular neurons in different and opposite ways, depending on its conversion to estrogenic or androgenic metabolites.

Sexual differences in adult body size [sexual size dimorphism (SSD)] and color (sexual dichromatism) are widespread, and both male- and female-biased dimorphisms are observed even among closely related species. A growing body of evidence indicates testosterone can regulate growth, thus the development of SSD, and sexual dichromatism. However, the mechanism(s) underlying these effects are conjectural, including possible conversions of testosterone to estradiol (E2) or <em>5α</em>-dihydrotestosterone (<em>DHT</em>). In the present study, we hypothesized that the effects of testosterone are physiological responses mediated by androgen receptors, and we tested two specific predictions: (1) that <em>DHT</em> would mimic the effects of testosterone by inhibiting growth and enhancing coloration, and (2) that removal of endogenous testosterone via surgical castration would stimulate growth. We also hypothesized that females share downstream regulatory networks with males and predicted that females and males would respond similarly to <em>DHT</em>. We conducted experiments on eastern fence lizards (Sceloporus undulatus), a female-larger species with striking sexual dichromatism. We implanted Silastic® tubules containing 150 µg <em>DHT</em> into intact females and intact and castrated males. We measured linear growth rates and quantified color for ventral and dorsal surfaces. We found that <em>DHT</em> decreased growth rate and enhanced male-typical coloration in both males and females. We also found that, given adequate time, castration alone is sufficient to stimulate growth rate in males. The results presented here suggest that: (1) the effects of testosterone on growth and coloration are mediated by androgen receptors without requiring aromatization of testosterone into E2, and (2) females possess the androgen-receptor-mediated regulatory networks required for initiating male-typical inhibition of growth and enhanced coloration in response to androgens.

Quisqualis indica (QI) has been used for treating disorders such as stomach pain, constipation, and digestion problem. This study was aimed to evaluate the therapeutic efficacy of QI extract on treating benign prostatic hyperplasia (BPH) in LNCaP human prostate cancer cell line and a testosterone-induced BPH rat model. LNCaP cells were treated with QI plus testosterone propionate (TP), and androgen receptor (AR) and prostate specific antigen (PSA) expression levels were assessed by Western blotting. To induce BPH, the rats were subjected to a daily subcutaneous injection of TP (3 mg/kg) for 4 weeks. The rats in treatment group were orally gavaged with QI (150 mg/kg) together with the TP injection. In-vitro studies showed that TP-induced increases in AR and PSA expression in LNCaP cells were reduced by QI treatment. In BPH-model rats, the prostate weight, testosterone in serum, dihydrotestosterone (<em>DHT</em>) concentration and <em>5α</em>-reductase type 2 mRNA expression in prostate tissue were significantly reduced following the treatment with QI. TP-induced prostatic hyperplasia and the expression of proliferating cell nuclear antigen (PCNA) and cyclin D1 were significantly attenuated in QI-treated rats. In addition, QI induced apoptosis by up-regulating caspase-3 and -9 activity and decreasing the B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein (Bax) ratio in prostate tissues of BPH rats. Further investigation showed that TP-induced activation of AKT and glycogen synthase kinase 3β (GSK3β) was reduced by QI administration. Therefore, our findings suggest that QI attenuates the BPH state in rats through anti-proliferative and pro-apoptotic activities and might be useful in the clinical treatment of BPH.

Steroid <em>5α</em>-reductase (S<em>5α</em>R) plays an important role in metabolizing testosterone into active androgen dihydrotestosterone (<em>DHT</em>) which is involved in many androgen dependent disorders, such as androgenic alopecia, benign prostatic hyperplasia and acne. The method for screening for S<em>5α</em>R inhibition is key in finding new antagonists. In this study, the label-free S<em>5α</em>R inhibitory assay using LC-MS was developed. S<em>5α</em>R type 1 enzyme was obtained from LNCaP prostate cancer cells. The enzymatic assay was optimised for enzyme-substrate (testosterone) concentration, NADPH-cofactor concentration, solvent tolerance, enzyme activity stability and incubation time. The developed assay was validated by measuring the signal to background ratio (S/B), the signal to noise ratio (S/N), the signal window (SW) and the zeta factor Z' in accordance with published bioassay guidelines. The enzymatic reaction was performed in 96-well plates and <em>DHT</em> formation was determined by LC-MS. S/B, S/N, SW and Z' factor were well above acceptable criteria and the reproducibility was good using Z' factor other 3days and further validated by dutasteride and finasteride inhibition. The method was successfully applied to quantify S<em>5α</em>R inhibitory activity of some Thai herbal extracts. Two plant extracts, Impatiens balsamina L. and Curcuma longa L. showed IC50 at 5.4±0.2 and 9.0±1.2μgmL-1 and are therefore promising sources of new S<em>5α</em>R inhibitors. The assay has high selectability and reproducibility and suited to medium throughput screening required by phytochemistry.

Surfactant protein A (SP-A) plays an important role in innate immunity. The sex-dependent survival of infected SP-A knockout (KO) mice has been observed. Our goal was to study the impact of ozone (O₃) and sex, as well as gonadal hormones, on the bronchoalveolar lavage (BAL) readouts and survival, respectively, of <i>Klebsiella pneumoniae-</i>infected SP-A KO mice. Male and female SP-A KO mice were exposed to O₃ or filtered air and infected with <i>K. pneumoniae</i>. We studied markers of inflammation and tissue damage at 4, 24, and 48 h, as well as the survival over 14 days, of gonadectomized (Gx) mice implanted with control pellets (CoP) or hormone (<em>5α</em>-dihydrotestosterone (<em>DHT</em>) in female gonadectomized mice (GxF) or 17β-estradiol (E₂) in male gonadectomized mice (GxM)). We observed: (1) an increase in neutrophil and macrophage inflammatory protein-2 levels as time progressed post-infection, and O₃ exposure appeared to increase this response; (2) an increase in lactate dehydrogenase, total protein, oxidized protein, and phospholipids in response to O₃ with no consistent sex differences in studied parameters; and (3) a reduction in survival of the GxM and CoP mice, the GxM and E₂ mice, and the GxF and <em>DHT</em> mice but not for the GxF and CoP mice after O₃. Without SP-A, (a) sex was found to have a minimal impact on BAL cellular composition and tissue damage markers, and (b) the impact of gonadal hormones on survival was found to involve different mechanisms than in the presence of SP-A.

Keywords: innate immunity; oxidative stress; pneumonia; sex differences; surfactant protein-A.

We have devised an efficient procedure for the synthesis of <em>5α</em>-dihydrotestosterone (<em>DHT</em>) (1) starting from 3β-hydroxy-<em>5α</em>-androstan-17-one, providing the product in unprecedented 82% yield. A reported method of using toxic Jones reagent is replaced by milder oxidizing agent (NMO/TPAP) in the synthesis of a key intermediate 17β-[(tert-butyldimethylsilyl)oxy]-<em>5α</em>-androstan-3-one (18). This new procedure is simple, does not require special apparatus/precautions or chromatographic purification in most of the steps.