Dihydrotestosterone Research Articles

Exercise-induced irisin improves follicular dysfunction by inhibiting IRE1α-TXNIP/ROS-NLRP3 pathway in PCOS

BackgroundExcessive production of androgen drives oxidative stress (OS) and inflammasome activation in ovarian granulosa cells (GCs). Therefore, the induced follicular developmental disorder is the major cause of infertility in women with polycystic ovary syndrome (PCOS). Exercise-induced upregulation of irisin is capable of regulating metabolism by reducing OS and inflammation. Exercise has been shown to alleviate a range of PCOS symptoms, including maintaining a normal menstrual cycle, in several clinical trials.MethodsFemale Sprague–Dawley (SD) rats and primary ovarian cells were treated with two different androgens, dehydroepiandrosterone (DHEA) and dihydrotestosterone (DHT), to simulate a hyperandrogenic environment, followed by eight weeks of exercise training and irisin intervention. The levels of reactive oxygen species (ROS), tissue inflammation and fibrosis were examined using hematoxylin and eosin (H&E) staining, western blot, quantitative real-time PCR (qRT-PCR), dichlorofluorescein diacetate (DCF-DA) probe detection, immunofluorescence staining, immunohistochemistry, and Sirius red staining.ResultsExercise for eight weeks improved polycystic ovarian morphology and decreased the levels of inflammation, OS, and fibrosis in PCOS rats. Hyperandrogen increased ROS production in ovarian cells by inducing endoplasmic reticulum stress (ERS) and activating the inositol-requiring enzyme 1α (IRE1α)-thioredoxin-interacting protein (TXNIP)/ROS-NOD-like receptor family pyrin domain containing 3 (NLRP3) signaling pathway, further enhancing the levels of inflammation. Irisin suppressed the expression of IRE1α and its downstream targets, thus improving the ovarian dysfunction of PCOS rats induced by hyperandrogen.ConclusionExercise can alleviate various phenotypes of PCOS rats induced by DHEA, and its therapeutic effect may be mediated by secreting beneficial myokines. IRE1α may be an important target of irisin for reducing OS and inflammation, thereby improving ovarian fibrosis.

Neuronal Ptpn1 and Socs3 deletion improves metabolism but not anovulation in a mouse PCOS model.

Polycystic ovary syndrome (PCOS) is one of the most common causes of infertility in women. Approximately half of diagnosed individuals also experience metabolic syndrome. Central and peripheral resistance to the hormones insulin and leptin have been reported to contribute to both metabolic and reproductive dysregulation. In PCOS and preclinical PCOS animal models, circulating insulin and leptin levels are often increased in parallel with the development of hormone resistance, however it remains uncertain whether these changes contribute to the PCOS state. In this study we tested whether central actions of protein tyrosine phosphatase 1B (PTP1B) and suppressor of cytokine signaling 3 (SOCS3), negative regulators of insulin and leptin signaling pathways respectively, play a role in the development of PCOS-like phenotype. A peripubertal dihydrotestosterone (DHT) excess PCOS-like mouse model was used, which exhibits both metabolic and reproductive dysfunction. Mice with knockout of the genes encoding PTP1B and SOCS3 from forebrain neurons were generated, and metabolic and reproductive function were compared between knockout and control groups. DHT treatment induced mild insulin resistance but not leptin resistance, so the role of SOCS3 could not be tested. As expected, DHT excess abolished estrous cycles and corpora lutea presence and caused increased visceral adiposity and fasting glucose levels. Knockout mice did not show any rescue of reproductive dysfunction, but did have reduced adiposity compared to control DHT mice. These data suggest that negative regulation of central insulin signaling by PTP1B is not responsible for peripubertal DHT excess-induced reproductive impairments, but may mediate its increased adiposity effects.

Effects of photobiomodulation at various irradiances on normal and dihydrotestosterone-treated human hair dermal papilla cells in vitro.

Androgenetic alopecia (AGA) is the most common type of hair loss caused by dihydrotestosterone (DHT) binding to androgen receptors in dermal papilla cells (DPCs). Photobiomodulation (PBM) is a promising treatment for AGA but suffers from inconsistent outcomes and inconsistent effective light parameters. This study investigated the impact of red light at various irradiances on normal and DHT-treated DPCs. Our results suggested that red light at 8 mW/cm2 was most effective in promoting DPCs growth. Furthermore, a range of irradiances from 2 to 64 mW/cm2 modulated key signaling pathways, including Wnt, FGF, and TGF, in normal and DHT-treated DPCs. Interestingly, 8 mW/cm2 had a greater impact on these pathways in DHT-treated DPCs and altered the Shh pathway, suggesting that the effect of PBM varies with the cellular environment. This study highlights specific factors that influence PBM effectiveness and provides insight into the need for personalized PBM treatment approaches.

Antioxidant mitoquinone suppresses benign prostatic hyperplasia by regulating the AR–NLRP3 pathway

Mitoquinone (MitoQ), a mitochondria-targeted antioxidant, has been used to treat several diseases. The present study aimed to investigate the therapeutic effects of MitoQ in benign prostatic hyperplasia (BPH) models and their underlying molecular mechanisms. In this study, we determined that MitoQ inhibited dihydrotestosterone (DHT)-induced cell proliferation and mitochondrial ROS by inhibiting androgen receptor (AR) and NOD-like receptor family pyrin domain-containing 3 (NLRP3) signaling in prostate epithelial cells. Molecular modeling revealed that DHT may combine with AR and NLRP3, and that MitoQ inhibits both AR and NLRP3. AR and NLRP3 downregulation using siRNA showed the linkage among AR, NLRP3, and MitoQ. MitoQ administration alleviated pathological prostate enlargement and exerted anti-proliferative and antioxidant effects by suppressing the AR and NLRP3 signaling pathways in rats with BPH. Hence, our findings demonstrated that MitoQ is an inhibitor of NLPR3 and AR and a therapeutic agent for BPH treatment.

The role of B cells in immune cell activation in polycystic ovary syndrome.

Variations in B cell numbers are associated with polycystic ovary syndrome (PCOS) through unknown mechanisms. Here we demonstrate that B cells are not central mediators of PCOS pathology and that their frequencies are altered as a direct effect of androgen receptor activation. Hyperandrogenic women with PCOS have increased frequencies of age-associated double-negative B memory cells and increased levels of circulating immunoglobulin M (IgM). However, the transfer of serum IgG from women into wild-type female mice induces only an increase in body weight. Furthermore, RAG1 knock-out mice, which lack mature T- and B cells, fail to develop any PCOS-like phenotype. In wild-type mice, co-treatment with flutamide, an androgen receptor antagonist, prevents not only the development of a PCOS-like phenotype but also alterations of B cell frequencies induced by dihydrotestosterone (DHT). Finally, B cell-deficient mice, when exposed to DHT, are not protected from developing a PCOS-like phenotype. These results urge further studies on B cell functions and their effects on autoimmune comorbidities highly prevalent among women with PCOS.

Screening the Chemical Composition and Bioactivity of Cerasus serrulata Flower Extracts Using Two Extraction Methods

Background Cerasus serrulata has an excellent antioxidative, antibacterial, and anti-alopecia effect, but its active ingredients and the underlying mechanism are currently unclear, in addition to the fact that the active products vary with the extraction methods. Aim Therefore, this study aims to clarify the active components and antioxidative property of C. serrulata extracted by two different methods (low-temperature vacuum extraction and ethanol extraction) and explore its potential biofunction. Materials and Methods First, the compositions in extracts were characterized by gas chromatographic mass spectrometry (GC-MS) and liquid chromatography-mass spectrography (LC-MS). Then, the antioxidative activity of both extracts was evaluated using free radical scavenging assay (ABTS) and ORAC. Next, the antibacterial activity of both extracts was measured via a bacteriostatic circle experiment. The anti-alopecia effects of both extracts were studied using a mouse model of alopecia. The hematoxylin and eosin (H&E) staining was used to evaluate hair follicles; vascular endothelial-derived growth factor (VEGF) and interleukin-6 (IL-6) expression in mouse skin were evaluated by immunohistochemistry, while β-catenin expression in tissues was used by immunofluorescence. The expression of FGFR and insulin-like growth factor 1 (IGF-1) in the skin was determined to evaluate using real-time quantitative polymerase chain reaction (RT-qPCR). The levels of DHT and 5α-reductase in the experimental mice were determined by ELISA. Results The GC-MS analysis results showed that the extracts were rich in alkanes and aromatic compounds, specifically benzaldehyde, which was the highest component in the cold vacuum extraction, while pipecolic acid was the main component in the ethanol extract. This indicates that C. serrulata has components that exhibit strong inhibitory ability against the growth of five common bacterial strains, expressing excellent free radical scavenging ability. In addition, the active ingredients in C. serrulata were found to significantly deregulate the protein levels in the skin of androgenetic alopecia (AGA) mice, with the levels of IL-6. They were also able to significantly inhibit VEGF reduction. The β-catenin was being significantly upregulated in skin tissue. In addition, FGFR and IGF-1 gene expression also increased. The 5α-reductase and dihydrotestosterone (DHT) can be reduced in the C. serrulata treatment. Conclusion Our data indicate that C. serrulata extract could be beneficial supplements of natural antioxidative, antibacterial, and anti-alopecia agents.

초록갈파래의 모발 성장기 활성화 효능

This study was conducted to evaluate the effects of Umbraulva japonica extract on the hair growth. Treatment with U. Japonica extract significantly increased the proliferation of dermal papilla cells (DPCs), a central regulator of the hair cycle. When isolated rat vibrissa follicles were treated with U. Japonica extract for 21 d, the hair-fiber lengths of the vibrissa follicles increased. When examined the activity of 5α-reductase, which converts testosterone to dihydrotestosterone (DHT), a main cause of androgenetic alopecia, U. Japonica extract did not inhibit the 5α-reductase activity. Moreover, we found that the U. Japonica extract also significantly accelerated anagen phase in vivo. These results suggest that the U. Japonica extract has the potential to treat alopecia via the stimulation of anagen phase.

Deciphering the Role of Phosphoglycerate Kinase 1 in Polycystic Ovarian Syndrome using Differential Gene Expression Analysis Approach

Polycystic Ovarian Syndrome (PCOS) is perhaps the most common Metabolic, Endocrine disorder characterized in females before menopause. PCOS majorly elevates Androgen (AR) levels and irregularities in menstrual cycles or can be morphologically seen as multiple cysts in the ovary. Patients with PCOS are more likely to develop obesity, cardiovascular disease, as well as insulin resistance. The etiology of this disease is still not known, but research suggests it can be due to environmental factors, lifestyle, or diet. According to a recent study, the glycolytic enzyme Phosphoglycerate Kinase 1 (PGK1) has an effective role in PCOS patients as it binds with AR, which is high gets expressed in PCOS patients. Thus, it can be a main causable factor for PCOS patients. The granulosa cells of the ovary provide nutrients to oocytes for development. The energy to the oocytes comes via cycle glycolysis. In PCOS patients, these granulosa cells show degradation. As a result, there may be a malfunction in the energy supply via granulosa cells, with Phosphoglycerate Kinase 1(PGK1) being one of the key glycolysis enzymes. And as the high levels of AR remain in proximity with disordered follicle development in PCOS. The actual significance of AR in the fertility of PCOS patients is still not known. Thus, we can have a close look at Androgen binding Receptors (ARs) and the role of PGK1. In this research, we analyzed single-cell RNA sequence data from Gene Expression Omnibus (GEO), including data from control Dihydrotestosterone (DHT) and PGK1-DHT of PCOS patients generated by deep sequencing in triplicate. Further, we explain the transcriptomic dynamics by co-expression network analysis and evaluate the differences between PCA and limmavoom analysis to discover which genes are differentially expressed. By calculating the p-value and performing gene ontology (GO) enrichment analysis, we were able to identify multiple genes with greater expression levels in the PGK1-DHT samples of PCOS patients, including insulin-related, glycolytic, tumor-associated, and apoptotic genes. We also carried out Functional enrichment and gene co-expression network analysis. Its biological, molecular, and cellular domain lies in the intracellular membrane-bound organelle. Gene co-expression network and functional enrichment found significant enrichment and highly differentially expressed genes in the intracellular cellular domain.

Dihydrotestosterone mediates the inflammation effect under lipopolysaccharides in bovine endometrial epithelial cells via AR blockading TLR4/MyD88 signaling pathway

Dihydrotestosterone (DHT) is a potent nonaromatizable 5α-reduced androgen with both positive and negative effect on inflammation process. However, it remains unknown whether DHT can regulate Lipopolysaccharides (LPS)-induced inflammation in bovine endometrial epithelial cells (bEECs). Here, we demonstrated that the DHT biosynthesis ability and androgen receptors (AR) expression is defective in bovine endometrial with endometritis, which aggravates endometrial inflammation. In vitro study, we established a LPS-induced inflammation model in bEECs, and found that DHT inhibited the TLR4 and MyD88 protein as well as TNF-α, IL-1β, and IL-6 mRNA of bEECs in a dose-dependent manner. Moreover, the anti-inflammation effect of DHT was blocked by AR inhibitor flutamide. Together, we demonstrated that supplementing DHT can alleviate the inflammation response of bEECs caused by LPS, which is associated with AR regulating the inhibition of TLR4/MyD88 signaling pathway.

Evaluation of the therapeutic effects of AGA drugs by measuring finasteride, dutasteride, and dihydrotestosterone in hair

BackgroundAndrogenetic alopecia (AGA) is treated by 5α-reductase inhibitors (5ARI) such as finasteride and dutasteride, which are widely used as therapeutic agents. However, their pharmacokinetics in target organs (scalp and hair follicles) have not yet been investigated. PurposeTo confirm the effective action of finasteride and dutasteride in the hair follicle tissues, we developed a method to measure these concentrations in hair. ResultsCompared to the non-detection (N.D.) group, the dihydrotestosterone (DHT) concentrations decreased significantly in both the finasteride and dutasteride groups. The dutasteride group showed significantly lower DHT concentrations among all groups. ConclusionsMeasurement of finasteride, dutasteride, and DHT concentrations in hair would aid in evaluating the drug pharmacokinetics and its therapeutic effects on AGA patients.

Seasonal expressions of COX-1, COX-2 and EP4 in the scent glands of muskrats (Ondatra zibethicus).

Prostaglandins (PGs) serve as signaling molecules that regulate various physiological processes, including inflammation, immune response, blood clotting and reproduction. The aim of this study was to investigate the immunolocalizations and expression patterns of prostaglandin-E2 (PGE2), cyclooxygenase (COX)-1 and COX-2, as well as its receptor subtypes 4 (EP4) in the scent glands of muskrats (Ondatra zibethicus) during the breeding and non-breeding periods. There were significant seasonal differences in the scent glandular mass, with higher values in the breeding season and relatively low in the non-breeding season. PGE2, EP4, COX-1 and COX-2 have been immunolocalized in the scent glandular and epithelial cells in both breeding and non-breeding seasons, whereas no immunostaining was observed in the interstitial cells. The protein and mRNA expression levels of EP4, COX-1 and COX-2 were higher in the scent glands of the breeding season than those of the non-breeding season. The mean mRNA levels of EP4, COX-1 and COX-2 were positively correlated with the scent glandular weights. The circulating follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T) and PGE2, as well as scent glandular PGE2 and dihydrotestosterone (DHT) concentrations were also significantly higher in the breeding season. Additionally, the transcriptomic study in the scent glands identified that differentially expressed genes might be related to fatty carboxylic monocarboxylic acid, steroidogenic-related pathway and prostanoid metabolic process. These findings suggested that prostaglandin-E2 might play an essential autocrine or paracrine role in regulating seasonal changes in the scent glandular functions of the muskrats.

O-222 Functional characterization and clinical implications of patient-specific iPSC-derived endothelial cells to unravel the pathogenesis associated with androgen-mediated endothelial dysfunction in polycystic ovarian syndrome

Abstract Study question Whether and how the endothelial cell (EC) dysfunction is involved in the pathogenesis of PCOS revealed by iPSC-based disease modeling? Summary answer PCOS-specific iPSC-derived ECs were successfully established and the results showed that EC proliferation and function were impaired in PCOS through the androgen receptor(AR)-mediated signaling pathway. What is known already ECs have been shown to play critical biological roles in the regulation of vascular tone and inflammatory process and any injury to ECs or disturbance in their homeostasis is called EC dysfunction, which has been recognized as an early marker of atherosclerosis and cardiovascular diseases. Several studies have also revealed indirect evidence of EC dysfunction among women with PCOS, including elevated circulatory markers of endothelial damage and impaired vascular structure. But whether and how EC dysfunction is involved in the pathogenesis of PCOS is still unclear. Study design, size, duration This is an experimental study at tertiary university hospital. iPSCs were established from skin fibroblasts and peripheral blood mononuclear cells from two patients with PCOS and two control participants. Participants/materials, setting, methods iPSCs were differentiated into ECs through monoculture with chemically defined conditions. Single cell RNA sequencing (scRNA-seq) was performed on the iPSCs-derived ECs to compare the differences between PCOS and control. iPSCs-derived ECs were treated with different dosages (1, 10 and 100 nM) of dihydrotestosterone (DHT). Cell cycle phases were analyzed by flow cytometry with BrdU incorporation assay. The expression of AR, cyclin-dependent kinase 1 (CDK1) and vascular endothelial growth factor (VEGF) were analyzed using rtPCR. Main results and the role of chance Up to 90% of iPSC-derived ECs successfully expressed EC markers, including CD31, CD144 and von Willebrand Factor in both PCOS and control groups, showing a high differentiation efficiency in the present study. The pathway enrichment analysis of transcriptomic data of iPSC-derived ECs showed functional differences involving cell cycle process, VEGF signaling and apoptotic process between PCOS and control. Analysis using sc-RNA seq revealed decreased cellular proliferation in the PCOS iPSC-derived ECs compared to control group, which was due to cell cycle arrest revealed by flow cytometry. The modulating effects of androgen on the proliferation and gene expression of iPSC-derived ECs were further investigated. DHT at doses ranging from 1 to 100 nM stimulated iPSC-derived ECs proliferation in the control group through upregulation of AR, CDK1 and VEGF gene expression. In contrast, DHT did not promote the cellular proliferation of PCOS iPSC-derived ECs at physiological concentrations of 1 and 10 nM and the expression of AR, CDK1, and VEGF remained unchanged. These data suggest that DHT-induced cellular proliferation and the expression of associated genes were more sensitive and dose-dependent in the control iPSC-derived ECs and blunted in the PCOS iPSC-derived ECs. Limitations, reasons for caution One of the major limitations of our study was the limited number of cases during the establishment of iPSCs, which was unable to reflect the heterogeneity and complexity of PCOS. Wider implications of the findings Impaired cellular proliferation and differentially expressed genes involved in cell proliferation, immune responses, cardiovascular functions, and apoptosis were observed among PCOS iPSC-derived ECs. Additionally, androgen-induced ECs proliferation was blunted in PCOS through a diminished AR/VEGF/cyclin D1 signaling pathway, which might hinder VEGF-dependent vascular repair and probably increase cardiovascular risks. Trial registration number not applicable

P-593 developmental programming by maternal androgen excess is mediated by androgen receptor pathways

Abstract Study question How does maternal hyperandrogenism featured PCOS-pregnancy affects the placental and embryo development and further contributes to the development of PCOS-like phenotypes in adult offsping? Summary answer Maternal hyperandrogenism compromises PCOS-pregnancy and embryo development due to placenta dysfunction, leading to the subsequent development of anxiety-like behavior and/or impaired metabolism in adult offspring What is known already Women with PCOS suffer an increased risk of having miscarriage, preterm delivery, and perinatal mortality. The hostile in utero environment because of maternal hyperandrogenism likely play a detrimental role in embryo development as well as the disease susceptibility later in life. As according to previous findings, daughters of women with PCOS have 5 fold increased risk of getting PCOS diagnosis, while sons of women with PCOS suffer from metabolic diseases. Study design, size, duration We used a PCOS-like mouse model induced by continuous exposure to dihydrotestosterone (DHT) from prepuberty, which developed obesity, anovulation, and abnormal ovarian morphology to study the effects of maternal hyperandrogenism during pregnancy. In comparison, we also simultaneously treated PCOS-like females with flutamide, an androgen-receptor blocker. We examined embryos at E10.5 and E13.5 focusing on placentas in relation to embryo development. Their male and female offspring is also phenotyped until 6 months of age. Participants/materials, setting, methods To explore molecular mechanisms contributing to the developmental defects, whole genome bisulfite and bulk RNA sequencing of placenta were performed. Human Trophoblast organoid culture under different conditions was applied to further evaluate the detrimental effects of maternal hyperandrogetism in placental development. With the help of trophoblast organoid culture system, possible treatment options are also going to be explored. Main results and the role of chance We found a lower pregnancy rate and impaired placentas together with defective embryonic development, which was partially prevented when co-treated with the androgen receptor blocker, flutamide. RNA sequencing of the placenta revealed that DHT severely interferes with placental and fetal development, which are prevented by the treatment with flutamide. In addition, the placenta from DHT-exposed dams showed impaired differentiation capacity of cell types located in the labyrinth. The hyperandrogenic maternal environment led to the development of anxiety-like behavior and impaired metabolism in adult male offspring and partial disturbance in the metabolism of female offspring later in life. The effect of hyperandrogenism on trophoblast differentiation and metabolic capacity is still under investigation. Limitations, reasons for caution Although androgen receptor pathway was found to be responsible for placental and fetal abnormalities and the development of adult phenotypes, and the treatment with flutamide prevented most of the unfavorable effects, the use of flutamide in clinic is tightly regulated and not suggested for women with PCOS. Wider implications of the findings Maternal hyperandrogenism greatly compromises PCOS-pregnancy and embryo development due to placenta dysfunction, leading to the subsequent development of unfavorable phenotypes in adult offspring. Such effects are mainly mediated by the androgen receptor pathway as the administration of flutamide partially prevents the compromised placenta and fetal development. Trial registration number not applicable

P-476 Metformin ameliorated testosterone-induced ferroptosis via inhibiting ferritinophagy in trophoblasts of polycystic ovary syndrome (PCOS) placentas

Abstract Study question Does trophoblasts of PCOS patients with aberrant iron metabolism, and how does metformin ameliorate androgen-induced ferroptosis in trophoblasts in vivo and in vitro? Summary answer Metformin ameliorates placental development and attenuates spontaneous miscarriage rate in early gestation by inhibiting ferritinophagy in trophoblasts of PCOS patients with hyperandrogenism. What is known already PCOS is a common reproductive endocrine disease in women of reproductive age, and a large proportion of patients present with hyperandrogenism. Studies have found that the incidences of spontaneous miscarriage and adverse pregnancy outcomes in PCOS patients are higher than those in healthy controls, which placental dysfunction is an important factor. It is related to mitochondrial dysfunction, oxidative stress and metabolic disorders. Ferroptosis is an iron-dependent and phospholipid peroxidation-mediated cell death. Studies have shown that iron ions participate in a variety of biochemical reactions in humans, and its abnormal metabolism may lead to some diseases especially in the reproductive system. Study design, size, duration Villi at gestational weeks of 6-8 were collected from 18 healthy controls and 18 women with PCOS. Human trophoblast stem cells (hTSCs) were isolated from fresh villi (6-8 weeks, n = 3) and cultured with testosterone, ferroptosis or autophagy inhibitors, metformin, or NCOA4 small interfering RNA. A dihydrotestosterone (DHT)-induced PCOS C57BL/6 mouse model was treated with or without metformin (3 months before mating) (n = 20 per group). Mouse placentas were obtained at embryonic days 11.5 (n = 50 per group). Participants/materials, setting, methods The levels of iron ions and lipid peroxidation were measured by PGSK staining, BODIPYC11 staining and MDA assay kit. The expressions of ferroptosis-related genes and proteins were assessed by qPCR, western blot and immunofluorescence. The binding of androgen receptor (AR) and NCOA4 to the putative androgen response element (ARE) was examined by ChIP, EMSA and luciferase assay. The influence of metformin on ferritinophagy in placentas of PCOS mice was confirmed by western blot and immunofluorescence. Main results and the role of chance The levels of iron ions and lipid peroxidation in villi at gestational weeks of 6-8 were higher in PCOS patients than in healthy controls. In vitro experiments, testosterone-induced ferroptosis was ameliorated by ferroptosis inhibitors, and ferritinophagy was mitigated by autophagy inhibitors or NCOA4 knockdown in primary cultured hTSCs. Testosterone induced ARs into the nucleus, where the recruitment of AR homodimers along with NCOA4 to the positive ARE on the SQSTM1 promoter, leading to autophagy induction and degradation of ferritin heavy chain 1 (FTH1) in autophagosomes, resulting in the release of ferritin bound irons as free ferrous irons. Metformin ameliorated testosterone-induced ferroptosis in hTSCs to a certain extent by inhibiting ferritinophagy. Compared to those of control mice, the placentas of DHT-treated PCOS mice showed increased levels of iron ions and lipid peroxidation, and altered expressions of ferroptosis-related proteins. Hyperandrogenism also induced ferritinophagy in labyrinthine zone of placentas in PCOS mice. Metformin ameliorated placental development was associated with decreased iron ions accumulation by inhibiting ferritinophagy in PCOS mouse placentas, which observably attenuated embryo absorption rate and spontaneous miscarriage incidence in the early gestation of PCOS mice. Limitations, reasons for caution The numbers of PCOS patients and mouse placentas in this study were small. The placental labyrinthine zone was not separated from the endocrine junctional zone and maternal decidua for molecular analyses. In addition, it would be better to isolated and cultured mouse TSCs to explore mechanisms on ferritinophagy in further. Wider implications of the findings This study provides further evidence for metformin as a PCOS treatment, which ameliorates placental development and attenuates spontaneous miscarriage rate in early gestation. Trial registration number not applicable

The alternate pathway of androgen metabolism and window of sensitivity.

Since the discovery in 1968 that dihydrotestosterone (DHT) is a major mediator of androgen action, a convincing body of evidence has accumulated to indicate that the major pathway of DHT formation is the 5α-reduction of circulating testosterone in androgen target tissues. However, we now know that DHT can also be formed in peripheral tissues by the oxidation of 5α-androstane-3α,17β-diol (adiol). This pathway is responsible for the formation of the male phenotype. We discuss the serendipitous discovery in the tammar wallaby of an alternate pathway by which adiol is formed in the testes, secreted into plasma and converted in peripheral tissues to DHT. This alternate pathway is responsible for virilisation of the urogenital system in this species and is present in the testes at the onset of male puberty of all mammals studied so far. This is the first clear-cut function for steroid 5α-reductase 1 in males. Unexpectedly, the discovery of this pathway in this Australian marsupial has had a major impact in understanding the pathophysiology of aberrant virilisation in female newborns. Overactivity of the alternate pathway appears to explain virilisation in congenital adrenal hyperplasia CAH, in X-linked 46,XY disorders of sex development. It also appears to be important in polycystic ovarian syndrome (PCOS) since PCOS ovaries have enhanced the expression of genes and proteins of the alternate pathway. It is now clear that normal male development in marsupials, rodents and humans requires the action of both the classic and the alternate (backdoor) pathways.

Combination of Lycopene and Curcumin Synergistically Alleviates Testosterone-Propionate-Induced Benign Prostatic Hyperplasia in Sprague Dawley Rats via Modulating Inflammation and Proliferation.

Benign prostatic hyperplasia (BPH) is a progressive urological disease occurring in middle-aged and elderly men, which can be characterized by the non-malignant overgrowth of stromal and epithelial cells in the transition zone of the prostate. Previous studies have demonstrated that lycopene can inhibit proliferation, while curcumin can strongly inhibit inflammation. This study aims to determine the inhibitory effect of the combination of lycopene and curcumin on BPH. To induce BPH models in vitro and in vivo, the BPH-1 cell line and Sprague Dawley (SD) rats were used, respectively. Rats were divided into six groups and treated daily with a vehicle, lycopene (12.5 mg/kg), curcumin (2.4 mg/kg), a combination of lycopene and curcumin (12.5 mg/kg + 2.4 mg/kg) or finasteride (5 mg/kg). Histologic sections were examined via hematoxylin and eosin (H&E) staining and immunohistochemistry. Hormone and inflammatory indicators were detected via ELISA. Network pharmacology analysis was used to fully predict the therapeutic mechanism of the combination of lycopene and curcumin on BPH. Combination treatment significantly attenuated prostate hyperplasia, alleviated BPH pathological features and decreased the expression of Ki-67 in rats. The upregulation of the expression of testosterone, dihydrotestosterone (DHT), 5α-reductase, estradiol (E2) and prostate-specific antigen (PSA) in BPH rats was significantly blocked by the combination treatment. The expression levels of inflammatory factors including interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α were strongly inhibited by the combination treatment. From the network pharmacology analysis, it was found that the main targets for inhibiting BPH are AKT1, TNF, EGFR, STAT3 and PTGS2, which are enriched in pathways in cancer. The lycopene and curcumin combination is a potential and more effective agent to prevent or treat BPH.

Nesfatin-1 in a reptile: its role and hormonal regulation in wall lizard testis

Nesfatin-1 is a pleiotropic hormone implicated in various physiological functions including reproduction. Studies though limited, have established an important role of the peptide in regulation of testicular functions in mammals and fishes. However, role of nesfatin-1 in regulation of spermatogenesis and testicular steroidogenesis remains completely unexplored in reptiles. Therefore, present study aimed to develop an insight into reproductive phase-dependent testicular expression, function and regulation of nucb2/nesfatin-1 in a reptile, Hemidactylus flaviviridis. Expression of nucb2/nesfatin-1 in testis of wall lizard varied significantly depending upon reproductive phase, being highest in the active phase while lowest during regressed phase. Further, in vitro treatment of wall lizard testis with nesfatin-1 showed a concentration- and time-dependent stimulatory effect of the peptide on expression of cell proliferation and differentiation markers like scf, c-kit and pcna suggesting a spermatogenic role of nesfatin-1 in wall lizard. Also, nesfatin-1 stimulated the anti-apoptotic marker, bcl-2 while inhibited the apoptotic marker, caspase-3, suggesting its role as an inhibitor of apoptosis of testicular cells. Further, treatment with nesfatin-1 resulted in significantly higher expression of star along with a concomitant increase in testosterone production by the lizard testis. The present study also demonstrates hormonal regulation of testicular nucb2/nesfatin-1 wherein follicle-stimulating hormone (FSH) inhibited while sex steroids like dihydrotestosterone (DHT) and 17β-estradiol-3-benzoate (E2) stimulated the mRNA expression of nesfatin-1. Observations from the current study for the first time provide comprehensive evidence of spermatogenic and steroidogenic role of nesfatin-1 as well as its hormonal regulation in the testis of a reptile, H. flaviviridis.

Androgen dihydrotestosterone promotes bladder cancer cell proliferation and invasion via EPPK1-mediated MAPK/JUP signalling

The incidence of bladder cancer (BLCA) in men is higher than that in women. Differences in androgen levels between men and women are considered the main causes of incidence rate differences. In this study, dihydrotestosterone (DHT) significantly increased the proliferation and invasion of BLCA cells. In addition, BLCA formation and metastatic rates were higher in N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-treated male mice than in female and castrated male mice in vivo. However, immunohistochemistry showed that androgen receptor (AR) was expressed at low levels in normal and BLCA tissues of men and women. The classical AR pathway considers that DHT binds to AR and induces it to enter the nucleus, where it functions as a transcription factor. Here, a non-AR combination pathway of androgen that promoted BLCA development was investigated. The EPPK1 protein was bombarded with DHT, as determined by biotinylated DHT-binding pull-down experiments. EPPK1 was highly expressed in BLCA tissues, and EPPK1 knockdown significantly inhibited BLCA cell proliferation and invasion promoted by DHT. Moreover, JUP expression was elevated in DHT-treated high-EPPK1 expressing cells, and JUP knockdown inhibited cell proliferation and invasion. EPPK1 overexpression increased tumour growth and JUP expression in nude mice. Furthermore, DHT increased the expression of the MAPK signals p38, p-p38, and c-Jun, and c-Jun could bind to the JUP promoter. However, the promotion of p38, p-p38, and c-Jun expression by DHT was not observed in EPPK1 knockdown cells, and a p38 inhibitor suppressed the DHT-induced effects, indicating that p38 MAPK may be involved in the regulation of DHT-dependent EPPK1-JUP-promoted BLCA cell proliferation and invasion. The growth of bladder tumours in BBN-treated mice was inhibited by the addition of the hormone inhibitor goserelin. Our findings indicated the potential oncogenic role and mechanism of DHT in BLCA pathogenesis through a non-AR pathway, which may serve as a novel therapeutic target for BLCA.

3βHSD activity saturates at physiological substrate concentrations in intact cells.

Conversion of adrenally produced dehydroepiandrosterone (DHEA) to the potent androgen dihydrotestosterone (DHT) is an important mechanism by which prostate cancer reaches castration resistance. At the start of this pathway is a branch point at which DHEA can be converted to Δ4 -androstenedione by the enzyme 3β-hydroxysteroid dehydrogenase (3βHSD) or to Δ5 -androstenediol by 17βHSD. To better understand this process, we studied the kinetics of these reactions in cells. Prostate cancer cells (LNCaP cell line) were incubated with steroids (DHEA and Δ5 -androstenediol) over a range of concentrations and the steroid metabolism reaction products were measured by mass spectrometry or by high-performance liquid chromatography to determine reaction kinetics. To confirm the generalizability of results, experiments were also performed in JEG-3 placental choriocarcinoma cells. The two reactions displayed very different saturation profiles, with only the 3βHSD-catalyzed reaction beginning to saturate within a physiological substrate concentration range. Strikingly, incubating LNCaP cells with low (in the ~10 nM range) concentrations of DHEA resulted in a large majority of the DHEA undergoing 3βHSD-catalyzed conversion to Δ4 -androstenedione, whereas high concentrations of DHEA (in the 100s of nM range) resulted in most of the DHEA undergoing 17βHSD-catalyzed conversion to Δ5 -androstenediol. Contrary to expectations from previous studies that used purified enzyme, cellular metabolism of DHEA by 3βHSD begins to saturate in the physiological concentration range, suggesting that fluctuations in DHEA concentrations could be buffered at the downstream active androgen level.

Effects of Androgen Treatment on Growth in Patients with 5-α-Reductase Type 2 Deficiency.

Patients with 5-α-reductase type 2 deficiency (5αRD2) require androgen treatment for the growth of normal male external genitalia. Since limited research has been conducted on the effects of androgen treatment on height in individuals with 5αRD2, we investigated the effect of androgen treatment on bone age (BA) and the height status in children with 5αRD2. Of the 19 participants who were followed up for an average of 10.6 years, 12 received androgen treatment. BA and height standard deviation scores (SDS) were compared between the treatment and non-treatment groups, as well as between the dihydrotestosterone (DHT) and testosterone enanthate (TE) treatment groups. Despite the above-average height of the 19 patients with 5αRD2, the height SDS relative to BA (htSDS-BA) was below average, particularly in the androgen treatment group. DHT treatment did not lead to an increase in BA or htSDS-BA, whereas TE treatment resulted in BA advancement and decreased htSDS-BA, especially in the prepubertal period. DHT treatment is more favorable for height than TE treatment in patients with 5αRD2, particularly during the prepubertal period. Therefore, age and the type of androgen used should be carefully considered to minimize the risk of height reduction in these patient groups.