Background: Testosterone is the predominant sex hormone in men and is increased in women with polycystic ovarian syndrome. These patients also experience an increased risk for cardiovascular diseases including hypertension and arterial stiffness. Since our previous work shows an important role for the G protein-coupled estrogen receptor (GPER) in arterial stiffness, we hypothesized that other hormones including androgens may impact arterial stiffness in female mice via regulation of GPER. Methods: The impact of the non-aromatizable androgen dihydrotestosterone (DHT), the glucocorticoid dexamethasone, and the progestin medroxyprogesterone acetate (all 100 nM for 24 h) on GPER and ERα expression was assessed in cultured vascular smooth muscle cells using droplet digital PCR (ddPCR). To assess the in vivo impact of the DHT-induced downregulation of GPER, female ovary-intact C57Bl/6 mice were treated with silastic capsules containing DHT for 4 weeks, one with a dosage expected to mimic human male DHT levels and another to double the expected human concentration (n=8-9/group). Results: GPER mRNA was only decreased by DHT (P=0.001), while ERα expression was significantly suppressed by all hormones (P<0.0001). While blood pressure was not different between groups (P= 0.59), there was a dose-dependent increase in body weight (control 22±2 g, single dose 24±2 g, double dose 26±2 g; P=0.0002). Intracarotid stiffness measured via pulse wave velocity showed a more than two-fold increase in both DHT-treated groups (control 1.9±0.3 m/s, single dose 4.3±0.8 m/s, double dose 4.8±1.0 m/s). Histological analysis of aortic sections using Masson's trichrome showed a significant decrease in collagen between the control group (24 ± 5%) and the double dose group (17 ± 3%, P=0.007), despite no changes in aortic wall thickness or smooth muscle content. Lastly, ddPCR showed that in vivo DHT treatment decreased aortic expression of both GPER (control 20±5, single dose 10.5 ± 5.6, double dose 10±4 copies/ng; P=0.001) and ERα (control 54±2, single dose 24±13, and double dose 23 ± 12 copies/ng; P=0.003). Conclusions: These findings indicate that testosterone promotes arterial stiffening and cardiovascular damage in female mice and is associated with decreased estrogen receptor expression. These data are important not only for polycystic ovarian syndrome patients but also women using testosterone for fitness, gender transitioning, or reduced libido.

Microglia-induced neuroinflammation plays a vital role in the etiology and progression of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease and multiple sclerosis. The neuroprotective role of androgens, including testosterone and its metabolite dihydrotestosterone (DHT), has been increasingly demonstrated in these diseases, but few studies investigated the effects of androgen on neuroinflammation. This study investigated the role of DHT in lipopolysaccharide (LPS)-induced neuroinflammation, neuronal damage and behavioral dysfunction, as well as underlying mechanisms. We showed that DHT inhibited LPS-induced release of proinflammatory factors, including TNF-α, IL-1β, IL-6; iNOS, COX-2, NO, and PGE2 in BV2 cells and primary microglia by suppressing the TLR4-mediated NF-κB and MAPK p38 signaling pathways, thus protecting SH-SY5Y neurons from inflammatory damage induced by activated microglia. In an LPS-induced neuroinflammation mouse model, endogenous DHT depletion by castration exacerbated inflammatory responses by upregulating the levels of TNF-α, IL-1β, IL-6, iNOS, and COX-2 in the serum and brain by increasing the LR4-mediated NF-κB and MAPK pathway activation, but these effects were restored by exogenous DHT supplementation. Moreover, DHT also regulated the mRNA levels of the anti-inflammatory cytokines IL-10 and IL-13 in the brain. In addition, DHT modulated the expression of Aβ, the apoptotic proteins caspase-3, Bcl-2, and Bax, and synaptophysin, as well as neuronal damage in LPS-treated mouse brains. Further behavioral tests revealed that DHT ameliorated LPS-induced spatial and learning impairment and motor incoordination, and partly improved the locomotor activity in LPS-injected mice. Therefore, this study suggests that DHT exerts anti-neuroinflammatory and neuroprotective effects; thus, androgen replacement therapy is a potential therapeutic strategy for improving cognitive and behavioral function in neuroinflammation-related diseases.

We have previously reported that hexamethylene bis-acetamide inducible protein 1 (HEXIM1) inhibits the activity of ligand-bound estrogen receptor α (ERα) and the androgen receptor (AR) by disrupting the interaction between these receptors and positive transcriptional elongation factor b (P-TEFb) and attenuating RNA polymerase II (RNAPII) phosphorylation at serine 2. Functional consequences of the inhibition of transcriptional activity of ERα and AR by HEXIM1 include the inhibition of ERα- and AR-dependent gene expression, respectively, and the resulting attenuation of breast cancer (BCa) and prostate cancer (PCa) cell proliferation and growth. In our present study, we determined that HEXIM1 inhibited AKR1C3 expression in BCa and PCa cells. AKR1C3, also known as 17β-hydroxysteroid dehydrogenase (17β-HSD) type 5, is a key enzyme involved in the synthesis of 17β-estradiol (E2) and 5-dihydrotestosterone (DHT). Downregulation of AKR1C3 by HEXIM1 influenced E2 and DHT production, estrogen- and androgen-dependent gene expression, and cell proliferation. Our studies indicate that HEXIM1 has the unique ability to inhibit both the transcriptional activity of the ER and AR and the synthesis of the endogenous ligands of these receptors.

Men are at a higher risk of developing bladder cancer than women. Although the urinary bladder is not regarded as an sex organ, it has the potential to respond to androgen signals. The mechanisms responsible for the gender differences remain unexplained. Androgen receptor (AR) after binding with 5α-dihydrotestosteron (DHT) undergoes a conformational change and translocates to nucleus to induce transcriptional regulation of target genes. However androgen/AR signaling can also be activated by interacting with several signaling molecules and exert its non-genomic function. The aim of present study was to explain whether the progression of bladder cancer in men is dependent on androgen/AR signaling. Studies were carried out on human bladder cancer cell lines: HCV29, T24, HT1376 and HTB9. Bladder cancer cells were treated for 48 h with 10 nM DHT or not, with replacement after 24 h. Expression of cell signaling proteins, was analyzed using Western Blot and RT-PCR. Subcellular localization of protein was studied using the ProteoExtract Subcellular Proteome Extraction Kit and Western blot analysis. We showed that DHT treatment significantly increased AR expression in bladder cell line HCV29. We also observed DHT-mediated activation of Akt/GSK-3β signaling pathway which plays a central role in cancer progression. Presented results also show that androgen/AR signaling is implicated in phosphorylation of eIF4E which can promote epithelial-mesenchymal transition (EMT). We indicate that AR plays an essential role in bladder cancer progression in male patients. Therefore, androgen-activated AR signaling is an attractive regulatory target for the inhibition or prevention of bladder cancer incidence in men.

Men exhibit a higher incidence of cardiovascular diseases than do women. The cardiovascular actions of sex steroids have been suggested as primary factors in mediating this sex difference. The mechanisms by which sex steroids, androgens and estrogens, mediate cardiovascular actions remain unclear. Excess aldosterone secretion has been associated with cardiovascular diseases. The hypothesis tested in this study was that at physiological concentrations, androgens stimulate and estradiol inhibits aldosterone secretion by human adrenal cells. In contrast to our hypothesis, physiological concentrations of sex steroids did not modify aldosterone secretion by H295R human adrenocortical cells. However, supraphysiological concentrations (300-1000 nM) of dihydrotestosterone (DHT) significantly stimulated basal and Angiotensin II-mediated aldosterone secretion. The stimulatory effect of DHT on aldosterone secretion was not blocked by the classical androgen receptor blocker flutamide. The stimulatory effect of DHT on aldosterone secretion was also independent of the intra-adrenal renin-angiotensin system since it was neither modified by treatment with the Angiotensin II receptor type 1 blocker losartan or the angiotensin converting enzyme inhibitor captopril. Inhibitors of the calmodulin/calmodulin-dependent protein kinase (CaMK) and protein kinase C intracellular signaling pathways abolished the DHT stimulatory effect on aldosterone secretion by H295R cells. In conclusion, physiological concentrations of sex steroids did not modify aldosterone secretion by human adrenal cells. However, supraphysiological concentrations of DHT-stimulated aldosterone secretion by human adrenal cells by the calmodulin/CaMK and protein kinase C intracellular signaling pathways but independently of the classical androgen receptor. Supraphysiological doses of androgen may promote cardiovascular diseases via stimulation of aldosterone secretion.

Anabolic-androgenic steroids (AAS) are drugs of abuse. Previous studies have shown that male and female hamsters self-administer testosterone (T) and other AAS, suggesting that androgens are reinforcing in a context where athletic performance is irrelevant. AAS are synthetic derivatives of T, which may be aromatizable to estrogen and/or reducible to dihydrotestosterone (DHT). However, we do not know which metabolites of T are reinforcing. To determine if DHT, estradiol (E(2)), or DHT + E(2) are reinforcing, we tested intracerebroventricular (icv) self-administration in male hamsters. The hypothesis was that androgen reinforcement is sensitive to both androgenic and estrogenic T metabolites. If so, hamsters would self-administer DHT, E(2), and DHT + E(2). Twenty four castrated male hamsters (n = 8/group) received icv cannulas and sc T implants for physiologic androgen replacement. One week later, hamsters self-administered DHT (0.1, 1.0, 2.0 microg/microl), E(2) (0.001, 0.01, 0.02 microg/microl), or DHT + E(2), each for 8 days in increasing concentration (4 h/day). Operant chambers were equipped with an active and inactive nose-poke. At the medium concentration, hamsters self-administered DHT (active nose-poke: 47.9 +/- 13.9 responses/4 h vs. inactive: 18.7 +/- 4.8), E(2) (active: 44.8 +/- 14.9 vs. inactive: 16.6 +/- 2.6), and DHT + E(2) (active: 19.1 +/- 2.4 vs. inactive: 10.4 +/- 2.4, P < 0.05). At the highest concentration, males self-administered DHT (active: 28.3 +/- 7.7 vs. inactive: 15.0 +/- 3.5, P < 0.05) and DHT + E(2) (active: 22.6 +/- 3.8 vs. inactive: 11.6 +/- 2.5, P < 0.05), but not E(2). Hamsters did not self-administer the lowest concentrations of DHT, E(2), or DHT + E(2). These results support our hypothesis that both androgenic and estrogenic T metabolites are reinforcing. Together, they do not exert synergistic effects.

Breast cancer is the most common cancer among women. Androgens, the male sexual hormones produced by ovary, act as protector of mammary gland. To elucidate the possible effects of dihydrotestosterone (DHT) on the transcriptome of mammary gland, serial analysis of gene expression was carried out on three groups of gonadectomized mice. After gonadectomy (GDX), DHT was injected 3 or 24h before sacrifice, whereas the control (GDX) group received vehicle solution. Approximately 42,000 tags were sequenced in each group. Genes involved in the cytoskeletal and extracellular matrix, such as troponin I skeletal fast 2 and keratin complex 1 acidic gene 14, were upregulated. In the immunity, complement component 1 q subcomponent gamma polypeptide and expressed sequence tag similar to lectin galactose binding soluble 3 were downregulated by DHT, whereas serine (or cystein) proteinase inhibitor clade A member 1a was upregulated. In the energy metabolism, the gene expression level of cytochrome c oxidase subunit I was upregulated by DHT, while NADH dehydrogenase subunit 2 was downregulated. In addition, transcripts involved in transport metabolism, such as apolipoprotein A-1, were upregulated by DHT, whereas retinol binding protein 4 plasma was downregulated. Several previously unknown sequence tags were identified, which may allow to characterize new molecules of interest. These results suggest the suppression of immune response in normal mammary gland after DHT injection. This study can assist in refining research on the role of androgens in mammary gland homeostasis and breast cancer.

A cross-talk between androgen/androgen receptor signaling and the AP-1 transcription factor has been proposed. In this study, we asked whether activation of AP-1 modifies androgen-responsive gene transcription, and whether androgens effect AP-1-regulated gene transcription. We show that activation of AP-1 via expression of a constitutively active mutant of mitogen-activated/extracellular signal responsive kinase kinase (MEK) kinase-1 did not increase the activity of the androgen-responsive probasin promoter. Likewise, expression of a constitutively active mutant of the transcription factor c-Jun, which is a major constitutent of AP-1, did not increase the activity of the probasin promoter. In contrast, 5α-dihydrotestosterone (DHT) activated both the probasin promoter and the AP-1-regulated collagenase promoter in LNCaP prostate cancer cells. The AP-1 binding site within the collagenase promoter was identified as DHT-responsive element. In line with this, DHT increased the activities of the c-Jun promoter and the tumor necrosis factor alpha promoter, which both contain AP-1 binding sites. The signal transduction pathway coupling DHT stimulation with AP-1 activation required c-Jun, MAP kinases and androgen receptors, but was independent of transient receptor potential melastatin-8 (TRPM8) channels, proposed to function as ionotropic testosterone receptors. Expression of the GTPase activating protein RGS2 attenuated DHT-induced activation of AP-1, indicating that the DHT-induced signaling cascade involves G proteins.

Testosterone (T) has been shown to cause vasodilation in rabbit coronary arteries through a nongenomic pathway. Part of this T-induced relaxation was shown to be mediated by opening voltage dependent K(+) channels. T infusion also reduces peripheral resistance in human males with heart failure. The effects of T or its active metabolite 5-alpha dihydrotestosterone (DHT) are not well studied. This study investigates the effect of T and DHT on contraction in guinea pig gallbladder strips. T or DHT induced a concentration-dependent relaxation of cholecystokinin octapeptide (CCK)-induced tension. Pretreatment of the strips with PKA inhibitor 14-22 amide myristolated had no significant effect on the relaxation induced by either T or DHT. Pretreatment of strips with 2-APB, an inhibitor of IP(3) induced Ca(2+) release, produced a significant (p<0.001) reduction in the T- or DHT-induced relaxation. Bisindolymaleimide IV and chelerythrine Cl(-) when used in combination had no significant effect on the amount of CCK-induced tension, but significantly (p<0.01) decreased the amount of T- or DHT-induced relaxation. The flavone chrysin, an aromatase inhibitor, and genistein, an isoflavone, each produced a significant (p<0.01) reduction in CCK-induced tension. Chrysin significantly (p<0.05) increased T-induced relaxation; however, genistein had no effect on T-induced relaxation. It is concluded that T and DHT inhibits gallbladder motility rapidly by nongenomic actions of the hormones. Multiple pathways that include inhibition of intracellular Ca(2+) release, inhibition of extracellular Ca(2+) entry, and the actions of PKC may mediate this effect.

Androgenetic alopecia involves the action of dihydrotestosterone (DHT) on dermal papilla cells (DPCs) that line the base of the hair follicle. However, the mechanism of DHT action is not completely understood. The effects of DHT on DPCs, regulatory cells that function in follicle growth and the hair cycle, were examined in immortalized cells derived from rat vibrissa follicles. DHT did not affect the proliferation of immortalized DPCs. However, flow cytometry analysis revealed that DHT increased cell-cycle arrest in these cells, which was accompanied by an increase in the p27(kip1) level and by decreases in cyclin E, cyclin D1, and cyclin-dependent kinase 2 levels. DHT treatment resulted in the phosphorylation and nuclear translocation of Smad2/3, a mediator of the transforming growth factor-β (TGF-β) signaling pathway, which leads to the catagen phase of the hair cycle. DHT also induced the phosphorylation and nuclear translocation of heat shock protein 27 (HSP27). Moreover, DHT decreased the levels of total and nuclear β-catenin, an important regulator of hair growth and proliferation, while lithium chloride, a glycogen synthase kinase-3β inhibitor, attenuated the DHT-induced downregulation of the β-catenin level. On the other hand, DHT increased the phosphorylation of mammalian target of rapamycin (mTOR), a regulator of proliferation, in immortalized DPCs. These results illustrate that DHT could shorten the duration of the hair growth cycle by initiating cell-cycle arrest, downregulating the β-catenin level, and upregulating the TGF-β/Smad and HSP27 level, whereas activation of mTOR by DHT could attenuate the inhibition of hair growth cycle in immortalized DPCs.

Dihydrotestosterone (DHT) causes the regression of human hair follicles in the parietal scalp, leading to androgenic alopecia (AGA). Sulforaphane (SFN) increases the expression of DHT degrading enzymes, such as 3α-hydroxysteroid dehydrogenases (3α-HSDs), and, therefore, SFN treatment may improve AGA. To determine the effects of SFN on hair growth, we administered SFN (10 mg/kg BW, IP) or vehicle (DMSO) to ob/ob mice for six weeks and examined hair regeneration and the plasma levels of testosterone and DHT. We also tested the effects of SFN on the expression of two forms of 3α-HSD, aldo-keto reductase 1c21 and dehydrogenase/reductase (SDR family) member 9, both in vitro and in vivo. SNF significantly enhanced hair regeneration in ob/ob mice. The mice treated with SFN showed lower plasma levels of testosterone and DHT than those treated with vehicle. SFN increased the mRNA and protein levels of the two forms of 3α-HSD in the liver of the mice and in cultured murine hepatocyte Hepa1c1c7 cells. These results suggest that SFN treatment increases the amount of 3α-HSDs in the liver, accelerates the degradation of blood DHT, and subsequently blocks the suppression of hair growth by DHT.

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 5α-dihydrotestosterone (DHT) by the enzyme 5α-reductase (5αR), but the role of this metabolite in GBM cells is unknown.

The polycystic ovary syndrome (PCOS) is a complex and heterogeneous endocrine condition characterized by hyperandrogenism, hyperinsulinemia, insulin resistance and chronic anovulation. Regulation and interaction of a multitude of genes required for follicular development are found to be altered in PCOS. MicroRNAs (miRNAs) mediate posttranscriptional gene regulation by binding to the 3´ untranslated region of mRNAs to either inhibit or enhance translation. However, the extent and regulation of miRNA expression in PCOS is poorly understood and the current study is the first to describe altered expression of miRNAs in PCOS.

Hyperandrogenism causes dysfunction of the hypothalamic-pituitary-gonadal (HPG) axis in reproductive women. In this study, we examined the effects of dihydrotestosterone (DHT) on characteristic changes in rat anterior pituitary gland samples. DHT was administered to ovary-intact 6-week postnatal female rats for 7 days, after which the anterior pituitary glands were examined and compared with those in control rats. Estrous cyclicity was not drastically disrupted by DHT treatment. Common gonadotropin α subunit (Cga), luteinizing hormone β subunit (Lhb), and follicle-stimulating hormone (FSH) β subunit (Fshb) gene expression levels were not modulated by DHT treatment, while prolactin (Prl) gene expression was significantly repressed by DHT. Gonadotropin-releasing hormone (GnRH) receptor (Gnrh-r) gene expression was significantly inhibited by DHT, whereas pituitary adenylate cyclase-activating polypeptide (PACAP) receptor (Pca1-r) gene expression was increased by DHT. Gene expression levels of the receptors encoded by thyrotropin-releasing hormone (Trh-r) and kisspeptin (Kiss1-r) genes were unchanged. Expression of inhibin α subunit (Inha) and activin βA subunits (Actba) within the pituitary was inhibited by DHT treatment, while activin B subunit (Actbb) and follistatin (Fst) gene expression was unchanged by DHT. In mouse pituitary gonadotroph LβT2 cells, DHT did not modulate the gene expression of Gnrh-r, but it inhibited the expression of Inha and Actba subunits within the LβT2 cells. In rat prolactin-producing GH3 cells, DHT did not modulate prolactin gene expression, but it increased Pac1-r gene expression. The present observations suggest that DHT directly or indirectly affects the anterior pituitary gland and induces characteristic changes in hormone-producing cells.

Although androgen deprivation therapy (ADT) and immunotherapy are potential treatment options in men with metastatic prostate cancer (CaP), androgen has conventionally been proposed to be a suppressor of the immune response. However, we herein report that DHT activates macrophages. When the murine macrophage cell line (RAW 264.7), human monocyte cell line (THP-1), and human peripheral blood monocytes were cultured with androgen-resistant CaP cell lines, DHT increased cytotoxicity of macrophages in a concentration-dependent manner. Further studies revealed that DHT induced M1 polarization and increased the expression levels of TNF-related apoptosis-inducing ligand (TRAIL) in macrophages and that this effect was abrogated when TRAIL was neutralized with a blocking antibody or small interfering RNA. Subsequent experiments demonstrated that induction of TRAIL expression was regulated by direct binding of androgen receptor to the TRAIL promoter region. Finally, an in vivo mouse study demonstrated that castration enhanced the growth of an androgen-resistant murine CaP tumor and that this protumorigenic effect of castration was blocked when macrophages were removed with clodronate liposomes. Collectively, these results demonstrate that DHT activates the cytotoxic activity of macrophages and suggest that immunotherapy may not be optimal when combined with ADT in CaP.

Androgen levels inversely correlate with the incidence, susceptibility and severity of asthma. However, whether male sex hormones such as 5α-dihydrotestosterone (DHT) have beneficial effects on asthma symptoms and/or could affect asthma susceptibility have not been investigated. DHT administration to female mice, during the sensitization phase, abrogates the sex bias in bronchial hyperreactivity. This effect correlates with inhibition of leukotriene biosynthesis in the lung. DHT significantly inhibits also other asthma-like features such as airway hyperplasia and mucus production in sensitized female mice. Conversely, DHT does not affect plasma IgE levels as well as CD3+CD4+ IL-4+ cell and IgE+c-Kit+ cell infiltration within the lung but prevents pulmonary mast cell activation. The in vitro study on RBL-2H3 cells confirms that DHT inhibits mast cell degranulation. In conclusion, our data demonstrate that immunomodulatory effects of DHT on mast cell activation prevent the translation of allergen sensitization into clinical manifestation of asthma.

The cardiovascular effects of testosterone and dihydrotestosterone are generally attributed to their modulatory action on lipid and glucose metabolism. However, no ex vivo studies suggest that circulating androgen levels influence the activation and reactivity of blood platelets - one of the main components of the haemostasis system directly involved in atherosclerosis. The levels of testosterone, dihydrotestosterone and oestradiol in plasma from men and women aged from 60 to 65 years were measured by LC-MS; the aim was to identify any potential relationships between sex steroid levels and the markers of platelet activation (surface membrane expression of GPII/IIIa complex and P-selectin) and platelet reactivity in response to arachidonate, collagen or ADP, monitored with whole blood aggregometry and flow cytometry. The results of the ex vivo part of the study indicate that the concentrations of testosterone and its reduced form, dihydrotestosterone are significantly negatively associated with platelet activation and reactivity. These observations were confirmed in an in vitro model: testosterone and dihydrotestosterone significantly inhibited platelet aggregation triggered by arachidonate or collagen. Our findings indicate that testosterone and dihydrotestosterone are significant haemostatic steroids with inhibitory action on blood platelets in older people.

Gonadal hormones play a vital role in driving motivated behavior. They not only modulate responses to naturally rewarding stimuli, but also influence responses to drugs of abuse. A commonality between gonadal hormones and drugs of abuse is that they both impact the neurocircuitry of reward, including the regulation of structural plasticity in the nucleus accumbens (NAc). Previous hormonal studies have focused on the mechanisms and behavioral correlates of estradiol-induced dendritic spine changes in the female NAc. Here we sought to determine the effects of androgens on medium spiny neuron (MSN) spine plasticity in the male NAc. Following treatment with the androgen receptor agonist dihydrotestosterone (DHT), MSNs in castrated male rats exhibited a significant decrease in dendritic spine density. This effect was isolated to the shell subregion of the NAc. The effect of DHT was dependent on mGluR5 activity, and local mGluR5 activation and subsequent endocannabinoid signaling produce an analogous NAc shell spine decrease. Somewhat surprisingly, DHT-induced conditioned place preference remained intact following systemic inhibition of mGluR5. These findings indicate that androgens can utilize mGluR signaling, similar to estrogens, to mediate changes in NAc dendritic structure. In addition, there are notable differences in the direction of spine changes, and site specificity of estrogen and androgen action, suggesting sex differences in the hormonal regulation of motivated behaviors.

Elevated serum chemerin levels correlate with increased severity of polycystic ovary syndrome (PCOS). However, the role of CMKLR1 signaling in ovarian biology under conditions of excess DHT remains unclear. In this study we compared the effects of continuous 90-day high dose DHT exposure (83.3 □g/day) on wild type and CMKLR1-deficient mice. DHT induced PCOS-like clinical signs in wild type mice as well as significant changes in the expression of hormone receptors, steroid synthesis enzymes, and BMPs and their receptors. In contrast, CMKLR1-deficient mice significantly attenuated DHT-induced clinical signs of PCOS and alterations in ovarian gene expression. To determine whether the BMP4 signaling pathway was involved in the pathogenic effects of CMKLR1 signaling in DHT-induced ovarian steroidogenesis, antral follicles were isolated from wild type and CMKLR1 knockout (KO) mice and treated in vitro with combinations of hCG, DHT, and BMP4 inhibitors. BMP4 inhibition attenuated the induction effects of hCG and DHT on estrogen and progesterone secretion in CMKLR1 KO mice, but not in WT mice, implicating the BMP4 signaling pathway in the CMKLR1-dependent response to DHT. In conclusion, CMKLR1 gene deletion attenuates the effects of chronic DHT treatment on ovarian function in experimental PCOS, likely via BMP4 signaling.

Hyperandrogenism is a key pathological feature of polycystic ovarian syndrome (PCOS). Excess androgen can lead to PCOS-like cell hypertrophy in the ovaries and adipose tissue of rodents. Here, we established a dihydrotestosterone (DHT)-induced hyperandrogenic mouse model to analyze the differences in gene expression and signaling pathways of the ovaries and gonad fat pads of mice treated with or without DHT by RNA microarray analysis. From the results, we focused on the overlapping differentially expressed gene-Col6a5-and the major differentially enriched signaling pathway-lipid metabolism. We employed DHT-induced mouse ovarian stromal cell, adipogenic 3T3-L1 cell and hepatic cell line NCTC1469 models to investigate whether androgens directly mediate lipid accumulation and hypertrophy. We found that DHT increased lipid droplet accumulation in ovarian stromal cells and adipogenic 3T3-L1 cells but not NCTC1469 cells. DHT significantly altered stromal cell cholesterol metabolism and steroidogenesis, as indicated by changes in cholesterol levels and the expression of related genes, but these effects were not observed in 3T3-L1 cells. Moreover, Col6a5 expression was significantly increased in ovaries and gonadal fat pads of DHT-treated mice, and Col6a5 inhibition alleviated DHT-induced excess lipid accumulation and hypertrophy of ovarian stromal cells and adipogenic 3T3-L1 cells, even improved lipid metabolism in overnourished NCTC1469 cells. Our results indicate that Col6a5 plays important roles in the pathogenesis of DHT-induced lipid metabolism disorder and the hypertrophy of ovarian stromal cells and adipocytes.