Androgen Receptor mRNA Research Articles

The prognostic significance of androgen receptor expression in gliomas

Androgen receptor (AR) overexpression has been identified in gliomas and its stem cells, suggesting that AR plays an important role in tumor carcinogenesis. The prognostic significance of AR overexpression in gliomas remains unknown. AR mRNA expression in gliomas and relevant clinical data were obtained from The Cancer Genome Atlas and Chinese Glioma Genome Atlas databases. AR expression levels were compared across gliomas of different histopathologic grades and molecular subtypes. Kaplan–Meier analyses in patients with different AR expression levels were investigated for the potential prognostic values of AR. Compared with normal brain tissue, gliomas show significantly higher AR mRNA expression (p < 0.01). AR mRNA expression was more prominent in higher grade disease and in worse prognostic molecular subtypes (p < 0.01). AR protein is more abundant in glioblastoma than in lower grade gliomas (LGG) (grade 2/3) (p < 0.0001). This is corroborated by a linear association between AR mRNA and protein expression (r = 0.65). In LGG, both higher AR mRNA and protein expression was associated with significantly worse overall survival (OS). Five-year OS for LGG patients with high versus low AR expression were 59.1% and 73.3%, respectively (p < 0.0001). AR expression is not prognostic for OS within glioblastoma patients. Gender was not associated with AR expression or prognosis. Higher AR expression levels are associated with higher grade disease and histopathologic features predicting poorer prognosis in lower grade gliomas. Higher gene expression in LGG patients is correlated with poor prognosis but not in the glioblastoma cohort suggesting saturated expression/functions of AR in glioblastoma.

Abstract C122: Androgen receptor-mediated regulation of innate immunity in prostate cancer in Black men

Abstract Black men are two times more likely to die from prostate cancer (PCa) than White men. We reported that innate immune cell infiltration varies between races, and others have reported differences in androgen receptor (AR) expression by race or ancestry. Herein, we evaluated AR status and immune cell infiltration in primary and metastatic PCa tissues obtained from Black and White men. RNA in situ hybridization (RISH) AR analysis was done on a tissue microarray (TMA) constructed from radical prostatectomy tissues from 120 Black men matched to 60 White men on age, grade and stage. RISH or immunohistochemistry (IHC) analysis of AR, NKX3.1, prostate specific antigen (PSA), mast cells (tryptase), neutrophils (CD66), and macrophages (CD206, CD68, CD80) was done on a TMA containing rapid autopsy tissues from metastatic sites (n=11) collected from Black (n=5) and White (n=16) men. Multiplexing IHC (mIHC) was used to assess AR, neutrophils (CD66ce), mast cells (tryptase), T-cells (CD4, CD8), and M2 macrophages (CD163) on radical prostatectomy whole-tissue sections obtained at Johns Hopkins Medicine, Baltimore, MD and biopsy tissues obtained from the University of Nairobi, Nairobi, Kenya. Tumor regions on TMAs were annotated by a pathologist and signal intensity per unit area measured using TMAJ and FrIDA software. HALO Image Analysis Platform (version 3.6.4134.137; Indica Labs) was used to analyze mIHC stained tissues. Statistical analysis and graphing were done using GraphPad Prism. There was increased AR mRNA expression in primary cancer tissues from Black men compared to White men (p&amp;lt;0.0001) after negative binomial regression with the adjustment of TMA set, age, grade and stage. There was a negative correlation between AR expression and CD66+ neutrophils in tumor and benign tissues from Black men (R = -0.318;p = 0.002) but this correlation was not significant among White men (R= -0.045; p = 0.765). Interestingly, we measured a positive correlation between AR mRNA expression and tryptase+ mast cells among White men (R= 0.320; p=0.016), but a trend toward a negative relationship among Black men (R= -0.252;p = 0.075) We also measured a positive correlation between AR and CD163+(R= 0.324; p&amp;lt;0.001), CD68+(R= 0.514;p&amp;lt;0.001), and CD80+(R= 0.386;p&amp;lt;0.001) macrophages in primary tumor tissues from all men. NKX3.1 and PSA protein were significantly increased in metastatic tissues from White compared to Black men. Conversely, CD68+ and CD80+ macrophages were increased in Black compared to White men. CD66+ neutrophils and CD206+ macrophages were increased in AR- compared to AR intact metastatic tissues. Immune cells appeared to be primarily void of AR expression in primary cancer tissues by mIHC, but there was a subset CD4+ and CD8+ T-cell clusters that express AR. Immune cell infiltration varies by the presence of AR in both primary and metastatic PCa tissues. We observed differences in AR mRNA and AR-related proteins between Black and White patients which may contribute to the differences observed in immune cell infiltration between the two races Citation Format: Kennedy Rains, Katie M. Farkouh, Taylor N. Godwin, Eric Erak, Aakash Parikh, Michael Considine, Elana Fertig, Jiayun Lu, Charles Waihenya, Valerie Odero-Marah, Karen S. Sfanos, Angelo M. De Marzo, Corinne E. Joshu, Janielle P. Maynard. Androgen receptor-mediated regulation of innate immunity in prostate cancer in Black men [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C122.

High seminal BPA in IVF patients correlates with lower sperm count and up-regulated miR-21 and miR-130a

Bisphenol A (BPA) is a widespread industrial chemical, used as the key monomer of polycarbonate plastics and epoxy resins. BPA has been detected in human seminal fluid and has been correlated with changes in sperm parameters, crucial in determining male fertility. In this study, semen samples were collected from 100 patients aged 29–47 years undergoing fertility assessment between 2021 and 2023 and analyzed according to WHO guidelines. BPA levels in the seminal plasma were then measured through an enzyme-linked immunosorbent assay (ELISA) and compared to sperm quality metrics. The relative mRNA/miRNA expression of key genes associated to male reproduction, including androgen receptor, miR-34c, miR-21, miR-130a, was then quantified and compared between groups with high or low BPA content. Our results revealed that BPA levels were increased with age and were negatively correlated with sperm counts (p<0.05). The negative correlation remained significant when patients were age-matched. No other relationships between seminal BPA and motility, morphology or DNA fragmentation levels were observed. qPCR analysis showed that androgen receptor mRNA expression was significantly greater in sperm with high seminal BPA (p<0.05). Moreover, we found that the expression of miR-21 and miR-130a was also upregulated in the higher BPA group (p<0.05). These results display a relationship between BPA content in the semen and male fertility parameters, and provide insights into the molecular mechanisms through which BPA may be affecting male reproductive capability. Ultimately, this research can potentially drive changes to guidelines and exposure limits for BPA exposure.

Novel Plasma Membrane Androgen Receptor SLC39A9 Mediates Ovulatory Changes in Cells of the Monkey Ovarian Follicle.

Follicular androgens are important for successful ovulation and fertilization. The classical nuclear androgen receptor (AR) is a transcription factor expressed in the cells of the ovarian follicle. Androgen actions can also occur via membrane androgen receptor SLC39A9. Studies in fish ovary demonstrated that androgens bind to SLC39A9 and increase intracellular zinc to regulate ovarian cell function. To determine if SLC39A9 is expressed and functional in the key cell types of the mammalian ovulatory follicle, adult female cynomolgus macaques underwent ovarian stimulation. Ovaries or ovarian follicular aspirates were harvested at 0, 12, 24, and 36 hours after human chorionic gonadotropin (hCG). SLC39A9 and AR mRNA and protein were present in granulosa, theca, and vascular endothelial cells across the entire 40-hour ovulatory window. Testosterone, bovine serum albumin-conjugated testosterone (BSA-T), and androstenedione stimulated zinc influx in granulosa, theca, and vascular endothelial cells. The SLC39A9-selective agonist (-)-epicatechin also stimulated zinc influx in vascular endothelial cells. Taken together, these data support the conclusion that SLC39A9 activation via androgen induces zinc influx in key ovarian cells. Testosterone, BSA-T, and androstenedione each increased proliferation in vascular endothelial cells, indicating the potential involvement of SLC39A9 in ovulatory angiogenesis. Vascular endothelial cell migration also increased after treatment with testosterone, but not after treatment with BSA-T or androstenedione, suggesting that androgens stimulate vascular endothelial cell migration through nuclear AR but not SLC39A9. The presence of SLC39A9 receptors and SLC39A9 activation by follicular androstenedione concentrations suggests that androgen activation of ovarian SLC39A9 may regulate ovulatory changes in the mammalian follicle.

AR loss in prostate cancer stroma mediated by NF-κB and p38-MAPK signaling disrupts stromal morphogen production.

Androgen Receptor (AR) activity in prostate stroma is required to maintain prostate homeostasis. This is mediated through androgen-dependent induction and secretion of morphogenic factors that drive epithelial cell differentiation. However, stromal AR expression is lost in aggressive prostate cancer. The mechanisms leading to stromal AR loss and morphogen production are unknown. We identified TGFβ1 and TNFα as tumor-secreted factors capable of suppressing AR mRNA and protein expression in prostate stromal fibroblasts. Pharmacological and RNAi approaches identified NF-κB as the major signaling pathway involved in suppressing AR expression by TNFα. In addition, p38α- and p38δ-MAPK were identified as suppressors of AR expression independent of TNFα. Two regions of the AR promoter were responsible for AR suppression through TNFα. FGF10 and Wnt16 were identified as androgen-induced morphogens, whose expression was lost upon TNFα treatment and enhanced upon p38-MAPK inhibition. Wnt16, through non-canonical Jnk signaling, was required for prostate basal epithelial cell survival. These findings indicate that stromal AR loss is mediated by secreted factors within the TME. We identified TNFα/TGFβ as two possible factors, with TNFα mediating its effects through NF-κB or p38-MAPK to suppress AR mRNA transcription. This leads to loss of androgen-regulated stromal morphogens necessary to maintain normal epithelial homeostasis.

Paxillin regulates androgen receptor expression associated with granulosa cell focal adhesions.

Paxillin is a ubiquitously expressed adaptor protein integral to focal adhesions, cell motility, and apoptosis. Paxillin has also recently been implicated as a mediator of nongenomic androgen receptor (AR) signaling in prostate cancer and other cells. We sought to investigate the relationship between paxillin and AR in granulosa cells (GCs), where androgen actions, apoptosis, and focal adhesions are of known importance, but where the role of paxillin is understudied. We recently showed that paxillin knockout in mouse GCs increases fertility in older mice. Here, we demonstrate that paxillin knockdown in human granulosa-derived KGN cells, as well as knockout in mouse primary GCs, results in reduced AR protein but not reduced mRNA expression. Further, we find that both AR protein and mRNA half-lives are reduced by approximately one-third in the absence of paxillin, but that cells adapt to chronic loss of paxillin by upregulating AR gene expression. Using co-immunofluorescence and proximity ligation assays, we show that paxillin and AR co-localize at the plasma membrane in GCs in a focal adhesion kinase-dependent way, and that disruption of focal adhesions leads to reduced AR protein level. Our findings suggest that paxillin recruits AR to the GC membrane, where it may be sequestered from proteasomal degradation and poised for nongenomic signaling, as reported in other tissues. To investigate the physiological significance of this in disorders of androgen excess, we tested the effect of GC-specific paxillin knockout in a mouse model of polycystic ovary syndrome (PCOS) induced by chronic postnatal dihydrotestosterone (DHT) exposure. While none of the control mice had estrous cycles, 33% of paxillin knockout mice were cycling, indicating that paxillin deletion may offer partial protection from the negative effects of androgen excess by reducing AR expression. Paxillin-knockout GCs from mice with DHT-induced PCOS also produced more estradiol than GCs from littermate controls. Thus, paxillin may be a novel target in the management of androgen-related disorders in women, such as PCOS.

Effects of crosstalk between steroid hormones mediated thyroid hormone in zebrafish exposed to 4-tert-octylphenol: Estrogenic and anti-androgenic effects

Alkylphenols, such as nonylphenol and 4-tert-octylphenol (OP), are byproducts of the biodegradation of alkylphenol ethoxylates and present substantial ecological and health risks in aquatic environments and higher life forms. In this context, our study aimed to explore the effect of OP on reproductive endocrine function in both female and male zebrafish. Over a period of 21 days, the zebrafish were subjected to varying concentrations of OP (0, 0.02, 0.1, and 0.5 μg/L), based on the lowest effective concentration (EC10 = 0.48 μg/L) identified for zebrafish embryos. OP exposure led to a pronounced increase in hepatic vitellogenin (vtg) mRNA expression and 17β-estradiol biosynthesis in both sexes. Conversely, OP exhibits anti-androgenic properties, significantly diminishes gonadal androgen receptor (ar) mRNA expression, and reduces endogenous androgen (testosterone and 11-ketotestosterone) levels in male zebrafish. Notably, cortisol and thyroid hormone (TH) levels demonstrated concentration-dependent elevations in zebrafish, influencing the regulation of gonadal steroid hormones (GSHs). These findings suggest that prolonged OP exposure may result in sustained reproductive dysfunction in adult zebrafish, which is largely attributable to the intricate reciprocal relationship between hormone levels and the associated gene expression. Our comprehensive biological response analysis of adult zebrafish offers vital insights into the reproductive toxicological effects of OP, thereby enriching future ecological studies on aquatic systems.

Androgen receptor and estrogen receptor variants in prostate and breast cancers

The androgen receptor (AR) and estrogen receptor alpha (ERα) are steroid receptor transcription factors with critical roles in the development and progression of prostate and breast cancers. Advances in the understanding of mechanisms underlying the ligand-dependent activation of these transcription factors have contributed to the development of small molecule inhibitors that block AR and ERα actions. These inhibitors include competitive antagonists and degraders that directly bind the ligand binding domains of these receptors, luteinizing hormone releasing hormone (LHRH) analogs that suppress gonadal synthesis of testosterone or estrogen, and drugs that block specific enzymes required for biosynthesis of testosterone or estrogen. However, resistance to these therapies is frequent, and is often driven by selection for tumor cells with alterations in the AR or ESR1 genes and/or alternatively spliced AR or ESR1 mRNAs that encode variant forms AR or ERα. While most investigations involving AR have been within the context of prostate cancer, and the majority of investigations involving ERα have been within the context of breast cancer, important roles for AR have been elucidated in breast cancer, and important roles for ERα have been elucidated in prostate cancer. Here, we will discuss the roles of AR and ERα in breast and prostate cancers, outline the effects of gene- and mRNA-level alterations in AR and ESR1 on progression of these diseases, and identify strategies that are being developed to target these alterations therapeutically.

Detecting androgen receptor (AR), AR variant 7 (AR-V7), prostate-specific membrane antigen (PSMA), and prostate-specific antigen (PSA) gene expression in CTCs and plasma exosome-derived cfRNA in patients with metastatic castration-resistant prostate cancer (mCRPC) by integrating the VTX-1 CTC isolation system with the QIAGEN AdnaTest

BackgroundTherapies for metastatic castration-resistant prostate cancer (mCRPC) include targeting the androgen receptor (AR) with androgen receptor inhibitors (ARIs) and prostate-specific membrane antigen (PSMA). Having the ability to detect AR, AR splice variant 7 (AR-V7), or PSMA in circulating tumor cells (CTCs) or circulating exosomal cell-free RNA (cfRNA) could be helpful to guide selection of the appropriate therapy for each individual patient. The Vortex Biosciences VTX-1 system is a label-free CTC isolation system that enables the detection of the expression of multiple genes in both CTCs and exosomal cfRNA from the same blood sample in patients with mCRPC. Detection of both AR-V7 and PSMA gene expression in both CTCs and cfRNA simultaneously has not yet been reported.MethodsTo characterize the combined VTX-1-AdnaDetect workflow, 22Rv1 cancer cells were spiked into blood from healthy donors and processed with the VTX-1 to mimic patient samples and assess performances (capture efficiency, purity, AR and AR-V7 expression). Then, we collected 19 blood samples from 16 patients with mCRPC and therapeutic resistance to androgen receptor inhibitors (ARIs). Plasma was separated and the plasma-depleted blood was processed further with the VTX-1 to collect CTCs. Both plasma exosomal cfRNA and CTCs were subsequently analyzed for AR, AR-V7, PSMA, and prostate-specific antigen (PSA) mRNA expression using the AdnaTest ProstateCancerPanel AR-V7 assay.ResultsAR-V7 expression could be detected in 22Rv1 cells spiked into blood from healthy volunteers as well as in CTCs and plasma-derived exosomal cfRNA from patients with mCRPC by processing blood with the VTX-1 CTC isolation system followed by the AdnaTest ProstateCancerPanel AR-V7 assay. 94.7% of patient blood samples (18/19) had detectable AR expression in either CTCs or exosomal cfRNA (16 in CTCs, 12 in cfRNA). 15.8% of the 19 patient blood samples (3/19) were found to have AR-V7-positive (AR-V7+) CTCs, one of which was also AR-V7+ in the exosomal cfRNA analysis. 42.1% of patient blood samples (8/19) were found to be PSMA positive (PSMA+): 26.3% (5/19) were PSMA+ in the CTC analysis and 31.6% (6/19) were PSMA+ in the exosomal cfRNA analysis. Of those 8 PSMA+ samples, 2 had detectable PSMA only in CTCs, and 3 had detectable PSMA only in exosomal cfRNA.ConclusionVTX-1 enables isolation of CTCs and plasma exosomes from a single blood draw and can be used for detecting AR-V7 and PSMA mRNA in both CTCs and cfRNA in patients with mCRPC and resistance to ARIs. This technology facilitates combining RNA measurements in CTCs and exosomal cfRNA for future studies to develop potentially clinically relevant cancer biomarker detection in blood.

The RNA secondary structure of androgen receptor-FL and V7 transcripts reveals novel regulatory regions.

The androgen receptor (AR) is a ligand-dependent nuclear transcription factor belonging to the steroid hormone nuclear receptor family. Due to its roles in regulating cell proliferation and differentiation, AR is tightly regulated to maintain proper levels of itself and the many genes it controls. AR dysregulation is a driver of many human diseases including prostate cancer. Though this dysregulation often occurs at the RNA level, there are many unknowns surrounding post-transcriptional regulation of AR mRNA, particularly the role that RNA secondary structure plays. Thus, a comprehensive analysis of AR transcript secondary structure is needed. We address this through the computational and experimental analyses of two key isoforms, full length (AR-FL) and truncated (AR-V7). Here, a combination of in-cell RNA secondary structure probing experiments (targeted DMS-MaPseq) and computational predictions were used to characterize the static structural landscape and conformational dynamics of both isoforms. Additionally, in-cell assays were used to identify functionally relevant structures in the 5' and 3' UTRs of AR-FL. A notable example is a conserved stem loop structure in the 5'UTR of AR-FL that can bind to Poly(RC) Binding Protein 2 (PCBP2). Taken together, our results reveal novel features that regulate AR expression.

Relationship between Androgens and Vascular and Placental Function during Pre-eclampsia.

Hypertensive disorders of pregnancy (HDP) represent a substantial risk to maternal and fetal health. Emerging evidence suggests an association between testosterone and pre-eclampsia (PE), potentially mediated through androgen receptors (AR). Nevertheless, the mechanism driving this association is yet to be elucidated. On the other hand, reports of transgender men's pregnancies offer a limited and insightful opportunity to understand the role of high androgen levels in the development of HDP. In this sense, a literature review was performed from a little over 2 decades (1998-2022) to address the association of testosterone levels with the development of HDP. Furthermore, this review addresses the case of transgender men for the first time. The main in vitro outcomes reveal placenta samples with greater AR mRNA expression. Moreover, ex vivo studies show that testosterone-induced vasorelaxation impairment promotes hypertension. Epidemiological data point to greater testosterone levels in blood samples during PE. Studies with transgender men allow us to infer that exogenous testosterone administration can be considered a risk factor for PE and that the administration of testosterone does not affect fetal development. Overall, all studies analyzed suggested that high testosterone levels are associated with PE.

Exercise-induced vitamin D receptor and androgen receptor mediate inhibition of IL-6 and STAT3 in muscle

BackgroundSkeletal muscle produces interleukin-6 (IL-6) during exercise as a myokine. Although IL-6 is required for skeletal muscle regeneration, its action increases the expression of myostatin and other proteins involved in muscle atrophy, resulting in skeletal muscle atrophy. In this study, we clarified the effects exercise-induced vitamin D receptor (VDR) and androgen receptor (AR) expression on IL-6 and signal transducer and activator of transcription 3 (STAT3) in vivo and in vitro. MethodC2C12 myotubes were subjected to electric pulse stimulation (EPS) in vitro. To evaluate VDR and AR function, a VDR/AR agonist and antagonist were administered before EPS to C2C12 myotubes. C57BL6 mice underwent 4 weeks of exercise. The expression levels of proteolytic-associated genes, including CCAAT/enhancer-binding protein delta (C/EBPδ) and myostatin, were measured by quantitative real-time polymerase chain reaction, and phosphorylated and total STAT3 levels were measured by Western blot analysis. ResultThe expression of VDR and AR mRNA was induced following EPS in C2C12 myotubes. IL-6 mRNA expression was also increased with a peak at 6 h after EPS and p-STAT3/STAT3 ratio reciprocally decreased. Although VDR/AR agonist administration decreased IL-6 mRNA expression and p-STAT3/STAT3 ratio, these two endpoints increased after treatment with VDR/AR antagonist, respectively. Exercise in mice also increased the expression of VDR/AR and IL-6 mRNA and decreased p-STAT3/STAT3 ratio. ConclusionExercise-induced VDR and AR expression results in the suppression of IL-6 mRNA and STAT3 phosphorylation in skeletal muscle.

The use of transcriptomic data in developing biomarkers in breast cancer

AbstractHER2 and hormone receptors are biomarkers for selecting breast cancer therapy and predicting outcomes. In the era of antibody‐drug conjugates (ADC), a relatively low HER2 expression level is adequate for targeting tumor cells. We explored the potential of RNA profiling, determined by next generation sequencing (NGS), to provide more flexible clinical biomarkers as compared with immunohistochemistry (IHC) or fluorescent in situ hybridization (FISH). Data from 57 breast cancers was used to study biomarker levels as detected by routine clinical transcriptomic tests. HER2 (ERBB2), estrogen receptor alpha (ESR1), and androgen receptor (AR) mRNA levels were compared with IHC and FISH results. There was a significant overlap in the levels of ERBB2 mRNA between cases scored by IHC as zero, 1+, and 2+. This variation correlated with progression‐free survival (PFS). Similarly, the ESR1 RNA accurately reflected estrogen receptor (ER) status. Patients with high AR mRNA had better PFS (p = 0.05). Patients expressing high ER and AR levels had better PFS than those expressing low ESR1 and AR (p = 0.03). These findings suggest that RNA analysis can be an alternative to IHC and FISH and provides continuous data that can better determine cut‐off points for predicting response to ADC.

RNA N6-methyladenosine-modified-binding protein YTHDF1 promotes prostate cancer progression by regulating androgen function-related gene TRIM68

PurposeThere is no report about the direct relationship between m6A modification and androgen receptor (AR)-related genes in prostate cancer (PC). We aimed to study the mechanisms of m6A methylation in regulating the pathogenesis of PC from the perspective of AR-related genes.MethodsqRT-PCR was applied to detect the expression of m6A-related genes in PC cell with or without AR inhibitor. The effects of YTHDF1 knockdown on PC cell viability, apoptosis, migration and invasion were investigated using flow cytometry, wound healing and transwell assays, respectively. The mechanism of YTHDF1 action was investigated using m6A RNA immunoprecipitation (MeRIP) sequencing. The biological functions of YTHDF1 were also explored through in vivo experiments.ResultsYTHDF1 was significantly down-regulated in AR inhibitor group. YTHDF1 knockdown significantly decreased AR level, viability and m6A methylation level of PC cells. TRIM68 was identified as a direct target of YTHDF1. Both YTHDF1 and TRIM68 knockdown increased apoptosis, and decreased cell viability, migration, and invasion of PC cells, while TRIM68 overexpression reversed the effects of YTHDF1 knockdown on PC cells. In addition, knockdown of YTHDF1 or TRIM68 significantly decreased the m6A methylation level, and mRNA and protein levels of YTHDF1, TRIM68 and AR in PC cells, while TRIM68 overexpression increased the expression levels above. Furthermore, subcutaneous xenografts of nude mice also revealed that TRIM68 could reverse the effects of YTHDF1 knockdown in PC in vivo.ConclusionThis study suggested the key role of YTHDF1-mediated m6A modification in PC progression by regulating androgen function-related gene TRIM68 in PC.

Differences in Androgen Receptor Expression in Human Heart Tissue in Various Types of Cardiomyopathy and in Aortic Valve Stenosis.

Background: Sex-specific differences in heart disease outcomes are influenced by the levels of the steroid hormones, estrogen and testosterone. While the roles of estrogen receptors in cardiac disease are well-studied in animals and humans, respective research on androgen receptors (AR) is limited. Here we investigate AR protein and mRNA expression in human myocardium of various cardiac diseases. Methods: AR expression was analyzed by western blotting in myocardium from human non-failing hearts (NF, n = 6) and patients with aortic stenosis (AS, n = 6), hypertrophic cardiomyopathy (HCM, n = 7), dilated cardiomyopathy (DCM, n = 7), and ischemic cardiomyopathy (ICM, n = 7). Using an AR45-specific antibody, a subsequent western blot assessed samples from male and female patients with HCM (n = 10) and DCM (n = 10). The same sample set was probed for full-length AR and AR45 mRNA expression. Immunohistochemistry (IHC) localized AR in myocardium from HCM and AS hearts. Results: Full-length AR was notably enriched in AS and HCM hearts compared to ICM, DCM, and NF. Similarly, AR45 was more abundant in HCM than in DCM. In contrast to the pattern observed for AR protein, full-length AR mRNA levels were lower in HCM compared to DCM, with no discernible difference for the AR45 isoform. Although gender differences in AR expression were not detected in western blots or qRT-PCR, IHC showed stronger nuclear AR signals in males than in females. Conclusions: Our findings indicate disease-specific regulation of AR mRNA and/or AR protein in cardiac hypertrophy, underscoring a potential role in this cardiac pathology.

Significantly shortened telomere length and altered androgen receptor level in cumulus cells from women with polycystic ovary syndrome

ObjectiveThe aim of this study was to investigate the correlation between hormone receptor levels and telomere length (TL) in infertile women with and without polycystic ovary syndrome (PCOS). Materials and methodsThis prospective cohort study recruited a total of 431 cumulus oocyte complex (COC) from 88 infertile women between July 2012 and June 2014. The participants were divided into three groups: young age (<38 years, n = 42 and 227 COC), advanced age (≥38 years, n = 33 and 107 COC) and PCOS patients (n = 13 and 97 COC). Cumulus cells were collected from individual follicle during oocyte pick-up, and the mRNA levels of hormone receptors and TL were measured using real-time PCR. ResultsThe cumulus cells of PCOS patients demonstrated lower mRNA levels of LH receptor (75.57 ± 138.10 vs. 171.07 ± 317.68; p < 0.01) and androgen receptor (1.13 ± 1.52 vs. 4.08 ± 9.57; p < 0.01), as well as a shorter TL (2.39 ± 2.58 vs. 3.96 ± 4.72; p < 0.01) compared to those of the young age group. In the young age group, only androgen receptor mRNA level showed a significant association with TL (rho = 0.148, p = 0.026), while FSH receptor mRNA level was the only factor associated with TL (rho = 0.247, p = 0.015) in PCOS patients. For advanced-aged patients, no significant relationship was observed between hormone receptor mRNA levels and TL. Alternative splicing of androgen receptors was identified in some PCOS patients but not in young age controls. ConclusionThe findings suggest that the androgen receptor level and function may be altered in the cumulus cells of PCOS patients, leading to a shorter TL in cumulus cells in PCOS patients.

THU573 Paxillin Regulates Androgen Receptor In Granulosa Cells

Abstract Disclosure: A. Weidner: None. A. Roy: None. K. Vann: None. O. Astapova: None. Paxillin is a ubiquitously-expressed adaptor protein that is integral in focal adhesions and plays an active role in the regulation of cell motility and apoptosis. Recently, paxillin was shown to mediate pro-tumorigenic androgen signaling in prostate cancer cells, where it is necessary for rapid cytoplasmic kinase signaling triggered by activation of membrane-bound androgen receptor. We sought to investigate the relationship between paxillin and androgen receptor (AR) in granulosa cells, where androgen actions, apoptosis and focal adhesions are of known importance, but the role of paxillin is understudied. When paxillin expression was silenced using siRNA in human granulosa-derived KGN cells, AR protein expression was significantly reduced, while its RNA expression was unaffected. Similarly, in granulosa cell-specific paxillin knockout mice, AR protein expression in granulosa cells was reduced. Further, we found that AR and paxillin co-localize in focal adhesions in mouse primary granulosa cells and in KGN cells, and their interaction requires the activity of focal adhesion kinase. Disruption of focal adhesion with a focal adhesion kinase inhibitor also leads to reduced AR protein levels. To further explore the mechanisms by which paxillin regulates AR expression, we created paxillin-null KGN cell lines using CRISPR-Cas9 gene editing. While paxillin loss resulted in reduced AR protein expression early (days) in the gene-editing process, this effect disappeared after weeks of clonal expansion from selected single cells. Expanded paxillin-null KGN clones showed normal AR protein expression, but increased AR mRNA expression, suggesting an adaptive increase in AR gene transcription to compensate for reduced AR protein levels in cells that survived paxillin deletion. Studies of AR protein degradation using cycloheximide to block protein synthesis showed that the half-life of AR was reduced from 6.7 to 4.5 hours (approximately 33%) by paxillin deletion, indicating accelerated AR protein degradation in cells lacking paxillin. Based on our results, we hypothesize that paxillin brings AR into focal adhesions in granulosa cells, thereby sequestering it from proteasomal degradation in the cytoplasm. Thus, paxillin may be a novel target in the management of androgen-related disorders in women, such as polycystic ovary syndrome. Presentation Date: Thursday, June 15, 2023

17β-Trenbolone activates androgen receptor, upregulates transforming growth factor beta/bone morphogenetic protein and Wnt signaling pathways, and induces masculinization of caudal and anal fins in female guppies (Poecilia reticulata)

Sexually mature female guppies (Poecilia reticulata) were exposed to environmentally relevant concentrations (20, 200, and 2000 ng/L) of 17β-trenbolone for four weeks. As evidenced by the increased caudal fin index and anal fins developing into gonopodium-like structures, exposed females displayed masculinized secondary sexual characteristics. Differential gene expression and subsequent pathway analysis of mRNA sequencing data revealed that the transcription of transforming growth factor beta/bone morphogenetic protein signaling pathway and Wnt signaling pathway were upregulated following 17β-trenbolone exposure. Enzyme-linked immunosorbent assays showed that the bone morphogenetic protein 7 protein content was elevated after 17β-trenbolone exposure. Finally, real-time PCR revealed that 17β-trenbolone treatment significantly increased androgen receptor mRNA levels, and molecular docking showed potent interaction between 17β-trenbolone and guppy androgen receptor. Furthermore, 17β-trenbolone-induced masculinization of caudal and anal fins in female guppies, concomitant to the upregulated expression of differentially expressed genes involved in the above-mentioned two signaling pathways, was significantly inhibited by flutamide (androgen receptor antagonist). These findings demonstrated that 17β-trenbolone masculinized fins of female guppies by activating the androgen receptor. This study revealed that 17β-trenbolone could upregulate signaling pathways related to fin growth and differentiation, and eventually cause caudal and anal fin masculinization in female guppies.

In Vitro Assessment of the Antioxidant and Anticancer Properties of Flammulina velutipes Stipe Extracts.

Flammulina velutipes (FV), also known as the golden needle mushroom, is an edible and medicinal fungus that contains bioactive substances regulating various physiological functions. While the fruiting bodies of FV are commonly consumed, their stipes are often discarded despite containing polysaccharides. In this study, the biological functions of FV stipes (FV-S) were investigated to reduce waste and pollution while increasing their value. The antioxidant activity of FV was evaluated using three methods: the DPPH radical-scavenging capacity assay, ferrous ion chelating assay, and reducing power analysis. The anti-cancer potential was assessed through MTT viability and immunoblotting analyses. Results showed that FV-S had higher polysaccharide and total phenolic contents and greater antioxidant abilities, particularly in ethanolic extracts. FV-S also exhibited significant anticancer properties, specifically in hot water extracts with high polysaccharide contents, and suppressed prostate cancer cell viability by inhibiting androgen receptor and PCa-specific antigen mRNA expression while inducing caspase-3/7 activation. FV-S is rich in bioactive components, possesses higher antioxidant and anticancer abilities, and has potential as an anticancer agent, which could enhance the value of FV.

P-045 Effects of seminal BPA levels on human sperm parameters and gene expression

Abstract Study question Are high BPA levels in human seminal fluid associated with low sperm metrics and changes in gene expression? Summary answer A trend towards an inverse relationship between seminal BPA and sperm count was observed. Expression of androgen receptor mRNA was down-regulated with high BPA exposure. What is known already Bisphenol A (BPA) is a widespread industrial chemical, used as the key monomer of polycarbonate plastics and epoxy resins. While some countries have introduced bans/stringent regulation for BPA, it continues to be widely used especially in North America, where its use is only limited in baby products. BPA has been detected in human seminal fluid and has been correlated with decreased sperm counts and motility - crucial factors in determining male fertility, which continue to decrease. We have demonstrated that in vitro BPA exposure can reduce motility, mitochondrial membrane potential, and capacitation in mature bovine spermatozoa. Study design, size, duration In collaboration with CCRM, Toronto, seminal fluid samples were collected from 90 patients undergoing fertility assessment between 2021 and 2023. Typical evaluations such as sperm counts, motility, and morphology were recorded by the clinic (following WHO guidelines). Samples were then shipped to the University of Guelph for further investigations. Information on outcomes for patients who underwent subsequent IVF cycles like fertilization rates and pregnancy rates were also recorded at the clinic. Participants/materials, setting, methods At the University of Guelph, seminal BPA levels were measured through an enzyme-linked immunosorbent assay (ELISA; My BioSource), then compared to sperm metrics obtained by the clinic. Next, a GeNorm analysis was performed to determine the most stable reference genes for subsequent qPCR analysis. The relative mRNA expression of three key genes related to male reproduction (androgen receptor (AR), cyp17a1 and cyp11a1) were quantified and compared between groups with high and low BPA content. Main results and the role of chance BPA concentrations in the seminal fluid samples ranged between 0-3000 pg/mL, with most samples containing 35-1000 pg/mL. When samples were graphed according to increasing BPA levels, no relationships were observed between BPA and motility or morphology rates. However, a negative trend between BPA and sperm count was detected, although this was not significant. Samples were then divided into high- (total 15 samples with &amp;gt;700 pg/mL) and low- (total 15 samples with &amp;lt;100 pg/mL) BPA content groups. The GeNorm M value indicated that HPRT1 and HMBS were the most stable reference genes across the groups. qPCR analysis on three biological replicates in technical triplicates showed that androgen receptor mRNA expression was significantly higher in sperm exposed to low seminal BPA compared to the high BPA exposure group (p = 0.03913). This finding is particularly interesting as AR expression has previously been positively correlated with sperm count, motility and morphology. cyp17a and cyp11a mRNA expression was undetectable in human spermatozoa under our experimental conditions. Future steps include increasing the sample size to increase the power of the correlation studies between sperm quality parameters and ART success rates. Furthermore, other fertility-related genes will be investigated at both the mRNA (qPCR) and protein levels (flow cytometry). Limitations, reasons for caution Relatively small sample size; duration of study must be extended to gain information on outcomes of ART cycles; many samples in the mid-range of BPA are excluded from gene expression studies. Wider implications of the findings These results can ultimately display a relationship between BPA exposure and male fertility and provide further insights into the molecular mechanisms through which BPA exerts its effects. Ultimately, this research can drive changes to guidelines/exposure limits for BPA, especially in North America. Trial registration number not applicable