Evaluation of androgen receptor markers in erectile dysfunction

Steroid hormones, such as testosterone, play a crucial role in modulating the development of male internal and external genitalia as well as secondary sex characteristics by binding to the androgen receptor. Once bound, androgen receptor operates as an inducible transcription factor, interacting with a multitude of co‐regulators to initiate various downstream signaling pathways. The androgen saturation hypothesis posits that beyond a specific threshold, androgen receptor binding and functionality remain unaltered despite an increase in serum testosterone levels.


INTRODUCTION
Androgens are steroid hormones produced by the testes and adrenal cortex that are responsible for the development and maintenance of internal and external male characteristics. 1 Testosterone (T) is carried by sex hormone binding globulin and albumin in the blood to reach target tissues, where the lipophilic molecule easily diffuses into cells. 2ile in some tissues (including male reproductive tissues such as the penis and prostate), the presence of 5a-reductase leads to the rapid and irreversible conversion of T to dihydrotestosterone (DHT), both T and DHT exert their physiological functions by binding to the androgen receptor (AR). 1 The AR is a nuclear receptor that acts as an inducible transcription factor to control the expression of protein coding genes and noncoding RNAs when activated. 3,4When bound by ligands and activated, AR binds to specific DNA sequences in the regulatory regions of target genes called androgen response elements. 2,5ARs are found throughout the body, with their highest density found in male reproductive tissues such as the prostate and penis, where they modulate the development of male secondary sex characteristics, spermatogenesis, and bone health. 6,7Importantly, AR-mediated T impacts in utero include the development and growth of the external penis, penile urethra, and corpus cavernosum, and a high density of ARs appears to remain in these structures into adulthood. 8like other transcription factors, the AR coregulators generally do not bind to DNA or affect the transcription rate.Because of the vast and diverse functions of the AR in normal physiology and pathophysiology, researchers have sought ways to measure its function.They have pursued the measurement of coregulators, downstream endothelial proteins, and induced gene expression such as vascular endothelial growth factor. 97][18] In this study, we sought to investigate the presence and signaling of ARs in the corpus cavernosum of men with severe ED in order to better elucidate the role of serum T on AR function and identify a potential saturation value in diseased penile tissue.

Patient selection
Patients undergoing surgical management for high-grade ED at the Lennar Clinic of the University of Miami between July 2022 and Jan-uary 2023 were invited to participate in the study.Patients were identified as having high-grade ED based on moderate to severe symptoms (International Index of Erectile Function score ≤16) and failure of response to medical management (oral phosphodiesterase inhibitors, intraurethral suppositories, intracavernosal injections) for their ED. 19,20Total T levels were obtained on the morning of surgery according to the institutional protocol.Patients who were undergoing revision or replacement of penile prosthesis or malleable penile prosthesis were excluded from the study.Demographic information, including prior urologic history and treatment, was obtained from electronic medical records.All the patients provided informed consent prior to study procedures.This study was approved by the University of Miami IRB (#20210887).Statistical analysis was performed with T test in Excel.

Tissue collection and preparation
During the surgery, corpus cavernosum biopsy was obtained.The tissue was immediately removed from the operating field and stored in a sterile specimen cup with 2 mL of sterile, injectable saline.
The tissue was transferred to a cryogenic vial and flash frozen using liquid nitrogen.It was then stored at −80 • C until processing.
When 12 samples were obtained, they were used for western blot analysis.

Western blot analysis
The tissue was homogenized using a mechanical homogenizer and RIPA lysis buffer.Lysis buffer contained protease inhibitor (Cat# P8340, Sigma-Aldrich).Protein concentration was measured using a standard Bradford assay according to manufacturer's protocol (Bio-Rad protein assay kit, Bio-Rad).Next, 250 µg of protein was loaded into two 10% polyacrylamide Tris-glycine extended mini gels (Bio-Rad), and electrophoresis was performed with SDS running buffer to separate proteins according to the manufacturer's protocol.Polyvinylidene difluoride membranes were used for protein transfer (Millipore).The membranes were blocked in a 5% non-fat dry milk and 1% Tween Trisbuffered saline solution.The western blots were incubated overnight at 4 • C with each primary antibody (Table 1) and then washed and incubated for 1.5 h with the secondary antibody (

Participant demographics and serum testosterone levels
A total of 12 men, aged between 47 and 75 years, agreed to participate in this study, with a median age of 61 years (interquartile range [IQR] 55, 66).Serum T levels were obtained from all participants on the morning of their respective surgeries.The recorded T levels varied from 2.5 to 724.3 ng/dL, with a median value of 300.15 ng/dL (IQR 190.95, 435.55).

Expression analysis of AR, HO, iNOS, and PDE-5
AR signaling was evaluated by measuring the expression levels of AR, HO, iNOS, and PDE-5 proteins.GAPDH was utilized for protein normalization (Figure 1).Full images of each blot may be seen in Supporting Information.

Influence of serum testosterone levels on protein expression
Our findings revealed a significant decrease in AR expression at serum T levels below 300 ng/dL when compared with to serum T levels above 300 ng/dL (p = 0.022).Similarly, HO expression levels were significantly lower at serum T concentrations below 200 ng/dL compared with levels above 200 ng/dL (p = 0.017).The expression of iNOS was also significantly reduced at serum T levels below 300 ng/dL relative to serum T levels above 300 ng/dL (p = 0.03).Last, we observed a significant decrease in PDE-5 expression at serum T levels below 200 ng/dL compared with serum T levels above 200 ng/dL (p = 0.014).
Together, we investigated the relationship between serum T levels and the expression of AR, HO, iNOS, and PDE-5 proteins in men with severe ED.Our results demonstrated a significant decrease in the expression of these proteins at lower serum T levels, suggesting a potential correlation between serum T concentration and AR signaling.
These findings contribute to our understanding of the role of serum T in regulating AR function and provide valuable insights for further research into potential saturation values.

DISCUSSION
The results of our study showed that among a wide range of serum T levels (2.5-724.3ng/dL), AR signaling was similar in diseased penile tissue.The lack of variation in downstream proteins suggests that, above a threshold value, excess T does not affect AR function in the corpus cavernosum.This suggests that there is a saturation value for penile ARs, in support of the previously described saturation hypothesis.Based on our data and in support of previously published research, this value appears to be between 200 and 300 ng/dL.
The saturation hypothesis was first introduced in prostate cancer research as a counterargument to the well-described model of prostate cancer progression dependent on T levels. 213][24] This hypothesis clarifies how serum T alterations near the castrate range produce profound effects on the prostate, while physiological and supraphysiological serum T levels appear to have little to no effect. 25ile the AR saturation hypothesis was originally developed to better characterize the impact of T on prostate cancer, because of its development, it has been applied to other functions of T. While hypogonadism alone is rarely the sole cause of ED, normal erectile function does appear to depend on androgens.26 In the case of erections, castration generally causes a decline in sexual function (including spontaneous erections, erectile strength, and duration of erections), which can be restored with even a relatively small amount of exogenous T therapy (TT).[27][28][29][30] It remains debatable what level of hypogonadism is required to induce or treat ED, but it appears that above a certain threshold, additional androgen stimulation does not change the frequency, amplitude, or rigidity of erections.27 Additionally, studies have shown that this threshold is likely to be below the normal physiological range, 31 thus supporting the previously described saturation hypothesis and our saturation value.
Understanding AR saturation and function in men with ED is of high clinical importance.ED affects 18% of adult men in the United States, and up to 40% of them demonstrate significant improvement in their ED symptoms during treatment with exogenous TT. 16,32 Importantly, our data highlight the role of serum T in AR function and help explain why some patients may not respond to medications targeting these pathways.While prior anecdotal evidence has suggested that patients with low T may not respond to pharmacotherapy for their ED such as PDE-5 inhibitors, our data show that this may be because of altered AR signaling in hypogonadal men, negating the impact of these drugs.
While American Urologic Association and European Urologic Association guidelines for the management of ED recommend measuring serum T and treating hypogonadism, these remain moderate evidence guidelines partially because of a lack of mechanistic data explaining exactly how T plays a role in erectile function. 33,34ditionally, this work is of high clinical importance because of the prostate and cardiovascular risks among those being treated with T.
Understanding the AR saturation hypothesis may provide context for appropriate prescription of TT to provide optimal patient outcomes and personalized patient care.
Our study is not without limitations.First, our small sample size of 12 participants introduces bias.We attempted to minimize these impacts through careful matching based on the T level, age, and medical history.We recognize that acute stress on the morning of surgery may artificially lower the serum T measurements.Second, while human and animal studies have suggested that the measurement of downstream AR mediators is an effective substitute for the direct measurement of AR-mediated T function 11,35 these experimental methods do not provide functional data.Rather, we show the quantity of each downstream protein and postulate that these markers reflect the AR function.
Within these experimental methods, we acknowledge difficulty extrapolating functional information about the AR from the western blot.For example, inherent variability in protein expression in each sample is present in human tissue (visible as varied control, GAPDH, expression despite equal protein concentration loading).Third, all participants in this study presented with severe ED, and therefore, we cannot draw conclusions about AR saturation in men with physiological erectile function or mild ED.While we hypothesize that these findings may be applicable to the general male population, at this time we are unable to conclude that AR signaling may behave as described in these experiments in any population besides those with high-grade ED.
Despite these limitations, this study confirms and expands on our prior work by suggesting an AR saturation value. 36There remains a shortage of experimental data published on the AR saturation hypothesis, as well as a lack of published work showing pathological changes in AR signaling in the corpus cavernosum of men with penile disease.
We hope that future work in our lab and others can (1) use larger sample sizes to confirm our findings, (2) involve castrate men to narrow down a more exact saturation value, and (3) perform functional studies involving AR and downstream mediators to better understand the mechanism by which AR saturation occurs.

CONCLUSION
Testosterone is a steroid hormone that has numerous impacts on normal male physiology through its interaction with the androgen receptor.The androgen receptor has numerous coregulators and instigates numerous intracellular processes as a transcription factor when activated by testosterone and dihydrotestosterone.Despite a prior understanding that increased testosterone leads to increased androgen receptor activity, the androgen receptor saturation hypothesis suggests a uniform serum testosterone level above which androgen receptor signaling is similar.This work presents the most comprehensive investigation into androgen receptor saturation in men with erectile dysfunction and demonstrates that above 200-300 ng/dL, androgen receptor signaling appears to be similar.Further investigation is necessary to understand the mechanisms by which androgen receptor saturation occurs and its downstream implications on disease and treatment options for men with erectile dysfunction.

Table 2
Western blot secondary antibody dilutions.
A). First, background noise was subtracted for the blot.Then, to compensate for human sample variability, AR intensity was normalized to the corresponding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) band intensity.Each blot was probed with one high molecular weight and one low molecular weight primary antibody for maximum visualization.TA B L E 1 Western blot primary antibodies.Abbreviations: AR, androgen receptor; GAPDH, glyceraldehyde-3phosphate dehydrogenase; HO, heme oxygenase; iNOS, inducible nitric oxide synthase; PDE-5, phosphodiesterase type 5; VEGF, vascular endothelial growth factor.