Androgen Receptor Antagonist – Diphenyleneiodonium

Testosterone up-regulates vacuolar ATPase expression and functional activities in vas deferens of orchidectomized rats

A B S T R A C T
Precise regulation of vas deferens ﬂuid pH is essential for sperm. However, the mechanisms underlying effect of testosterone on vas deferens ﬂuid pH have never been identiﬁed, which could involve changes in expression and functional activity of vacoular (V)-ATPase.Methods: Orchidectomized, adult male Sprague-Dawley rats were treated subcutaneously with 125 mg/ kg/day and 250 mg/kg/day testosterone with or without ﬂutamide (androgen receptor blocker) and ﬁ- nasteride (5a-reductase inhibitor) for seven (7) days. Following treatment completion, in vivo perfusion of vas deferens lumen was performed and changes in ﬂuid secretion rate, pH and HCO—3 content were measured with and without baﬁlomycin, a V-ATPase inhibitor. Rats were then sacriﬁced and vas deferenswere harvested and subjected for V-ATPase A1 and B1/2 protein expression and distribution analysis by western blotting and immunohistochemistry, respectively.Results: In sham-operated and testosterone-treated orchidectomized rats, higher ﬂuid secretion rate, which was not antagonized by baﬁlomycin but lower HCO—3 content and pH which were antagonized by baﬁlomycin were observed when compared to orchidectomized-only and orchidectomized, testosterone-treated rats receiving ﬂutamide or ﬁnasteride, respectively. Baﬁlomycin had no effect on ﬂuid secretion rate, HCO3— content and pH in orchidectomized and testosterone-treated orchidectomized rats receiving ﬂutamide and ﬁnasteride. V-ATPase A1 and B1/2 proteins were expressed at high levels in vas deferensand were highly distributed at the apical membrane of luminal epithelium and in muscle layer of this organ, mainly in sham and testosterone-treated orchidectomized rats.Conclusions: V-ATPase is involved in acidiﬁcation of vas deferens ﬂuid under testosterone inﬂuence.

1.Introduction
Testosterone plays important role in the development and function of the male reproductive tract. However, its role in the regulation of vas deferens ﬂuid pH remains unknown. Vas deferens plays important role in transporting the sperm from the epididymis to the ejaculatory ducts with pH and electrolytes content of its ﬂuid were precisely regulated. These regulation is crucial for sperm survival [1]. The pH of epididymal and vas deferens ﬂuid is reported to be acidic [2]. Acidiﬁcation of the luminal ﬂuid pH in general is reported to involve the membrane transporter proteins including vacuolar (V)-ATPase [2,3]. The acidic pH of vas deferens ﬂuid is necessary to maintain the sperm quiescence and to preventpremature activation of sperm’ acrosomal enzymes during their storage in cauda epididymis and vas deferens [4].V-ATPase is a complex enzyme that is composed of several subunits, which are assembled into two domains: trans-membrane (V0) and cytosolic (V1) domains [3,5]. Epididymal epithelial cells of rats has been reported to contain V-ATPase which is distributed at the apical pole of the narrow and clear cells [2]. These cells are known to be involved in generating low epididymal ﬂuid pH [6]. Some of V-ATPase subunits have more than one isoform [7]. As an example, four “a” isoform and two “b” isoform of V-ATPase subunit are present in the human genome [8,9]. Subunit a1 was expressed ubiquitously while subunit B1 was found in the epididymis and other tissues, while B2 was found to be ubiquitously expressed in the intracellular organelles [9].

The importance of V-ATPase to male reproduction has been demonstrated in mice in which lack of transcription factor, Foxil, a master regulator of V-ATPase in clear cells of epididymis affects male fertility due to inability of the sperm to move up the female genital tract to fertilize the oocyte [10e12].In epididymal clear cells, V-ATPase function is under hormonal controls with hormone known to inﬂuence V-ATPase activity include angiotensin II (AngII). Ang II stimulates V-ATPase function via binding to Ang II type II receptors (AGTR2), which are found in the adjacent basal cells [13]. Once binding to AGTR2, nitric oxide (NO) is released from the basal cells which then diffuses out to stimulate proton secretion in clear cells via activation of cGMP pathway [14]. In vas deferens, the role of hormones in controlling V-ATPase expression and functional activity is largely unknown. Therefore, in this study, we hypothesized that testosterone affects V-ATPase expression and functional activity in vas deferens through which this hormone control ﬂuid pH of this organ. Thus the objectives of this study were to identify the functional activity, expression level and distribution of V-ATPases in vas deferens un- der testosterone inﬂuence. The ﬁnding of this study could help to explain the mechanisms underlying testosterone-mediated effect on vas deferens ﬂuid pH where dysregulation could adversely affect male fertility.

2.Materials and methods
All experimental protocols were approved by the Institutional Animal Ethics Committee, University of Malaya with ethics num- ber: 201405-07/PHYSIO/R/NS (2014/85). Adult male Spra- gueeDawley rats weighing 200e225 g were obtained from Animal House, Faculty of Medicine, University of Malaya and were housedunder standardized housing condition (temperature: 23 ± 2 ◦C, 12/12-h lightedark cycle, 30%e70% humidity). The rats had free access to rodent diet (Harlan, Rossdoff, Germany) and tap water ad libitum. Orchidectomy was performed following the previously described method [15,16]. Following orchidectomy, rats were given intra- muscular injection of 0.1 ml Kombitrim antibiotic to avoid post- surgical wound infection.Testosterone propionate (SigmaeAldrich, MO, USA) was dis- solved in ethanol prior to mixing with peanut oil. Three weeks after orchidectomy, drugs were subcutaneously administered for seven(7) days at the neck scruff. The doses of testosterone were selected on the basis of the previously reported doses [17e19]. Animals were 24 h after the last drug injection, in-vivo perfusion of vas def- erens lumen was performed with the rats under anesthesia. Following completion of the perfusion experiment, rats were sacriﬁced via cervical dislocation and vas deferens were immedi- ately removed and then stored in an appropriate medium prior to analyses of protein expression and distribution. Following perfusion experiment, histology was performed to determine the integrity of vas deferens epithelium, where the epithelial lining was found to be intact (data not shown). In the meantime, blood was collected via direct heart puncture in order to determine the plasma testosterone levels using ELISA kit (ALPCO Diagnostic, Salem, NH, USA).In order to investigate the changes in secretion rate, pH and HCO3— content of vas deferens ﬂuid, in vivo perfusion of vas deferens lumen was performed according to the methods as previously described [20,21], but with a slight modiﬁcation. In brief, anes-thetized rats were placed on a heat pad to maintain their constant body temperature.

An incision was made in the genital area in order to expose the cauda epididymis and vas deferens. Then, a blunt-end 27G needle, connected to a 3 cc syringe attached to a perfusion pump (Harvard apparatus, MS, USA) was inserted into the proximal end of vas deferens. The distal end of vas deferens (at the junction with epididymis) was cut open and directly placed above the opening of an eppendorf tube. Perfusion was conducted over the period of 3 h, at a rate of 0.25 ml/h.The perfusate contains the following compositions: 50mosmol/l NaCl, 50mosol/l K gluconate, 1.2 mosmol/l MgSO4, 0.6 mosmol/l CaCl2, 4 mosmol/l Na acetate, 1 mosmol/l trinatrium citrate, 6.4 mosmol/l NaH2 PO4 and 3.6 mosmol/l Na2HPO4. The pH of the perfusate was adjusted to 6.8, using 350e360 mosmol (kg H20)—1rafﬁnose In order to detect the functional activity of V-ATPase,baﬁlomycin A1 (Santa Cruz, CA, USA), a V-ATPase inhibitor was dissolved in the perfusate at 2 mM, and then perfused. To determine the changes in ﬂuid secretion rate, net weight of the collected ﬂuids was divided by the total perfusion time (180 min). All tubes were weighed by using an electronic balance prior to and after perfusate collection.The difference between tube weight before and after perfusate collection was considered as the rate of ﬂuid secretion. pH of the perfusate was directly measured by using aquatwin pH meter (Horiba Scientiﬁc, Japan). HCO—3 concentration was determined by using enzymatic assay of phosphoenolpyruvate carboxylase (PEPC)and malate dehydrogenase (MDH) where the end products were measured by using a spectrophotometer at wavelength 405 or 415 nm. Changes in the colour were directly proportional to the HCO—3 concentration in the samples.Immunohistochemistry was performed following the methods as previously described [22].

Firstly, immediately after harvesting, vas deferens was ﬁxed in 10% formalin overnight prior to pro- cessing, following then, tissues were dehydrated through increasing concentrations of ethanol, cleared in chloroform and blocked in parafﬁn wax. Tissues were then sectioned into 5-mm thickness, deparafﬁnized in xylene followed with rehydration in reducing concentrations of ethanol. Antigen retrieval was per- formed by using Tri-EDTA buffer (10-mM Tris base, 1-mM EDTA, 0.05% Tween 20, pH 9.0). Endogenous peroxidase was neutralize by using 1% H2O2 in methanol. The sections were then blocked with a blocking serum to prevent the non-speciﬁc binding. This was per- formed prior to incubation with VATPase A1 (sc-28801, Santa Cruz, CA, USA) and VATPase B1/2 (sc-20943, Santa Cruz, CA, USA) anti- bodies, at 1:100 dilution. Sections were incubated with these an-tibodies at 4 ◦C overnight. 24 hrs later, sections were rinsed withPBS, three times, 5 min each, prior to incubation with biotinylated secondary antibody for 1 h at room temperature. Proteins were localized by diaminobenzidine HCL (Santa Cruz Biotechnology, CA, USA) staining where dark brown precipitate will appear at the site of primary antibody binding to horseradish peroxidase. Sections were then rinsed with deionized water for 5 min and counter- stained with hematoxylin to visualize the nuclei. Slides were then dehydrated with different dilutions of ethanol and xylene before adding a drop of mounting medium.Protein extraction and western blotting were performed following the methods as previously described [23].

Firstly, vasdeferens was snapped frozen in liquid nitrogen and stored at —80 ◦C. Protein extraction was performed by using PRO-PREP solution (Intron, Korea). Brieﬂy, 40-mg proteins were mixed withloading dye, separated, transferred onto polyvinylidene diﬂuoride membranes (Bio-Rad, Hercules, CA, USA). The proteins were then blocked with 5% BSA for 90 min at room temperature. The mem- branes were then exposed overnight to V-ATPases primary anti- bodies (as above) at a dilution of 1:1000 in PBS containing 1% BSA and Tween-20 overnight, then rinsed three times in PBS-T, 5 min each.The membranes were then incubated with anti-rabbit horse- radish peroxidaseeconjugated secondary antibody (Santa Cruz, CA, secretion rate was higher in sham-operated rats as compared to orchidectomized control rats. Baﬁlomycin A1 administration had no effect on ﬂuid secretion rate in all groups.Vas deferens ﬂuid pH was lowest in rats receiving 250 mg/kg/day testosterone (Fig. 1B) Orchidectomy caused ﬂuid pH to increase (p < 0.05 as compared to sham-operated rats). In testosterone- treated rats receiving ﬂutamide and ﬁnasteride, vas deferens ﬂuid pH was higher than the rats receiving testosterone-only treatment (p < 0.05). Administration of baﬁlomycin A1 caused signiﬁcant in- crease in vas deferens ﬂuid pH in sham and orchidectomized testosterone-treated rats (both 125 mg/kg/day and 250 mg/kg/day testosterone) (p < 0.05). However, baﬁlomycin had no effect on ﬂuid pH in orchidectomized rats and in testosterone-treated rats concomitantly receiving ﬂutamide and ﬁnasteride.HCO—3 content was lowest in rats receiving 250 mg/kg/day testosterone (Fig. 1C). In orchidectomized control rats, HCO—3 con- tent was higher than sham-operated rats (p < 0.05). Meanwhile, co-administration of 125 mg/kg/day and 250 mg/kg/day testosterone with ﬂutamide and ﬁnasteride caused HCO—3 content to increase. USA) at a dilution of 1:500, for 90 min. Membranes were rinsed and Baﬁlomycin had no effect on vas deferens ﬂuid HCO—3 content subjected to Opti-4CN Substrate Kit (Bio-Rad) to visualize the protein bands. Images of the bands were captured by using a gel documentation system, and density of each band was determined by ImageJ software (version 1.46j; National Institutes of Health, Bethesda, MD, USA). The ratio of each protein/b-actin was calcu- lated and was considered as the expression level of the target.One-way ANOVA was used to evaluate the statistical differences. A probability level of less than 0.05 (P < .05) was considered as signiﬁcant. Tukey's post hoc analysis was performed, and all values were greater than 0.8, which indicates adequate sample size. Sha- piroeWilk test was performed, and all values were greater than 0.05, which indicates data normality.For protein expression and immunoﬂuorescence, mean value in each group was obtained from four (4) different rats receiving same treatment, while for functional study, data were obtained from six(6) rats receiving similar treatment. 3.Results Plasma testosterone level following orchidectomy (0.5 ± 0.43 nmol/l) was markedly reduced when compared to its level in sham-operated rats (6.2 ± 0.43 nmol/l) (p < 0.05). Plasma level of testosterone signiﬁcantly increased in orchidectomized rats following treatment with 125 mg/kg/day testosterone (7.5 ± 0.43 nmol/l) and further increased following treatment with 250 mg/kg/day testosterone (18.3 ± 0.43 nmol/l) (p < 0.05).Fluid secretion rate was highest in rats receiving 250 mg/kg/day testosterone, followed by rats receiving 125 mg/kg/day testosterone (Fig. 1A). In rats receiving 125 mg/kg/day and 250 mg/kg/day testosterone, co-administration of ﬂutamide and ﬁnasteride resulted in decreased in ﬂuid secretion rate (p < 0.05). Fluid except in sham and testosterone-treated orchidectomized rats where the HCO3— content was signiﬁcantly increased (p < 0.05).Immunohistochemistry images showed V-ATPase A1 protein was mainly distributed at the apical membrane of the epithelium lining the vas deferens lumen (Fig. 2A). V-ATPases A1 was also found to be distributed in the muscles of vas deferens. However, its distribution in the epithelium was higher than the muscle. Rela- tively higher distribution of V-ATPase A1 could be seen in sham- operated rats and in orchidectomized rats receiving testosterone- only treatment (as compared to orchidectomized rats and testosterone-treated orchidectomized rats receiving ﬂutamide and ﬁnasteride, respectively) and for the latter, the distribution was relatively higher following treatment with 250 mg/kg/day testos- terone as compared to 125 mg/kg/day testosterone.Expression level of V-ATPase A1 protein was higher in vas def- erens of sham-operated rats and in rats receiving testosterone-only treatment, as compared to orchidectomized control rats (p < 0.05) (Fig. 2B). Higher amount of V-ATPase protein was observed following treatment with 250 mg/kg/day testosterone (20-fold) as compared to 125 mg/kg/day testosterone. In 125 mg/kg/day and 250 mg/kg/day testosterone-treated orchidectomized rats, concomitant administration of ﬂutamide or ﬁnasteride resulted in V-ATPase protein expression level to decrease (p < 0.05).Immunohistochemistry images showed V-ATPases B1/2 was distributed at the apical membrane of the luminal epithelium and muscles of vas deferens (Fig. 3A). In sham-operated rats, V-ATPases B1/2 distribution was relatively higher that orchidectomized rats. Relatively higher V-ATPases B1/2 distribution was also observed in orchidectomized rats following 125 mg/kg/day and 250 mg/kg/day testosterone treatment. However, in testosterone-treated orchid- ectomized rats, co-administration of ﬂutamide and ﬁnasteride caused distribution of V-ATPases B1/2 in vas deferens to relatively decrease.Higher V-ATPase B1/2 protein expression level was also observed in sham-operated rats as compared to orchidectomized rats (p < 0.05). Meanwhile in orchidectomized rats, expression levelof V-ATPase B1/2 protein increased with increasing doses of testosterone (Fig. 3B). Treatment with 125 and 250 mg/kg/day testosterone resulted in 7 and 11- fold higher V-ATPase B1/2 pro- tein expression level in vas deferens as compared to orchid- ectomized control rats, respectively. In testosterone-treated orchidectomized rats, co-administration of ﬂutamide or ﬁnasteride resulted in V-ATPase B1/2 protein expression level to decrease (p < 0.05). 4.Discussion In this study, orchidectomy was performed in order to minimize the inﬂuence of endogenous testosterone on vas deferens morphology and functions. Our ﬁndings, which found that plasma testosterone level in rats decreased following orchidectomy, was consistent with the report by previous study [19], which indicate that the main source of testosterone in males is the testes. In males, no other organs were able to compensate for the decrease in testosterone production by the testis, and this was in contrast to the female where elevation in estrogen levels following ovariectomy was due to compensatory hyperplasia of the adrenal gland [24].Our ﬁndings further demonstrated that testosterone was able to induce secretion of ﬂuid in vas deferens, as well as causing the ﬂuid pH and HCO—3 content to decrease. A report showed that testos-terone was able to induce ﬂuid and electrolyte secretion inandrogen-sensitive male reproductive organs including seminal vesicles and prostate [25]. The ﬁndings from this study which found that vas deferens ﬂuid secretion rate, pH and HCO—3 content couldbe inﬂuence by testosterone further added information withregards to the role of this hormone in controlling vas deferens ﬂuid parameters. In this study, in vivo perfusion technique was used, where changes in the secretion rate, pH and electrolyte content of vas deferens ﬂuid were monitored in life animal model. Therefore, this technique ideally reﬂects changes in the internal milieu of vas deference ﬂuid environment [20].The ﬁndings from this study have shown that V-ATPase is achannel that is involve in testosterone-induced acidiﬁcation of vas deferens ﬂuid. A speciﬁc V-ATPase inhibitor, baﬁlomycin A1 was added to conﬁrm the functional activity of V-ATPase where upon its administration, testosterone-induce decrease in the pH and HCO—3 content of vas deferens ﬂuid was reversed. The effect of baﬁlomycinconﬁrms the role of V-ATPase in mediating these changes. Baﬁlo- mycin A1 is a macrolide antibiotic isolated from Streptomyces spe- cies and has been reported able to inhibit V-ATPase via binding to the V0 sector subunit c of V-ATPase complex which results in in- hibition of Hþ translocation, thus inducing accumulation of Hþ inthe cytoplasm of the cells [26]. However, baﬁlomycin A1 has noeffect on vas deferens ﬂuid secretion rate in rats which either possess endogenous testosterone or those treated with exogenous testosterone. These suggested that testosterone-induced ﬂuid secretion in vas deferens could involve other protein transporter such as aquaporins, which is reported to be present in vas deferens [27]. The role of aquaporins in vas deferens ﬂuid secretion need further elucidation. The ﬁnding that V-ATPase is involve in luminal ﬂuid acidiﬁcation is consistent with its role in causing low luminal ﬂuid pH in many organs [28]. This effect is important since maintaining the low pH of vas deferens ﬂuid is crucial for sperm survival as well as help to inhibit sperm activation [4]. Failure of vas deferens ﬂuid acidiﬁcation could result in poor sperm maturation and this can also cause premature motility of the sperm [6]. It is also reported that in vas deferens, V-ATPase contributes to approxi- mately 80% of proton secretion into the lumen [29], implying that this channel is indeed essential for vas deferns ﬂuid acidiﬁcation. In this study, signiﬁcant reduction in the HCO—3 content in sham-operated and testosterone-treated rats following baﬁlomycin A1 administration suggested that V-ATPase could be involved in mediating the decrease in vas deferens ﬂuid HCO—3 content, mostprobably due to the increase in Hþ secretion that neutralizes theHCO3—. It is generally known that besides increased in Hþ content, HCO—3 content of the luminal ﬂuid can also be affected by intra- cellular pH, CO2 partial pressure as well as glucose [30e34]. Besidesits ability to stimulate proton secretion, testosterone was also re- ported able to modulate the transport of ions across the vas def- erens epithelium [35], which might also affect the luminal HCO—3content. Other possibilities were that testosterone could modulatethe HCO—3 trsnporters such as Cystic Fibrosis transmembrane regulator (CFTR), Naþ/HCO3— co-transporters (NBC) and Cl—/HCO—3exchanger. The possible role of anion transporter/exchanger in mediating changes to vas deferens ﬂuid pH was supported by a report which indicated a concerted action between V-ATPase andNaþ—HCOe cotransporter in regulating acidiﬁcation and/or HCO— parameters. AR has a direct role in the up-regulation of V-ATPase A1 and B1/2 expression by testosterone. Besides, the observed testosterone effects were most probably mediated via DHT as V- ATPase expression decreased following administration of 5a-reductase inhibitor which inhibits testosterone conversion to DHTreabsorption of the excurrent ductal ﬂuid of the testis [36]. Besides, the presence of Naþ—HCOe cotransporter (SLC4A4) has also been reported in the vas deferens in mice [37]. Nevertheless, the physical and functional relationship between these transporters in pH regulation in vas deferens have yet to be identiﬁed.In the meantime, this study has shown the direct involvement of androgen receptor (AR) and dihydrotestosterone (DHT) in medi- ating testosterone effect on vas deferens ﬂuid secretion, its pH and [38]. The involvement of AR suggests that genomic mechanism is likely to be involved, where upon binding of the ligand to AR, transcription factor will be activated which results in transcription of androgen-responsive genes [39]. The role of AR in the male reproductive organs have been reported. Cultured vas deferens epithelial cells have been found to contain high levels of AR, which expression can be induced by androgen [40]. There were evidences which showed that DHT can bind directly to AR in vas deferens of HCO—3 content. Higher pH and HCO3— content were observed humans [41]. Further, Jean Faucher et al. [42] reported that the following concomitant administration of testosterone with ﬂuta- mide and ﬁnasteride, indicating that blocking testosterone action or DHT action/formation could have reversal effect on the studied proximal region of vas deferens contains higher amounts of an- drogens (testosterone þ DHT) as compared to caput epididymidis. Besides, the important role of DHT is reﬂected by high levels of 5a- reductase in vas deferens and other male accessory reproductive organs such as epididymis, where this enzyme is involve in DHT synthesis from testosterone [43,44].Distribution of V-ATPase further conﬁmed its role in vas defer- ens ﬂuid acidiﬁcation. V-ATPases is present at the apical membrane of vas deferens and its relatively higher distribution under testos- terone inﬂuence suggested its direct involvement in establishing the acidic vas deferens ﬂuid pH. We have also shown that V-ATPase was also distributed in the smooth muscle of vas deferens, however its role is unknown. Kurauchi-Mito, Ichihara [45] suggested that there might be interaction between smooth muscle cell speciﬁc pro (renin) receptor (PRR) and V-ATPase which is required for V- ATPase-dependent autophagy, a process essential for smooth muscle survival [46e48].In conclusions, testosterone has been shown to play a critical role in acidiﬁcation of vas deferens ﬂuid via Androgen Receptor Antagonist modulating V-ATPase expression and functional activity. This modulation is essential for sperm survival and subsequently in preserving male fertility.