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Testosterone induces and maintains prostaglandin endoperoxide synthase 2 (PTGS2, also known as cyclooxygenase 2) expression in vas deferens epithelial cells, but it remains unknown whether this has a physiological role in the context of male reproductive biology. Prostaglandins induce concentration-dependent increases in anion secretion in porcine vas deferens epithelial cell (1°PVD) monolayers, where bicarbonate contributes to cAMP-stimulated anion secretion. Moreover, bradykinin induces anion secretion across 1°PVD monolayers that is indomethacin sensitive, and both PTGS2 and PTGS1 are expressed in this model system. Therefore, it was hypothesized that testosterone modulates anion secretion across vas deferens epithelia via PTGS-dependent pathways and prostaglandin synthesis. Porcine vas deferens epithelial cells were isolated and cultured as monolayers on permeable supports until assayed in modified Ussing chambers. RNA and protein were isolated concurrently for semiquantitative expression analysis. Testosterone upregulated basal and bradykinin-induced short-circuit current across 1°PVD monolayers, indicative of anion secretion. Testosterone also induced greater transepithelial electrical resistance. Increases in anion secretion were associated with preferential upregulation of PTGS2 at the mRNA and protein levels. In addition, testosterone induced greater basal and bradykinin-induced anion secretion across vas deferens epithelial cells isolated from the distal segment of the duct. Taken together, these results suggest that testosterone upregulates epithelial responsiveness to acute modulations of anion secretion (likely bicarbonate secretion), which ultimately modifies the environment to which sperm are exposed.
Early studies [1, 2] reported that testosterone upregulated prostaglandin synthesis in various segments of the male reproductive duct and that this occurred most intensively in the vas deferens. The prostaglandin concentration in the vas deferens was shown to increase with puberty, and an increase in prostaglandin concentration was induced by testosterone [2–4]. Furthermore, it was shown that vas deferens tissue derived from intact rats converted arachidonic acid into prostaglandins at twice the rate compared with vas deferens tissue isolated from castrated animals and that testosterone administration to castrated rats rescued the prostaglandin synthesis rate . Subsequently, it was demonstrated that testosterone induced and sustained prostaglandin endoperoxide synthase 2 (PTGS2, also known as cyclooxygenase 2) expression in epithelial cells lining the adult rat vas deferens . Castration eliminated PTGS2 expression in a time-dependent pattern, and testosterone supplementation rescued that expression in the distal vas deferens of castrated rats . In addition, testosterone modulates PTGS2 expression in human fetal and adult vas deferens epithelial cells .
Vas deferens epithelia exhibit cAMP-stimulated anion secretion that requires bicarbonate in the basolateral medium and depends on sodium bicarbonate cotransporter activity in the basolateral membrane . Bicarbonate exchangers and the cystic fibrosis transmembrane conductance regulator are also thought to participate in bicarbonate secretion across vas deferens epithelia . Moreover, prostaglandins induce anion secretion across vas deferens epithelial cells via activation of prostaglandin E receptor 4 (PTGER4) and PTGER2, which are known to initiate cAMP-generating pathways . In addition, bradykinin (BK)-induced anion secretion is sensitive to the nonselective PTGS blocker indomethacin in vas deferens epithelia, where PTGS2 and PTGS1 are highly expressed .
Bicarbonate was recently shown to be required for the development of sperm fertilizing capacity and male fertility . Other data suggest that, in addition to the epididymis, the vas deferens also functions as a sperm storage site . Thus, regulated bicarbonate transport across epididymal and/or vas deferens epithelium likely affects sperm activity and viability.
Experiments were designed to investigate whether testosterone modifies ion transport and/or electrophysiological properties across primary monolayer cultures of porcine vas deferens epithelial cells (1°PVDs). Results reported herein support the conclusion that testosterone increases both basal and BK-induced anion secretion across porcine vas deferens epithelia in vitro. Moreover, increases in anion secretion are associated with upregulation of PTGSs at both the mRNA and protein levels. Data are also provided to suggest that testosterone upregulation of anion secretion is of greater magnitude at the distal segment of the boar vas deferens.
Porcine vas deferens were surgically excised immediately postmortem from sexually mature boars at a local swine production facility, placed in ice-cold Hanks buffered salt solution (137 mM NaCl, 5.4 mM KCl, 0.4 mM KH2PO4, 0.6 mM Na2HPO4, and 5.5 mM glucose) and transported to the laboratory, where isolation of epithelial cells for primary culture was performed as described previously . This same protocol was used to isolate epithelial cells from porcine proximal and distal vas deferens. A single vas deferens was transected in three segments of equal length, namely, proximal, middle, and distal. Thereafter, proximal and distal vas deferens segments were subjected to the cell isolation procedure, while the middle segment was discarded.
Vas deferens epithelial cells isolated from the entire duct, as well as proximal and distal vas deferens epithelial cells, were seeded on 25-cm2 tissue culture flasks and grown in Dulbecco modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (FBS; Atlanta Biologicals, Atlanta, GA) and 1% penicillin and streptomycin (Invitrogen), which is hereafter referred to as basal medium. Cell isolates had media changes every other day for 2–5 days. Cells were lifted subsequently with PBS containing trypsin and ethylene-diaminetetraacetic acid (Invitrogen), suspended in basal medium, and seeded on 1.13-cm2 Snapwell permeable supports (Corning-Costar, Cambridge, MA). Monolayers were kept in culture in either the absence or presence of testosterone cypionate (TC [100 μM]; Pharmacia & Upjohn Company, Kalamazoo, MI), with media changes every other day until assay. In a separate subset of experiments, vas deferens epithelial cells were isolated from the entire porcine duct, but the isolation and cell culture procedures were conducted with phenol red-free (PR-free) DMEM supplemented with 4500 mg/L l-glutamine (Invitrogen) to match this nutrient's concentration to that of basal medium, 10% basal FBS or charcoal-stripped FBS (CS-FBS), and 1% penicillin and streptomycin. Fetal bovine serum either was used as provided by the supplier or was charcoal stripped as described previously . Medium containing CS-FBS was partitioned, and one of the parts was supplemented with TC.
Epithelial cell monolayers were mounted in modified Ussing flux chambers (model DCV9; Navicyte, San Diego, CA; and model P2300; Physiologic Instruments, San Diego, CA), bathed symmetrically in Ringer solution (120 mM NaCl, 25 mM NaHCO3, 3.3 mM KH2PO4, 0.83 mM K2HPO4, 1.2 mM CaCl2, and 1.2 mM MgCl2), maintained at 39°C, and bubbled with 5% CO2-95% O2 to provide mixing and pH stability. Monolayers were clamped to 0 mV, and short-circuit current (ISC) was measured continuously with a voltage clamp apparatus (model 558C; Department of Bioengineering, University of Iowa, Iowa City, IA; and model VCCMC8; Physiologic Instruments). Data were acquired digitally at 0.1–1 Hz with an Intel-based computer (Intel Corporation, Santa Clara, CA) using either an MP100A-CE interface and AcqKnowledge software (version 3.7.3; BIOPAC Systems, Santa Barbara, CA) or Acquire and Analyze software (version 2.3.159; Physiologic Instruments). Once recordings began, this system periodically generated a bipolar voltage pulse. The resulting change in ISC was used to calculate transepithelial electrical resistance (RTE) according to the Ohm law. While in chambers, monolayers were exposed to lysyl-BK (LBK; Bachem, King of Prussia, PA) and prostaglandin E2 (PGE2; Sigma-Aldrich, St. Louis, MO).
Total RNA was isolated from epithelial cell monolayers following Ussing chamber protocols using an RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's recommendations. RNA quality was assessed with a 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA). RNA concentrations were determined using an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE), and all samples were diluted to 100 ng/μl. The RT-PCR was performed using the One-Step RT-PCR kit (Qiagen) according to the manufacturer's recommendations with 100 ng of RNA per reaction. Single-target reactions were conducted using primers for porcine glyceraldehyde-3-phosphate dehydrogenase (GAPDH), PTGS1, or PTGS2 as described previously . Briefly, SYBR Green I (Molecular Probes, Eugene, OR) fluorescence signals were detected and quantified by a SmartCycler (Cepheid, Sunnyvale, CA). Transcript abundance measurements in any given RNA sample were performed by reactions carried out in duplicate. Melting analyses were performed, and reaction products were subjected to agarose gel electrophoresis. Acquired threshold cycle values for target and reference genes were normalized according to the amplification efficiency method . Means ± SEMs were derived from normalized results within each condition. Results were expressed as the fold-change of PTGS expression in cells cultured in TC-supplemented medium compared with the paired cells cultured in basal medium.
Epithelial cell monolayers cultured on permeable supports were lysed and solubilized in buffer containing protease inhibitors (Calbiochem, San Diego, CA). After centrifugation at 6000 rpm at 4°C for 15 min, the supernatant was transferred to a fresh tube, and protein was quantified with the Micro BCA protein assay kit (Pierce, Rockford, IL). Protein sample aliquots were prepared for electrophoresis by addition of loading buffer (Boston Bioproducts, Worcester, MA) and β-mercaptoethanol (Sigma-Aldrich) and were heated to 95°C for 5 min. Polyacrylamide 4%–20% gradient precast gels (Pierce) were loaded with a set of protein samples isolated from 1°PVD monolayers originating from the same cell isolation and cultured in paired conditions. Gel lanes received either 40 or 20 μg of protein. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was conducted at 100 V for ~45 min or until optimal separation of prestained protein standards (Bio-Rad Laboratories, Hercules, CA) was observed. Electrotransfer onto polyvinylidene fluoride membranes (Millipore, Billerica, MA) was carried out in methanol-containing transfer buffer (20% v/v) at 30 V for 3 h. Gels were stained by GelCode Blue Stain (Pierce) for assessment of protein transfer efficiency. Two blots were obtained from each 1°PVD isolation in a paired manner. Blocking was conducted with blotting-grade nonfat dry milk (5% w/v; Bio-Rad Laboratories). Antibody treatment of each blot was conducted so that within each blot pair one was exposed to 0.5 μg/ml monoclonal anti-human PTGS2 (Cayman, Ann Arbor, MI), while the other was exposed to 0.5 μg/ml monoclonal anti-mouse Ptgs1 (Abcam, Cambridge, MA). None of these PTGS antibodies exhibit PTGS isoform cross-reactivity as tested by their respective manufacturers. Specificity of the anti-PTGS2 sera was demonstrated previously . Secondary detection of both PTGS1 and PTGS2 was conducted with 0.14 μg/ml anti-mouse Enhanced Chemiluminescence antibody (GE Healthcare, Piscataway, NJ), the substrate SuperSignal West Femto (Pierce), and a FluorChem HD2 imager (Alpha Innotech, San Leandro, CA). After PTGS detection, each blot was stripped with Restore Plus Western blot stripping buffer (Pierce), washed, and incubated with 0.16 μg/ml anti-ACTIN (Sigma-Aldrich) for 1 h. Secondary detection of ACTIN was conducted with the same reagents and equipment as for PTGSs, except that 0.05 μg/ml anti-rabbit Enhanced Chemiluminescence antibody (GE Healthcare) was used.
Densitometry was carried out on the band of expected mobility for each of these targets, resolved on lanes where 40 μg of protein had been loaded, using the AlphaEase FC software (version 6.0.0; Alpha Innotech). Each PTGS band density value was divided by the density of the respective ACTIN band within each condition and cell isolation so that an ACTIN-normalized PTGS density was determined. Means ± SEMs were derived from ACTIN-normalized PTGS densities within each condition. Results are presented as the fold-change of PTGS expression in TC-supplemented cells compared with the paired untreated cells.
Paired and unpaired Student t-tests and ANOVA were performed as appropriate. These tests and the calculation of means ± SEMs were performed with Excel Microsoft Office Suite 2003 (Microsoft Corporation, Redmond, WA). All graphs were made with SigmaPlot (version 6.0; Systat Software Inc., Point Richmond, CA).
Initial experiments to determine whether testosterone modulates ion transport across 1°PVDs were performed using monolayers derived as reported previously . Paired 1°PVD monolayers were cultured in either the absence of TC (basal medium) or the presence of TC (TC-supplemented medium). Monolayers were maintained in these conditions for 11–19 days and then assayed in modified Ussing flux chambers. Monolayers cultured in TC-supplemented medium exhibited greater basal ISC and RTE than monolayers maintained in basal medium (Fig. 1, A and B). Basal ISC was almost doubled in TC-supplemented cells, while RTE was ~20% greater. These results indicate that testosterone exposure modifies the composition and/or volume of secretion and that the ability of these cells to separate fluid compartments of different compositions is enhanced.
Once exposed to LBK (1 nM) in both the apical and basolateral compartments, all monolayers responded with rapid increases in ISC and decreases in RTE that were suggestive of anion secretion (Fig. 2A). The TC-supplemented monolayers exhibited greater maximal changes in ISC (ΔISC-MAX) than those of monolayers cultured in basal medium (Fig. 2B). Changes in ISC were also assessed over 900 sec to test independently for differences in net ion flux, in addition to a difference in the maximal rate of flux. Like in ΔISC-MAX, net ion flux across TC-supplemented monolayers over 900 sec was ~65% greater compared with that of monolayers cultured in basal medium (Fig. 2C). Monolayers responded to LBK stimulation in a pattern whereby large decreases in RTE occurred rapidly in both conditions. Maximal changes in RTE (ΔRTE-MAX) were also greater in TC-supplemented monolayers (Fig. 2D).
To test if the lack of testosterone impairs the capacity of epithelial cell monolayers to respond to prostaglandins, monolayer pairs (n = 4) were exposed symmetrically to PGE2 (0.5 or 1 μM). PGE2 induced ΔISC-MAX of 4.6 ± 0.9 and 4.7 ± 0.6 μA*cm−2 in basal medium and TC-supplemented medium, respectively, indicating that the underlying mechanisms accounting for the ion transport were not changed by TC exposure (data not shown).
Additional experiments were conducted in conditions optimized to minimize steroid effects in basal medium. Phenol red, a pH indicator present in most culture media preparations, reportedly has estrogenic effects , and steroid hormones can be present in FBS. Thus, experiments were conducted in which PR-free medium and CS-FBS or basal FBS were used to formulate the following three experimental conditions: PR-free + CS-FBS (hereafter PR-free + CS-FBS), PR-free + CS-FBS + TC (100 μM) (hereafter PR-free + CS-FBS + TC), and PR-free + FBS (hereafter PR-free + FBS). Paired 1°PVDs were cultured in these conditions for 19–21 days and then assayed in modified Ussing flux chambers. Consistent with the initial observations, testosterone upregulated basal ISC (Fig. 3A). However, no statistically significant differences in RTE were observed (Fig. 3B). The PR-free + CS-FBS + TC exhibited statistically greatest LBK-induced net ion flux (Fig. 4B). ΔISC-MAX and ΔRTE-MAX were also greater in PR-free + CS-FBS + TC compared with PR-free + CS-FBS, but no statistically significant differences were noted between these experimental groups (Fig. 4, A and C). All monolayers were exposed subsequently to symmetrical PGE2 (1 μM). PGE2 induced ΔISC-MAX of 3.9 ± 0.9, 3.5 ± 0.7, and 5.2 ± 1.8 μA*cm−2 across PR-free + CS-FBS, PR-free + CS-FBS + TC, and PR-free + FBS, respectively, with no statistically significant differences among these responses. This outcome suggests that in a condition in which steroid presence is minimal (such as in PR-free + CS-FBS) 1°PVD monolayers still preserve their capacity to respond to PGE2 at a magnitude comparable to that in which slightly greater levels of steroids are present.
Taken together, these results support the notion that testosterone increases basal ISC and RTE of 1°PVDs in vitro. The findings also indicate that testosterone upregulates BK-induced ion transport.
Testosterone was shown to induce and maintain PTGS2 expression in vas deferens epithelial cells of rats and men [6, 7]. In addition, PTGS2 and PTGS1 mRNAs are expressed in 1°PVDs, where they are thought to increase anion secretion by synthesizing prostaglandins upon BDKRB2 activation . To determine if the 1°PVD testosterone-induced upregulation of anion secretion reported herein is caused by a transcriptional testosterone effect that upregulates PTGS2 and/or PTGS1 mRNA copy numbers and (subsequently) the amount of the respective proteins, total RNA and protein samples were isolated from the monolayers used in the functional assays.
RNA isolates derived from paired epithelial cell monolayers cultured in the absence or presence of TC were used in SYBR Green-based quantitative RT-PCR targeting the PTGS2, PTGS1, and GAPDH transcripts. GAPDH-normalized results revealed a certain degree of PTGS2 upregulation in the TC-supplemented group (Fig. 5A). Further quantitative analysis of these transcripts was sought in RNA isolates derived from monolayers cultured in steroid-free conditions as already described. Outcomes from these experiments were similar to those previously acquired, as PTGS2 mRNA was approximately one and a half times more abundant in PR-free + CS-FBS + TC than in PR-free + CS-FBS. Alternatively, PTGS1 mRNA abundance did not change or was slightly decreased in PR-free + CS-FBS + TC (Fig. 5B).
PTGS2 and PTGS1 protein expression levels were measured in 1°PVDs cultured in the presence or absence of TC supplementation by Western blot analysis. The anti-PTGS2 sera used in these experiments were previously shown to selectively detect human PTGS2  and have no cross-reactivity with PTGS1 as reported by the manufacturer. Results from immunoblots generated with 1°PVD protein and probed with anti-PTGS2 revealed either a single band of mobility at ~75 kDa or two prominent bands that included this band at 75 kDa and a second band with a mobility at ~210 kDa (Fig. 6A). The 75-kDa band is consistent with published data for porcine PTGS2 . This band was subjected to densitometric analysis that included ACTIN immunoreactivity as a reference for normalization (Fig. 6A). PTGS2 was upregulated by 2-fold in monolayers cultured in the presence of TC supplementation both in regular media conditions and in the optimized cell culture conditions that included PR-free medium and CS-FBS (Fig. 6A). Although the mobility of porcine PTGS1 was previously demonstrated at 69 kDa , immunoblots generated with 1°PVD protein exhibited a band at ~60 kDa as the signal of greatest molecular weight. One or two bands at the range of 35–55 kDa were also present in lanes loaded with 1°PVD protein both in TC-supplemented medium or not. The 60-kDa band was detected in six of eight Western blots conducted for PTGS1. The ~60-kDa band was also the form of greatest apparent molecular weight detected when 1°PVD protein was subjected to electrophoresis side by side with protein isolated from porcine kidney and a platelet-enriched porcine blood cell preparation (Fig. 6B). Densitometry conducted with the signals derived from the ~60-kDa band and normalized to ACTIN revealed that PTGS1 expression was unchanged in TC-supplemented monolayers compared with basal medium and/or CS-FBS.
These results suggest that vas deferens epithelial cells cultured in the presence of testosterone supplementation exhibited a greater number of PTGS2 transcripts. The findings also indicate that the vas deferens epithelial cells showed increased levels of PTGS2.
Testosterone was shown to induce and support PTGS2 expression in the rat distal vas deferens , although the physiological implications of this site-specific expression are unknown. To test whether TC supplementation has differential effects in proximal versus distal vas deferens, epithelial cells were isolated from the proximal and distal thirds of the porcine vas deferens. Paired proximal and distal cell isolates derived from the same duct were cultured as epithelial monolayers in basal medium or TC-supplemented medium for 18–19 days and assayed in modified Ussing flux chambers. Basal ISC was greatest in TC-supplemented distal epithelial cell monolayers, and distal monolayers maintained in basal medium had the lowest basal ISC (Fig. 7A). RTE was high and similar across the different experimental groups (Fig. 7B).
Exposure to LBK (1 nM) induced greatest ISC changes in distal epithelial cell monolayers that had been supplemented with TC. Net ion flux over 900 sec was significantly greater in this group, suggesting a response to LBK that was more sustained (Fig. 8, A and B). ΔRTE-MAX were 3800 ± 270, 4400 ± 700, 3800 ± 900, and 4600 ± 600 Ω*cm2 in the proximal basal condition, proximal TC-supplemented condition, distal basal condition, and distal TC-supplemented condition, respectively, with no significant differences being observed.
Outcomes from these assays suggest that epithelial cells lining the distal vas deferens present greater basal net anion secretion, which is testosterone dependent. In addition, these results suggest that anion secretion elicited by BK is upregulated to greater levels by testosterone acting in distal vas deferens epithelia.
Results presented herein demonstrate that testosterone modulates ion transport across vas deferens epithelial cells. Functional data derived from 1°PVD monolayers reveal that testosterone upregulates the basal levels of net anion secretion across vas deferens epithelia. Moreover, these data suggest that rapid increases in anion secretion induced by BK are of greater magnitude if vas deferens epithelial cells are maintained in the presence of testosterone supplementation. Results from molecular assays that quantified PTGSs at both the mRNA and protein levels indicate that modulation of ion transport by testosterone is exerted through upregulation of PTGS2. These data support that, in addition to its widely known effects on the male reproductive tract and other tissues, testosterone has both direct and indirect roles in modifying the luminal environment to which sperm cells are exposed. It was recently shown that bicarbonate is required for the development of sperm cell fertilizing capacity . In addition, vas deferens epithelia anion secretion, demonstrated herein to be upregulated by testosterone, was shown previously to be bicarbonate dependent . Thus, these data provide evidence for an additional pathway by which testosterone can modify male fertility.
Monolayers of 1°PVD, whether maintained in basal medium or PR-free medium, exhibited a consistently greater level of basal ISC when cultured in the presence of testosterone supplementation. This is expected to generate greater amounts of solute and water transported onto the lumen of the postpubertal vas deferens. Assuming that the porcine vas deferens luminal diameter is ~1 mm and that epithelial secretion is ~150 mM in monovalent salts, then the basal ISC level (0.8 μA) carried out by epithelia maintained without testosterone supplementation would add ~0.2% per day to luminal contents. Following testosterone exposure, the contribution of vas deferens epithelial cells to luminal content would double. Testosterone supplementation was also associated with an increase in basal RTE. The elevated RTE likely results in a decrease in passive ion and water fluxes driven by concentration or electrical gradients. Thus, the combination of increased basal ISC and RTE likely empowers this epithelial system to more efficiently separate and modify the luminal compartment. Such observation of concomitantly increased ISC and RTE contrasts with the classic scenario in which increased ISC is usually detected along with lower levels of RTE, which reflects a greater number of open ion channels in one or both cell membranes. No data are yet available to offer an explanation as to how testosterone is upregulating both ISC and RTE in 1°PVDs, but it has been demonstrated that testosterone has an androgen receptor-independent antagonistic effect on voltage-gated calcium channels expressed in vascular smooth muscle . Thus, testosterone could be inducing increases in RTE through either direct action on specific ion transporting proteins or through signaling pathways that are not investigated herein. In a previous study  in which 1°PVD cells were cultured in the presence or absence of 100 nM testosterone, no upregulation of basal ISC or RTE was detected, which suggests the possibility of a concentration-dependent factor in these observations.
Bradykinin induces rapid and profound changes in ISC and RTE that are largely inhibited by indomethacin in 1°PVDs, where PTGS2 and PTGS1 are expressed and proposed to be activated upon binding of the BDKRB2 . In addition, it was reported that testosterone induces expression of PTGS2 in the rat vas deferens . Data reported herein demonstrate that testosterone upregulates BK-induced anion secretion across 1°PVD monolayers. Upregulation of responses to LBK by testosterone took place not only in terms of the magnitude of rapid and maximal ISC changes (ΔISC-MAX) but also over the course of 900 sec. Thus, it is proposed that greater levels of BK-induced anion secretion, including that occurring over an extended period, is brought about in 1°PVDs by greater expression of PTGSs. Greater abundance of PTGSs would take place through a genomic effect and could account for greater levels of prostaglandin synthesis. Increased concentration of prostaglandins in this system ultimately would be responsible for greater or longer-lasting anion secretion. Corroborating this, at least to some extent, are data revealing that prostaglandins induce changes in ISC in a concentration-dependent fashion in 1°PVDs . Consistent with greater ISC changes, testosterone also induced greater reductions in RTE elicited by LBK. As previously reported, BK-induced changes in RTE in this system tend to be maximal at the onset of ISC responses and return over the course of ~15 min to levels similar to those of baseline . Such a response profile is consistent with a rapid and robust channel-opening process that might be triggered by increased cAMP levels once prostaglandins increase in abundance and bind to PTGER4 and/or PTGER2, which are expressed in 1°PVDs . Similarly, it is proposed that greater levels of prostaglandin synthesis elicited by LBK and brought about by greater PTGS abundance in testosterone-treated monolayers are factors contributing to greater reductions in RTE.
It was proposed initially that BK, acting on the basolateral membrane of epididymal epithelia, induces or stimulates anion secretion through a prostanoid-producing pathway, as opposed to a prostanoid-independent pathway when BK is applied to the apical membrane [21–23]. It was reported subsequently that the prostanoid-producing pathway resides in epididymal basal cells, where LBK induces activity of Ptgs1 only . However, a recent study  from another laboratory indicates that BDKRB2 is localized at the apical membrane of principal cells of the cauda epididymis. These aspects regarding BK receptor localization, as well as the involvement of specific PTGSs and their localization, have particular importance herein whereby an attempt to correlate testosterone modulation of anion secretion in epididymal epithelia  versus that in vas deferens epithelia is made. In epididymal epithelia, testosterone supplementation failed to alter responses to apical LBK but induced substantial LBK responsiveness when LBK was applied basolaterally . Confocal microscopy with immunodetection showed that BDKRB2 is localized at the apical membrane of principal cells in porcine vas deferens epithelium . Moreover, immunocytochemistry derived from the intact rat and human vas deferens epithelium reveals that PTGS2 is expressed widely throughout principal cells of the distal vas deferens [6, 7]. PTGS2 is more abundant than PTGS1 in 1°PVDs at the mRNA level . Taken together, these data suggest that the testosterone modulation of anion secretion in vas deferens epithelia reported herein might occur via an apically expressed BDKRB2 that activates PTGS2 and PTGS1.
PTGS2 expression in human and rat vas deferens epithelial cells was shown to be testosterone dependent [6, 7]. Epithelial cell monolayers that were derived from the entire extent of the porcine vas deferens and used in the functional assays reported herein were also subjected to gene and protein expression analysis that targeted PTGSs. The outcomes reveal that testosterone upregulated expression of PTGS2 at the mRNA and protein levels. Thus, these data suggest that the functionally detected testosterone effect on 1°PVDs was carried out, at least in part, through transcriptional and translational upregulation of PTGS2. Greater PTGS2 abundance would most likely contribute to greater prostaglandin synthesis and ultimately greater anion secretion. Testosterone was reported to upregulate both Ptgs1 and Ptgs2 expression in rat epididymal epithelia . Those data, combined with the results reported herein, are apparently a first indication that PTGS expressions are differentially regulated by testosterone in the epididymis and vas deferens.
Results reported herein also show that basal anion secretion is enhanced to a greater extent by testosterone in the distal porcine vas deferens compared with the proximal segment. Testosterone supplementation upregulated basal anion secretion in distal epithelia, and the absence of testosterone supplementation brought basal anion secretion to a significantly lower level than that of proximal vas deferens epithelia. This suggests that net anion secretion is quantitatively increased in more distal portions of the duct. In addition, responses to LBK were greater in testosterone-supplemented distal porcine epithelia. From a fundamental perspective, these outcomes align with expectations, as PTGS2 is a major component of the BK-stimulated pathway that culminates in anion secretion by 1°PVDs , considering that PTGS2 expression is induced and supported by testosterone at the distal rat vas deferens  and that prostaglandins induce anion secretion in 1°PVDs . These outcomes from segmental 1°PVD isolates might also serve as further evidence that increases in anion secretion observed in the experiments using whole vas deferens cell isolates are due, in large part, to testosterone-induced PTGS2 upregulation. Early investigation on the ultrastructure of epithelial cells lining the rat vas deferens revealed marked differences between the proximal and distal segments and suggested that steroidogenic capacity would be present at the distal segment . It was shown subsequently that the vas deferens of rats, dogs, and men are capable of generating several testosterone metabolites, including 5α-dihydrotestosterone [27, 28]. In addition, the proximal and distal segments of the rat vas deferens were shown to present equivalent rates of testosterone metabolism . Thus, it seems unlikely that the effect of testosterone supplementation, which induced greater anion secretion in distal 1°PVDs, is due to different and intrinsic capacities in proximal and distal cells to process TC into metabolites of greater affinity for the androgen receptor.
Data reported herein are from an in vitro system and largely represent electrophysiological measurements. However, these data might well be of relevance for further understanding of male fertility regulation. For instance, the vas deferens lumen has been shown to function as a sperm storage site . More than 20% of sperm cells are found in the vas deferens, and close to half of these are found at the middle and distal thirds of its length . Sperm cells stored in the vas deferens lumen, located distally from a vasectomy site, and/or seminal vesicles constitute a contraceptive concern that requires patients to observe a period of several weeks until sperm clearance is complete [29, 30]. These observations demonstrate that sperm cells remain in the vas deferens lumen, exposed to its luminal composition, for much longer than is generally thought. Obviously, the vas deferens luminal environment reflects activity of the epithelia lining this compartment, as well as contents arriving from more proximal portions of the duct. As indicated previously, vas deferens epithelia carry out anion secretion that is largely bicarbonate dependent , and expression of a subset of bicarbonate transporters has been demonstrated in vas deferens epithelial cells [8, 9]. Moreover, sperm cells take up bicarbonate from the extracellular environment in a process that is required for the development of fertilizing capacity . Hence, there is great likelihood that testosterone participates in sperm maturation by modifying anion secretion across vas deferens epithelia.
In conclusion, our results show that testosterone upregulates both basal and BK-induced anion secretion across 1°PVDs in vitro. Moreover, distal vas deferens epithelial cells are more responsive to testosterone modulation and exhibit greater anion secretion in the presence of testosterone supplementation. Furthermore, upregulation of PTGSs, markedly PTGS2, is associated with testosterone supplementation, which suggests that testosterone induces the effects described herein via increased prostaglandin synthesis. Increased anion secretion is expected to modify the luminal content to which sperm are exposed and to modulate male fertility.
The authors extend sincere thanks to the KSU-COBRE for Epithelial Function in Health and Disease for resources provided through its Molecular Biology Core facilities and to Dr. Lisa Freeman for directing the K-State Veterinary Research Scholars Program. Appreciation is also expressed to Henrys Ltd, KSU Swine Teaching and Research Center, and Dr. Pradeep Malreddy for their assistance with tissue procurement.
1Supported by Cystic Fibrosis Foundation SCHULT06PO, National Institutes of Health RR-17686, National Institutes of Health R01 HD058398, National Institutes of Health T35RR007064, and the Merck-Merial Veterinary Scholars Program. This manuscript represents contribution number 09–212-J from the Kansas Agricultural Experiment Station.