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BOR - Papers in Press, published online ahead of print April 18, 2007.
Biol Reprod 2007, 10.1095/biolreprod.106.056762
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BIOLOGY OF REPRODUCTION 77, 416–424 (2007)
DOI: 10.1095/biolreprod.106.056762
© 2007 by the Society for the Study of Reproduction, Inc.

Oxytocin and Vasopressin Stimulate Anion Secretion by Human and Porcine Vas Deferens Epithelia1

Travis M Hagedorn, Ryan W Carlin, and Bruce D Schultz 2

Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas 66506

ABSTRACT

Experiments were conducted to characterize the effects of oxytocin (OT) and vasopressin (VP) on epithelial cells isolated from human (1°HVD) and porcine (1°PVD) vas deferens and an immortalized epithelial cell line derived from porcine vas deferens (PVD9902 cells). Cultured monolayers were assessed in modified Ussing flux chambers and the OT- or VP-induced change in short circuit current (ISC) was recorded. All cell types responded to basolateral OT or VP with a transient increase in ISC that reached a peak of 3–5 µA cm–2. Concentration-response curves constructed with 1°PVD and PVD9902 cells revealed that the apparent KD (kapp) for OT was ~100-fold less than the kapp for VP. Amplicons for the OT receptor (OXTR) and vasopressin type 2 and type 1a receptors (AVPR2 and AVPR1A) were generated with RT-PCR and the identification of each amplicon confirmed by sequence analysis. A selective antagonist for OXTR and AVPR1A fully blocked the effects of OT and partially blocked the effects of VP when assessed in both 1°PVD and PVD9902 monolayers. APVR2 antagonists blocked the effects of low (≤30 nM) but not high concentrations of VP, indicating that VP was affecting both AVPR2 and a second receptor subtype, likely OXTR or AVPR1A. Experiments employing chelerythrine demonstrated that OT stimulation of vas deferens monolayers requires PKC activity. Alternatively, VP (but not OT) increased the accumulation of cytosolic cAMP in vas deferens epithelial cells. Results from this study demonstrate that OT and VP can modulate ion transport across vas deferens epithelia by independent mechanisms. OT and VP have the potential to acutely change the environment to which sperm are exposed and thus, have the potential to affect male fertility.

male reproductive tract, mechanisms of hormone action, oxytocin, vas deferens, vasopressin

INTRODUCTION

The deferent duct possesses ion transport mechanisms that change the environment to which sperm are exposed and thus can contribute to male fertility. Mammalian sperm leaving the testis are incapable of fertilization [1, 2]. Substantial maturation occurs in the efferent ducts and epididymes [3, 4], where it appears that the environment must typically be acid [5–9], although exposure to an alkaline environment is required for sperm to show hyperactivated motility [10] and to induce other cytosolic processes associated with capacitation [11, 12]. Epithelial cells lining the epididymis possess H+ and HCO3– transporters that can acidify the duct lumen [6–9] and anion channels that are modulated by cAMP and Ca2+ [13, 14]. Nonetheless, only a limited amount of work has been done to characterize the regulatory cascades that modulate vas deferens epithelial ion transport.

Infertility affects 15% of couples worldwide, with male factors contributing to approximately half of all cases [15–19]. Diseases of epithelial ion transport contribute a portion of these cases. For example, cystic fibrosis (CF) is an inherited disease of anion transport that has long been associated with male infertility [20]. Greater than 97% of men diagnosed with classical CF exhibit congenital bilateral absence of the vas deferens [21, 22]. Thus, the cystic fibrosis transmembrane conductance regulator (CFTR; the gene that is mutated to cause CF) is critical to maintain the anatomical structure of the distal male duct in humans (mice lacking CFTR exhibit normal reproductive anatomy and function). Some CFTR mutations that are associated with milder forms of airway and pancreatic disease have been associated with both obstructive and nonobstructive male infertility [23–26]. Clearly, there is a link between CFTR-associated ion transport disorders and male infertility, even in the presence of a patent deferent duct [27]. In addition to CFTR, vas deferens epithelial cells expresses various ion transporters including the epithelial Na+ channel (ENaC), Na+-HCO3– cotransporters, and various anion exchangers [28–30] that may also be affected in disease states. Although progress has been made in characterizing the ion transport properties of the vas deferens, there are many aspects of this tissue that remain unexplored.

Neurohypophyseal hormones affect the male reproductive tract. For example, oxytocin (OT), a hormone that is classically associated with smooth muscle contraction in the uterus and mammary gland [31], stimulates or potentiates smooth muscle contraction in the male duct [32–34]. Previous studies have shown that OT receptors (OXTR) are present in male reproductive tissues [35, 36] and that plasma levels of OT increase in males during sexual arousal [37–39], suggesting that this hormone may play an acute critical role in male fertility. Vasopressin (VP), typically associated with its effects on either vascular tone or water and salt balance [40], likewise stimulates male duct smooth muscle contraction and potentiates smooth muscle contraction that is induced by adrenergic agonists [32, 41, 42]. A survey for effects of agents that are reportedly present in vas deferens revealed that VP directly stimulates ion transport across cultured porcine vas deferens epithelia [30]. Thus, neurohypophyseal hormones appear to play an important, although not fully understood, role in male fertility. Any effect that either hormone has on vas deferens ion transport has not been fully defined.

The goal of this study was to characterize the effects of OT and VP on vas deferens epithelial ion transport. Particular focus was placed on determining the membrane receptor location and molecular identity that correlates with functional effects that would be expected to modify sperm delivery, activity, and viability due to changes in the luminal environment. The outcomes from these studies suggest that neurohypophyseal hormones play an integral regulatory role that likely contributes to male fertility.

MATERIALS AND METHODS

Tissue Acquisition and Epithelial Cell Isolation

The distal portions of porcine reproductive ducts were removed immediately postmortem from 3- to 5-month-old boars at a local swine production facility. Tissues were placed in ice-cold Hanks buffered salt solution (HBSS; composition in mM: 137 NaCl, 5.4 KCL, 0.4 KH2PO4, 0.6 Na2HPO4, 5.5 glucose) supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 20 µg/mL amphotericin B for transport to the laboratory.

Epithelial cells lining the vas deferens were isolated as described previously [30]. Briefly, the distal epididymis and transitional vas deferens were discarded to insure that only the vas deferens was employed to obtain cells for the reported studies. The remaining duct was stripped of connective tissue and flushed with HBSS supplemented with antibiotics. After being filled to distension with PBS containing trypsin, EDTA, and collagenase, the ducts were incubated at 37°C for 35 minutes. Following incubation, each duct was massaged gently and epithelial cells were flushed from the duct with 5 mL of growth medium (Dulbeccos modified Eagle medium, DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Atlanta Biological, Atlanta, GA), 100 U/mL penicillin and 100 µg/mL streptomycin. The resulting suspensions of porcine vas deferens epithelial cells for primary culture (1°PVD) were seeded onto 25 cm2 tissue culture flasks (Corning, Corning, NY).

Segments of distal human vas deferens were obtained from a local hospital using procedures approved by both the University and hospital Institutional Review Boards. The isolation of human vas deferens epithelial cells for primary culture (1°HVD) is similar to that of 1°PVD cells and has been described previously [43]. 1°HVD cells were grown initially on 24-well tissue culture plates (Cellstar; Marsh Biomedical Products, Rochester, NY).

Cell Culture

1°PVD, 1°HVD, PVD9902 (an immortalized epithelial cell line derived from porcine vas deferens [28]), and LLC-PK1 cells (American Type Culture Collection, Manassas, VA) were seeded and grown on 25-cm2 tissue culture flasks or 24-well tissue culture plates and maintained in DMEM supplemented with 10% FBS and 1% penicillin and streptomycin with media changes every other day. LLC-PK1 cells, which were originally derived from porcine kidney, are epithelial in nature and have been shown to express receptors for both oxytocin and vasopressin [44]. Upon reaching confluency, cells were lifted with PBS containing trypsin and EDTA, suspended, and seeded onto 1.13 or 0.33 cm2 Transwell permeable supports (Corning-Costar; Cambridge, MA) with media replacement every other day until used (typically ~14 days) for electrophysiology, RNA isolation, or cAMP assays.

Electrophysiology

Epithelial cell monolayers on permeable supports were placed into individual modified Ussing flux chambers (model DCV9, Navicyte; San Diego, CA) and bathed symmetrically in Ringer solution (composition in mM: 120 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.83 K2HPO4, 1.2 CaCl2, 1.2 MgCl2) maintained at 39°C for 1°PVD and PVD9902 or 37°C for 1°HVD cells and continuously bubbled with 5% CO2–95% O2. Monolayers were then clamped to 0 mV and ISC was measured continuously with a voltage-clamp apparatus (model 558C, University of Iowa, Dept. of Bioengineering, Iowa City, IA). Monolayers were periodically exposed to a 5 mV bipolar pulse and the resulting change in current was used to calculate transepithelial electrical resistance (RTE) according to Ohm's law. Data were acquired digitally at 1 Hz with an Intel-based computer using an MP100A-CE interface and AcqKnowledge software (ver. 3.7.3, BIOPAC Systems, Santa Barbara, CA).

RNA Isolation

1°PVD, PVD9902, and LLC-PK1 cells were grown on 4.5-cm2 Transwell permeable supports (Corning-Costar) for 12–16 days with media changes every other day, and total RNA was isolated using an RNeasy kit (Qiagen, Valencia, CA). Cells were exposed to a lysis buffer supplied with the kit, transferred to a QIAshredder column (Qiagen) and centrifuged to homogenize the preparation. The supernatant was moved to an RNeasy column and, following a set of appropriate washings, the RNA was eluted from the column using 30 µL of nuclease-free water. Liver and pituitary were also dissected from pigs <30 min postmortem, and samples were immediately snap frozen in liquid nitrogen until used for RNA extraction, at which time the tissue was ground with a mortar and pestle and the RNA was isolated with the protocol outlined above. All RNA samples were treated with Turbo DNA-free (Ambion, Austin, TX) to remove genomic DNA, quantified on an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE), and finally stored at –80°C until used for molecular studies.

Reverse Transcription-PCR

Reverse transcription followed by amplicon generation of OXTR and the vasopressin receptor subtypes (AVPR1A, AVPR1B, AVPR2) was performed using a one-step RT-PCR kit (Qiagen). Primers specific for porcine OXTR and AVPR2 and the porcine homologs of AVPR1A and AVPR1B were designed with Beacon software (version 4.0, Premier Biosoft International; Palo Alto, CA) and are presented in Table 1. RT-PCR reactions were assembled using 100–300 ng of total RNA, 400 µM dNTPs, 300 nM sense and antisense primers, 0.75 µL enzyme mix and 5 µL buffer in a 25-µL reaction volume. The following thermocycler protocol was performed: 30 min reverse transcription step at 50°C, 15 min RT inactivation step at 95°C followed by 40 cycles of a denature step at 94°C for 1 min, annealing step for 1 min and elongation at 72°C for 1 min, with a final extension at 72°C for 10 min on a Techne Touchgene thermocycler (Krackler Scientific, Albany, NY). Reactions conducted with ExTaq (TaKaRa, Otsu, Shiga, Japan) in place of the OneStep RT-PCR enzyme mix were run in parallel. PCR products were resolved in a 1% agarose gel containing 1 µg/mL ethidium bromide. Images were captured on a FluorChem 8900 imaging system (Alpha Innotech, San Leandro, CA) and processed for publication using CorelDRAW (version 10.0) and Paint Shop Pro (version 5.01, Corel Corp.; Ottawa, Ontario, Canada).


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  		TABLE 1 Primer set characteristics.

Complementary DNA Sequencing

Amplicons from RT-PCR reactions were cut from gels, and the cDNA was purified using a QIAquick gel extraction kit (Qiagen). Samples of cDNA were then sequenced with a CEQ8000 (software version 8.0.52, instrument version 6.0.2, sequence analysis algorithm version 2.3.13, fragment analysis algorithm version 2.2.1; Beckman Coulter; Fullerton, CA) according to the manufacturer's specifications.

Cyclic AMP Measurements

Intracellular cAMP generation was measured in PVD9902 epithelial monolayers using an immunoassay kit (Biotrak; Amersham Pharmacia Biotech, Inc.; Piscataway, NJ) as described previously [43]. Briefly, PVD9902 cells were seeded to permeable supports (0.33 cm2) and maintained for 13–15 days with media changes every other day. Media in both the apical and basolateral compartments was replaced by warmed PBS 1 h prior to assay. Monolayers were then exposed to one of several treatments in PBS for 6 min prior to cell lysis using reagents supplied in the kit. Results are initially expressed as pmol cAMP per Transwell (0.33 cm2) as determined from a standard curve and were then normalized to standard treatments within each assay.

Chemical Sources

Forskolin (Coleus forskohlii) was purchased from Biomol (Plymouth Meeting, PA). Penicillin, streptomycin, amphotericin B, collagenase, gentamicin and trypsin-EDTA (0.5% trypsin, 5.3 mM EDTA, 10X liquid) were purchased from Invitrogen-Life Technologies (Carlsbad, CA). Oxytocin, Lys8-vasopressin, Arg8-vasopressin, isoproterenol, chelerythrine, [ß-Mercapto-ß,ß-cyclopentamethylene-propionyl1, O-Me-Tyr2, Orn8]-oxytocin (selective OXTR antagonist that also blocks AVPR1A designated by Sigma catalog # O6887), and [Adamantaneacetyl1, O-Et-D-Tyr2, Val4, Aminobutyryl6, Arg8,9]-vasopressin (selective AVPR2 antagonist designated by Sigma catalog # V2381) were purchased from Sigma-Aldrich, Inc. (Saint Louis, MO). P9, a scrambled nonapeptide (sequence: [H]-Met-Ala-Glu-Glu-Gly-Ala-Gly-Gly-Cys-[OH]), was generously provided by Dr. John Tomich (Kansas State University, Manhattan, KS). desGly-NH2, d(CH2)5[D-Tyr2, Thr4]ornithine-vasotocin (ST-11-61; selective OXTR antagonist [45]) was generously provided by Dr. Maurice Manning (Medical College of Ohio, Toledo, Ohio). (±)-5-dimethylamino-1-[4-(2-methylbenzoylamino)benzoyl]-2,3,4,5-tetrahydro-1H-benzazepine monohydrochloride (OPC-31260; selective AVPR2 antagonist [46]) and 1-[1-[4-(3-acetyalaminopropoxy)benzoyl]-4-piperidyl]-3, 4-dihydro-2(1H)-quinolinone (OPC-21268; selective AVPR1A antagonist [46]) were gifts provided by Dr. Koji Komuro (Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan).

Statistical and Regression Analysis

A paired Student t-test or ANOVA was conducted using SAS (version 9.1, SAS Institute Inc., Cary, NC) to compare means between different treatments. Probability of type I error < 0.05 was considered statistically significant. A modified Hill equation, y = [axb]/[cb + xb], was used in which y is the measured {Delta}ISC, a is the maximum {Delta}ISC ({Delta}ISC-MAX), x is the concentration of agonist, b is the Hill coefficient, and c is the concentration required for a half-maximal response (i.e., the apparent KD or kapp). The equation was fitted to each set of data collected to test for concentration dependency. A Hill coefficient not different from unity was confirmed for each fit, and the data were subsequently refitted with the Hill coefficient constrained to unity to allow for a more direct comparison of the kapp values derived for OT and VP in the presence and absence of receptor antagonists and with different cell sources. Curve fitting was conducted and all graphs were created using SigmaPlot (version 6.0, Systat Software, Inc., Point Richmond, CA). Mean and SEM were calculated using a Microsoft Excel spreadsheet.

RESULTS

OT Increases Anion Secretion Across Cultured Vas Deferens Epithelial Cell Monolayers

Experiments were conducted to test the hypothesis that OT stimulates ion transport across human vas deferens epithelia. 1°HVD epithelial monolayers exhibited a baseline ISC and RTE of 4.6 ± 0.6 µA cm–2 and 1750 ± 300 {Omega} cm2, respectively (n = 12). A subset of these monolayers was then exposed to 1 µM OT, a concentration expected to be maximally effective. Apical exposure to OT caused no discernable effect while basolateral exposure stimulated a rapid increase in ISC, indicating an increase in anion secretion [29, 30], that subsequently relaxed toward baseline (Fig. 1). In all experiments, 1°HVD monolayers responded to basolateral OT exposure and the peak {Delta}ISC was 5.7 ± 0.9 µA cm–2 (n = 4). These results suggest that OXTRs are present in the basolateral aspect of human vas deferens epithelia and that OT exposure is linked to acute stimulation of anion secretion.


Figure 01
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  		FIG. 1 1°HVD epithelial cell monolayers respond to oxytocin (OT) with increased anion secretion as indicated by elevated ISC. Apical and basolateral aspects of a 1°HVD monolayer were exposed to OT (1 µM) as indicated by the bars. Results are typical of two experiments conducted with cells derived from two individuals. Dashed line represents zero net current.

OT stimulates a change in ISC across 1°PVD epithelial cell monolayers that is similar to the response of 1°HVD. 1°PVD epithelial cells were employed to more fully characterize the response due to their ready availability and ease of use. Cells used in the present studies were derived from 13 different animals. Cultured monolayers used throughout all experiments exhibited a baseline ISC and RTE of 0.7 ± 0.1 µA cm–2 and 3400 ± 200 {Omega} cm2 (n = 90 wells), respectively. A subset of these monolayers was employed in experiments to characterize the concentration-dependent effect of OT on vas deferens epithelial ion transport. Typical results are presented in Figure 2, A and B. The basolateral aspect of 1°PVD monolayers were exposed to OT (1 nM or 100 nM, as indicated) that resulted in rapid rise in ISC followed by a return toward baseline. Data for {Delta}ISC from Figure 2, A and B are included with similar observations that are summarized in Figure 2C, where each data point represents the average response of 1°PVD monolayers to the given OT concentration. The solid line represents the best fit of a modified Hill equation to the data set, which predicts a {Delta}ISC-MAX of 3.1 ± 0.4 µA cm–2 and a kapp of 1.8 ± 1.2 nM.


Figure 02
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  		FIG. 2 Porcine vas deferens epithelial cell monolayers respond to oxytocin (OT) with changes in ISC that indicate anion secretion. A, B) Typical response of 1°PVD monolayers to basolateral OT exposure as indicated by the bars. The typical responses illustrated in A and B were observed in 4–5 experiments. C) Summary of data from 1°PVD monolayers where each data point represents 3–5 observations and vertical bars indicate SEM. D) Responses of PVD9902 monolayers to basolateral OT exposure are summarized. Each data point represents 3–6 observations, vertical bars indicate SEM. Solid lines in C and D represent the best fit of a modified Hill equation to the data sets. Parameters of the fits are included in the text.

PVD9902 epithelial cells also exhibited OT-induced increases in anion secretion. PVD9902 have been described recently [28] and were used extensively for the present studies. Baseline parameters are similar to 1°PVD with baseline ISC and RTE of 0.5 ± 0.1 µA cm–2 and 4600 ± 200 {Omega} cm2, respectively (n = 226 wells). A subset of these PVD9902 monolayers was exposed to various concentrations of OT, and the resultant {Delta}ISC were virtually indistinguishable from responses of 1°PVD cells. Data are summarized in Figure 2D, where each data point indicates the average response of monolayers to the given concentration of OT, and the fit of the modified Hill equation to the data revealed a {Delta}ISC-MAX of 4.8 ± 0.4 µA cm–2 with a kapp of 0.2 ± 0.1 nM. When compared with observations with 1°PVD monolayers (Fig. 2C), additional observations were made at lower concentrations to document the full concentration-response profile and conformity to Michalis-Menten kinetics. These results indicate that OT stimulates a concentration-dependent increase in anion secretion across vas deferens epithelia, and that stimulation occurs at concentrations that have been measured in serum from sexually aroused men [47] and from rams [48].

OXTR Antagonists Reduce OT-Stimulated Anion Secretion Across Cultured Porcine Vas Deferens Epithelial Cell Monolayers

The receptor(s) that mediates the effect of OT on anion secretion across vas deferens epithelia was evaluated utilizing pharmacological methods. OXTR antagonists ST-11–61 (80 nM) and O6887 (20 nM) had no effect on ISC when added to the basolateral compartment. However, the subsequent response to OT (3 or 30 nM) was reduced by both antagonists when compared to untreated controls (Fig. 3A). Similar studies were performed with PVD9902 monolayers and both ST-11–61 (80 nM) and O6887 (20 nM) reduced the magnitude of OT-stimulated {Delta}ISC. Data analysis revealed that ST-11–61 caused a right-shift in the concentration-response when compared to untreated monolayers where the derived kapp values were 15.2 ± 20.6 nM and 1.3 ± 1.2 nM, respectively (Fig. 3B). Additionally, O6887 caused a right-shift in the concentration-dependent response compared to untreated monolayers where the derived kapp values were 12.7 ± 21.3 nM and 0.5 ± 1.1 nM, respectively (Fig. 3C). These results suggest that OXTR is expressed in porcine vas deferens epithelia and functionally linked to the stimulation of anion secretion.


Figure 03
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  		FIG. 3 Oxytocin receptor antagonists competitively inhibit oxytocin-stimulated anion secretion. A) 1°PVD monolayers pretreated with ST-11–61 (80 nM) or O6887 (20 nM) exhibit a significant reduction in {Delta}ISC compared with untreated monolayers following exposure to 30 nM OT (gray bars) or 3 nM OT (cross-hatched bars). B, C) Summary of data demonstrating the concentration-dependent response of PVD9902 monolayers to OT in the absence (white triangle) or presence (black circle) of ST-11–61 (B) or in the presence (black square) of O6887 (C). Each data point represents the mean and SEM of 3–4 observations. Each solid line represents the best fit of a modified Hill equation to each data set. Parameters of each fit are included in the text. Asterisk (*) indicates significant difference (P < 0.05) from response in monolayers not exposed to OXTR antagonists.

OXTR, AVPR2, and AVPR1A mRNAs Are Present in Porcine Vas Deferens Epithelia

Experiments were conducted to test for the presence of mRNA for all of the receptors that OT is known to bind and activate. Primers designed to detect transcripts of OXTR, AVPR2, AVPR1A, and AVPR1B were used to probe total RNA isolated from 1°PVD epithelial cells from 6 to 12 different pigs and 3 to 4 different PVD9902 cell passages. Primer information, including probe sequence, annealing temperature, and expected product size is presented in Table 1. Reactions using OXTR, AVPR2, and AVPR1A primers and total RNA isolated from 1°PVD and PVD9902 epithelial monolayers yielded bands of expected mobility (Fig. 4). Parallel reactions were conducted using RNA from alternative tissues (LLC-PK1 cells and porcine liver) shown previously to express mRNA for each receptor of interest, and amplicons of expected mobility were obtained using the same primer sets to generate amplicons from OXTR, AVPR2, and AVPR1A transcripts. Primers designed to amplify AVPR1B failed to generate a product from 1°PVD or PVD9902 total RNA. However, a parallel reaction conducted with the same primer set and RNA isolated from porcine pituitary tissue produced a band of expected mobility. Each amplicon represented in Figure 4 was sequenced and BLAST (NCBI) analysis revealed that each product had a sequence identity corresponding to each receptor that was studied. The molecular analysis for expression of various receptors in 1°PVD and PVD9902 cells indicate that mRNA for OXTR, AVPR2, AVPR1A, but not AVPR1B is present in porcine vas deferens epithelia.


Figure 04
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  		FIG. 4 Gel images demonstrate the expression of mRNA for various receptors in 1°PVD monolayers (first column), PVD9902 monolayers (second column), and alternative tissues (third column). Amplicons for OXTR and AVPR2 with expected mobilites (525 bp and 353 bp, respectively) were generated via RT-PCR utilizing RNA isolated from 1°PVD, PVD9902, and LLC-PK1 monolayers. Amplicons for the AVPR1A (expected mobility 340 bp) were generated from RNA isolated from 1°PVD monolayers and porcine liver tissue. An amplicon for the AVPR1B was generated from RNA isolated from porcine pituitary tissue. All reactions utilizing total RNA from 1°PVD and PVD9902 monolayers were repeated in at least three individual experiments, and each band illustrated in this figure was sequenced with a confirmed identity for the given receptor. Arrowheads denote 500 bp.

Vasopressin Increases ISC in Cultured Vas Deferens Epithelial Cell Monolayers

The presence of AVPR2 and AVPR1A mRNA in porcine vas deferens epithelial cells suggested that these receptors may also be functionally expressed and linked to changes in ion transport. Therefore, experiments were conducted to test the effect of VP on cultured porcine vas deferens epithelial monolayers. 1°PVD monolayers were exposed to various concentrations of VP that resulted in a concentration-dependent increase in ISC that returned toward baseline values (Fig. 5, A and B). Data are summarized in Figure 5C, where the {Delta}ISC-MAX and kapp values derived from a Hill equation that was fitted to the data were 2.9 ± 0.4 µA cm–2 and 200 ± 100 nM, respectively. PVD9902 epithelial monolayers also responded to VP and the data are summarized in Figure 5D, where the values derived for {Delta}ISC-MAX was 4.3 ± 0.6 µA cm–2 and kapp was 54 ± 35 nM. Experiments performed using 1°HVD monolayers revealed that apical VP (1 µM) was without effect, but that basolateral exposure to VP (0.1 µM) resulted in a rapid increase in ISC (Fig. 6). For 1°HVD monolayers, the average {Delta}ISC induced by 100 nM VP was 5.3 ± 2.0 µA cm–2 (n = 7). These results indicate that VP has a clear effect on both human and porcine vas deferens epithelia, but the identity of the receptor(s) mediating this effect was unclear.


Figure 05
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  		FIG. 5 Vasopressin (VP) stimulates ISC across porcine vas deferens epithelial cell monolayers. A, B) Typical responses of 1°PVD monolayers to basolateral VP exposure as indicated by the bars. The typical responses illustrated in A and B were observed in four experiments. C) Summary of data for VP-stimulated {Delta}ISC where each data point represents 3–4 observations, vertical bars indicate SEM, and the solid line shows the best fit a modified Hill equation to the data set. D) Summarized data demonstrating response of PVD9902 monolayers to VP where each data point represents 3–7 observations, vertical bars indicate SEM, and the solid line represents the best fit a modified Hill equation to the data set. Parameters of the fitted lines are included in the text.


Figure 06
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  		FIG. 6 Vasopressin (VP) stimulates ISC across human vas deferens epithelial cell monolayers. The apical and basolateral aspects of a 1°HVD monolayer were exposed to VP as indicated by the bars. Responses are typical of results from two experiments.

OT and VP Receptor Antagonists Reduce VP-Stimulated Anion Secretion Across Cultured Porcine Vas Deferens Epithelial Cell Monolayers

A set of pilot experiments was performed to test for the receptor(s) that mediates VP-stimulated {Delta}ISC across porcine vas deferens epithelia. PVD9902 monolayers were exposed to one of three concentrations of VP in the absence or presence of O6887, a selective OXTR antagonist that also has blocking activity at AVPR1A. Monolayers that were not exposed to O6887 exhibited a concentration-dependent increase in VP-stimulated {Delta}ISC with a maximal effect observed at 300 nM (Fig. 7, A–C). Alternatively, monolayers that were pretreated with O6887 exhibited a reduced {Delta}ISC that was of the same magnitude regardless of the concentration of VP used for stimulation (Fig. 7, D–F). Additionally, the magnitude of {Delta}ISC in all monolayers pretreated with O6887 was similar to that observed in the monolayer that had no O6887 exposure and was stimulated with 30 nM VP (Fig. 7C). These results suggested that, at concentrations in excess of 30 nM, VP may be stimulating anion secretion by interactions with an OXTR or AVPR1A. However, the lower concentration of VP used in this study stimulates a {Delta}ISC that is mediated by a receptor other than OXTR or AVPR1A in porcine vas deferens epithelia. Additional experiments were conducted to identify this receptor.


Figure 07
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  		FIG. 7 O6887, an OXTR and AVPR1A antagonist, precludes the stimulatory effect of high but not low concentrations of VP on PVD9902 monolayers. PVD9902 monolayers were exposed to three different concentrations of VP, as indicated, in the absence (A–C) or presence (D–F) of O6887 (300 nM).

Pharmacological analysis suggests that the lowest concentration of VP used in these experiments, 30 nM, interacts with AVPR2 to stimulate an increase in anion secretion. Therefore, PVD9902 epithelial monolayers were exposed to 30 nM VP in the absence or presence of one of several receptor subtype selective antagonists (V2381, OPC-31260, O6887, OPC-21268, or ST-11–61). Monolayers exposed to VP in the presence of AVPR2-selective antagonists (V2381 or OPC-31260) exhibited a significantly lower {Delta}ISC compared with paired monolayers that had no antagonist pretreatment (Fig. 8). Alternatively, pretreating PVD9902 monolayers with antagonists selective for OXTR (ST-11–61), for AVPR1A (OPC-21268), or mixed OXTR/AVPR1A (O6887) resulted in a VP-stimulated {Delta}ISC that was indistinguishable from untreated monolayers (Fig. 8). These outcomes provide compelling evidence that lower concentration of VP used in these experiments stimulates anion secretion across vas deferens epithelial cells by interacting with an AVPR2.


Figure 08
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  		FIG. 8 AVPR2 antagonists inhibit VP-stimulated {Delta}ISC across porcine vas deferens epithelia. PVD9902 monolayers were exposed to selective receptor antagonists or remained untreated as indicated and subsequently were exposed to VP (30 nM). When compared to vehicle-treated control, the response to VP was less with AVPR2 antagonists (V2381, 10 µM; OPC-31260, 10 µM), but unaffected by oxytocin or AVPR1A antagonists (O6887, 300 nM; OPC-21268, 100 µM; ST-11–61, 1 µM). Each bar represents mean and SEM for 4–6 observations from a total of six experiments. Asterisks indicate significant difference (P < 0.05) from response in paired control conditions.

OT- and VP-Stimulated Anion Secretion Requires PKC Activity

Functional, pharmacological, and molecular analysis indicated the presence of OXTR and AVPR2 in vas deferens epithelia. Thus, experiments were conducted to explore the intracellular pathways that might be associated with activation of these receptors in porcine vas deferens epithelia.

The effects of OT, VP, and forskolin on {Delta}ISC were assessed in PVD9902 monolayers in the absence and presence of chelerythrine, a broad-spectrum PKC antagonist. Monolayers were mounted in Ussing chambers and exposed to chelerythrine (4 µM) for 20 min prior to stimulant exposure. OT stimulated a maximal {Delta}ISC that was, on average, 65% less than the response in paired monolayers that were not exposed to chelerythrine (Fig. 9). Additionally, the response to VP was, on average, 40% less in the presence of chelerythrine, although statistical significance was not achieved. Alternatively, chelerythrine had no discernable effect on forskolin-stimulated {Delta}ISC. These results demonstrate that OT stimulation of anion secretion across porcine vas deferens epithelia requires PKC activity, as expected for OXTR mediated responses. Additionally, the data suggest that PKC contributes to a portion of VP-induced anion secretion, likely due to activation of OXTR, or perhaps an AVPR1A.


Figure 09
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  		FIG. 9 Chelerythrine, a PKC antagonist, reduces oxytocin (OT)-induced ion transport across porcine vas deferens epithelial monolayers. Paired monolayers were exposed to chelerythrine (4 µM) or vehicle control for 20 min before exposure to OT, vaspressin (VP), or forskolin (Fsk). Each bar represents the mean and SEM from 4–6 paired observations. Asterisk indicates significant difference (P < 0.05) from response in paired untreated conditions.

VP but Not OT Stimulates an Increase in Intracellular cAMP

PVD9902 monolayers were exposed to one of several treatments, and following cell lysis, the resulting generation of intracellular cAMP was measured. In order to establish a baseline level of cAMP, one monolayer in each experimental block was exposed to water as a vehicle treatment. Another monolayer was exposed to forskolin, a receptor independent activator of adenylyl cyclase, in order to determine a standard level of cAMP generation in PVD9920 monolayers. The level of cAMP generated from all other treatments (OT, VP, Isoproterenol, P9) was measured and expressed as a percent of the average forskolin-stimulated response. Monolayers that were exposed to OT generated levels of cAMP that were no different from the levels associated with water or scrambled nonapeptide (P9; Fig. 10). Alternatively, monolayers treated with various concentrations of VP exhibited incremental increases in cAMP accumulation. At the highest concentration of VP tested, 3 µM, the amount of cAMP was greater than that observed in the forskolin treated cells and lower than that observed in isoproterenol treated cells (Fig. 10). These data suggest that VP-stimulated anion secretion across porcine vas deferens epithelia is coupled to cAMP generation, while OT-stimulated anion secretion occurs thru a cAMP-independent mechanism.


Figure 10
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  		FIG. 10 Vasopressin stimulates cAMP generation in porcine vas deferens epithelia. PVD9902 cell monolayers were exposed to the indicated treatments for 6 min prior to cell lysis and assay. Results are expressed as the percent of the forskolin-stimulated change in cAMP. Treatments: P9, a nonapeptide lacking disulfide bonds; OT, oxytocin; VP, vasopressin; Fsk, forskolin; Iso, isoproterenol. Each bar represents the mean and SEM from 3–5 observations that were included in a total of seven individual assays.

DISCUSSION

The results of this study provide functional evidence to demonstrate that OT and VP stimulate ion transport across vas deferens epithelia. Key experiments were conducted with cells derived from human vas deferens and the results show that OT and VP act exclusively at the basolateral membrane to cause a rapid increase in ISC that we interpret as HCO3– and/or Cl– secretion. Additional studies with cells derived from porcine ducts (1°PVD and PVD9902 epithelial monolayers) provide functional, pharmacological, and molecular evidence to support the conclusion that OT and VP modify ion transport across vas deferens epithelium through interactions with OXTR and AVPR2, respectively, although AVPR1A may also be present. Finally, the results show that stimulation of anion secretion by OT, and to a lesser extent VP, requires PKC activity. Alternatively, VP acts through AVPR2 to increase cAMP generation. All of these observations have a significant impact on our understanding of vas deferens physiology as it participates to normal reproductive function.

Vas deferens epithelia are capable of secreting both HCO3– and Cl– [28–30]. Results from previous studies in this laboratory have documented, with functional assays, CFTR and ENaC in apical membranes and SLC4A4 (formerly known as pNBC), Na+/K+ ATPase, Na+/K+/2Cl– cotransporter, and K+ channel(s) in the basolateral membranes of cells derived from porcine or human vas deferens [28–30, 43]. A recent report provided molecular evidence indicating expression of additional transporters that might contribute to HCO3– secretion, including SLC26A3 (the epithelial protein that is mutated to cause congenital chloride diarrhea, also known as downregulated in adenoma, DRA), SLC26A4 (pendrin), and SLC26A6 (also known as putative anion transporter 1, PAT1, and as a Cl–/formate exchanger, CFEX) [28]. AQP2 and AQP9 are reportedly expressed in rat epididymis and/or vas deferens [49, 50] and mRNA coding for these proteins has been detected in 1°HVD (BDS, unpublished observation). These components can be configured in theoretical cell models to secrete preferentially either HCO3– or Cl–, or some combination of these anions while maintaining an isotonic luminal milieu. Secretion associated with OT and VP stimulation of the vas deferens could incorporate one or both of these anions that would osmotically affect water movement through aquaporins that are reportedly resident in the epithelial cell membranes [49, 51]. Thus, neurohypophyseal hormones have the potential to modify the fluid volume in the duct lumen as well as changing the pH due to HCO3– secretion, outcomes that would be expected to affect sperm activity or viability.

OT and VP are now firmly placed among a list of physiological agents that are documented to increase anion secretion across epithelial cells derived from the vas deferens. Norepinephrine, vasoactive intestinal peptide (VIP), histamine, adenosine, and VP were reported previously to stimulate anion secretion across 1°PVD [30] and PVD9902 cells [28], and adenosine was shown to stimulate anion secretion by 1°HVD cells [43]. A clearer picture of anion secretion regulation emerges as these observations are combined. Norepinephrine, VIP, VP, and OT exhibit activity exclusively from the basolateral aspect of the cells, whereas adenosine effects on 1°HVD are observed only with apical exposure [43]. Adrenergic agonists, adenosine and VP increase adenylyl cyclase activity to promote cAMP generation, whereas OT (and VP likely acting at OXTR) appears to act via a PKC-dependent mechanism. These results suggest that vas deferens epithelial cells integrate signals from a variety of sources to produce a fine-tuned response in the rate and composition of secretions. Submucosal neurons in pig and human vas deferens reportedly contain noradrenalin, VIP, neuropeptide Y, somatostatin, calcitonin gene-related peptide, substance P, galanin, and nitric oxide synthase [52–56]. There may, however, be some species to species differences in OXTR-mediated function, as strong OXTR immunoreactivity has been reported for epithelial cells lining the vas deferens of rams [57] while little OXTR immunoreactivity was observed in marmoset vas deferens [36]. There is clearly a response to OT and VP by cells derived from both the human and pig vas deferens when grown in culture. The source of either VP or OT that would affect vas deferens function in vivo is uncertain. Some reports have suggested that OT is synthesized in the testis [36] and that sexual arousal or orgasm may be associated with elevated serum OT levels [37–39, 47], presumably from the pituitary. Likewise, substantial amounts of VP are also present in testis [38], which suggests local synthesis, and serum levels of VP increase prior to ejaculation [39]. Regardless of source, the present results show that concentrations of OT and VP at or near the physiological range can modify the activity of epithelial cells derived from the vas deferens that, in vivo, would be expected to modify the volume and composition of the luminal environment.

Contributions of the vas deferens to male fertility are poorly defined. Mammalian sperm can neither swim nor fertilize an egg when they leave the testis, but acquire these abilities as they traverse the epididymis [1, 2]. The efferent ducts concentrate sperm by reducing fluid volume by ~96% while maintaining constant Na+ and Cl– concentrations [3, 4]. Approximately 50% of the remaining fluid is absorbed in the epididymes [3]. Luminal pH is reportedly reduced from neutral in the rete testis to ~6.5 in the caput epididymis, a pH that maintains sperm in a quiescent state during storage [58]. HCO3– exposure activates sperm by directly stimulating soluble adenylyl cyclase [11, 12] and by the activation of Ca2+ channels resulting in hypermotility [10]. It was proposed previously that adrenergic stimuli during the pre-ejaculatory arousal period would be expected induce HCO3– secretion that would raise luminal pH and initiate the activation process in sperm [30]. Current results suggest that OT and/or VP may have a similar effect. The serum concentration of both hormones increases during sexual arousal [39] and the results show that direct effects on epithelial anion secretion can occur near the range of concentrations reported in serum. Taken together, these observations strongly suggest that vas deferens epithelium is acutely modulated by OT and VP, in addition to locally released neurotransmitters.

Infertility is a pervasive problem throughout the world. Using the World Health Organization definition for infertility, ~15% of couples worldwide experience primary or secondary infertility, with male factors contributing to approximately half of all cases [15–19]. That epithelial ion transport diseases contribute to male infertility, including obstructive azoospermia, is well-documented with the recessively inherited disease of CF. Ninety-seven percent of CF men have impaired reproductive function [59] with reduced sperm quality in some cases [23–27, 60] and gross changes in vas deferens anatomy in others [20, 60–63]. Furthermore, numerous studies have shown that a disproportionately high percentage of men seeking assistance at reproductive health centers have at least one affected CFTR allele [23–26]. These observations suggest that, in the vas deferens or the epididymis, CFTR plays a role that is critical to normal male reproductive function. In addition to this well-documented population, an unacceptably high proportion of male infertility cases are diagnosed as idiopathic [16, 22, 27]. Such diagnoses provide no logical therapeutic options and research to determine root causes is lagging [15]. Outcomes from the current studies suggest that OT or VP contribute to normal male reproductive function and thus, aberrant hormone release or function may contribute to infertility in some individuals. Additionally, the current outcomes suggest that therapies for other conditions may impact function in the male reproductive tract incidentally. Indeed, consideration must be given to current clinical treatments that target OXTR or AVPR2. OT has been utilized to treat a variety of disorders including autism, Asperger's syndrome, and obsessive compulsive disorder [64–66]. Additionally, VP analogs such as OPC-31260, SR-121463, and OPC-41061 (tolvaptan) have been developed to antagonize AVPR2 in order to treat a variety of conditions including congestive heart failure, hypertension, hyponatremia, and nephritic syndrome [67, 68]. Although the prospect of treating these illnesses is promising, the effects of OT and VP analogs on the normal function of vas deferens epithelium must be considered as one seeks to optimize patient quality of life.

In conclusion, the results from this study have expanded the working model that describes ion transport across vas deferens epithelial cells. Neurohypophyseal hormones, OT and VP, have a clear stimulatory effect on anion secretion across porcine and human epithelial cells, and these effects are mediated though OXTR and AVPR2, respectively. Additionally, OT and VP stimulation of vas deferens epithelia requires PKC activity, and the effects of VP, but not OT, are coupled to the generation of cAMP. These results could provide insight necessary to develop treatments for male infertility as well as male contraceptives. Finally, treatment regimens that exploit OXTR and AVPR2 may need to be reviewed in light of their potential effects on male fertility.

ACKNOWLEDGMENTS

The authors extend special thanks to Dr. Fernando Pierucci-Alves, Dr. Rebecca Quesnell, and Ms. Natalee Holt for technical assistance. Thanks are extended to Mr. Roy Henry, Dr. John Devine, Mr. Dick Allen, Henrys Limited, and the Via Christi Health System for assistance in tissue procurement. Finally, the authors extend their appreciation to Dr. Frank Blecha and Dr. Lisa Freeman for coordinating research opportunities for professional students in the veterinary medicine curriculum.

FOOTNOTES

1Supported by National Institutes of Health R21 DK064001, National Institutes of Health P20 RR017686, National Institutes of Health T32 RR017497, and Cystic Fibrosis Foundation SCHULT06P0. This manuscript represents contribution no. 07-41-J from the Kansas Agricultural Experiment Station. Back

Correspondence: 2Bruce D. Schultz, Department of Anatomy and Physiology, Kansas State University, 1600 Denison Ave., Coles Hall 228, Manhattan, KS 66506. FAX: 785 532 4557; e-mail: bschultz{at}vet.ksu.edu

Received: 24 August 2006.

First decision: 27 September 2006.

Accepted: 5 April 2007.

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