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BOR - Papers in Press, published online ahead of print July 20, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.044123
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BIOLOGY OF REPRODUCTION 73, 1057–1063 (2005)
DOI: 10.1095/biolreprod.105.044123
© 2005 by the Society for the Study of Reproduction, Inc.

Chloride Channels in Physiological Volume Regulation of Human Spermatozoa1

C.H. Yeung 2, 3, J.P. Barfield 3,4 , and T.G. Cooper 3

Institute of Reproductive Medicine of the University,3 D-48129 Münster, Germany Department of Biological Sciences,4 University of New Orleans, New Orleans, Louisiana 70148


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
As with other mammalian species, human spermatozoa experience a decrease in extracellular osmolarity in cervical mucus upon ejaculation, which requires the efflux of osmolytes and water to counteract swelling that hinders mucus penetration. Recent evidence for the operation of K+ channels in the process of volume regulation suggests parallel involvement of Cl–/anion channels for electro-neutrality as in somatic cells. This was studied using ejaculated spermatozoa washed at seminal osmolality and incubated for 30 min in a medium of mucus osmolality in the presence of Cl– channel blockers. Increases in cell size measured as laser forward-scatter by flow cytometry were detected in the presence of 100 µM 5-nitro-2(3-phenylpropylamino) benzoic acid, 400 µM diisothiocyanato-stilbene-2,2'-disulphonic acid, and 20 µM tamoxifen. No volume changes were found with 400 µM 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid, 200 µM verapamil, or niflumic acid, whereas 1 mM niflumic acid induced shrinkage. Among the candidate channel proteins, Western blotting revealed the presence of ClC-3 (CLCN3) at 87 kDa, but the absence of ClC-2 (CLCN2) from sperm proteins in all samples tested. ICln (CLNS1A) was found in only one of eight samples. Immunocytochemistry localized CLCN3 to the sperm tail. To confirm molecular identities, sperm mRNA was extracted and checked for quality by the presence of protamine 2 transcripts and the absence of sperm DNA and leukocyte mRNA using reverse transcription-polymerase chain reaction. Transcripts of Clcn3 were found in all samples and that of Clns1a in some but not all samples. Clcn3 was therefore considered the most likely candidate of Cl– channel involved in volume regulation of human sperm.

gamete biology, male reproductive tract, sperm, sperm motility and transport


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Before ejaculation, mammalian spermatozoa are stored in the distal segment of the epididymis where the luminal fluid has an osmolality above that of serum. Upon ejaculation, spermatozoa encounter fluids in the female tract with an osmolality close to that of serum (see [1]). In humans, this represents a decrease from 342 mmol/kg, as measured in the vas deferens [2], to 287 mmol/kg in cervical mucus [3]. This hypotonic challenge should lead to a volume increase that would activate mechanisms for regulatory volume decrease (RVD). Inhibition of RVD by the ion-channel blocker quinine results in cell swelling and failure to penetrate and migrate through surrogate mucus [4]. In transgenic mouse models, a defect in RVD causes sperm swelling in the uterus, which hinders migration into the oviduct, causing male infertility [5, 6]. In boars and bulls, there is an association of sperm volume with fertility [7, 8]. Volume regulation is therefore an important sperm function.

In somatic cells, RVD involves efflux of K+, Cl– and organic osmolytes. The former can be effected by the potassium-chloride cotransporter (KCC) or by separate K+ and Cl– channels. Whereas the KCC plays an important role in RVD in erythrocytes of all mammalian species studied, its major role in nonerythrocytes is in other functions such as K+ and Cl– homeostasis, cell growth, and apoptosis (see [9]). In erythrocytes, KCC is activated only by swelling caused by an increase in intracellular ionic strength, but not by swelling caused by extracellular hypotonicity, which activates separate K+ and Cl– channels as well as taurine efflux [10]. In human spermatozoa, the involvement of K+ channels in RVD is indicated by their swelling in quinine and its reversal by the K+ ionophore valinomycin [4]. K+ efflux leads to hyperpolarization, which would limit further K+ loss; therefore, effective volume regulation requires a parallel efflux of Cl–/anion for electro-neutrality (see [11]).

Although the role of Cl– channels in RVD is well established in somatic cells, their molecular identities are not. Among the candidates are ClC-2 (CLCN2) and ClC-3 (CLCN3) (see [12, 13]). CLCN2 has been accepted, at least in some cell types, as contributing to RVD [12], yet some of its electrophysiological features are at odds with the typical swelling-induced Cl– channels [14]. CLCN3 has been studied as a molecular identity of volume-sensitive chloride channel for almost 2 decades [15] but consensus is still to be reached (see [14]). Recent positive evidence has been provided by experiments involving inhibition of volume regulation by antisense oligonucleotide transfection and injection [16]. The lack of differences in volume-sensitive chloride currents between the wild-type and Clcn3-nullified mice cast doubt on the role of CLCN3 [17], but this has been counteracted by reports of possible compensatory changes in such mutant tissues [18]. ICln (CLNS1A) as another candidate has been a controversial issue since its proposal [19] because of its constitutive cytoplasmic location and channel properties in lipid bilayer studies (see [13]). On the other hand, positive evidence includes functional correlation of RVD in transfected cells with overexpression and inhibition with antibody or antisense oligodeoxynucleotides, and the translocation from cytosol to the plasma membrane stimulated by hypotonicity [12].

The molecular identities of K+ as well as anionic channels for sperm RVD have been studied in the mouse [20, 21] and boar [22]. The present study investigated the involvement of potential Cl– channels in the physiological process of RVD in human spermatozoa using channel blockers, and their identification at the protein and mRNA levels. The test substances and their dosages employed in the present study have been reported to be effective in blocking the volume-sensitive anion channels in somatic cells (see review in [12]).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All ejaculates used in this study were obtained from donors with written consent, by masturbation at the institute after abstinence of 2–5 days, and were analyzed by routine procedures according to guidelines [23]. The use of semen samples for the investigation was approved by the Ethics Committee of the University Medical Faculty and the Chamber of Physicians of Westfalen-Lippe.

Sperm Preparation and Incubation

For volume regulation experiments, ejaculates were obtained from 20 healthy volunteers. The average spermiogram (mean ± SEM) was as follows: semen volume, 4.0 ± 0.4 ml; sperm concentration, 41 ± 7 million/ ml; normal morphology, 15% ± 1%; and motility (grades a+b+c), 57% ± 2%. After liquefaction, spermatozoa were washed through a Percoll gradient (40%/80%) with osmolality adjusted to that of each ejaculate, which was measured in a vapor pressure osmometer (Wescor Vapro; Kreienbaum Messsystem, Langenfeld, Germany). Aliquots of washed sperm were incubated at 37°C in 5% CO2 in air, in media with or without channel blockers.

Incubation medium was Biggers, Whitten, and Whittingham (BWW) [24] modified to contain 20 mM Hepes in addition to NaHCO3 and 4 mg/ ml BSA at pH 7.4, with osmolality adjusted to 290 mmol/kg by addition of deionized water or 1 M NaCl. Blockers of volume-sensitive anion channels tested included 5-nitro-2(3-phenylpropylamino benzoic acid (NPPB; Calbiochem, Darmstadt, Germany), diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS; disodium salt), 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid (SITS; disodium salt; Molecular Probes, Leiden, the Netherlands), tamoxifen, verapamil, nifedipine, and niflumic acid (Sigma, Taufenkirchen, Germany). All test chemicals were made up in stock solutions in dimethylsulfoxide (DMSO), except for verapamil, which was prepared in deionized water. In each experiment, the control media contained 0.1%–0.4% (v/v) DMSO, which was equivalent to the final concentrations of the vehicle for the test substances.

Measurement of Cell Size by Flow Cytometer

Changes in sperm cell volume were measured by a previously validated method [25]. After 30 min of incubation, 20–40 µl of sperm suspension was added to 200 µl of the same medium lacking BSA and containing 3 µl of propidium iodide (PI; final concentration 6 µg/ml). Forward and side scatter signals from laser excitation at 488 nm were measured by a flow cytometer (version 3.0, Coulter Epics XL; Krefeld, Germany). From 6000 events recorded in each sample, excluding cell debris, nonviable sperm were gated out by the detection of PI fluorescence. Mean values of the forward scatter of viable sperm, which reflect cell volume, were analyzed and the drug-treated sperm were compared to the control from the same ejaculates. Effects of channel blockers (P < 0.05) were statistically tested using a one-way repeated-measured analysis of variance followed by the Dunnett test against the controls.

Analysis of Sperm Channel Proteins by Western Blotting

For the study of channel proteins, ejaculates were obtained with written consent from 28 volunteers and normozoospermic patients attending the Andrology Clinic and analyzed as described above. The average spermiogram (mean ± SEM) was as follows: semen volume, 4.0 ± 0.3 ml; sperm concentration, 36 ± 2 million/ml; normal morphology, 14% ± 1%; motility, 60% ± 2%. The washed sperm pellet was taken up in either a CHAPS buffer (10 mM CHAPS in 100 mM Tris buffer, pH 7.4) or another lysis buffer (125 mM NaCl, 25 mM Hepes, 10 mM EDTA, 10 mM sodium-pyrophosphate, 10 mM NaF, 0.1% [w/v] SDS, 0.5% [w/v] deoxycholate, 1% [v/v] Triton X-100 at pH 7.3), with both buffers containing 10 µl of protease inhibitor cocktail (Sigma) and 1 mM Na3VO4, as the phosphatase inhibitor for 1 h on ice with frequent vortexing. The suspension was centrifuged for 20–30 min at 15 000–20 000 x g at 4°C. As positive controls, murine kidney, testis, and cardiac atrium were snap-frozen, ground to powder using a Micro-dismembrantor (Braun Biotech International, Melsungen, Germany), and extracted as described above. Tissue extracts were estimated for protein concentrations using the bicinchoninic acid protein assay (Bio-Rad Laboratories, München, Germany) and stored at –80°C. Polyacrylamide gel electrophoretic separation of proteins was carried out using 4%–12% Bis-Tris precast gels (8 x 8 cm, 1 mm thick; NuPage, Invitrogen, CA) according to the manufacturer's instructions, with 40 µg of protein sample in each lane after heating at 65°C for 10 min in the absence of dithiothreitol (DTT).

For Clcn3 analysis, samples were heated at 100°C with DTT. Separated proteins were transferred onto Hybond-enhanced chemiluminescence (ECL) membranes at 150 mA for 3 h and stained with Ponceau Red to check protein loading and to visualize the molecular weight markers. After blocking with StartingBlock (Pierce, Perbio Science, Bonn, Germany) for 1 h at room temperature, the membrane was incubated with a purified antibody against CLCN2 (Alomone labs, Jerusalem, Israel) or CLCN3 (Santa Cruz Biotechnology, CA) at final dilutions of 1:2500 overnight at 4°C. For adsorption, the primary antibody was mixed with a 10-fold concentration of the peptide antigen and incubated overnight at 4°C with rotation, in parallel with the nonadsorbed aliquot. The antibody solutions were centrifuged at 20 000 x g for 20 min at 4°C before use for incubation with the blotted membrane. After washing, the membrane was incubated with the appropriate secondary antibodies (peroxidase-conjugated goat anti-rabbit immunoglobulin G [IgG], Pierce; at 1:1000 dilution; or bovine anti-goat IgG, Santa Cruz; at 1:100 000 dilution for 1 h). Peroxidase-bound protein bands were visualized using the ECL-Plus method (Femto-signal; Pierce).

For CLNS1A (ICln) analysis, primary antibody from Santa Cruz (N-20) was used at 1:100 after blocking with 5% skim milk, and the Pico-signal ECL system (Pierce) for visualization was used. To check for specificity of the ECL signals, parallel gel electrophoresis and blotted membranes were processed with primary antibody adsorbed with the corresponding antigen peptide from the same manufacturer. The molecular weights of the signal bands were analyzed using line densitometer software (ChemImage System, IS4400; Alpha Innotech Corp., San Leandro, CA).

Immunolocalization of Chloride Channels on Spermatozoa

Air-dried smears of Percoll-washed and PBS-washed spermatozoa as described above were prepared on polylysine-coated slides and fixed in 4% paraformaldehyde for 30 min and blocked by incubation in PBS containing 3% BSA for 30 min. Cells were incubated with the antibody against CLCN3 (Santa Cruz; 1:50) overnight at room temperature in a humid chamber and stained with fluorescein isothiocyanate-conjugated rabbit anti-goat IgG (Sigma; 1:100) for 1 h. Slides were washed for 20 min, mounted with Vectashield antifade medium (Vector Laboratories, Burlingame, CA), and examined with a Zeiss Axiovert 200 fluorescence microscope.

Analysis of Sperm mRNA by Reverse Transcription-Polymerase Chain Reaction

For the study of mRNA, ejaculates were obtained from six volunteers and normozoospermic patients attending the Andrology Clinic and analyzed as described above. The averaged spermiogram (mean ± SEM) was as follows: semen volume, 4.9 ± 0.8 ml; sperm concentration, 108 ± 35 million/ml; normal morphology, 15% ± 3%; motility, 62% ± 2%; and no observable leukocytes. Sperm were washed through Percoll gradient (35%/ 70%). The pellet containing 20–60 x 106 spermatozoa was resuspended in 0.5 ml BWW medium and incubated for 20 min with 5 x 106 anti-CD45-coated latex magnetic beads (Dynabeads; Dynal Biotech, Hamburg, Germany) to remove any possible presence of leukocytes, following the manufacturer's instruction.

For RNA extraction using the QIA shredder column and RNeasy mini kit (Qiagen, Heidelberg, Germany), the purified sperm pellet of 20–60 x 106 cells was resuspended in 600 µl of lysis buffer with 1% ß-merceptoethanol and stored at –70°C until further processing. Homogenization, isolation, precipitation, and purification of RNA were carried out according to the manufacturer's procedure, with an extra step of DNase treatment for removal of DNA contamination. For reverse transcription-polymerase chain reaction (RT-PCR), 15 µl of the extracted RNA was used for the synthesis of the first-strand cDNA using the SuperScript II reverse transcriptase (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instruction. The other 15-µl RNA extract was processed in parallel without the transcriptase as negative control for DNA contamination. Primers pairs used for PCR are shown in Table 1, and the products were separated in 2% agarose gel and identified against molecular weight markers (DNA-HaeIII Digest; BioLabs, Frankfurt, Germany). The quality of each sperm RNA sample was checked by RT-PCR for a clear strong signal of protamine 2 (Prm2, 147 base pairs [bp]) without any intron expression (309 bp, an indication of DNA content) and for the absence of the leukocyte-derived chemokine receptor Cxcr4 to ensure no contamination by leukocyte mRNA. Only samples with such proven qualities were used for the analysis of Clcn3 and Clns1a mRNA. For nucleotide sequencing, the signal bands of the PCR products from Clcn3 and Clns1a were each extracted and purified, and ligated into and cloned from the Escherichia coli XL1 Blue plasmid using the pGEM-T Easy Kit (Promega, Mannheim, Germany) by standard procedures. The cloned product was purified and sequenced.


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  		TABLE 1. Sequences of the forward (F) and reverse (R) oligonucleotide primers used for PCR in the 5'–3' direction, the sizes of the reaction products for each gene studied, and the conditions of the PCR reactions


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Volume-Sensitive Anion Channel Blockers on Sperm Volume

The osmolality of the semen samples used was measured to be 317 ± 5 mmol/kg. Incubation of the washed sperm in a medium of 290 mmol/kg mimicking cervical mucus osmolality, in the presence of the most versatile Cl– channel blockers NPPB and DIDS, resulted in cell swelling as reflected by the increase in laser forward-scatter compared to the control, whereas SITS had no significant effect (Fig. 1). Tamoxifen also blocked RVD, leading to significant swelling, whereas verapamil and nifedipine did not influence cell volume. Niflumic acid at 200 µM had no effect but caused cell shrinkage at 1 mM (Fig. 1). None of the drugs tested had any significant effect on the viability of sperm cells.



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  		FIG. 1. Effects of volume-sensitive Cl–/anion channel blockers on the size of human ejaculated sperm after incubation for 30 min in physiologically hypotonic medium in the absence (control) or presence of each blocker (concentrations in abscissa). The relative sizes of the treated sperm as a ratio of the control laser forward scatter (FS) are given on the y-axis. Values are mean ± SEM; n = 7. *Significant differences from the control value

Western Blotting of the Chloride Channel Proteins CLCN2, CLCN3, and CLNS1A

From a total of 18 ejaculate samples, including 2 from recent fathers examined in 6 experiments, none showed the presence of protein bands specifically stained by the antibody against CLCN2, although a specific band of the expected molecular size of 97 kDa was detected in proteins from the mouse testis used as positive control (Fig. 2). For the expression of CLCN3, a specific positive band of 87 kDa was found in all 10 sperm samples tested, which was of identical size to that of mouse heart tissue serving as a positive control (Fig. 3).



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  		FIG. 2. Western blotting analysis of CLCN2 channel protein performed on proteins from murine testis (lane 1, as positive control) and four different human sperm samples (lanes 2–5) in the absence (left panel) and presence (right panel) of the antigen peptide for antibody adsorption. A specific band of 97 kDa is evident in the control tissue but not in the spermatozoa



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  		FIG. 3. Western blotting analysis of CLCN3 channel performed on proteins from murine atrium (lane 5, as positive control) and four different human sperm samples (lanes 1–4) in the absence (left panel) and presence (right panel) of the antigen peptide for antibody adsorption. A specific band of 87 kDa is evident in the heart tissue and in all the sperm samples. An additional weak band of 97 kDa is visible in the first two sperm samples

The expression of CLNS1A was examined in eight ejaculates, including two from recent fathers. In the positive control tissues, a specific band of 32 kDa was evident in the kidney and 42 kDa in the heart, which were absent when the antibody was adsorbed with the antigenic peptide before use. From the eight sperm samples tested, only one sample, not from a recent father, showed a specific band of 40 kDa (Fig. 4). There were no remarkable differences in the semen parameters between this positive sample and the negative ones.



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  		FIG. 4. Western blotting analysis of CLNS1A channel protein performed on proteins from murine kidney (lanes 1 and 8) and heart (lane 2) as positive controls, and five different human sperm samples (lanes 3–7) in the absence (left panel) and presence (right panel) of the antigen peptide for antibody adsorption. A specific band of about 32 kDa was evident in the kidney, 42 kDa in the heart tissue, and 40 kDa in one of the five sperm samples

Analysis of mRNA of Chloride Channels Clcn3 and Clns1a

Using normal RT-PCR amplification, mRNA of Clcn3 and Clns1a could be detected only as very weak signal bands, or it was nondetectable, whereas further amplification using nested RT-PCR produced much stronger signals (Fig. 5). To verify the quality of the extracted mRNA samples and the absence of DNA as well as leukocyte contamination, RT-PCR for expression of the sperm-specific Prm2 and for the leukocyte-expressed Cxcr4 were also performed. Strong signals of the intron-excluding Prm2 amplicon with absence of the intron-including amplicon indicated the absence of DNA, whereas absence of Cxcr4 expression indicated no leukocyte contamination in the mRNA samples tested for the expression of the chloride channels (Fig. 6). All samples tested expressed Clcn3, whereas four of the six samples expressed Clns1a.



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  		FIG. 5. Conventional RT-PCR (left panels) of the Cl– channel Clcn3 (upper) and Clns1a (lower panel) showing detection of the mRNA in some but not all of four human sperm samples (lanes 1, 2, 4, and 6), which became detectable using nested RT-PCR (right panels). Lanes 3, 5, and 7 are negative controls using the same amount of RNA extracts as in lanes 2, 4, and 6, but with the reverse transcriptase omitted. The starting lanes indicate the molecular size markers and the arrows indicate the targeted PCR products



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  		FIG. 6. Sperm RNA extracts were reverse transcribed (lanes 1, 3, 5, 7, and 9), or not reverse transcribed (i.e., reverse transcriptase was omitted as the negative control for each sample (lanes 2, 4, 6, 8, and 10, respectively) to cDNA, which was used for normal PCR (Prm2 for RNA quality and Cxcr4 for confirming no contamination with leukocyte RNA) and nested PCR (Clcn3 and Clns1a). Leukocyte DNA (lane 11, Prm2) and RNA (lane 11, lower panels) were used as positive controls. The starting lanes indicate the molecular size references and the arrows indicate the targeted PCR products

The cloned PCR products from Clcn3 (597 bp) and Clns1a (738 bp) both revealed nucleotide sequences that were 100% identical to those published in the GenBank data base (accession numbers NM001829 and NM001293, respectively).

Immunolocalization of Chloride Channel on Spermatozoa

Using immunocytochemistry, CLCN3 was clearly detectable with similar staining patterns in sperm smears from all three donor ejaculates examined. The entire sperm tail was stained by the antibodies with high intensity at the neck and on the midpiece (Fig. 7). Sperm heads showed no fluorescence stronger than that of the negative control when secondary antibodies were used without the primary antibody (Fig. 7).



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  		FIG. 7. Fluorescence (A and C) and the corresponding phase-contrast (B and D) micrographs of washed, ejaculated spermatozoa incubated with (A and B) or without (C and D) the primary antibody against the chloride channel protein CLCN3, showing specific staining of the entire tail, especially the neck and midpiece. Arrows indicate cytoplasmic droplet. Bar = 10 µm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Unlike the study of K+ channels, in which blockers specific to certain type of channels can be employed in an attempt to identify the channels involved in volume regulation [21], no specific blockers can be used to identify different chloride/anion channels. For instance, besides the swelling-activated Cl– channels, NPPB can also inhibit Ca2+-activated Cl– channel [26], Ca2+-activated K+ channel [27], or L-type Ca2+ current [28]. Nevertheless, the induction of swelling by the standard volume-sensitive Cl– channel blockers NPPB, DIDS, and tamoxifen during incubation in medium of cervical mucus osmolality (290 mmol/kg) indicates the involvement of such channels in physiological volume regulation of human sperm. Although the extents of volume changes were very small, similar changes have been demonstrated to drastically hinder mucus penetration and reduce the linearity (LIN) of swim-path by 50% without decreasing curvilinear velocity [4]; LIN changes have been shown to correlate with volume changes [25].

Whereas 400 µM DIDS in the present study inhibited human sperm RVD, the same concentration of SITS did not. Volume regulation or swelling induced Cl– currents are inhibited by SITS at concentrations <100 µM or <1 mM in many types of somatic cells [12]. Although DIDS and SITS are very similar in their chemical components, they are very different in their 3-D structures. May be the allosteric environment of the sperm Cl– channel is less susceptible to SITS. The effectiveness of NPPB and the ineffectiveness of SITS in sperm RVD have been shown in other species, but there are differences in their sensitivity to DIDS and tamoxifen (mouse, [20]; bull, [29]; boar, [22]; cynomolgus monkey, [30]). Another difference between murine and human sperm is the effectiveness of verapamil and nifedipine in the former but not the latter. Verapamil can act as an antagonist to P-glycoprotein, which is phosphorylated by protein kinase C upon volume changes, and switches its function from a multidrug-resistance transporter to a Cl– channel [31], whereas nifedipine at 100 µM has been reported to inhibit the swelling-activated Cl– current [32]. However, in murine sperm RVD, these two drugs are more likely to act via Ca2+ [20].

In the family of chloride channels, CLCN2 and CLCN3 have been suggested to be involved in cell volume regulation. Because there is no specific channel blocker to identify these different channels, in the present study, sperm protein was analyzed using Western blotting. Murine testicular tissue was used as the positive control for CLCN2, because its expression has been localized in the Sertoli cells, but not germ cells [33]. No specific protein band could be detected in human sperm proteins. On the other hand, expression of CLCN3 channel protein was found in all the samples tested. The failure in obtaining positive signals for Clcn3 mRNA in some samples using conventional RT-PCR methods is presumably due to the low amounts of extracted material. This was overcome using the nested RT-PCR technique for further amplification, which demonstrated the presence of the mRNA of Clcn3 in all samples. This channel protein of 87 kDa determined in the present study compares well with the reported molecular sizes in pulmonary artery and cardiac smooth muscles (85–92 kDa, [34, 35]). The minor band of 97 kDa in some samples could represent another differently glycosylated form, because CLCN3 has four potential N-glycosylation sites, and a double band in sizes of 90–100 kDa has been expressed by transfected HeLa cells [16]. The 597-bp amplification product of RT-PCR shared 100% identity with the published sequence. The expression of CLCN3 on sperm cells was localized all along the sperm tail, especially in the neck and midpiece region. This clear demonstration of Clcn3 and CLCN3 expression in human sperm may support the suggestion of this protein in porcine sperm [22], but it is in clear contrast to its absence from murine sperm [20], again highlighting species differences.

Another molecular candidate for the volume-sensitive chloride channel is Clns1a, (see [12]). The candidacy of CLNS1A is controversial because it is uncertain whether the protein is a component of a channel or a cytosolic regulatory protein of the chloride channel [36]. Lipid bilayer studies also cast doubt on whether it is strictly an anion channel, because it also displays cation channel properties [37]. This protein has a molecular weight of 26 kDa estimated from the deduced amino acid sequence [38], but the protein has been reported to be of various sizes among different cell types ranging from 35 to 47 kDa [39, 40]. In human sperm in this study, it was detectable only in one of eight samples investigated, with a molecular weight of 40 kDa. Using the highly sensitive method of nested RT-PCR for the detection of mRNA, it was absent from one-third of the samples, although they showed strong signals for the presence of protamine 2. The significance for the presence of sperm Clns1a in some but not all men is unclear, and the possibility of contribution by early germ cells that could be present in the ejaculate samples could not be excluded. Nevertheless, these findings do not support the candidacy of CLNS1A for such a general physiological function of sperm cell volume regulation.

In somatic cells in which volume increases induced by hypotonic environments activate both K+ and Cl– efflux through independent K+ and Cl–/anion channels, these fluxes are nevertheless constrained to a certain extent to achieve electro-neutrality via the membrane potential. In many cell types, especially when intracellular Cl– concentration is not high, organic osmolyte effluxes also occur through yet undefined channels [11, 41]. In addition to blocking volume-sensitive Cl– channels, the effective RVD blockers presently found (DIDS, NPPB, and tamoxifen) can also inhibit the efflux of amino acids [42, 43] and other organic osmolytes such as myo-inositol during RVD [11, 41]. Some of these organic osmolytes are found in high concentrations in the epididymal lumen, including myo-inositol, glutamate, and taurine [44].

In the mouse, incubation of cauda epididymidal spermatozoa in an osmolality that reflects uterine fluid, which normally activates RVD, leads to cell swelling in the presence of potential organic osmolytes added to eliminate concentration gradients across the sperm membrane [45]. However, these osmolytes, which include glutamate, taurine, carnitine, and myo-inositol, had no effect on RVD of human spermatozoa at the concentrations tested [25]. Such ineffectiveness suggests either the concentration gradients of these substances in human sperm are much greater than in murine sperm, or other organic osmolytes are involved in human sperm RVD. It is interesting that human spermatozoa exhibited a smaller volume than the control in the presence of 1 mM niflumic acid. It has been reported that niflumic acid at 0.5 mM stimulates efflux of swelling-activated sorbitol release from inner medullary collecting duct cells [46]. Whereas many potential osmolytes, including myo-inositol, are secreted and accumulated in high concentrations in the mammalian epididymis, and could be taken up by the maturing spermatozoa via isovolumetric regulation during their epididymal sojourn to be used as osmolytes for RVD in the female genital tract, as suggested by Cooper and Yeung [1], there are no data regarding sorbitol in epididymal fluid.

In conclusion, physiological volume regulation of human spermatozoa is sensitive to Cl– channel blockers DIDS, NPPB, and tamoxifen, suggesting efflux of Cl– for RVD complementary to K+ efflux through K+ channels [4]. Among the three channel proteins investigated as molecular candidates, CLCN2 was ruled out, whereas CLNS1A was questionable because it was not detectable in all samples. ClC-3 was identified consistently at both the protein and the mRNA levels and localized on the tail, suggesting its possible involvement in human sperm volume regulation. The only other Cl– channel identified in mammalian spermatozoa at present is the glycine receptor involved in the zona pellucida-induced acrosome reaction [47]. Although the cystic fibrosis transductance regulator (CFTR) channel is reported in the rat spermatids, it is absent from spermatozoa [48]. The CFTR channel does not play a role in RVD; defective RVD in tracheal cells in cystic fibrosis is caused by failure of the Ca2+-dependent K+ efflux [49, 50]. Further elucidation of the mechanisms of sperm volume regulation and the involvement of other osmolytes are needed to understand this physiological process for clinical application in infertility diagnosis and development of a male contraceptive.


   ACKNOWLEDGMENTS
 
We thank Barbara Hellenkemper and Jolanta Körber for technical assistance, and Raphaele Kürten, Sabine Rehr, and Daniela Hanke for semen analysis.


   FOOTNOTES
 
1 Supported by the Deutsche Forschungsgemeinschaft, grant YE37/6-1, and by a Crescent City doctoral scholarship from the University of New Orleans to J.P.B. Back

2 Correspondence: C.H. Yeung, Institute of Reproductive Medicine, Domagkstrasse 11, D-48129 Münster, Germany. FAX: 49 251 835 6093; chinghei.yeung{at}ukmuenster.de Back

Received: 25 May 2005.

First decision: 5 July 2005.

Accepted: 20 July 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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