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Male Reproductive Tract |
Institute of Reproductive Medicine of the University,4 Münster, Germany
College of Medicine,5 Wuhan University, Wuhan, People's Republic of China
Institute of Physiology,6 University of Tübingen, Tübingen, Germany
Department of Physiology,7 University of Zürich, Zürich, Switzerland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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26 mM and 30 µM, respectively) were measured by microelectrodes to be identical in the caudal fluid from both genotypes.
epididymis, gamete biology, male reproductive tract, sperm maturation, sperm motility and transport
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Comparative gene expression profiling has been used in the initial screening of candidate genes for the relevant epithelial function that may result in an abnormal epididymal milieu in the infertile animals. One of these is the sodium-dependent glutamate transporter EAAC1, which is down-regulated in the c-ros KO mouse proximal epididymis and could partially contribute to the sperm defect in cell volume regulation [7, 8]. Another candidate gene is the sodium-inorganic phosphate cotransporter (NaPi-IIb), which was found to be overexpressed in the proximal region of the epididymis of the c-ros homozygous KO mice compared to the fertile, heterozygous (HET) animals. The NaPi-II membrane transporters are highly regulated systems employed by epithelia for inorganic phosphate (Pi) homeostasis. Whereas the isoform NaPi-IIa is expressed only in the kidney and the brain, the phylogenically older isoform NaPi-IIb was first cloned from the small intestine [9] and is expressed in several other epithelia (for review, see [10]). In the epididymis, the control of Pi level would be crucial in view of its very high concentrations in the luminal fluid reported in the literature. These amount to 16 mM in the distal end of the tubule in rats [11] and 24 mM in the vas deferens of men [12], presenting a risk for the precipitation of calcium phosphate. Because NaPi-II could serve as a backup system for Na+ reabsorption in the proximal kidney tubule [13], it is possible that NaPi-IIb is also involved in the highly active Na+ reabsorption of the epididymis.
In this report of NaPi-IIb in the epididymis, we have quantified the gene as well as protein expression profiles, and we have established the immunological localization with regional differences within the organ. Concentrations of Na+, Ca2+, and Pi in the epididymal fluid bathing the fertile and infertile murine spermatozoa have also been measured to gain insight regarding the potential role of epididymal NaPi-IIb in male fertility regulation.
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MATERALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male and female mice that are heterozygous for targeted deletions of c-ros [2, 14] were generously donated as breeding pairs by Dr. E. Riethmacher and Prof. Dr. C. Birchmeier from the Max-Delbrück Centre of Molecular Medicine (Berlin, Germany), where the transgenic animals were generated. The transgenic colony was maintained at 22°C under a 12L:12D photoperiod. Experimental studies were conducted according to the German Federal Law on the Care and Use of Laboratory Animals (licenses A38/2002 and G61/2001). Because HET male mice showed normal fertility [2] and sperm motility [4] without any difference in phenotype compared to the wild-type mice, they were used as normal controls.
Each epididymis from the HET and KO mice was dissected clean of connective tissue and divided into four segments: the initial segment (or the equivalent gross anatomical region in the KO mice) and the remaining caput, corpus, and cauda regions. The tissues were snap-frozen in liquid nitrogen and stored at -80°C until use. For some gene-chip array experiments, the most proximal part of the epididymis, consisting in the HET mice of the initial segment and proximal caput region and in the KO mice of the equivalent gross anatomical region, were isolated, frozen in liquid nitrogen, and stored at -80°C. For other array experiments, whole epididymides from both genotypes were divided into the caput, corpus, and cauda regions for comparison.
RNA Extraction, Reverse Transcription-Polymerase Chain Reaction, and Northern Blot Analysis
From 50 to 100 mg of frozen tissue of various segments were homogenized by a microdismembrator (Braun Biotech International, Melsungen, Germany), and total RNA was extracted using the Qiagen RNeasy Midi kit (Qiagen, Heidelberg, Germany) and quantified spectrophotometrically (BioPhotometer; Eppendorf, Hamberg, Germany). For the detection of NaPi-IIb mRNA and the generation of cDNA as template for Northern blot analysis, conventional reverse transcription-polymerase chain reaction (RT-PCR) was performed as previously described [15] using the forward oligo-primer 5'-CGGACAGTTCTTCAGCAACA-3' and the reverse primer 5'-CAGAATGTCTGGGGCATCTT-3', resulting in an amplicon of 403 base pairs (bp). Northern blot hybridization of the extracted total RNA, using 15 µg of each sample, was performed similar to the method described previously [15].
Gene Expression Analysis by AffyMetrix Gene Chip
With each gene chip (MG U74A; Affymetrix, Santa Clara, CA), 20 µg of total RNA from each epididymal region were analyzed according to procedures recommended by Affymetrix. Double-stranded cDNA reverse-transcribed from the RNA sample was used as template to transcribe biotin-labeled cRNA. After hybridization of the labeled probe onto the gene chip, the marker was visualized by conjugate incubation in a Fluidics station 400 (Affymetrix) and scanned with the GeneArray scanner. Quantification of the digitized fluorescence signals generated mean values, and the differences between four pairs of gene chips (KO and HET samples processed in parallel in each experiment) were analyzed by the GeneChip Suite Analysis Software (Affymetrix).
Western Blot Analysis and Quantification
Frozen tissues were ground to powder in 100 µl lysis buffer using the microdismembrator and taken up in a total of 0.5 ml of 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, and 1% (v/v) Triton X-100 at pH 7.3 containing 10 µl of protease-inhibitor cocktail [Sigma, St. Louis, MO] and 1 mM Na3VO4). The suspension was vortexed and centrifuged for 30 min at 15 000 x g at 4°C. The tissue extract was estimated for its protein concentration using the bicinchoninic acid (BCA) protein assay and stored at -20°C.
Polyacrylamide gel electrophoretic separation of proteins was carried out using 10% Bis-Tris precast gels (8 x 8 cm; thickness, 1 mm; NuPage; Invitrogen, Carlsbad, CA) according to the manufacturer's instructions with 30 µg of protein sample in each lane after heating at 70°C for 10 min in the presence of dithiothreitol. Separated proteins were transferred onto ECL-Hybond membranes (Amersham Biosciences, Little Clalfont, UK) at 35 V for 100 min. After blocking with 5% milk powder at 4°C overnight, the membrane was incubated with the purified rabbit antibody against NaPi-IIb (produced as described in Hilfiker et al. [9]) at a final dilution of 1:2400 for 1 h at room temperature and with the secondary antibody (peroxidase-conjugated goat anti-rabbit immunoglobulin [Ig] G; Sigma) at 1:12 000 dilution for 1 h. Peroxidase-bound protein bands were visualized using the ECL-Plus method (Amersham Biosciences). Intensities of bands on films were quantified using the line densitometer software (ChemImage System, IS4400; Alpha Innotech Corp., San Laendro, CA).
To check for specificity of the enhanced chemiluminescence signals for NaPi-IIb, blotted membranes were processed with normal rabbit IgG (8 µg/ml) replacing the primary antibody or with primary antibody adsorbed with the antigen peptide (60 µl of a 10 mg/ml concentration with 5 µl of antibody diluted 1:1 in glycerol).
Immunohistochemistry
Paraformaldehyde-fixed and paraffin-embedded tissues were cut into sections (thickness, 4 µm). After deparaffinization and rehydration, tissue sections were heated in a microwave oven for 25 min at 80°C in 0.05 M glycine buffer (pH 6.0) for antigen retrieval. After blocking with 5% normal goat serum in 1% bovine serum albumin in Tris-buffered saline, sections were incubated for 1 h at room temperature with the primary antibody (see above) at a dilution of 1:100 and for 60 min at room temperature with secondary antibody (1:100, goat anti-rabbit IgG conjugated with alkaline phosphatase; Sigma). Bound antibody conjugates were visualized by fuchsin substrate-chromogen (DAKO Corporation, Carpinteria, CA). Sections were counter-stained with Mayer hematoxylin and mounted in Faramount (DAKO). Negative controls for the immunostaining included replacement of the primary antibody with normal rabbit IgG (10 µg/ml) or with primary antibody after absorption with the antigen peptide (12 µl of a 10 mg/ml concentration with 1 µl of antibody diluted 1:1 in glycerol in a final volume of 100 µl of staining solution).
Fluorimetric Assay of Pi
The proximal vas deferens of freshly isolated epididymis was cannulated with a drawn-out polyvinylchloride catheter (Critchley Electrical Products Pty Ltd., Silverwater, Australia). The perfusion solution used had osmolalities of approximately 400 mmol/kg to mimic native epididymal fluid [4] and consisted of either sucrose with 10 mM Tris (pH 7.4) or NaCl with 100 mM Tris (pH 7.5). Luminal contents from both caudae epididymidum (3 µl in total, approximately half the volume comprising sperm cells according to values from other rodents [16]) were exuded from a cut end of the tubule in the proximal cauda region by retrograde perfusion from the vas deferens and transferred via a positive displacement pipette into 100 µl of the same solution. Sperm cells were removed by centrifugation at 4°C at 2000 x g for 2 min, and the supernatant was centrifuged again for 5 min before storage of the sperm-free fluid at -20°C. Protein concentrations of the samples were measured using the BCA assay.
Inorganic phosphate in samples diluted 1:50 with the assay buffer was measured in 96-well plates by a commercial kit (Kit A-12216; Molecular Probes, Leiden, The Netherlands). This is an enzymatic assay measuring resorufin as an end product after conversion of maltose into glucose by maltose phosphorylase using the Pi available in the sample. Emission at 590 nm with excitation at 530 nm was read in a fluorimetric plate reader (spectroMAX Gemini; Molecular Devices GmbH, Munich, Germany) after incubation for 45 min at 37°C. The same dilution of the perfusion solution was added to the assay blank and Pi standards (0.125–50 µM) to correct for any interference of the assay. Endogenous glucose, if any, did not contribute to any reading, because measurements made in the absence of maltose phosphorylase did not give any positive absorbance. Because the volume of the neat epididymal fluid was too small to be measured accurately, results were expressed as the amount of Pi per milligram of protein in each sample.
Measurements of Na+ and Ca2+ in Epididymal Fluids Using Ion-Selective Electrodes
Ion-selective microelectrodes were pulled, coated, and filled with either the sodium ionophore I-cocktail A or the calcium ionophore II cocktail A (Fluka Chemicals, Deisenhofen, Germany) as previously described [17]. The electrode was then backfilled with a solution of 100 mM NaCl and 10 mM Hepes at pH 7.4 for determination of Na+ or with 10 mM CaCl2 and 10 mM Hepes at pH 7.4 for measurement of Ca2+. Droplets of standards (5 µl) and samples were placed on a Petri dish surrounded by wet tissue paper. Both the reference and the ion-selective microelectrodes were inserted into the drop to measure the potential difference. Signals were recorded with an electrometer (Model FD223, World Precision Instruments, Sarasota, FL). Only electrodes with a linear slope and stable calibration were used. For each sample from one epididymis, a new standard curve was prepared just before collection of the sample (see below). Ion concentrations were calculated from linear regression of the potential response of microelectrodes using Microsoft Origin 6.0 (Microsoft, Redmond, WA).
The mice were anesthetized using Avertin (0.4 ml of a 2.5% [w/v] solution). One epididymis was isolated, and the luminal contents of the cauda epididymidis were collected neat as exudate by retrograde perfusion from the cannulated vas deferens as described above. The perfusion solutions contained Indian ink or trypan blue to mark any mixing with the native luminal contents; were made up in 10 mM Hepes with the target ion at concentrations close to those reported in rats (30 mM Na+ and 0.1 mM Ca2+ [18]) to reduce any diffusion gradient, if any; and had osmolalities of approximately 400 mmol/kg adjusted with sucrose. Uncontaminated luminal exudate was collected into a positive displacement pipette and immediately measured as described above. Samples from the infertile homozygous KO and the fertile HET control mice were measured alternately. After closing the wound, the animal was allowed to recover for a day or two, and the remaining epididymis was used to measure another electrolyte.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Comparison of the proximal head of the epididymis from KO and HET animals in four pairs of Affymetrix chips indicated an overexpression of NaPi-IIb by 9.9 ± 4.0-fold (mean ± SD) in the KO mice. On the other hand, no difference was observed in the corpus region, and expression was nondetectable (registered as absence by the analysis program) in the cauda regions of both genotypes. The corpus region expressed more NaPi-IIb than the caput region in HET tissues, whereas the same expression levels were detected in the KO tissues, probably because of overexpression in the proximal segment.
Of the other sodium-inorganic phosphate cotransporters analyzed using the gene chips, the level of NaPi-I (solute carrier family 17, member 1) expression was nondetectable in any region of either genotype investigated. Expression of NaPi-IIa (solute carrier family 17, member 2) was present in the most proximal region as well as in the caput and corpus and was only marginally detected in the cauda epididymidis, without any significant differences between the genotypes. In both the caput and corpus regions, expression of NaPi-IIb was more than 5-fold higher than expression of NaPi-IIa.
RNA Expression of NaPi-IIb Using Northern Blot Analysis
Using RNA extracted from the caput region, a single signal band of cDNA, with the expected size 403 bp of the amplicon, was obtained for both genotypes by RT-PCR (Fig. 1). Northern blot analysis of tissues from various regions revealed a strong signal band of 4.3 kilobases (kb; similar to the size of intestinal NaPi-IIb cloned in mouse), with similar intensities in RNA samples from the caput and corpus regions of both genotypes and from the KO region equivalent to the initial segment of the HET (Fig. 2). However, the HET initial segment and the cauda region of both genotypes showed extremely weak signals. Quantification of signal intensities after normalization against total rRNA of each applied sample confirmed the findings in the gene-chip experiments (Fig. 3). Another strong, but very small, signal band of approximately 0.8 kb was also present in all regions except the cauda.
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Protein Expression of NaPi-IIb Using Western Blot Analysis
The NaPi-IIb antibody recognized two protein bands that were absent from the negative controls (Fig. 4), one in the size of 80 kDa (as expected from the amino acid sequence) and a 67-kDa band. Despite some variability between experiments, probably because of the lack of clear, three-dimensional demarcation for dividing the regions during tissue preparation, a general trend was observed of strongest expression in the corpus region; very weak, if any, expression in the cauda of both genotypes; and higher expression in the KO compared to the HET tissues in the most proximal epididymal region (n = 7) (Fig. 5).
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Localization of NaPi-IIb Protein in the Epididymis
In the HET epididymis, no immunostaining was observed in the initial segment. The protein was localized on the brush border of the epithelium in the more distal regions up to the cauda, from which it was absent. Staining in the lumen was apparently nonspecific, because it was also present after antibody adsorption. In the KO tissues, staining was obvious even in the most proximal region, where the epithelium had not differentiated into the tall columnar cells as in the HET initial segment (Fig. 6). No apparent difference was found between genotypes in the more distal regions. In both genotypes, the efferent ducts showed specific positive staining in the apical membrane of the ciliated cells and in some cilia (micrograph not shown).
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Levels of Pi in Cauda Epididymidal Fluid
Luminal fluids flushed out of the cauda epididymidal tubules contained 1.14 ± 0.13 mmol (mean ± SEM) of Pi per 1 g of protein (n = 17) in the HET and 0.83 ± 0.05 mmol/g protein in the KO tissues (n = 16), which showed a decrease of 27% (P = 0.037).
Concentrations of Na+ and Ca2+ in Cauda Epididymidal Fluids
Both the HET and KO animals showed similar concentrations of Na+ (mean ± SEM: 26.3 ± 2.4 and 26.8 ± 2.6 mM, respectively; n = 8 and 7, respectively) and Ca2+ (30 ± 5 and 35 ± 3 µM, respectively; n = 8 for both genotypes).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The lack of NaPi-IIb in the initial epididymal segment of c-ros HET mice more likely reflects down-regulation by factors present in the differentiated epithelium than it does a direct down-regulation by c-ros, because NaPi-IIb was also absent from the cauda, where c-ros is not expressed [2]. Factors involved in the documented developmental down-regulation of NaPi-II discussed above are unclear. Epidermal growth factor (EGF) administration in developing rats decreases NaPi-II at the protein level in kidney tubules [22], presumably via inhibition of the gene promoter [23]. Both EGF and EGF receptor are expressed in the epididymis [24, 25]. Another candidate is vitamin D, which stimulates NaPi-IIb protein expression [26] as well as increases its mRNA in intestinal epithelium [20]. High amounts of vitamin D are known to be present in the epididymis from the detection of its metabolites [27], and vitamin D-binding sites are demonstrated in the epithelium of the caput epididymides [28]. Any differences of these candidate genes or proteins between the c-ros HET and KO mice are unknown.
Data regarding NaPi-IIb gene expression obtained using the gene arrays and Northern blot analysis were in agreement. The transcript of 4.3 kb was similar in size to those reported in the literature (4 kb in the mouse intestine [9] and 5.0 kb in human tissues [19]). However, another small transcript of 0.8 kb was also detected in abundance in the proximal epididymal regions but only weakly in the corpus and not at all in the cauda regions. This transcript could be generated by alternative splicing. However, any translation into a functional protein is doubtful, because no specific, low-molecular-size protein band was detected in Western blot analyses using the antibody raised against peptide near the C-terminus. On the other hand, the two specific bands of 80 and 67 kDa were smaller than the 108-kDa protein in the adult mouse intestine [9, 29] and lung [30] but were similar to the major band of 78 kDa found in the intestine of prepubertal rats. Interestingly, the extent of glycosylation of the intestinal protein is age dependent, such that an 88-kDa band is expressed in prepubertal mice instead of the 110-kDa band in adults [29], whereas a size of Mr 78 can be deduced from the mRNA sequence [9]. In the epididymis, the 80-kDa molecule could differ from the intestinal protein in glycosylation and likely is the functional isoform, whereas the 67-kDa protein might be a product of posttranslational processing. On the other hand, such differences between tissues as a result of different methods of protein extraction and treatment for Western blot analysis cannot be ruled out.
The concentration of Pi in the luminal fluid from the distal end of the epididymis is very high in the two species studied (16 mM in rats [11] and 24 mM in humans [12]). The present measurement in fertile mice would be equivalent to approximately 18–40 mM, assuming a dilution factor of approximately 75 during sample collection (see Materials and Methods), or a protein concentration of 35 mg/ml of neat luminal fluid (values from other rodents [16, 31]). The level in infertile c-ros KO mice was significantly reduced by 27% (equivalent to 5–11 mM) assuming no change in the protein concentration. However, whether this is a result of the abnormal patterns of gene and protein expression is uncertain. Some degree of Pi reabsorption along the rat epididymis is indicated by the concentrations of Pi measured in the caput, corpus, and cauda regions and the extent of concentration of luminal contents by water reabsorption along the tubule length, as estimated either from the increases in sperm number [32] or in spermatocrit values [11]. The very high level of Pi is compatible with the very low concentration of Ca2+ measured in the fluid, suggesting the importance of tight control of both Pi and Ca2+ transport in the epididymis to avoid crystallization of calcium phosphate, which may block the passage of sperm. Nevertheless, despite a difference in the Pi concentrations, no difference was observed in the Ca2+ concentrations between the fertile and infertile mice.
The physiological implication of a lower Pi concentration in the epididymal lumen of infertile KO mice is unclear, and the site of this decrease remains undefined. High Pi concentrations may be required for the sperm maturation processes taking place in the caput and corpus regions and for the maintenance of the stored spermatozoa in the cauda segment, where NaPi-IIb was no longer expressed. It may be essential for the development of sperm function that no phosphate reabsorption should occur in the initial segment, which is missing in the KO mice. If the presently measured 27% (estimated to be 5–11 mM) decrease were to occur in this proximal region, it could have crucial consequences for the maturation of testicular spermatozoa coming into the epididymis, because only 6.4 mM Pi is present in the fluid of the proximal caput epididymides of rats (40% of the concentration in caudal fluid [11]).
Because the coupling ratio of NaPi-II is 3 Na+ to 1 Pi [10], it is possible that NaPi-IIb is also involved in Na+ reabsorption. In this respect, renal NaPi-II is markedly up-regulated when one of the major Na+-transporter genes, either the Na+-H+ exchanger NHE3 or the Na+-Cl- cotransporter NCC, is nullified [13], and the upregulation was interpreted as one of the compensatory mechanisms. The epididymal epithelium is very active in sodium reabsorption (for review, see [33]), with luminal concentrations decreasing from greater than 100 mM to approximately 30 mM along the entire length of the epididymal tubule [31]. However, despite a decrease in the concentration of Pi, no difference was detectable in the concentration of Na+ present in the cauda epididymidal fluid, which was at levels as low as those reported for other species. Therefore, NaPi-IIb is unlikely to be involved in Na+ reabsorption in the epididymis.
In conclusion, the present work established and quantified the regional expression profiles of epididymal NaPi-IIb and differences between the fertile and infertile genotypes. High expression of NaPi-IIb was found in the epithelial brush border in regions where sperm maturation occurs, except in the initial segment. In the infertile c-ros KO mouse, the cotransporter was already highly expressed at both mRNA and protein levels in this most proximal region, which does not undergo the normal differentiation at puberty. The resultant lower contents of Pi in the epididymal luminal fluid, without changes in the Na+ or Ca2+ concentrations, may have implications in the normal maturation of spermatozoa into functional, potent cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Ching-Hei Yeung, Institute of Reproductive Medicine, Domagkstrasse 11, Münster, D-48129 Germany. FAX: 49 251 8356093; yeung{at}uni-muenster.de
3 Current address: Schering AG, 13342 Berlin, Germany
Received: 9 April 2003.
First decision: 7 May 2003.
Accepted: 14 May 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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