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BOR - Papers in Press, published online ahead of print October 10, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.062760
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BIOLOGY OF REPRODUCTION 78, 342–351 (2008)
DOI: 10.1095/biolreprod.107.062760
© 2008 by the Society for the Study of Reproduction, Inc.

Male Reproductive Tract; 

Alterations in Gene Expression in the Caput Epididymides of Nonobstructive Azoospermic Men1

Evemie Dubé 3, Louis Hermo 4, Peter T.K Chan 5, and Daniel G Cyr 2 3 4

INRS-Institut Armand Frappier,3 Université du Québec, Laval, Québec, Canada H7V 1B7 Department of Anatomy and Cell Biology,4 McGill University, Montreal, Québec, Canada H3A 2B2 Department of Urology,5 Royal Victoria Hospital, McGill University, Montreal, Québec, Canada H3A 1A1

ABSTRACT

Spermatozoal maturation in the epididymis is dependent on proteins secreted by the epithelium and those that create the proper ionic composition and pH of the lumen as well as the blood-epididymal barrier. For the human epididymis, little information exists about the regulation of these proteins in male infertility. Our objectives were to assess gene expression profiles in the caput epididymidis from men with normal spermatogenesis and men with nonobstructive azoospermia. With microarrays, we identified 414 genes in the caput epididymidis that were differentially regulated in infertile men by at least 2-fold compared with the fertile men. They were mostly involved in transcription, intracellular signaling, immunity, and fertility. Although the expression of genes encoding tight junctional proteins was not affected, the localization of CLDN10 and TJP1, but not CLDNs 1, 3, and 8, was altered in infertile patients, suggesting that there are changes in the paracellular functions of the blood-epididymal barrier. Differentially regulated genes included several encoding proteins involved in spermatozoal maturation, water and ion channels, and beta-defensins: CRISP1, SPINLW1, FAM12B, and DEFB129 were upregulated, whereas CFTR, AQP5, KCNK4, KCNK17, SLC6A20, SLC13A3, DEFB126, and DEFB106A were downregulated. Furthermore, the immunolocalization of AQP5, but not of CFTR or CRISP1, varied in infertile and fertile patients. The observation that the expression of genes involved in water and ion transport were repressed in infertile patients suggests that these genes are regulated by the presence of testicular products or spermatozoa in the epididymal lumen or are part of a broader syndrome associated with nonobstructive azoospermia.

beta-defensins, epididymal junctions, epididymis, gene regulation, genomics, infertility, male reproductive tract, male sexual function, water and ion channels

INTRODUCTION

Infertility is defined as the inability to conceive after 1 yr of unprotected intercourse and affects about 13%–18% of consulting couples. About 40%–50% of infertility cases in couples involve male factor infertility. Male infertility can occur either as an isolated disease or as part of a complex syndrome. In more than half of infertile men, the cause of their infertility is unknown [1–3]. Azoospermia, or the absence of spermatozoa in the ejaculate, may be caused by obstruction of the excurrent ductal system (obstructive azoospermia) or testicular failure (nonobstructive azoospermia), with the latter being the most severe form of male infertility. Various conditions, including congenital or developmental defects of the reproductive system, genetic anomalies, acquired testicular insults, and toxin exposures, can cause nonobstructive azoospermia [4–6]. Recent progress in the treatment of male infertility has enabled the recovery of testicular sperm from nonobstructive azoospermic patients for intracytoplasmic sperm injection to allow these men to father children. Unfortunately, the recovery of testicular sperm is not always successful in these patients. Moreover, the fertilization and pregnancy rates are significantly reduced in patients with nonobstructive azoospermia compared with other types of infertility [7–9]. It is therefore important to better understand the molecular genetics of male infertility to find new therapeutic approaches to alleviate human infertility.

Evidence for the involvement of the epididymis in infertility has been known for several years. The epididymis is a long single convoluted tubule that is generally divided into three segments: the caput, corpus, and cauda (Fig. 1). Its main functions are the maturation, transport protection, and storage of mammalian spermatozoa. Spermatozoal protection is dependent on proteins secreted or reabsorbed by the epithelium and those that create the proper ionic composition and pH of the lumen as well as the blood-epididymal barrier. The blood-epididymal barrier is composed of apical tight junctions between principal cells forming an impenetrable seal, forcing movement of molecules across these cells by specific receptors, ion and water channels, and solute carrier proteins [10–13]. Tight junctional proteins include several peripheral membrane proteins, such as tight junction protein (TJP) 1, TJP2, and TJP3, and transmembrane proteins, such as claudins (CLDNs). Although these proteins are well defined in rodents, their identification in the human epididymis and their regulation in male infertility are relatively unknown [11, 14, 15].


Figure 1
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  		FIG. 1 Schematic representation of the human epididymis. This organ is generally divided into three regions: the caput, the corpus, and the cauda.

In the male reproductive tract, gene profiling studies have been done on spermatozoa [16], on the epididymis from fertile men [17, 18] and rodents [19–21], on testicular germ cell tumorogenesis [22], on FSH-stimulated Sertoli cells [23], and on testicular development [24]. Elucidation of the transcriptional profiles of epididymal segments in infertile men is a crucial step toward understanding the causes of male infertility and could help diagnose male infertility [25]. In the present study, given the extensive modifications associated with spermatozoal maturation that occur in the caput epididymidis, we have investigated possible changes in epididymal gene expression in the caput region of men suffering from nonobstructive azoospermia.

MATERIALS AND METHODS

Tissue Preparation

Epididymal tubules from caput epididymides were obtained microsurgically from four patients (29–50 yr old) undergoing radical orchidectomy for localized testicular cancer (confined within testicular tunica albuginea) [18] and from four nonobstructive azoospermic patients (32–42 yr old) undergoing microsurgical sperm extraction surgery. All patients undergoing orchidectomy had active spermatogenesis. Tissues were received in cold culture medium containing antibiotics (Dulbecco modified Eagle and Ham F-12 medium with penicillin-streptomycin) and were processed as described in the following sections within 1 h of surgery. This study was conducted with the approval of the McGill University ethics committee for research on human subjects, and informed consent was obtained from each patient.

Electron Microscopy

Pieces of tissue (1 mm3) were fixed by immersion in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 24 h and then washed in cacodylate buffer and postfixed in potassium ferrocyanide-reduced osmium tetroxide for 1 h to enhance the staining of membranes, as described by Hermo and Jacks [26]. After fixation, tissues were rinsed in cacodylate buffer, dehydrated in ethanol and propylene oxide, and embedded in Epon 812. Thin sections were cut with a diamond knife, mounted on copper grids, counterstained with uranyl acetate and lead citrate, and examined with an FEI Tecnai 12 electron microscope (FEI Company, The Netherlands).

Microarray Processing and Analysis

Total cellular RNA was isolated with an Absolutely RNA RT-PCR miniprep kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The quality of the RNA was verified with an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Wilmington, DE). The Low RNA Input Linear Amplification Kit (Agilent) was used to amplify and label 500 ng of total RNA with either cyanine 3 or cyanine 5 (Perkin-Elmer Inc., Woodbridge, ON, Canada). Human oligo microarrays (20 174 human genes; Agilent) were then hybridized according to the manufacturer's instructions with the In Situ Hybridization kit Plus (Agilent). After hybridization, microarrays were scanned with a ScanArray Express scanner (Perkin-Elmer). Fluorescence ratios for array elements were extracted by ScanArray Express Software (Perkin-Elmer) and imported into GeneSpring 6.1 software (Agilent) for further analysis. Expression analysis was done accordingly to MIAME (minimum information about a microarray experiment) standards [27]. Genes were considered enriched if the expression was 2-fold lower or higher in the caput epididymides of infertile patients compared with the same epididymal segment of fertile patients. Statistical analyses were performed by a one-way ANOVA (significance level set at P < 0.05).

Real-Time PCR

Real-time PCR was used to confirm microarray data. Five hundred nanograms of total RNA was reverse transcribed with an Oligo d(T)16 primer. Forward and reverse primers for the genes of interest (Table 1) were designed by Oligo Primer Analyses Software (Molecular Biology Insights, Cascade, CO) based on sequences published in GenBank. Real-time PCR was performed with a Rotor-Gene RG3000 (Corbett Research, Cambridgeshire, United Kingdom). A 2-µl aliquot of the RT reaction was amplified in a 15-µl solution containing 1x Platinum SYBR Green qPCR SuperMix UDG (Invitrogen, Burlington, ON, Canada) and 0.3 µM (both) reverse and forward primers. The PCR cycling protocols were optimized to maximize the reaction efficiency and ensure that only the target product was contributing to the SYBR Green fluorescence signal. For each quantification, a standard curve was created with suitably appropriate cDNA. Amplification consisted of 40 cycles at 95°C for 15 sec, melting temperature for 30 sec, and 72°C for 30 sec. Primers for the housekeeping gene, GAPDHS, were used to normalize values for each sample. Samples were done in duplicate, and identical samples were run in each assay to calibrate for interassay variation. After the PCR amplification, melting curve analysis was performed to ensure the accuracy of quantification.


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  		TABLE 1 Sequences of primers used in real-time PCR.

Immunocytochemistry

Small pieces of epididymal tissue were fixed at the time of surgery by immersion in Bouin fixative (Fisher Scientific, Ottawa, ON, Canada) for 24 h; they were then dehydrated and embedded in paraffin. Thick sections (5 µm) were cut and mounted on glass slides. For immunostaining, the tissue sections were rehydrated through graded ethanol, including 70% alcohol with 1% lithium carbonate for 5 min to remove residual picric acid. The sections were then incubated in 300 mM glycine for 5 min to block free aldehydes and washed in 1 M PBS (pH 7.4). Heat-induced epitope retrieval was done by boiling the slides for 10 min in citrate buffer (1.8 mM citric acid and 8.2 mM sodium citrate) for the immunolocalization of CLDNs 1, 3, 8, and 10, TJP1, and aquaporin-5 (AQP5) and for 20 min for the immunolocalization of the cystic fibrosis transmembrane conductance regulator (CFTR). Immunolocalization was performed according to the manufacturer's instructions (DAKO Catalyzed Signal Amplification System; DAKO, Carpenteria, CA). The primary antibodies used in this study were a rabbit polyclonal antibody against human CLDN1 (5 µg/ml; Zymed Laboratories, San Francisco, CA), a rabbit polyclonal antibody against mouse CLDN3 (2.5 µg/ml; Zymed), a rabbit polyclonal antibody against human CLDN8 (3 µg/ml; Genetex Inc., San Antonio, TX), a rabbit polyclonal antibody against human CLDN10 (2 µg/ml; Abcam, Cambridge, MA), a rabbit polyclonal antibody against human TJP1 (0.5 µg/ml; Zymed), a mouse monoclonal antibody against human CFTR (8 µg/ml; Neomarkers, Fremont, CA), a rabbit polyclonal antibody against rat AQP5 (20 µg/ml; Calbiochem, San Diego, CA), and a rabbit polyclonal antibody against human cysteine-rich secretory protein 1 (CRISP1, 20 µg/ml; Santa Cruz, CA). Incubations with the primary antibodies were done overnight at 4°C (CLDN8, CLDN10, and AQP5) for 15–30 min (TJP1, CLDN1, and CLDN3) to 2 h (CFTR) at room temperature or for 2 h (CRISP1) at 37°C. Incubations with the secondary antibodies were done for 15 min at room temperature, except for CFTR, AQP5, and CRISP1, for which the incubations were done for 1–2 h at room temperature. Epididymal sections were counterstained for 2 min with 0.1% methylene blue, dehydrated in ethanol, immersed in Histoclear (Fisher), and mounted in Permount (Fisher). Sections were examined with a Leica DMRE microscope (Leica Microsystems, Inc., Bannockburn, IL).

RESULTS

Ultrastructure of Caput Epididymides of Fertile and Infertile Men

Caput epididymidis of fertile and infertile patients, subsequently used for gene analysis, retained normal ultrastructural features. A pseudostratified epithelium underlying a basement membrane and a muscular coat composed of several layers of smooth muscle cells was observed (Fig. 2, A–C). Columnar principal cells were the main cell type of the epithelium. These cells contained large oval or elongated nuclei located near the base of the epithelium with dispersed chromatin clumps. Principal cells had long microvilli (Fig. 2, A and C). Their cytoplasm contained pale endosomes, dense lysosomes, mitochondria, endoplasmic reticulum, and Golgi apparatus (Fig. 2, A and C). Basal cells were round to ovoid and rested on the basement membrane (Fig. 2, B and C). No apparent changes could be seen in either the epithelial cell height or the distribution of cell types between fertile and infertile patients (Fig. 2, D and E).


Figure 2
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  		FIG. 2 Electron and light micrographs of the human epididymis. A) Apical region of the caput epididymidis of men with proven fertility. Original magnification x2550. B) Basal region of the caput epididymidis of men with proven fertility. Original magnification x2550. C) Epithelium of the caput epididymidis of infertile men. Original magnification x1250. D) Epididymal tubules of the caput epididymidis of men of proven fertility. Original magnification x100. E) Epididymal tubules of the caput epididymidis of infertile men. Original magnification x100. Mu, Muscular coat; P, principal cells; B, basal cells; N, nucleus; L, lumen; Mv, microvilli; Jc, junctional complex; ER, endoplasmic reticulum; M, mitochondria; Ly, lysosomes; V, vesicles; Spz, spermatozoa; IT, interstitial space.

Gene Expression Profiling

The human oligonucleotide microarray used in the present study consisted of 20 174 genes. Most genes encoded matrix/structural proteins and proteins dealing with signal transduction, synthesis/translational control, energy/metabolism, and transcription/chromatin. The array was hybridized with probes of the caput epididymidis of fertile and infertile patients to identify genes that were either down- or upregulated in infertile tissues. The data discussed in this publication have been deposited in the National Center for Biotechnology Information's Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE9194. In these analyses, 414 genes were differentially expressed in the caput epididymidis of infertile men by a 2-fold change when compared with the caput epididymidis of fertile patients (Fig. 3). There were 251 genes that were downregulated and 163 that were upregulated by a 2-fold change. Most of those genes for which the function was known encoded proteins implicated in transcription, intracellular signaling, fertility, and immunity (Fig. 4). We selected three genes for real-time PCR analysis to authenticate the results from the microarray data. Differential expression patterns of DEFB126 (beta-defensin 126) and SPINLW1 (serine protease inhibitor-like, with Kunitz and WAP domains 1, also known as EPPIN) and CRISP1 all showed consistency between microarray and real-time PCR analysis (Fig. 5). For CRISP1, the ratio detected in real-time PCR was slightly lower than the ratio detected by microarray analysis. This may be because of the differences in the sensitivity of the assays as well as the specificity of the primers used in real-time PCR. Further analyses were done on genes that were differentially expressed by a 4-fold change (Fig. 6): 7 genes were upregulated, and 10 were downregulated. Most of these genes are implicated in transcriptional regulation (Tables 2 and 3). Otherwise, genes whose expression was altered are involved in immunity, fertilization, sperm motility, DNA packaging, and proteolysis. Genes that are involved in various aspects of fertility were mostly downregulated. Further analysis revealed that several genes encoding for sperm antigens, such as SPINLW1, were upregulated by at least 2-fold (Fig. 5).


Figure 3
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  		FIG. 3 Scatterplot comparing genes expressed in the caput epididymidis of infertile patients with fertile patients. Axes of the scatterplot represent the log scale of the mean (n = 4) fluorescence intensity value minus the background intensity values for each region. The middle line indicates values that represent a ratio of 1.0 (similar levels of expression in both epididymal regions). The outer lines represent a ratio of 2.0 (upper line; 2-fold greater expression in the caput epididymidis of infertile compared with fertile) and 0.5 (lower line; 2-fold greater expression in the caput epididymidis of fertile compared with infertile).


Figure 4
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  		FIG. 4 Functional classification of differentially expressed genes by at least 2-fold in the human caput epididymidis of infertile versus fertile patients. The genes were grouped based on their biological function. The bars in white represent the upregulated genes, whereas the bars in black represent the downregulated genes.


Figure 5
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  		FIG. 5 Confirmation of microarray results by quantitative real-time PCR with selected genes known to be expressed in the epididymis. Messenger RNA expression levels of DEFB126, SPINLW1, and CRISP1 in the caput epididymidis of infertile and fertile men were investigated and compared with both microarray results and real-time PCR data. Data are expressed as the ratio of the mRNA levels of infertile in relation to fertile patients. Values represent the mean relative expression ± SEM (n = 4).


Figure 6
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  		FIG. 6 Scatterplot comparing genes differentially expressed by at least 4-fold in the caput epididymidis of infertile compared with fertile patients. Axes of the scatterplot represent the log scale of the mean (n = 4) fluorescence intensity value minus the background intensity values for each region. The middle line indicates values that represent a ratio of 1.0 (similar levels of expression in both epididymal regions). The outer lines represent a ratio of 2.0 (upper line; 2-fold greater expression in the caput epididymidis of infertile compared with fertile) and 0.5 (lower line; 2-fold greater expression in the caput epididymidis of fertile compared with infertile).


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  		TABLE 2 Upregulated genes in the caput epididymidis of infertile as compared to fertile patients by at least a 4-fold change.


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  		TABLE 3 Downregulated genes in the caput epididymidis of infertile as compared to fertile patients by at least a 4-fold change.

Genes Encoding for Tight Junctional Proteins

Several genes present on the microarray encoded tight junctional proteins, such as CLDNs 1 to 12, 14 to 19, and 23 and TJP1, TJP2, and TJP3. None of these genes were differentially regulated in the caput epididymidis of infertile patients compared with the same segment of fertile patients (Fig. 7, A and B). Electron microscopy detected intact apical junctional complexes between principal cells in the caput epididymidis of infertile patients (Fig. 7C).


Figure 7
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  		FIG. 7 Ratio of expression of genes encoding for CLDNs (A) and TJPs (B) in the caput epididymidis of infertile versus fertile men (n = 4; ±SEM). C) Electron micrograph of an apical tight junctional complex (arrowhead) in the caput epididymidis of infertile men (x43 000). L, Lumen.

Genes Encoding for Water Channels, Ion Channels, and Solute Carriers

Of the AQPs on the array, all, except for AQP5, were expressed at similar levels in the caput epididymidis of infertile and fertile patients. AQP5 was downregulated by at least 2-fold in infertile patients compared with fertile patients (Fig. 8A). Other genes encoding potassium ion channels, KCNK4 (also known as TRAAK) and KCNK17 (also known as TALK2 or TASK4), were also downregulated by at least a 2-fold change in infertile patients compared with fertile patients. Genes encoding two solute carriers, SLC6A20 (amino acid transporter) and SLC13A3 (anion transporter, also known as NADC3), as well as CFTR, which encodes for a cAMP-activated chloride channel, were also downregulated (Fig. 8B).


Figure 8
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  		FIG. 8 Ratio of expressed genes encoding for AQPs (A) and other ionic channels (B) in the caput epididymidis of infertile versus fertile patients (n = 4; ±SEM).

Genes Encoding for Beta-Defensins

The expression of several genes encoding for beta-defensins was changed by at least 2-fold in infertile patients compared with fertile patients. DEFB129 was upregulated, whereas DEFB126 and DEFB106A were both downregulated (Fig. 9).


Figure 9
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  		FIG. 9 Ratio of expression of genes encoding for beta-defensins. The ratio corresponds to the level of expression of these genes in the caput epididymides of infertile compared with fertile patients (n = 4; ±SEM).

Immunolocalization

Immunocytochemistry performed with anti-CLDN 1, 3, 8, and 10 antibodies showed that they were localized to the area of tight junctions between adjacent principal cells in the caput epididymidis of fertile and infertile patients (Fig. 10). CLDNs 1 and 3 were also localized along the lateral margins of adjacent principal cells as well as between basal and principal cells (Fig. 10, A–F), and CLDN8 was localized to the lateral margins of principal cells in the caput epididymidis of both fertile and infertile patients (Fig. 10, G and H). In contrast with CLDNs 1, 3, and 8, the immunolocalization of CLDN10 in the caput epididymidis of infertile patients was different from fertile patients. In the caput epididymidis of infertile patients, CLDN10, in addition to being localized to the apical tight junctional complex, was localized along the lateral plasma membrane between principal cells (Fig. 10K). The same observation was made for TJP1: in the caput epididymidis of fertile patients, TJP1 was exclusively localized to the apical tight junctional complex, but in infertile patients, the protein was also observed along the lateral margins between adjacent principal cells (Fig. 10, M and N). AQP5 was localized to the apical border of a subpopulation of principal cells in fertile and infertile patients (Fig. 11, A and B), but in infertile patients, AQP5 was also localized along the lateral plasma membrane of principal cells (Fig. 11B). CFTR was localized to the apical membrane of a subpopulation of principal cells in both fertile and infertile patients as well as in the cytoplasm of the narrow cells but with a weaker signal in infertile patients (Fig. 11, D–G). CRISP1 was localized to the apical border of principal cells of the epididymis, and its immunolocalization was the same in both fertile and infertile patients (Fig. 11, H and I).


Figure 10
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  		FIG. 10 Immunolocalization of CLDN1 (A–C), CLDN3 (D–F), CLDN8 (G–I), CLDN10 (J–L), and TJP1 (M–O) in the caput epididymides of fertile men (A, D, G, J, M) and infertile men (B, E, H, K, N). Negative controls with no primary antibodies are shown (C, F, I, L, O). Results indicate that CLDNs 1, 3, 8, and 10 and TJP1 were localized (arrowheads) to the area of tight junctions between adjacent principal cells in the caput epididymidis of fertile and infertile patients. In both types of tissues, CLDNs 1 and 3 were localized along the lateral margins of adjacent principal cells as well as between basal and principal cells, whereas CLDN8 was localized to the lateral margins of principal cells. In the caput epididymidis of infertile patients, CLDN10 and TJP1, in addition to being localized to the apical tight junctional complex, were localized along the lateral plasma membrane between principal cells. Original magnification x640. P, Principal cells; B, basal cells; IT, intertubular space; Lu, lumen.


Figure 11
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  		FIG. 11 Immunolocalization of AQP5 (A–C), CFTR (D–G), and CRISP1 (H–J) in the caput epididymides of fertile men (A, D, E, H) and infertile men (B, F, I). Negative controls with no primary antibodies are shown (C, G, J). Results indicate that AQP5 and CFTR were localized (arrowheads) to the apical border of a subpopulation of principal cells in fertile and infertile patients. In addition, CFTR was localized in the cytoplasm of the few narrow cells that could be seen in fertile and infertile patients. In infertile patients, AQP5 was also localized along the lateral plasma membrane of principal cells. CRISP1 was localized (arrowheads) to the apical border of principal cells of the epididymis of fertile and infertile patients. Original magnification in A x1000; B, C, and F, x640; D and G, x400; E, H–J, x480. P, Principal cells; B, basal cells; N, narrow cells; IT, intertubular space; Lu, lumen.

DISCUSSION

Microarrays constitute a powerful tool to study the molecular basis of male infertility and to highlight genes that may be important in posttesticular spermatozoal maturation, especially because the molecular basis of male infertility is heterogeneous and poorly understood. In the present study, samples from infertile patients were compared with samples originating from four fertile males with histologic confirmation of normal spermatogenesis and intact epididymis [18].

The epididymis displays a complex pattern of gene expression in several species, including mice, rats, and humans, with a larger number of reproductive and somatic genes expressed in the caput epididymidis than in the other epididymal segments [17–19, 28]. Indeed, the caput epididymidis is known to be the most active segment for protein synthesis and secretion in several species and is the region of the epididymis where spermatozoal maturation is initiated [10, 14]. Therefore, it is interesting that 414 genes were differentially expressed in the caput epididymidis of infertile men by at least a 2-fold change when compared with the caput epididymidis of fertile patients (Fig. 3). Although none of the genes encoding tight junctional proteins were changed in nonobstructive azoospermic patients, it is nevertheless interesting that the immunolocalization of CLDN10 and TJP1 is altered. It has been previously shown that CLDN10 is differentially expressed along the different segments of the human epididymis. Indeed, CLDN10 is mainly expressed in the cauda epididymidis [18]. Furthermore CLDN10 has been reported to play a role in the paracellular transport of cations across epithelial tight junctions [29]. It has also been suggested that TJP1 is important for the structural integrity of the tight junction [30]. Thus, alterations in the localization of CLDN10 and TJP1 may be suggestive of effects on the permeability of the blood-epididymal barrier and more particularly associated with paracellular cationic transport. Furthermore, several cell adhesion genes are upregulated (Fig. 4). The formation of tight junctions involves different cell adhesion signaling components [31]. Interestingly, one of the upregulated genes in the caput epididymidis of infertile patients is CASK (calcium/calmodulin dependent serine protein kinase), a member of the MAGUK (membrane associated guanylate kinase) family, which is involved in protein targeting and cell polarity in MDCK (Mardin-Darby canine kidney) cells [32]. It is possible that alterations in cell adhesion-mediated intracellular pathways influence, or regulate, the localization of TJPs.

Interestingly, our results also show altered expression of several genes encoding for different water and ion channels, such as CFTR and AQPs, which could result in a major alteration of the specific luminal microenvironment, which is essential for spermatozoal maturation in the human epididymis. Several studies of patients with cystic fibrosis have observed that the male reproductive system is highly dependent on CFTR for normal function. Indeed, more than 95% of men with cystic fibrosis are infertile. Furthermore, CFTR mRNA levels are high in the proximal caput region of the human epididymis. That CFTR expression is reduced could alter the ionic exchange and fluid content within the epididymal lumen, and this could lead to excessive viscosity of the epididymal fluid and the progressive blocking of different segments of the epididymis [33]. CFTR has been demonstrated to be implicated in the transepithelial secretion of electrolytes and water [34, 35] but also to act as a regulator of other membrane transport proteins, such as the epithelial Na+ channels [36] and AQP3 [37]. Several AQPs, which are involved in the transport of water, are expressed in the rat and human epididymis [17, 38–41]. Therefore, the fluidity of the epididymal lumen appears to be fine-tuned by the presence of both AQPs and CFTR, resulting in a microenvironment that is conductive for spermatozoal maturation. In the human caput epididymidis, it is also possible that CFTR is implicated in the water permeability of AQP5, as suggested for AQP9 [42]. Furthermore, studies have suggested that the lack of expression of several AQPs in the male reproductive system, AQP7 in human sperm [43] and AQP9 in rat epididymal epithelium [42], are important contributors to male infertility. However, the study of various AQP knockout mice (AQP7 and AQP8), which are fertile, indicates that a compensation mechanism exists among the AQPs in rodents [44, 45]. Alteration in the immunostaining pattern of AQP5 suggests that not only the expression but also the targeting and binding of AQP5 to the apical plasma membrane are affected in nonobstructive azoospermic patients. Furthermore, it has been observed that in AQP5–/– mice, there is a decrease in the expression of several TJPs in the salivary glands, suggesting interactions between transcellular and paracellular water transport pathways [46].

Other genes encoding ionic channels, such as KCNK4, KCNK17, SLC6A20, and SLC13A3, are also downregulated by at least a 2-fold change in the caput epididymidis of infertile patients compared with fertile patients. Potassium channels play a role in many cellular processes, including maintenance of the osmotic regulation and ion flow [47]. Furthermore, transport of small hydrophilic substances across cell membranes is mediated by substrate-specific transport proteins. SLC6A20 is a member of the subgroup of transporters with unidentified substrates within the Na+ and Cl– coupled transporter family [48], whereas SLC13A3 is implicated in the handling of citrate [49]. Studies have suggested that another solute carrier, SLC26A3, acts in conjunction with CFTR as a ductal HCO3– secretor and as an absorber of NaCl based on the coimmunolocalization of SLC26A3 and CFTR in the human epididymis [50, 51]. Both of these respective functions have previously been proposed in vitro for the pancreas [52–54] and intestine [55]. The loss or decrease in the number of different solute carriers and water and ionic channels could be responsible for the poor reabsorption of luminal fluid. Whether or not this is related to the absence of spermatozoa in the lumen or is part of a general syndrome associated with infertility is unknown. Although it is tempting to speculate that the presence of spermatozoa alone can regulate gene expression in the epididymis, we cannot discount the possibility that other testicular factors are also missing in nonobstructive patients and that these are also implicated in the regulation of these different water and ion channels.

Interestingly, another group of genes differentially regulated in the caput epididymidis of infertile patients compared with fertile patients encode for proteins that have been known to play a role in spermatozoal maturation, such as CRISP1 [56], SPINLW1 [57], DEFB129, DEFB126, and DEFB106A [58]. CRISP1 has been suggested to play a role in sperm-egg interaction and in the inhibition of the uptake of ions, such as Ca2+, required for capacitation by the spermatozoa [56]. Furthermore, SPINLW1 has been shown to bind to semenogelin in seminal plasma and on human spermatozoa after ejaculation. This complex is believed to be part of a larger network of protein complexes on the sperm surface that provides a protective shield before capacitation in the female reproductive tract [59]. Beta-defensins are also believed to play a role in both fertility and host defense [60–63]. Such sperm-coating proteins function in innate immunity, antimicrobial activity, and inhibition of proteases that may directly attack the sperm plasma membrane. Furthermore, genes downregulated by at least a 4-fold change included several genes that are implicated in spermatogenesis, sperm motility, and fertilization, such as STK22B, AKAP4, PRM1, and TNP1 (Table 3). Several studies have linked AKAP4, PRM1, and TNP1 to different types of male infertility [64–67]. The absence of spermatozoa in the lumen may explain the lack of expression of these genes. Altered expression of the different families of genes could also be due to the absence of testicular factors. However, in contrast with rodents who have been extensively studied [28, 68, 69], it has not been shown in the human epididymis whether or not testicular regulation of gene expression occurs. Some of our results support previous studies of rodents on the testicular regulation of epididymal gene expression. For example, FOS, RAB27A, and EGR1 are upregulated in the caput epididymides of the infertile patients (Fig. 6). This gene has also been shown to be upregulated in the mouse caput epididymidis after orchidectomy [28]. As well, PRM1 is downregulated in the caput epididymides of the infertile patients (Fig. 6) and has been shown to be downregulated in the mouse caput epididymidis after orchidectomy [28]. This correlation cannot be made for all the differently regulated genes in the infertile versus fertile caput epididymidis. For example, CLDN14 has been demonstrated to be downregulated by orchidectomy in the mouse caput epididymidis [28], whereas in the present study, its expression is not altered. Note, however, that there are important differences between an orchidectomized animal model and nonobstructive azoospermic patients. In nonobstructive azoospermic patients, testicular luminal factors that can act on the epididymis are still present as are factors acting from the testis on the epididymis through circulation, including androgens. This is not the case for an orchidectomized animal, where there are no luminal and no testicular bloodborne factors that can act on the epididymis. It is likely that these differences account for some of the differences between different animal models and observations in the present study.

In conclusion, we have shown that several families of genes are downregulated in the caput epididymidis of infertile nonobstructive azoospermic patients. Many of these genes appear to be implicated in the regulation of spermatozoal maturation and the regulation of ions in the epididymal lumen. Although there are no spermatozoa in the epididymis of these patients, it is unknown whether or not the presence of spermatozoa in the epididymis regulates the expression of these genes or if alterations in their expression are representative of a larger effect, or syndrome, associated with nonobstructive azoospermia. Furthermore, there is no information about the expression of these genes after reconstructive surgery or if these contribute to the lower-quality sperm in patients after vasoepididymostomy.

ACKNOWLEDGMENTS

The assistance of Julie Dufresne, Mary Gregory, Jeannie Mui, and Alexandra Lacroix during the course of this study was greatly appreciated.

FOOTNOTES

1Supported by a National Sciences and Engineering Research Council of Canada-Canadian Institutes of Health Research collaborative grant and a Canadian Institutes of Health Research operating grant to D.G.C. and P.T.K.C. E.D. is the recipient of a studentship from the Armand-Frappier Foundation and the FRSQ (Fonds de la Recherche en Santé du Québec). The data discussed in this publication have been deposited in the National Center for Biotechnology Information's Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE9194. Back

Correspondence: 2Daniel G. Cyr, INRS-Institut Armand Frappier, Université du Québec, 531 boul. des Prairies, Laval, QC, Canada H7V 1B7. FAX: 450 686 5309; e-mail: daniel.cyr{at}iaf.inrs.ca

Received: 10 May 2007.

First decision: 31 May 2007.

Accepted: 3 October 2007.

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New insights into epididymal biology and function
Hum. Reprod. Update, March 1, 2009; 15(2): 213 - 227.
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