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BOR - Papers in Press, published online ahead of print February 7, 2007.
Biol Reprod 2007, 10.1095/biolreprod.106.059246
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BIOLOGY OF REPRODUCTION 76, 1034–1044 (2007)
DOI: 10.1095/biolreprod.106.059246
© 2007 by the Society for the Study of Reproduction, Inc.

research-article

Gene Expression Profiling and Its Relevance to the Blood-Epididymal Barrier in the Human Epididymis1

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

INRS-Institut Armand Frappier,3 Université du Québec, Pointe Claire, Québec, Canada H9R 1G6 Department of Urology,4 Royal Victoria Hospital, McGill University, Montreal, Québec, Canada H3A 1A1 Department of Anatomy and Cell Biology,5 McGill University, Montreal, Québec, Canada H3A 2B2

ABSTRACT

The luminal environment along the epididymal duct is important for spermatozoal maturation. This environment is unique and created by the blood-epididymal barrier, which is formed by tight and adhering junctions. For the human epididymis, little information exists on the proteins that comprise these junctions. Our objectives were to assess the gene expression profiles in the different segments of the human epididymis and to identify the proteins that make up the blood-epididymal barrier. Using microarrays, we identified 2980 genes that were differentially expressed by at least 2-fold between the various segments. Of the many genes involved in diverse functions, were those that encoded adhesion proteins (cadherins and catenins) and tight junctional proteins (claudins [CLDN] and others). PCR analyses confirmed the microarray data. Immunolocalization of CLDNs 1, 3, 4, 8, and 10 revealed that the localization of CLDNs differed along the epididymis. In all three segments, CLDNs 1, 3, and 4 were localized to tight junctions, along the lateral margins of adjacent principal cells, and at the interface between basal and principal cells. CLDN8 was localized to tight junctions in all three segments, in addition to being localized in the caput along the lateral margins of principal cells, and in the corpus, at the interface between principal and basal cells. CLDN10, tight junction protein 1, and occludin were localized exclusively to tight junctions in all three epididymal segments. These data indicate that the epididymis displays a complex pattern of gene expression, which includes genes that are implicated in the formation of the blood-epididymal barrier, which suggests complex regulation of this barrier.

cadherin, catenin, claudin, epididymal junctions, epididymis, gene regulation, genomics, male reproductive tract

INTRODUCTION

The epididymis is a highly specialized tissue that is involved in the maturation, transport, protection, and storage of mammalian spermatozoa. This organ is a long, single, convoluted tubule that is morphologically divided into three main segments: the caput, corpus, and cauda epididymidis (Fig. 1). During epididymal transit, spermatozoa acquire progressive motility and the abilities to bind and fertilize the oocyte [1–4]. Spermatozoal maturation involves the remodeling of the sperm plasma membrane by the addition, removal or modification of cell surface molecules, which is in part due to the interaction of spermatozoa with molecules found in the luminal microenvironment [5–7]. The luminal microenvironment of the epididymis is comprised of specific ions, small organic molecules, and proteins that are secreted or absorbed by the epididymal epithelium [1, 8, 9]. The blood-epididymal barrier, which is composed of apical tight junctions between principal cells, forms an impenetrable seal and forces the movement of molecules across these cells by specific receptors, ion and water channels, and solute carrier proteins [1, 10, 11]. Thus, the barrier creates specific environments between the lumen and circulation and within the epithelial cells [12, 13].


Figure 01
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  		FIG. 1. Schematic representation of the human epididymis. The epididymis is a long convoluted tubule that connects the efferent ducts from the rear of each testicle to the vas deferens. The typical pattern of epididymal segmentation, as used in the present study, is shown.

The ultrastructure of the blood-epididymal barrier was first described by Friend and Gilula [14], who also reported that the tight junctions varied in terms of the number of strands and complexity along the epididymis, being more extensive in the caput. To date, tight junctions have been shown to be composed of a variety of peripheral membrane proteins, including tight junction proteins 1, 2, and 3 (TJP1, 2, and 3, also known as ZO proteins), symplekin, cingulin, 7H6 antigen, cytoskeletal elements (fodrin and actin), as well as integral transmembrane proteins, such as occludin and claudins (CLDNs) [15–18]. In particular, CLDNs form a multigene family that contains at least 20 members with variable tissue distributions [19–22]. CLDNs are transmembrane proteins that are essential for both the barrier function of tight junctions [23] and specific paracellular ion transport [24–26]. In the rat epididymis, occludin, TJP1, and several CLDNs are present in the tight junctions [13, 27–31]. However, there are few studies that deal with the localization and functions of CLDNs in the human epididymis.

Several genes unrelated to junctional protein genes have been characterized and shown to be expressed differentially along the epididymis [32, 33]. However, the roles of these genes and their products in the formation of the complex luminal fluid and in sperm maturation are not well understood. It is known that the epididymis has a highly region-specific gene expression pattern [34]. However, the molecular mechanisms that contribute to the formation of a specific luminal microenvironment that is crucial for sperm maturation via segment-specific gene expression are unknown [35]. Recently, microarrays have enabled the global study of gene expression and the rapid discovery of novel genes throughout the body. In the human male reproductive tract, gene profiling studies have been performed with microarray analyses of the spermatozoa from fertile men [36], the epididymis of one fertile man [37], testicular germ cell tumorigenesis [38], FSH-stimulated Sertoli cells [39], and testicular development [40]. Several studies of epididymal gene and protein expression patterns have also been carried out in rodents, and it has been reported that gene expression in the epididymis can be affected by androgens and ageing [41–44]. A number of regionally expressed genes have been identified that are unique to the epididymis, which suggests specific roles in epididymal function, whereas other genes are not tissue-specific, which implies broader functions. The latter encode secretory proteins (such as proteases, protease inhibitors, antioxidant enzymes, modifying enzymes, growth factors, neuropeptides, and transporters), intracellular proteins (transcription factors, signaling molecules, receptors, and kinases), as well as proteins of unknown function. Together, these studies provide important information on the molecular events underlying posttesticular sperm maturation. Nevertheless, gene profiling of the human epididymis has been limited due to difficulties in obtaining sufficient biological material. Elucidation of the transcriptional profiles of the different human epididymal segments is a critical step towards understanding not only the process of sperm maturation but also the causes of clinical idiopathic male infertility.

The objectives of the present study were to investigate the global gene expression patterns in different segments of the epididymis, to determine the intrasegmental differences in gene expression, particularly of genes associated with adherens and tight junctions, and to assess the localization of different tight junctional proteins in cell type- and segment-specific manners along the human epididymis.

MATERIALS AND METHODS

Tissue Preparation

Human epididymides were obtained from four patients (29–50 years of age) who were undergoing radical orchidectomy for localized testicular cancer (confined within the testicular tunica albuginea). Informed consent was obtained from each patient. This study was conducted with the approval of the Ethics Committee for Research on Human Subjects of McGill University. Epididymides were subdivided into three separate segments (caput, corpus, and cauda epididymidis). All patients had active spermatogenesis. Tissues were received in cold culture medium that contained antibiotics (Dulbecco modified Eagle medium and Ham F-12 with penicillin-streptomycin) and were transported to the laboratory within 1 h of surgery. Tissues were either frozen in liquid nitrogen for RNA preparation or fixed for light and electron microscopy.

Electron Microscopy

Small pieces of tissue (1 mm3) from each segment were fixed immediately at the time of surgery by immersion in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 24 h, after which they were washed in cacodylate buffer, and then postfixed in potassium ferrocyanide-reduced osmium tetroxide for 1 h, to enhance the staining of membranes as described by Hermo and Jacks [45]. Subsequently, the epididymal tissue was rinsed several times 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 under the FEI Tecnai 12 electron microscope.

Microarray Processing

Total cellular RNA was isolated using the Absolutely RNA RT-PCR miniprep kit (Stratagene, La Jolla, CA) according to the manufacturers instructions. The quality of the total RNA was verified using the Agilent 2100 Bioanalyzer (Agilent Technologies). Gene expression profiling was performed with commercially available Human oligonucleotide microarrays (20 174 human genes; Agilent Technologies). Amplification and labeling of 500 ng of total RNA were performed using the Low RNA Input Linear Amplification Kit (Agilent Technologies). The cRNA was labeled with either cyanine 3 or cyanine 5 (Perkin Elmer, Woodbridge, Canada). Arrays were hybridized according to the manufacturers instructions using the In Situ Hybridization kit Plus (Agilent Technologies). Following hybridization, microarrays were scanned with a ScanArray Express scanner (Perkin Elmer). Fluorescence ratios for array elements were extracted using the ScanArray Express Software (Perkin Elmer) and imported into the GeneSpring 6.1 software (Agilent Technologies) for further analysis.

Microarray Analysis

Expression analysis of all the microarray experiments was performed with GeneSpring 6.1 (Agilent Technologies). The data were normalized using a locally weighted regression Lowess method. Genes were considered to be enriched in a specific segment of the epididymis if their expression was 2-fold lower or higher in that segment than in other segments for at least three patients. Statistical analyses were performed with one-way ANOVA (significance level set at P < 0.05). Analyses were carried out in accordance with the MIAME standards.

Real-Time PCR

Real-Time PCR was used to confirm the differences in transcription that were observed in the microarray analysis. Total RNA (500 ng) was reverse-transcribed using an oligo(dT)16 primer. Forward and reverse primers for the genes of interest were designed using the Oligo Primer Analyses software (Molecular Biology Insights, Cascade, CO) based on sequences published in GenBank. The primers are listed in Table 1. Real-Time PCR was performed with a Rotor-Gene RG3000. A 2-µl aliquot of the RT reaction was amplified in a 15-µl solution that contained 1x Platinum SYBR Green qPCR SuperMix UDG (Invitrogen, Burlington, ON, Canada) and 0.3 µM of each of the reverse and forward primers. The PCR cycling protocols were optimized to maximize reaction efficiency and to ensure that only the target product contributed to the SYBR Green fluorescence signal. For each quantification, a standard curve was created using the appropriate cDNA. Amplification consisted of 40 cycles at 95°C for 15 sec, melting temperature (Tm) for 30 sec, and 72°C for 30 sec. Primers for the housekeeping gene, GAPDHS, were used to normalize the values for each sample. Samples were assayed in duplicate and identical samples were run in each assay, to calibrate for interassay variations. Following PCR amplification, melting curve analysis was performed to ensure the accuracy of quantification.


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  		TABLE 1. Sequences of the primers used in PCR.

Reverse Transcription-Polymerase Chain Reaction

RT-PCR was used to confirm the presence of transcripts for the different CLDN genes. Total RNA (500 ng) was obtained from two different individuals using Biochain (Hayward, CA) and reverse-transcribed using an oligo(dT)16 primer. Forward and reverse primers for the genes of interest were designed with the Oligo Primer Analyses software (Molecular Biology Insights) based on the GenBank sequences. The primers are listed in Table 1. PCR amplification was carried out as follows: 94°C for 5 min, followed by 30 cycles of 94°C for 30 sec, Tm for 30 sec, 72°C for 1 min, and a final step of cooling to 4°C. The PCR products were separated on a 2% agarose gel and visualized with ethidium bromide using a Fluor-S Multi-Imager densitometer (Bio-Rad Laboratories, Mississauga, ON, Canada).

Immunocytochemistry

Small pieces of epididymal tissue were fixed at the time of surgery by immersion in Bouin fixative (Fisher Scientific, Pittsburgh, PA) for 24 h, 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 (HIER) was performed by boiling the slides for 10 min in citrate buffer (1.8 mM citric acid, 8.2 mM sodium citrate). Immunolocalization was performed with the DAKO Catalyzed Signal Amplification System (DAKO, Carpenteria, CA). The primary antibodies used in this study were: rabbit polyclonal anti-CLDN1 (5 µg/ml; Zymed Laboratories, San Francisco, CA); rabbit polyclonal anti-CLDN3 (2.5 µg/ml; Zymed Laboratories); mouse monoclonal anti-CLDN4 (2.5 µg/ml; Zymed Laboratories); rabbit polyclonal anti-CLDN8 (3 µg/ml; Genetex, San Antonio, TX); rabbit polyclonal anti-CLDN10 (2 µg/ml; Abcam, Cambridge, MA); rabbit polyclonal antibody anti-TJP1 (2.5 µg/ml; Zymed Laboratories); and rabbit polyclonal anti-occludin (2.5 µg/ml; Zymed Laboratories). Incubations with the primary antibodies were done either overnight at 4°C (CLDN8 and CLDN10) or for 30–60 min at room temperature (CLDN1, CLDN3, CLDN4, TJP1, and occludin). Omission of primary antibodies served as negative controls. Epididymal sections were counterstained for 2 min with 0.1% methylene blue, dehydrated in ethanol, immersed in Histoclear (Fisher Scientific), and mounted in Permount (Fisher Scientific). Sections were examined under a Leica DMRE microscope.

RESULTS

Ultrastucture of the Human Epididymis

The human epididymis was found to consist of a pseudostratified epithelium underlying a basement membrane and muscular coat that comprised several layers of smooth muscle cells (Fig. 2, A–F). The epithelium was composed mainly of tall columnar principal cells with long microvilli and occasional apical blebs that extended into the lumen (Fig. 2, A–F). The basal cells resided basally (Fig. 2, B, D, and F), while the halo cells were scattered sporadically in the epithelium (Fig. 2D). Principal cells had large oval or elongated nuclei located near the base of the epithelium, with dispersed chromatin clumps (Fig. 2, B, D, and F). In their cytoplasm, pale endosomes, dense lysosomes, mitochondria, endoplasmic reticulum, and the Golgi apparatus were evident (Fig. 2, A, C, E, and F). Apically, tight junctional complexes connected adjacent principal cells to one another (Fig. 2, A, C, and E). Basal cells were round to ovoid in appearance and rested on the basement membrane (Fig. 2, B, D, and F). The lateral intercellular spaces between adjacent cells were at times dilated, suggestive of movement of fluid through the epithelium (Fig. 2D). These results confirm that the tissues that were subsequently used for gene analysis were normal.


Figure 02
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  		FIG. 2. Electron micrographs of the human epididymis. A) Apical region of the caput epididymidis. B) Basal region of the caput epididymidis. C) Apical region of the corpus epididymidis. D) Basal region of the corpus epididymidis. E) Apical region of the cauda epididymidis. F) Basal region of the cauda epididymidis. Mu, Muscular coat; P, principal cells; B, basal cells; N, nucleus; L, lumen; Mv, microvilli; Jc, junctional complex; ER, endoplasmic reticulum; M, mitochondria; E, endosomes; Ly, lysosomes; V, vesicles. Original magnification x9900 (A, C), x1700 (B, F), x4200 (D), and x6000 (E).

Segmental Gene Expression Profiling

The human oligonucleotide microarray used in the present study consisted of 20 174 genes. The majority of these genes encoded matrix/structural proteins, as well as proteins that are involved in signal transduction, synthesis/translational control, energy/metabolism, and transcription/chromatin. The array was hybridized with probes representing two different epididymal segments, to identify genes that were highly enriched in each. Genes were considered to be expressed when they were detected in at least three individuals. The number of genes detected in each segment varied. While 98% (19 875) of the genes studied were expressed in the caput epididymidis, 79% (16 036) were expressed in the corpus epididymides, and 88% (17 573) in the cauda epididymidis (Fig. 3). Only 138 genes had no detectable signal, as evidenced by their negative intensity values with either of the probes (data not shown).


Figure 03
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  		FIG. 3. Regional distributions of the genes detected in each segment of the human epididymis. Overall, 99% (19 875 out of 20 036) of the genes studied are expressed in the caput epididymidis, 80% (16 036 out of 20 036) in the corpus epididymidis, and 87.7% (17 573 out of 20 036) in the cauda epididymidis. Different subsets of genes have been defined subsequently. Of the genes detected, 75% (15 184 out of 20 036) are expressed in all segments, whereas some genes are expressed in more than one, but not all, of the segments, i.e., 4% (810 out of 20 036) in the caput and the corpus, 0.1% (20 out of 20 036) in the corpus and the cauda, and 12.7% (2550) in the cauda and caput. Some genes are also segment-specific, i.e., 6.6% in the caput, 0.1% in the corpus, and 0.6% in the cauda. Only 138 genes have no detectable signal.

Several genes that were expressed in the epididymis were highly characterized with respect to their levels of expression in each epididymal segment. We selected three of these genes for Real-Time PCR analysis, to authenticate the results from the microarray. The expression patterns of CRISP1 (cysteine-rich secretory protein 1), DEFB126 (ß-defensin 126), and SPINLW1 (serine peptidase inhibitor-like, with Kunitz and WAP domains 1, also known as EPPIN) all showed consistency between the microarray and Real-Time PCR analyses (Fig. 4).


Figure 04
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  		FIG. 4. Confirmation of microarray results by quantitative Real-Time PCR using selected genes that are known to be expressed in the epididymis. The mRNA expression levels of CRISP1 (A), SPINLW1 (B), and DEFB126 (C) in the three segments of the human epididymis were investigated and compared using both microarray and Real-Time PCR (Q-PCR) data. The data are expressed as the ratios of the mRNA levels in relation to GAPDHS. Values represent the mean relative expression ± SEM; n = 4.

Segment Specificity of Gene Expression

All three segments of the epididymis showed expression of the highly expressed genes (Fig. 3). Of the 20 174 genes studied, 6.6% (1331) were expressed exclusively in the caput, whereas only 0.1% (22) and 0.6% (119) were expressed exclusively in the corpus and cauda epididymides, respectively (Fig. 3). Overall, these 1475 segment-specific genes comprised 7.3% of the transcripts expressed in the epididymis (Fig. 3). Genes expressed in a segment-specific manner were classified into diverse entities based on biological function (Table 2). The products of these genes were primarily involved in fundamental processes, such as apoptosis, signal transduction, transcription, protein modification, host defense, cell growth and differentiation, cell adhesion, and cell signaling. Several genes that have been detected previously in other parts of the male reproductive system were found to be expressed in the epididymis, i.e., NPM2 (nuclear chaperone), MOV10L1 (RNA helicase), DNMT3L (DNA methyltransferase), ADAM18 and ADAM20 (a disintegrin and metalloprotease), HIST1H1T (histone), OVOL1 (zinc finger protein), DAZL, DMRTC2, and DMRT3 (DNA methyltransferase) in the caput, and KRTAP4–7 (keratin-associated protein) in the corpus. Most of these genes have not been reported previously in the human epididymis. In the cauda epididymidis, no uniquely expressed genes related to processes implicated in fertility were detected. In the human epididymis, 15.3% of the expressed genes were present in only two segments (Fig. 3). Indeed, 4% (810) of the genes were expressed only in the caput and corpus, while 0.1% (20) were found only in the corpus and cauda, and 11.2% (2250) in the caput and cauda (Fig. 3).


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  		TABLE 2. Functional classification of region-specific genes in the human epididymis.

Differential Gene Expression in the Human Epididymis

The majority of the genes exhibited an expression ratio of 1.0 when comparisons were made between two segments, which suggests that their expression levels are similar in all the epididymal regions (data not shown). A number of genes were differentially expressed 2-fold. In the corpus, 971 genes exhibited a 2-fold difference in expression level compared to their expression in the caput. Of those, 596 genes exhibited an increase, whereas 375 genes showed a decrease in expression level. In the cauda, 280 genes exhibited an increase in expression level as compared to the corpus, whereas 593 genes showed a decrease. Comparison of the cauda and caput revealed 1136 genes with a 2-fold difference in expression level; 484 genes had higher expression in the caput, whereas 652 genes were more highly expressed in the cauda.

In order to acquire additional information on the selective regional gene expression pattern along the epididymis, genes that showed at least a 4-fold difference in expression in one segment compared to an adjacent segment were examined (Supplementary Tables, available online at www.biolreprod.org). In the caput, the expression levels of 65 genes were enriched by a 4-fold difference as compared to the corpus epididymidis. These genes included DMRTB1, RSBN1, FAM12A (HE3 ALPHA), FAM12B (HE3 BETA), GGTL4, RNASE9, DEFB129, and CRISP1, as well as genes involved in transcriptional regulation, cell signaling, and cell growth. In the corpus, the expression levels of 90 genes were enriched 4-fold as compared to the caput. Most of these genes were implicated in transcriptional regulation, cell adhesion, metabolism, and ion transport. Several genes that have previously been detected in the male reproductive tract were also expressed, including CST9L, AQP9, CLDN10, SPAG11 (HE2), CLDN2, DEFB119, DEFB123, DEFB106A, SULT1E1, PTGDS, and OSTbeta. In the cauda, the expression levels of 27 genes were enriched 4-fold as compared to the corpus epididymidis. These genes included DMRTB1 and RSBN1. Other genes implicated in transport, transcriptional regulation, cell adhesion, and cell signaling were also expressed.

Genes Implicated in Adhesion and Tight Junctions

Of the genes expressed in the human epididymis, several encoded tight junctional proteins, such as CLDNs 1 to 12, 14 to 19, and 23, and TJPs 1, 2, and 3, and adhesion proteins, such as E-cadherin (CDH1), P-cadherin (CDH3), {alpha}-catenin (CTNNA1) and ß-catenin (CTNNB1). Of the twenty CLDN genes on the array, with the exceptions of CLDN16 and CLDN22, all were expressed in all three segments of the human epididymis (Fig. 5). CLDN16 was expressed only in the caput and cauda epididymidis and CLDN22 was not expressed at all. CLDN4 and CLDN7 were highly expressed, with relative intensities of more than 1000 (Fig. 5A), whereas CLDNs 2, 5, and 10 had relative intensities of 500–1000 (Fig. 5B). All the other CLDN genes were weakly expressed, with relative intensities of less than 300 (Fig. 5C). While most CLDN genes were expressed at similar levels along the human epididymis, there were some notable exceptions, e.g., CLDNs 2, 8, 10, 16, and 23 (Fig. 5). CLDN2 and CLDN10 were mainly expressed in the cauda epididymidis (Fig. 5B), whereas CLDN8 and CLDN23 were mainly expressed in the caput epididymidis (Fig. 5C). RT-PCR, which was performed for two different individuals using specific primers for each human CLDN, confirmed the microarray data (see Fig. 7). TJPs 1, 2, and 3 were all expressed at similar levels along the human epididymis. TJP2 was expressed at higher levels, with relative intensities of 150–200, as compared to TJP1 and TJP3, which had relative intensities of 50–100 (Fig. 6A). Of the different cadherin genes on the array, CDH1 and CDH3 were expressed in all three segments of the human epididymis. CDH1 was expressed at higher levels than CDH3 with at least a 3-fold difference (Fig. 6B). Three other cadherin genes, CDH16, CDH22, and CDH24, were highly expressed (Fig. 6B) at similar levels along the epididymis, except for CHD16, which had lower expression in the caput epididymidis. Several catenin genes were also detected. CTNNA1 and CTNNB1 were expressed in all three segments of the human epididymis at similar levels (Fig. 7C). However, CTNNA1 was expressed at higher levels than CTNNB1 and the two p120 catenins (CTNND1, CTNND2) by more than a 4-fold difference (Fig. 6C). The {alpha}-catulin gene (CTNNAL1) was expressed at similar levels to CTNNA1 (Fig. 6C).


Figure 05
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  		FIG. 5. Expression patterns of CLDN genes in the human epididymis. The CLDN genes are classified into three groups: highly expressed (A), moderately expressed (B), and weakly expressed (C). Values represent the mean relative intensity ± SEM; n = 4.


Figure 07
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  		FIG. 7. RT-PCR analysis of different CLDN genes in the human epididymis. The primers and PCR conditions are listed in Table 1. Left lane, water control; right lane, total epididymal RNA obtained from a normal 24-yr-old man; bp, 100-bp ladder.


Figure 06
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  		FIG. 6. Expression patterns in the human epididymis of members of the tight junction protein family (A), cadherin family (B), and catenin family (C). Values represent the mean relative intensity ± SEM; n = 4.

Immunolocalization

Immunocytochemistry performed with antibodies against CLDNs 1, 3, 4, 8, and 10 revealed that these proteins were localized to the area of tight junctions between adjacent principal cells in all three segments (Figs. 8–10). The intensity of CLDN10 immunoreactivity was higher in the corpus and cauda epididymidis than in the caput (Fig. 10, A, D, and G). Furthermore, in all three segments of the epididymis, CLDNs 1, 3, 4, and 8 showed various reactions in other cellular domains. In all three segments, CLDNs 1, 3, and 4 were localized along the lateral margins of adjacent principal cells, as well as between basal and principal cells (Figs. 8 and 9). In contrast, CLDN8 in the caput epididymidis was localized to the lateral margins of principal cells (Fig. 9B), while in the corpus, it was localized to the interface between principal and basal cells (Fig. 9D). Immunocytochemistry for TJP1 and occludin revealed that both proteins were localized exclusively to the apical tight junctional complex (Fig. 10).


Figure 08
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  		FIG. 8. Immunolocalization of CLDN1 (A, C, E) and CLDN3 (B, D, F) in the human epididymis. CLDN1 and CLDN3 are localized (arrowheads) to the apical tight junctional complex and along the lateral margins of epithelial cells, as well as between the basal and principal cells in the caput (A, B), corpus (C, D), and cauda (E, F) epididymidis. P, Principal cells; B, basal cells; IT, intertubular space; Lu, lumen. Original magnification x640.


Figure 10
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  		FIG. 10. Immunolocalization of CLDN10 (A, D, G), TJP1 (B, E, H), and occludin (C, F, I) in the human epididymis. CLDN10, TJP1, and occludin are exclusively localized (arrowheads) to the apical tight junctional complex in the caput (A, B, C), corpus (D, E, F), and cauda (G, H, I) epididymidis. P, Principal cells; B, basal cells; IT, intertubular space; Lu, lumen. Original magnification x640.


Figure 09
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  		FIG. 9. Immunolocalization of CLDN4 (A, C, E) and CLDN8 (B, D, F) in the human epididymis. CLDN4 is localized (arrowheads) to the apical tight junctional complex and along the lateral margins of epithelial cells, as well as between the basal and principal cells in the caput (A), corpus (C), and cauda (E) epididymidis. CLDN8 is localized to the lateral margins of principal cells and to the apical tight junctions in the caput epididymidis (B). In the corpus epididymidis, CLDN8 is localized to the tight junctional complex and to the interface between principal and basal cells (D). In the cauda epididymidis (F), CLDN8 is localized exclusively to apical tight junctions. P, Principal cells; B, basal cells; IT, intertubular space; Lu, lumen. Original magnification x640.

DISCUSSION

Microarrays constitute a powerful and efficient tool to establish segment-specific gene expression and to highlight genes that may be important in epididymal functions. While several studies have been done on gene profiling in the epididymis, most of them have focused on rodents [41–44, 46, 47].

In the present study, the samples originated from four healthy males with histological confirmation of normal spermatogenesis within the seminiferous tubules and a structurally intact epididymis, as revealed by electron microscopy (Fig. 2), with results that were comparable to those described in the literature [1, 48, 49]. Thus, our present results can be considered to be representative of relatively normal physiological conditions. The microarray data generated in the present study were validated by Real-Time PCR (Fig. 4) and by the published expression patterns of well-studied epididymal transcripts, such as CRISP1 [50], SPINLW1 [51, 52], and DEFB126 [53, 54]. However, in the microarray analysis, there was a difference in the regional distribution of CRISP1 in the caput epididymidis relative to other regions. These differences were not confirmed by Real-Time PCR. The reasons for these differences are unknown but they may be related to the sensitivities of the different methods used.

The epididymis displays a complex pattern of gene expression. Indeed, many of the genes detected in the present study are either highly or differentially expressed along the human epididymis. Approximately 15% of the genes differed by at least 2-fold in their levels of expression in different segments. Similar findings have been reported in the mouse [47] and human epididymis [37]. These data suggest that the events in the epididymis that are related to sperm maturation, transport, and storage are complex. Epididymal secretion varies along the length of the duct, resulting in sequential changes to sperm as they move down the duct [7]; this may explain the continuous alterations in the protein composition of the epididymal fluid. Furthermore, the caput epididymidis is known to be the most active segment for protein synthesis and secretion in several species [1, 9, 55]. Therefore, it is not surprising that many of the reproductive and somatic genes were either exclusively expressed (Table 2) or exhibited the highest level of expression in this segment (Supplementary Table 1, available online at www.biolreprod.org). Interestingly, the caput and the cauda epididymidis expressed the largest number of differentially regulated genes. Similar observations have been made by Johnston et al. [47] and Zhang et al. [37] in the mouse and human epididymal transcriptomes, respectively. For example, the gene that encodes 5{alpha}-reductase type 2, which is involved in the conversion of testosterone to 5{alpha}-dihydrotestosterone (DHT), an essential metabolite for spermatozoal maturation, was expressed at higher levels (2-fold difference; data not shown) in the caput epididymidis as compared to the other segments. Indeed, the caput and cauda epididymidis are morphologically very different and have specialized roles in sperm maturation and storage, respectively, as suggested by our microarray results. Therefore studies on epididymal gene expression profiles can help us elucidate specific functions along the various segments of the epididymis by analyzing the differential expression of specific genes. However, differences were noted between the results obtained in the present study and those obtained by Zhang et al. [37] for specific transcripts, such as AQP1 and AQP9. Zhang et al. [37] have shown that AQP1 and AQP9 are most abundant in the cauda and caput epididymidis, respectively, in contrast to our present findings that AQP9 is mostly expressed in the corpus epididymidis and that AQP1 is not differentially expressed along the epididymal duct. These differences can be due to genetic variability and low sample size, since their results were generated with samples from a single individual.

Genes expressed along the human epididymis include several that encode adhesion proteins (cadherins, protocadherins, and catenins) and tight junctional proteins (CLDNs and TJPs 1, 2, and 3), which are involved in the formation and integrity of the rodent blood-epididymal barrier [27, 56–58]. Cdh1 and Cdh3 are also expressed in the rat epididymis [27, 59, 60], which suggests a degree of conservation in the composition of epididymal adhering junctions and similar roles for these particular cadherins, especially since Cdh1 is also expressed in the mouse epididymis [47]. The two other cadherins, CDH22 (also known as rat PB-cadherin) and CDH24, which are highly expressed in the human epididymis, have been shown to have two transcripts. The longer isoforms are active in cell adhesion and retain their catenin-binding sites for both p120 catenin and ß-catenin, whereas the shorter isoforms do not [61]. The identification of catenins that bind to the cytoplasmic domains of cadherins have been shown to be important for the formation of cadherin-catenin complexes, which are essential for cadherin-mediated cell adhesion and intracellular signaling [62, 63]. CDH16 (also known as kidney-specific cadherin), which is the only cadherin to be highly differentially expressed along the human epididymis, has been shown to be expressed in the mouse epididymis during embryonic development [64]. Furthermore, in the adult mouse, Cdh16 is also differentially expressed along the epididymis but with the highest levels of expression in the caput and proximal corpus epididymidis [47]. Interestingly, it has a short intracellular domain and lacks the ß-catenin-binding site, which is crucial for the adhesive functions of classical cadherins [65]. It has been proposed that Cdh16, in concert with other cadherins, may be required for the differentiation of kidney, lung, and sex duct epithelia, as well as in maintaining the integrity of collecting duct epithelia [64]. In the human epididymis, as in the mouse and rat [47, 57], different catenins are expressed. However, the high-level expression of {alpha}-catulin, which is involved in the signal transduction pathway of small G-proteins, such as Rho, suggests that these signaling pathways may be important for the regulation of cadherin-mediated intracellular signaling and epididymal functions. Interestingly, in the mouse epididymis, Ctnnb1, and not Ctnnal1, is the most highly expressed catenin [47], which suggests the existence of different mechanisms of regulation of cadherin-mediated intracellular signaling in mice and humans.

Tsukita and Furuse [66] have suggested that the combination and mixing ratio of CLDNs may determine the physiological nature of tight junctions. In the human, rat, and mouse epididymis, similar CLDN genes are expressed [29- 31, 47], suggesting a high degree of conservation of the composition of epididymal tight junctions and similar roles for each particular CLDN within the tight junctional complex. However, some differences exist between rodents and human in terms of the expression patterns of certain CLDN genes. In the rat, Cldn16 (also known as paracellin-1) and Cldn10 are expressed exclusively in the initial segment of the epididymis [31]. In the mouse, Cldn16 is expressed all along the epididymis, albeit at very low levels. In the human epididymis, some CLDN genes (2, 8, 10, 16, and 23) also show segment-specific expression, evoking different roles for these CLDN genes along the duct. Similar observations have been made in the mouse epididymis for several Cldn genes (2, 3, 8, and 23), although their expression patterns are different in the mouse than in the human epididymis [47]. Interestingly, these CLDN proteins seem to be part of the cation barrier [31, 30]. Indeed, CLDN2 in MDCK cells increases the permeability of sodium [67], and CLDN16 is implicated in paracellular transport of magnesium, potassium, and sodium [68]. Furthermore, it has been suggested that the declining sodium:potassium ratio in the epididymis is important for sperm maturation [11].

Immunohistochemistry revealed that not only the expression pattern, but also the localization of CLDNs differed along the human epididymis. All the CLDNs studied were expressed in the apical tight junctional complex. Thus, it appears that CLDN1, CLDN3, and CLDN4, are implicated in epididymal tight junctions and cell adhesion based on their localizations along the lateral plasma membranes between principal cells, as well as between principal and basal cells. This immunostaining pattern resembles that of cell adhesion molecules [69, 70]. Similar observations have been made for CLDN1 in the rat epididymis [29]. Previous studies have also shown that CLDN4 is associated with decreased membrane permeability to sodium ions in MDCK cells [67], which suggests that its increased expression is related to a decrease in cation permeability across the blood-epididymal barrier. In contrast to CLDNs 1, 3, 4, and 10, CLDN8 exhibited segment-specific localization. The localization of CLDN8 suggests that, like CLDN1, this protein may mediate the adherence of principal cells to basal cells in the corpus segment of the human epididymis, whereas in the caput and cauda epididymidis, CLDN8 plays a role primarily in epididymal tight junctions. Recently, it has been reported that CLDN8 expression in MDCK cells reduces the paracellular permeability of protons, ammonium, and bicarbonate, suggesting a role in limiting the passive leakage of these ions via paracellular routes [71]. In the rat epididymis, the decline in pH is dependent upon bicarbonate and sodium reabsorption and the lowest pH is seen in the corpus epididymidis [11]. CLDN8 may play an indirect role in the control of intraluminal pH by regulating bicarbonate permeability. Furthermore, bicarbonate plays a key role in triggering modification of the architecture of the sperm plasma membrane during capacitation, a process that eventually leads to sperm death [72]. Therefore, initiation of premature capacitation in vivo would represent a threat for sperm survival during storage in the epididymis. The establishment of a low bicarbonate concentration in the lumen of the epididymis may thus contribute to maintaining an optimal environment for sperm storage and viability.

The localizations of TJP1 and occludin in the human epididymal tight junctional complex, as in the rat and mouse [27, 28], suggest similar roles for these proteins in humans and rodents. However, in the human epididymis, the expression of TJP2 and TJP3 reveals that the junctional complex may be different from that in the rat. Johnston et al. [47] have found that in the mouse epididymis, Tjp1, Tjp2, and Tjp3 are expressed at similar levels along the duct, as in the human. Previous studies have suggested that TJP1 is involved in the targeting of CLDN proteins to the area of tight junctions in the epididymis, indicating that the regulatory mechanisms for the formation of the blood-epididymal barrier include not only the expression of specific genes but also their targeting to the tight junctional complex. In the rat epididymis, TJP1 also interacts with ß-catenin, especially during the formation of epididymal tight junctional strands [57].

In conclusion, the human epididymis displays a complex gene expression pattern, including several genes that are implicated in the formation and integrity of the blood-epididymal barrier, suggesting complex regulation of this barrier. The fact that human epididymal adhering and tight junctions resemble those found in rodents indicates a high degree of conservation of composition. However, regional differences in the expression and localization of CLDNs along the human epididymis reveal different roles for each CLDN in the formation of the luminal environment by contributing to the creation of specific ionic balances. Further studies on the regulation of these genes in fertile versus infertile patients will allow us to understand better how these genes may be altered in infertile patients.

ACKNOWLEDGMENTS

The assistance of Julie Dufresne, Mary Gregory, Jeannie Mui, Bardia Moosavi, and Alexandra Lacroix during the course of this work is greatly appreciated.

FOOTNOTES

1Supported by an NSERC-CIHR collaborative grant. E.D. is the recipient of a studentship from the Armand-Frappier Foundation. Back

Correspondence: 2Daniel G. Cyr, INRS-Institut Armand Frappier, Université du Québec, 245 Hymus Boul., Pointe Claire, QC, Canada H9R 1G6. FAX: 514 630 8850; e-mail: Daniel.cyr{at}iaf.inrs.ca

Received: 1 December 2006.

First decision: 7 January 2007.

Accepted: 6 February 2007.

REFERENCES

  1. Physiology of Reproduction. Robaire B and Hermo L. Efferent duct, epididymis, and vas deferens: structure, functions, and their regulation. 1988:New York: Raven Press;999–1080. In:
  2. Turner TT. On the epididymis and its role in the development of the fertile ejaculate. J Androl 1995; 16:292–298[Free Full Text]
  3. The Epididymis: from Molecules to Clinical Practice. Yeung C-H and Cooper TG. Acquisistion and development of sperm motility upon maturation in the epididymis. 2002:New York: Plenum Press;417–434. In:
  4. Knobil and Neill's Physiology of Reproduction, 3rd ed. Robaire B, Hinton B, Orgebin-Crist MC. The epididymis. 2006:New York: Elsevier;1071–1148. In:
  5. Sullivan R. Male fertility markers, myth or reality. Anim Reprod Sci 2004; 82–83:341–347
  6. Dacheux JL, Dacheux F, Paquignon M. Changes in sperm surface membrane and luminal protein fluid content during epididymal transit in the boar. Biol Reprod 1989; 40:635–651[Abstract]
  7. Dacheux JL, Belghazi M, Lanson Y, Dacheux F. Human epididymal secretome and proteome. Mol Cell Endocrinol 2006; 250:36–42[CrossRef][Medline]
  8. Hermo L, Oko R, Morales CR. Secretion and endocytosis in the male reproductive tract: a role in sperm maturation. Int Rev Cytol 1994; 154:106–189[Medline]
  9. The Epididymis: From Molecules to Clinical Practice. Dacheux JL and Dacheux F. Protein secretion in the epididymis. 2002:New York: Plenum Press;151–168. In:
  10. The Epididymis: From Molecules to Clinical Practice. Hermo L and Robaire B. Epididymal cell types and their functions. 2002:New York: Plenum Press;81–102. In:
  11. The Epididymis: From Molecules to Clinical Practice. Turner TT. Necessity's potion: Inorganic ions and small organic molecules in the epididymal lumen. 2002:New York: Plenum Press;131–150. In:
  12. Cyr DG, Robaire B, Hermo L. Structure and turnover of junctional complexes between principal cells of the rat epididymis. Microsc Res Tech 1995; 30:54–66[CrossRef][Medline]
  13. The Epididymis: From Molecules to Clinical Practice. Cyr DG, Finnson KW, Dufresne J, Gregory M. Cellular interactions and the blood-epididymal barrier. 2002:New York: Plenum Press;103–118. In:
  14. Friend DS and Gilula NB. Variations in tight and gap junctions in mammalian tissues. J Cell Biol 1972; 53:758–776[Abstract/Free Full Text]
  15. Fanning AS, Mitic LL, Anderson JM. Transmembrane proteins in the tight junction barrier. J Am Soc Nephrol 1999; 10:1337–1345[Abstract/Free Full Text]
  16. Mitic LL, Van Itallie CM, Anderson JM. Molecular physiology and pathophysiology of tight junctions I. Tight junction structure and function: lessons from mutant animals and proteins. Am J Physiol Gastrointest Liver Physiol 2000; 279:G250–254[Abstract/Free Full Text]
  17. Denker BM and Nigam SK. Molecular structure and assembly of the tight junction. Am J Physiol 1998; 274:F1–F9[Medline]
  18. Van Itallie CM and Anderson JM. Claudins and epithelial paracellular transport. Annu Rev Physiol 2006; 68:403–429[CrossRef][Medline]
  19. Morita K, Furuse M, Fujimoto K, Tsukita S. Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands. Proc Natl Acad Sci U S A 1999; 96:511–516[Abstract/Free Full Text]
  20. Rahner C, Mitic LL, Anderson JM. Heterogeneity in expression and subcellular localization of claudins 2, 3, 4, and 5 in the rat liver, pancreas, and gut. Gastroenterology 2001; 120:411–422[CrossRef][Medline]
  21. Furuse M and Tsukita S. Claudins in occluding junctions of humans and flies. Trends Cell Biol 2006; 16:181–188[CrossRef][Medline]
  22. Hewitt KJ, Agarwal R, Morin PJ. The claudin gene family: expression in normal and neoplastic tissues. BMC Cancer 2006; 6:186–194[CrossRef][Medline]
  23. Furuse M, Sasaki H, Fujimoto K, Tsukita S. A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occludin in fibroblasts. J Cell Biol 1998; 143:391–401[Abstract/Free Full Text]
  24. Van Itallie C, Rahner C, Anderson JM. Regulated expression of claudin-4 decreases paracellular conductance through a selective decrease in sodium permeability. J Clin Invest 2001; 107:1319–1327[Medline]
  25. Amasheh S, Meiri N, Gitter AH, Schoneberg T, Mankertz J, Schulzke JD, Fromm M. Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells. J Cell Sci 2002; 115:4969–4976[CrossRef][Medline]
  26. Muller D, Kausalya PJ, Claverie-Martin F, Meij IC, Eggert P, Garcia-Nieto V, Hunziker W. A novel claudin 16 mutation associated with childhood hypercalciuria abolishes binding to ZO-1 and results in lysosomal mistargeting. Am J Hum Genet 2003; 73:1293–1301[CrossRef][Medline]
  27. Levy S and Robaire B. Segment-specific changes with age in the expression of junctional proteins and the permeability of the blood-epididymis barrier in rats. Biol Reprod 1999; 60:1392–1401[Abstract/Free Full Text]
  28. Cyr DG, Hermo L, Egenberger N, Mertineit C, Trasler JM, Laird DW. Cellular immunolocalization of occludin during embryonic and postnatal development of the mouse testis and epididymis. Endocrinology 1999; 140:3815–3825[Abstract/Free Full Text]
  29. Gregory M, Dufresne J, Hermo L, Cyr D. Claudin-1 is not restricted to tight junctions in the rat epididymis. Endocrinology 2001; 142:854–863[Abstract/Free Full Text]
  30. Guan X, Inai T, Shibata Y. Segment-specific expression of tight junction proteins, claudin-2 and –10, in the rat epididymal epithelium. Arch Histol Cytol 2005; 68:213–225[CrossRef][Medline]
  31. Gregory M and Cyr DG. Identification of multiple claudins in the rat epididymis. Mol Reprod Dev 2006; 73:580–588[CrossRef][Medline]
  32. Krull N, Ivell R, Osterhoff C, Kirchhoff C. Region-specific variation of gene expression in the human epididymis as revealed by in situ hybridization with tissue-specific cDNAs. Mol Reprod Dev 1993; 34:16–24[CrossRef][Medline]
  33. Clauss A, Lilja H, Lundwall A. A locus on human chromosome 20 contains several genes expressing protease inhibitor domains with homology to whey acidic protein. Biochem J 2002; 368:233–242[CrossRef][Medline]
  34. Cornwall GA and Hsia N. Cres (cystatin-related epididymal spermatogenic) gene regulation and function. Zhonghua Nan Ke Xue 2002; 8:313–318[Medline]
  35. Rodriguez CM, Kirby JL, Hinton BT. Regulation of gene transcription in the epididymis. Reproduction 2001; 122:41–48[Abstract]
  36. Ostermeier GC, Dix DJ, Miller D, Khatri P, Krawetz SA. Spermatozoal RNA profiles of normal fertile men. Lancet 2002; 360:772–777[CrossRef][Medline]
  37. Zhang JS, Liu Q, Li YM, Hall SH, French FS, Zhang YL. Genome-wide profiling of segmental-regulated transcriptomes in human epididymis using oligo microarray. Mol Cell Endocrinol 2006; 250:169–177[CrossRef][Medline]
  38. Skotheim RI, Monni O, Mousses S, Fossa SD, Kallioniemi OP, Lothe RA, Kallioniemi A. New insights into testicular germ cell tumorigenesis from gene expression profiling. Cancer Res 2002; 62:2359–2364[Abstract/Free Full Text]
  39. McLean DJ, Friel PJ, Pouchnik D, Griswold MD. Oligonucleotide microarray analysis of gene expression in follicle-stimulating hormone-treated rat Sertoli cells. Mol Endocrinol 2002; 16:2780–2792[Abstract/Free Full Text]
  40. Rodriguez S, Jafer O, Goker H, Summersgill BM, Zafarana G, Gillis AJ, van Gurp RJ, Oosterhuis JW, Lu YJ, Huddart R, Cooper CS, Clark J, et al. Expression profile of genes from 12p in testicular germ cell tumors of adolescents and adults associated with i(12p) and amplification at 12p11.2-p12.1. Oncogene 2003; 22:1880–1891[CrossRef][Medline]
  41. Hsia N and Cornwall GA. DNA microarray analysis of region-specific gene expression in the mouse epididymis. Biol Reprod 2004; 70:448–457[Abstract/Free Full Text]
  42. Chauvin TR and Griswold MD. Androgen-regulated genes in the murine epididymis. Biol Reprod 2004; 71:560–569[Abstract/Free Full Text]
  43. Ezer N and Robaire B. Gene expression is differentially regulated in the epididymis after orchidectomy. Endocrinology 2003; 144:975–988[Abstract/Free Full Text]
  44. Sipila P, Pujianto DA, Shariatmadari R, Nikkila J, Lehtoranta M, Huhtaniemi IT, French FS, O'Rand M. Differential endocrine regulation of genes enriched in initial segment and distal caput of the mouse epididymis as revealed by genome-wide expression profiling. Biol Reprod 2006; 75:240–251[Abstract/Free Full Text]
  45. Hermo L and Jacks D. Nature's ingenuity: bypassing the classical secretory route via apocrine secretion. Mol Reprod Dev 2002; 63:394–410[CrossRef][Medline]
  46. Jervis KM and Robaire B. Dynamic changes in gene expression along the rat epididymis. Biol Reprod 2001; 65:696–703[Abstract/Free Full Text]
  47. Johnston DS, Jelinsky SA, Bang HJ, DiCandeloro P, Wilson E, Kopf GS, Turner TT. The mouse epididymal transcriptome: transcriptional profiling of segmental gene expression in the epididymis. Biol Reprod 2005; 73:404–413[Abstract/Free Full Text]
  48. Yeung CH, Cooper TG, Bergmann M, Schulze H. Organization of tubules in the human caput epididymidis and the ultrastructure of their epithelia. Am J Anat 1991; 191:261–279[CrossRef][Medline]
  49. Yeung CH, Nashan D, Sorg C, Oberpenning F, Schulze H, Nieschlag E, Cooper TG. Basal cells of the human epididymis–antigenic and ultrastructural similarities to tissue-fixed macrophages. Biol Reprod 1994; 50:917–926[Abstract]
  50. Jalkanen J, Huhtanieni I, Poutanen M. Mouse cysteine-rich secretory protein 4 (CRISP4) exclusively expressed in the epididymis in an androgen-dependent manner. Biol Reprod 2005; 72:1268–1274[Abstract/Free Full Text]
  51. Richardson RT, Sivashanmugam P, Hall SH, Hamil KG, Moore PA, Ruben SM, Poulanen M. Cloning and sequencing of human Eppin: a novel family of protease inhibitors expressed in the epididymis and testis. Gene 2001; 270:93–102[CrossRef][Medline]
  52. Sivashanmugam P, Hall SH, Hamil KG, French FS, O'Rand MG, Richardson RT. Characterization of mouse Eppin and a gene cluster of similar protease inhibitors on mouse chromosome 2. Gene 2003; 312:125–134[CrossRef][Medline]
  53. Perry AC, Jones R, Moisyadi S, Coadwell J, Hall L. The novel epididymal secretory protein ESP13.2 in Macaca fascicularis. Biol Reprod 1999; 61:965–972[Abstract/Free Full Text]
  54. Rodriguez-Jimenez FJ, Krause A, Schulz S, Forssmann WG, Conejo-Garcia JR, Schreeb R, Motzkus D. Distribution of new human beta-defensin genes clustered on chromosome 20 in functionally different segments of epididymis. Genomics 2003; 81:175–183[CrossRef][Medline]
  55. Viger RS and Robaire B. Gene expression in the aging brown Norway rat epididymis. J Androl 1995; 16:108–117[Abstract/Free Full Text]
  56. Cyr DG, Hermo L, Robaire B. Developmental changes in epithelial cadherin messenger ribonucleic acid and immunocytochemical localization of epithelial cadherin during postnatal epididymal development in the rat. Endocrinology 1993; 132:1115–1124[Abstract]
  57. DeBellefeuille S, Hermo L, Gregory M, Dufresne J, Cyr DG. Catenins in the rat epididymis: their expression and regulation in adulthood and during postnatal development. Endocrinology 2003; 144:5040–5049[Abstract/Free Full Text]
  58. Troxell ML, Chen YT, Cobb N, Nelson WJ, Marrs JA. Cadherin function in junctional complex rearrangement and posttranslational control of cadherin expression. Am J Physiol 1999; 276:C404–C418[Medline]
  59. Cyr DG and Robaire B. Developmental regulation of epithelial- and placental-cadherin mRNAs in the rat epididymis. Ann N Y Acad Sci 1991; 637:399–408[CrossRef][Medline]
  60. Cyr DG, Hermo L, Blaschuk OW, Robaire B. Distribution and regulation of epithelial cadherin messenger ribonucleic acid and immunocytochemical localization of epithelial cadherin in the rat epididymis. Endocrinology 1992; 130:353–363[Abstract]
  61. Katafiasz BJ, Nieman MT, Wheelock MJ, Johnson KR. Characterization of cadherin-24, a novel alternatively spliced type II cadherin. J Biol Chem 2003; 278:27513–27519[Abstract/Free Full Text]
  62. Takeichi M. Cadherins: a molecular family important in selective cell-cell adhesion. Annu Rev Biochem 1990; 59:237–252[CrossRef][Medline]
  63. Behrens J. Cadherins and catenins: role in signal transduction and tumor progression. Cancer Metastasis Rev 1999; 18:15–30[CrossRef][Medline]
  64. Wertz K and Herrmann BG. Kidney-specific cadherin (cdh16) is expressed in embryonic kidney, lung, and sex ducts. Mech Dev 1999; 84:185–188[CrossRef][Medline]
  65. Aberle H, Schwartz H, Kemler R. Cadherin-catenin complex: protein interactions and their implications for cadherin function. J Cell Biochem 1996; 61:514–523[CrossRef][Medline]
  66. Tsukita S and Furuse M. Pores in the wall: claudins constitute tight junction strands containing aqueous pores. J Cell Biol 2000; 149:13–16[Abstract/Free Full Text]
  67. Van Itallie CM, Fanning AS, Anderson JM. Reversal of charge selectivity in cation or anion-selective epithelial lines by expression of different claudins. Am J Physiol Renal Physiol 2003; 285:F1078–F1084[Abstract/Free Full Text]
  68. Hou J, Paul DL, Goodenough DA. Paracellin-1 and the modulation of ion selectivity of tight junctions. J Cell Sci 2005; 118:5109–5118[Abstract/Free Full Text]
  69. Tiwari-Woodruff SK, Buznikov AG, Vu TQ, Micevych PE, Chen K, Kornblum HI, Bronstein JM. OSP/claudin-11 forms a complex with a novel member of the tetraspanin super family and beta1 integrin and regulates proliferation and migration of oligodendrocytes. J Cell Biol 2001; 153:295–305[Abstract/Free Full Text]
  70. Berman AE, Kozlova NI, Morozevich GE. Integrins: structure and signaling. Biochemistry (Mosc) 2003; 68:1284–1299[CrossRef][Medline]
  71. Angelow S, Kim KJ, Yu AS. Claudin-8 modulates paracellular permeability to acidic and basic ions in MDCK II cells. J Physiol 2006; 571:15–26[Abstract/Free Full Text]
  72. Gadella BM and Van Gestel RA. Bicarbonate and its role in mammalian sperm function. Anim Reprod Sci 2004; 82–83:307–319



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