Mol. Hum. Reprod. Advance Access originally published online on September 19, 2007
Molecular Human Reproduction 2007 13(10):691-704; doi:10.1093/molehr/gam051
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Region-specific gene expression profiling along the human epididymis
1Département d’Obstétrique-Gynécologie, Centre de Recherche en Biologie de la Reproduction, Centre de Recherche du CHUL, Université Laval, 2705 Boul. Laurier, Sainte Foy, Québec, Canada G1V 4G2 2Faculté de Médecine, Oncologie and Endocrinologie Centre de Recherche du CHUL, Université Laval, 2705 Boul. Laurier, Sainte Foy, Québec, Canada G1V 4G2
3 Correspondence address. Tel: +418-656-4141; Fax: +418-654-2765; E-mail: robert.sullivan{at}crchul.ulaval.ca
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During their transit through the epididymis, spermatozoa undergo many biochemical modifications necessary to acquire flagellar motility and fertilizing ability. These modifications, collectively called sperm maturation, are well orchestrated along the epididymis and depend on highly regionalized gene expression patterns. Based on clinical observations, the role of the epididymis in human sperm maturation has been questioned. To further understand the function of the excurrent duct in humans, we analysed gene expression of three donors on ‘Affymetrix human GeneChip U133 plus 2’ representing 47 000 transcriptional variants. More than 50% of transcripts were detected in each epididymal region. The analysis of hierarchical clustering performed from 2274 modulated qualifers between the three regions revealed that 1184, 713 and 269 were highly expressed in the caput, corpus and cauda region, respectively, in a very specific manner. The expressed qualifers were grouped according their similarity by Gene Ontology to give an overview of the functional features of the encoded proteins and to elucidate their potential roles in the epididymis. Northern blot analysis of eight gene transcripts predicted by microarray data to be highly expressed in the human epididymis was performed. All the transcript expression patterns confirmed the microarrays results. The data generated in this study demonstrate a region-specific gene expression pattern along the human epididymis that seems to coincide with the morphologically distinctive features of the excurrent duct.
Key words: epididymis/gene expression/transcriptome
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The epididymis is a long, single convoluted tubule through which spermatozoa must transit to acquire progressive motility and fertilizing ability. In humans, it can be morphologically divided into three principal regions: the caput and corpus regions, involved in the acquisition of fertilizing ability, and the distal cauda epididymidis, responsible for sperm storage. The caput region in man also contains multiple small ductules called efferent ducts (Yeung et al., 1991; Yeung and Cooper, 2002). There is no evidence for the existence of a differentiated initial segment in humans (Legare and Sullivan, 2004). The morphology of the human epididymis is distinguishable from the excurrent duct in other mammalian species: the cauda region is less developed and has a poor sperm reservoir capacity (Bedford, 1994), whereas the caput region has a rather bulbous shape consisting of vasa efferentia (Yeung et al., 1991; Bedford, 1994; Turner, 1995). The rapid transit in the human epididymis (2–6 days) (Amann and Howards, 1980), compared with the other species (10–13 days) (Robaire and Hermo, 1988) is another peculiarity in human epididymis suggesting that spermatozoa are rapidly processed in men (Turner, 1995) or that biochemical modifications underlying sperm maturation in humans are simpler than in other animal species.
Sperm maturation requires the interaction of spermatozoa with proteins that are synthesized and secreted in a region-specific manner by principle cells of the epididymal epithelium (Sullivan, 1999; Gatti et al., 2004). Each epididymal region is characterized by its own gene expression pattern encoding its specific secretome sequentially interacting with the maturing spermatozoa (Kirchhoff, 1999). To date, knowledge at molecular level of the complex process of sperm maturation along the human epididymis is limited owing to the scarcity of normal human tissue. Some transcripts which are highly expressed in the human epididymis have been identified using subtractive cDNA library strategies (Kirchhoff, 2002). Some human epididymal (HE) specific proteins, such as P34H, have been identified using different approaches (Legare et al., 1999). What we know about region-specific genes expression patterns along the human epididymis is very limited and results obtained using animal models should be extrapolated to human with caution (Jervis and Robaire, 2001; Hsia and Cornwall, 2004; Johnston et al., 2005).
In this study, DNA microarray technology was used to analyse regionalized gene expression along the human epididymis to identify the relative abundance of HE transcripts expressed in the three principal epididymal regions. RNAs were isolated from healthy human caput, corpus and cauda epididymidal tissues and used to hybridize ‘Affymetrix Human U133 Plus 2.0’ oligonucleotide microarrays. These arrays contain over 55 000 probe sets representing >47 000 transcripts derived from 
39 500 human genes. Genes encoding the major epididymal-specific human secretory proteins shown to be implicated in the human sperm maturation (Kirchhoff et al., 1990,1998; Kirchhoff, 2002; Cooper and Yeung, 2006) were identified as well as new gene families expressed at relatively high levels in the epididymis. Subsequent pathway analysis revealed signalling events in each epididymal region that are associated with high expression of a gene group or gene families presenting similar patterns.
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Biological material and RNA extraction
Human epididymides were obtained through our local organ transplantation program (Québec, QC, Canada) after obtaining family permission. Three donors of 26–50 years of age with no medical pathologies that could affect reproductive function were used for this study. Tissues were obtained and processed as previously described (Legare et al., 1999). Briefly, tissues were collected under optimum conditions, while artificial circulation was maintained to preserve organs assigned for transplantation. Epididymides were dissected into the caput, corpus and cauda epididymidis, immediately frozen in liquid nitrogen and stored at –80°C until used for RNA extraction.
Total RNA was extracted by Trizol reagent (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions, and the RNA quantity was determined by absorbance at 260 nm. Total RNA was further purified using RNeasy mini kit column (Qiagen, Mississauga, ON, Canada), and the integrity of the RNA was assessed by denatured agarose gel electrophoresis and the purity was determined using an Agilent Bioanalyser.
Microarray processing
The samples were processed following the Small Sample Labelling Protocol version II from Affymetrix (http://www.affymetrix.com/support/technical/technotes/smallv2_technote.pdf). This protocol was based on the principle of performing one cycle of cDNA synthesis and in vitro transcription reactions for target amplification. Briefly, 10 µg of total RNA of each epididymal segment were converted to cDNA by incubation with 400 units of Superscript II reverse transcriptase (Invitrogen), a T7 oligonucleotide –d(T)24 as a primer, combined with 1 mM dNTPs in 1X first-strand buffer at 42°C for 1 h. Second strand cDNA synthesis was performed using 40 units of DNA polymerase I (Invitrogen), 10 units of Escherichia coli DNA ligase (Invitrogen), 2 units of RNasse H (Invitrogen) and 0.2 mM dNTPs in 1X reaction buffer at 16°C for 2 h. Each cDNA sample was blunt ended by incubation with of 10 units of T4 polynucleotide kinase (Invitrogen) at 16°C for 5 min. cDNA samples were purified using GeneChips sample clean-up columns (Affymetrix, Santa Clara, CA, USA). cDNA was transcribed in vitro using the GeneChip labelling kit (Affymetrix) to produce biotinylated cRNA. Labelled cRNA was isolated using GeneChips sample clean-up columns (Affymetrix). Purified cRNA was fragmented to 200–300mer cRNA using a fragmentation buffer (100 mM potassium acetate, 30 mM magnesium acetate, 40 mM Tris-acetate pH 8.1) for 20 min at 94°C. The quality of cRNA amplification and cRNA fragmentation was monitored by micro-capillary electrophoresis (Bioanalyser 2100, Agilent Technologies, Santa Clara, CA, USA).
Hybridization, scanning and analysis
The cRNA was hybridized to human oligonucleotide array U133 Plus 2.0 (Genechip, Affymetrix). The array comprised 55 000 oligonucleotide features covering over 47 000 transcripts and variants, which represent 39 000 of the best characterized human genes. A 15 µg sample of fragmented cRNA was hybridized for 16 h at 45°C with constant rotation (60 rpm). After hybridization, chips were processed by using the Affymetrix GeneChip Fluidic Station 450 (protocol EukGE-WS2Av4). Staining was performed with streptavidin-conjugated pycoerythrin (SAPE), followed by amplification with a biotinylated anti-streptavidin antibody (Vector Laboratories, Burlingame, CA, USA), and by a second round of SAPE. The signal intensities of ß-actin and glyceraldehydes-3-phosphatedehydrogenase (GAPDH) genes were used as internal quality controls. The ratio of fluorescent intensities for the 5' and 3' of these housekeeping genes was <2. Chips were scanned using a Genechip Scanner 3000 G7 (Affymetrix). Images were extracted with the GeneChip Operating Software (Affymetrix GCOS v1.4). Quality control of microarray chips was performed using the AffyQCReport software (Bioconductor, Berkeley).
The background subtraction and normalization of probe set intensities was performed using the method of robust multiarray analysis described by Irizarry et al. (2003). To identify differentially expressed genes, gene expression intensity was compared using a moderated t-test and a Bayes smoothing approach developed for a low number of replicates (Smyth, 2004). To correct for the effect of multiple testing, the false discovery rate was estimated from P-values derived from the moderated t-test statistics (Benjamini et al., 2001). The analysis was performed using the AffylmGUI Graphical User Interface for the Limma microarray package (Wettenhall et al., 2006). Note that our primary microarray data described in this manuscript have been submitted to Gene Expression Onmiburs.
The following link has been created to allow review of GSE7808 [NCBI GEO] : <http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=flktdmggsqkkovg&acc=GSE7808>http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=flktdmggsqkkovg&acc=GSE7808.
Microarray analysis using genespring
Expression analysis of all replicate experiments was visualized, and ordered with GeneSpring v7.2 (Sillicon Genetics, Redwood City, CA, USA). Normalized mean values for the epididymal regions of the three donors were generated for the experimental analysis. An unsupervised clustering algorithm, group hierarchical genes clustering (Gene Tree), was applied to median normalized expression of genes differentially expressed among the three epididymal regions. The lists of genes highly expressed in each epididymal region and presenting a similar distribution pattern were selected from this clustering algorithm.
Northern blot analysis
Samples of 20 µg of total RNA isolated from the caput, corpus and cauda epididymidis of each donor were denatured in 50% formamide at 65°C for 15 min and electrophoresed on 1% agarose gels containing 2.2 M formaldehyde. The RNAs were transferred to a nylon membrane (Hybond N+, Amersham Biosciences, Baie d’Urfé, PQ, Canada) by overnight capillary blotting in 20X saline sodium citrate (SSC; 3 M NaCl, 0.3 M sodium citrate) and cross-linked by exposure for 30 s under ultraviolet light.
The membrane was stored at –20°C until prehybridization. The probes used for AK1, AQP9, HE1, HE3, HE12, WAP9, CRISP-1 and ADAM7 detection were partial sequences amplified by reverse transcription–polymerase chain reaction (RT–PCR) using primers deduced from human sequences (Table 1). The cDNA probes were random prime labelled using the T7 Quick Prime kit (Pharmacia Biotech, Baie D’Urfé, PQ, Canada) and purified with the QIAquick Purification Kit (Qiagen). The cDNA probes were labelled with [
-32P] dCTP (Megaprime II, Amersham Biosciences). The RNA was prehybridized at 42°C for 4 h in a prehybridization solution [50% formamide, 5X Denhart solution, 1% sodium dodecyl sulphate (SDS), 5X SSC, 100 µg/ml heat-denatured salmon sperm DNA (ICN Biomedicals, Aurora, OH, USA)]. The hybridization was performed overnight at 42°C in hybridization solution (prehybridization buffer with 5% dextran sulphate to which 1–2.106 cpm/[-32-P] dCTP labelled probe was added). The membrane was then washed twice for 30 min at 42°C with 2X SSC and 0.1% SDS followed by two incubations of 30 min at 65°C in 0.1 SSC and 0.1% SDS. The membranes were exposed on Kodak Bio-Mak Ms Films (Eastman Kodak, Rochester, NY, USA) with intensifying screens at –80°C. An RNA ladder (1.6–7.4 kb; Gibco-BRL, Burlington, ON, Canada) was used to determine the length of the transcript. GAPDH was used as a constitutive internal control.
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Segmental microarrays analysis
Gene expression profiling of each epididymal region was analysed from provided data at P-value <0.01. The analysis revealed that 1839, 201 and 1265 qualifiers were differentially expressed between the caput and corpus, the corpus and cauda and the caput and cauda HE region, respectively. The union of these three clusters of probe sets revealed that 2274 were modulated along the total epididymis.
In order to obtain an overview of genes differentially expressed between the three regions, an unsupervised clustering algorithm performed with GeneSpring V7.2 Software was applied using the median normalized expression data of these 2274 modulated probe sets in the three epididymal regions. It is a schematic reordering of the data table, which is useful for its ability to arrange genes according to similarity in the pattern of expression. This tree view graphical representation illustrates that each epididymal region displayed different gene groups showing a relatively high level expression. Among the 2274 probe sets analysed, the caput is the region that displays the most dominant probe sets cluster highly expressed with 51.2% (1184 qualifiers) of the genes expressed along the epididymis compared with 31.4% (713 qualifiers) in the corpus, and 11.2% (269 qualifiers) in the cauda epididymidis (Fig. 1). This cluster analysis demonstrates clearly that the gene expression pattern along the human epididymis is highly regionalized. The comparison between these probe sets clusters exhibiting a high level of expression revealed that the caput region shares only 5 common qualifiers with the corpus and 8 with the cauda, whereas the corpus and the cauda regions share 55 qualifiers. It is noticeable that there is no common gene highly expressed along the three epididymal regions (Fig. 2).
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Region-specific transcripts
Each of these qualifier groups was then analysed. Given the vast amount of information generated by these analyses, only selected qualifiers are presented according to the ratio of their expression between each epididymal region. In the caput epididymidis, up-regulated qualifiers presenting >10-fold differences with the corpus were selected (Table 2). In the corpus segment, selected up-regulated qualifiers with >20-fold differences with the caput are presented (Table 3) and in the cauda, selected up-regulated qualifiers with >5-fold differences with the corpus were considered (Table 4). Analysis of the expression pattern of these selected transcripts in each epididymal region showed that the specific caput transcripts were down-regulated in the corpus (Fig. 3A), while the most specific corpus transcripts were up-regulated in the corpus and down-regulated in the cauda (Fig. 3B). However, a few specific corpus transcripts presented an expression level higher in the cauda region, such as CRISP-1, WFDC9 (WAP9), MUC15, DEFB129, CAMP and DEFB 126 (Fig. 3B). The specific cauda transcripts, highly expressed in the distal epididymal segment were weakly expressed in the two other epididymal regions (Fig. 3C). In spite of the greatest number of qualifiers highly expressed in the caput region, very few of them have been previously reported to be expressed in the epididymis (1%), compared with the corpus region. However, the cauda epididymidis is the region that displayed the fewer genes encoding proteins known to be involved in sperm maturation.
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In order to explore these large sets of data, qualifiers were organized according to common functional features using Gene Ontology (GO) categories (biological process, molecular function and cellular component) using WebGestalt (web-based gene set analysis toolkit) software. These softwares are integrated data mining systems for the organization, visualization and statistical analysis of large set of genes. Clusters of qualifiers were analysed in each epididymal region; the different GO categories generated for each set of qualifiers results from the GO Tree Analysis (Zhang et al., 2005). In the caput, among the 1184 highly expressed qualifiers, 53% (624) are known and have thus been analysed. Using all genes in the geneCHIP ‘U133 plus 2’ as reference, the bar chart illustrating genes biological process GO showed that the number of expressed genes of the ‘cell–cell adhesion’ category is increased in the proximal region of the epididymis with P < 0.01 (Fig. 4A, Table 5). This category is essentially composed of different cadherin transcripts (CDH; CDH2, 6, 16) encoding the family members of cadherin, catenin delta 2 transcripts (CTNND2) and claudin transcripts (CLDN; CLDN 2, 10). Moreover, most of the highly expressed caput transcripts seem to be implicated in fundamental cellular metabolism and the signal transduction process. In this cluster of qualifiers highly expressed in the caput epididymidis, a few were previously reported to be expressed in the epididymis, such as: aquaporin 9 (AQP9) (Pastor-Soler et al., 2001), encoding a water channel involved in the transcellular transport of water in a wide variety of tissues; estrogen receptor-
(ESR1) (Hess et al., 2000), encoding the estrogen receptor highly expressed in the vasa efferentia; CD52 (HE5) (Kirchhoff et al., 1993), a major HE protein GPI anchored to sperm plasma membrane and RNASE4, a ribonuclease, member of the RNAse A family (Castella et al., 2004). Two adenylate kinase genes, AK1 and AK7, were also identified. AK1 is known to be localized in the flagella and is proposed to be involved in sperm motility control (Cao et al., 2006).
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The corpus region was the epididymal segment from which the highest number of transcripts previously reported to be expressed in the human epididymis were identified. We detected transcription of DCXR gene encoding P34H; an epididymal-originating acrosomal sperm protein previously identified by our laboratory and known to be involved in sperm-zona pellucida interaction (Boue et al., 1994; Legare et al., 1999). Genes encoding other major HE proteins such as HE1, HE3 (constituted in two genes transcripts; FAM12A and FAM12B) and HE12 were identified. The bar chart relative to biological processes, performed with 344 qualifiers known (48%) among the 713 expressed at a high level in the corpus highlighted an enriched gene category involved in defence response (Fig. 4B, Table 6). In this category, several members of the beta defensin (DEFB) genes family (DEFB32, 129, 127, 126, 108, 107, 123 and 119) and cathelicidin (CAMP) are the principal represented transcripts. The molecular function GO emphasized the protease inhibitor activity category composed essentially of SPINWL1 (Eppin) and SPINK 2–5, three transcripts encoding serine peptidase inhibitors (Moritz et al., 1991; Jalkanen et al., 2006), and three WFDC (Whey acid protein, WAP) transcripts (Fig. 4C, Table 7). The WAP transcripts, members of a new gene family characterized by the presence of 1 or 2 four-disulphide-core (FDC) domains (Clauss et al., 2002), showed high levels of expression in the human corpus epididymidis. The transcripts WAP8, WAP9 and WAP10 were the predominant WAP members found in this study (Table 3). The endoribonuclease activity is another enriched category under the molecular function GO (not shown). In this GO category are classified the two isoforms of HE3 transcripts, FAM12A (HE3
) and FAM12B (HE3ß), and the RNASE 11, A, ribonuclease, transcripts. Furthermore, ADAM7 and CRISP-1 transcripts, previously reported to be expressed in the epididymis, and encoding secretory proteins involved in gamete physiology (Hayashi et al., 1996; Lin et al., 2001), were detected in the corpus epididymidis transcriptome.
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In the cauda segment, very few transcripts previously reported to be expressed in the epididymis and involved in the fertility processes were identified by the microarray analysis. The bar chart illustrating genes biological processes highlighted mainly cluster of transcripts implicated in muscular contraction process genes. For example, this cluster is composed of transcripts encoding for proteins such as actin (ACTG2), calponin 1 (CNN1), tropomyosin (TPM1) and desmin (DESM1) and adrenergic alpha-1A-receptor (ADRA1A) (Table 4) (Fig. 4D, Table 8). The Actin and tropomyosin cytoskeletal proteins have been localized in the apical junctional complex of the principal cells of epididymal epithelium and are also associated to the neck region of the bovine spermatozoa (Yagi and Paranko, 1992). This biological process GO emphasized also a large cluster of qualifiers involved in the establishment of localization process, essentially composed of different collagen (COL) member family transcripts. Moreover, CLND 2, 10 and the CTNND2 transcripts were retrieved in the human cauda epididymal transcriptome.
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Northern blot analysis of selected transcripts
To confirm the region-specific gene expression pattern along the human epididymis, nine transcripts were selected: AK1, AQP9, two genes highly expressed in the caput region; HE1, HE3 and H12 encoding for major human secretory proteins and WAP9, ADAM7 and CRISP-1 that displayed relative high intensity levels in the transcriptome of HE tissues. The probes used in northern blots were generated by RT–PCR and the blotted RNAs were the same than those used to hybridize microarrays.
Northern blots analysis of AK1 and AQP9 transcripts showed a high level of expression in the caput epididymidis confirming the microarray results. The 5-fold down-regulation of AK1 transcript from the caput to the corpus epididymidis agreed with the 10-fold decrease observed in microarray data (Figs. 5 and 6A and B). Microarray detection of another gene, AQP9, which is expressed in the caput epididymidis, was also confirmed with northern blot analysis (Fig. 6A and B).
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In order to validate microarray results, northern blot analyses were also performed on selected genes known to be expressed in the human epididymis. Microarray showed that, compared with the caput signal intensity, HE1 corpus epididymal expression is up-regulated by a factor of 7.1, whereas northern blots revealed a 13-fold increase. The two techniques also give consistent results regarding HE1 expression in the cauda epididymidis where it is down-regulated compared with the two other epididymal regions. According to the microarray experiment, HE3 was up-regulated by a >100-fold factor in the corpus and down-regulated in the cauda epididymidis by a 2.9-fold factor. Northern blot estimations of HE3 are consistent with these results (Figs. 5 and 6A and B). According to microarray data, HE12 was up-regulated in the corpus 85-fold and down-regulated by a 2.3-fold factor in the cauda epididymidis. Northern blot analysis confirmed these data showing that HE12 is 33-fold up-regulated in the corpus followed by a 4-fold decrease in the cauda epididymidis. Consistent results between microarray data and northern blot estimations were also obtained for WAP9, ADAM 7, and CRISP-1. Taken together, these results validate microarray determination of patterns of gene expression along the human epididymis.
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Through our local transplantation program we have been able to obtain HE tissues dissected under optimal conditions. Tissues are processed within 1 h of surgery allowing extraction of high-quality RNAs. We successfully used these tissues in the past to study the human epididymis at the transcriptional and translational levels. Microarrays were performed using epididymides from three different men of reproductive age who died by accident, and generated consistent results between samples. Furthermore, microarray results regarding selected genes showing region-specific patterns of expression were confirmed by northern blot analyses. We noted that few transcripts showed a different expression pattern in one donor when compared with the two others. The causes of these few differences are unknown. Taken together, these results generated reliable information regarding pattern of gene expression along the human epididymis.
Over 50% of transcripts were detected in each HE segment, suggesting that these tissues are highly active in protein synthesis and secretory activity (Cornwall and Hann, 1995; Kirchhoff et al., 2003). Clustering algorithm of the 2274 modulated qualifiers (P-value 0.01) along the total epididymis, representing 11% of the genes expressed in each epididymal segment, shows that gene expression is highly regulated along the human epididymis as shown for other species as well as in humans. The caput epididymidis is the most active segment with the largest set of transcripts highly expressed, an observation similar to what has been shown in mouse and human (Johnston et al., 2005; Zhang et al., 2005; Dube et al., 2007). Very few highly expressed transcripts are found to be expressed in two epididymal regions, with a maximum of 55 genes highly expressed in both the corpus and cauda epididymidis. It is noticeable that no gene has been detected as being highly expressed all along the epididymis (Fig. 2). Thus, as for rodent models, the pattern of gene expression is highly regulated along the human epididymis, with each region being characterized by its own set of highly expressed genes.
Despite the large number of genes expressed at high levels in the caput epididymidis, few of them have been already described when referring to published epididymal transcriptome in different species (Cornwall et al., 2002; Hsia and Cornwall, 2004; Sipila et al., 2006). Two transcripts localized in the proximal epididymis, AQP9 and the ESR1, were found highly expressed in our microarryay data. AQP9 encodes a member of the water channel family that allows transcellular transport of water in the efferent duct (Pastor-Soler et al., 2001). AQP9 is regulated by estrogen (Oliveira et al., 2005), which is present at high concentrations in rete testis and efferent duct fluids (Hess et al., 1997; Hess, 2003). Considering that the human caput epididymidis is mainly formed by efferent (Yeung et al., 1991) ducts, it can be expected that AQP9 and ESR1 expression would be higher in this region. However, as noticed by Dube et al. (2007), the expression pattern of AQP9 revealed by our microarray data, and validated in the northern blot analysis, shows that AQP9 transcript is more abundant in the human corpus than in the caput epididymis (Figs. 5 and 6). Another cluster of genes of particular interest in the caput segment, also implicated in transport processes in the epididymis, was emphasized in our results. This gene group is essentially represented by CLDN, CDH and CTNN transcripts encoding proteins involved in cell–cell adhesion. The two claudin transcripts, CLDN 10 and CLDN 2, were first detected in the rat epididymis according to a regionalizing expression pattern (Guan et al., 2005). As described in the rat epididymis, our microarray data show that CLDN 2 occurs with a high expression level principally in the caput, whereas the CLDN 10 is highly expressed in the caput and corpus epididymidis (data not shown). Many SERPIN transcripts were also identified in the human caput epididymidis. SERPINA1, 5, 6, SERPINI2 and SERPINE2, encode novel members of the SERPIN family with a serine protease inhibitory putative function. Different members of the SERPIN family are localized in various tissues and are thought to be involved in fundamental biological processes, such as blood coagulation, inflammation, apoptosis, etc. In the epididymis, several genes with SERPIN domains are expressed and have been shown to be involved in sperm maturation and fertility processes (Hu et al., 2002; Yamazaki et al., 2006). This illustrates the importance of the proximal portion of the human epididymis in sperm maturation processes.
The corpus is the HE segment with the largest number of highly expressed genes previously reported to be transcribed in epididymal tissues. The corpus epididymidal transcriptome reveals a new gene family encoding for the putative protease inhibitors: WAP characterized by the presence of 1 or 2 four FDC domains (Clauss et al., 2002). The principal WAP genes first identified in the epididymis include the leucocyte protease inhibitor (SLPI) (Thompson and Ohlsson, 1986), HE4 (Kirchhoff et al., 1991) and SPINWL1 (Eppin) (Richardson et al., 2001). Among this gene family, WAP8, WAP9 and WAP10 are the predominant WAP transcripts detected in our microarrays. The biological function of these genes is not yet elucidated, but SLPI has been demonstrated to be a potent antimicrobial agent (Tomee et al., 1998). A recent study suggests that WAP10 may also be involved in defence against microbial infection in the monkey epididymis (Shayu et al., 2006). Two other transcripts encoding for protease inhibitor proteins, SPKINK 2 and SPINK 5 characterized by a Kazal domain, are expressed in corpus epididymidis. They are known as putative regulators of the proteolytic processing of some sperm surface proteins (Moritz et al., 1991; Jalkanen et al., 2006). For example, proteolytic processing was reported to be involved in the release of germinal form of converting enzyme localized at the sperm surface (Thimon et al., 2005). Also of interest, are the different genes encoding for the HE proteins highly expressed in the human corpus epididymidis. The function of these HE proteins in sperm maturation is however not completely elucidated (Kirchhoff et al., 1998). The NPC2 (HE1) transcript, the most abundant transcript in the human epididymis (Kirchhoff et al., 1996), is highly expressed in all segments of the human epididymis, with the exception of the proximal caput containing the efferent ducts. It encodes for the HE1 protein, i.e. proposed to play a role in cholesterol transport during sperm maturation (Legare and Sullivan, 2004; Legare et al., 2006). The others HE transcripts such as HE3 and HE12 display high expression levels in the corpus epididymidis but their functions remain to be elucidated. HE3 is a secretory protein associated with the ribonuclease family, while HE12, according to its sequence homology to the canine Ce12 cDNA with a highly conserved fibronectin type II (Fn-2)-module protein, may play a role in capacitation (Saalmann et al., 2001; Kirchhoff et al., 2003). Finally the corpus segment was enriched in transcripts encoding for proteins known to be involved in sperm interactions with the zona pellucida or with the oocyte’s plasma membrane such as P34H (Boue et al., 1994; Legare et al., 1999), ADAM7 (Lin et al., 2001) and CRISP-1 (AEGL1) (Hayashi et al., 1996; Ellerman et al., 2006). As for the caput, the corpus transcriptome strongly supports the concept that the human epididymis plays a major role in the acquisition of sperm fertilizing ability.
The cauda epididymidis is the region showing the fewest number of transcripts and the lowest level of total transcriptional activity (Fig. 1). Very few transcripts highly expressed in the human cauda epididymidis have been reported to play a potential role in sperm maturation. In fact, expression of genes involved in muscular contraction is the main signature of the human cauda epididymidis transcriptome. Most transcripts retrieved in this biological process, such as actin (ACTG2), tropomyosin (TPM1), myosin (MYH11), calponin, (CNN1), smothelin (SMTN), etc., encode for cytoskeletal-binding proteins. This probably reflects the predominance of smooth muscular tissues in the distal excurrent duct involved in sperm expulsion at ejaculation. The two cytoskeletal proteins, actin and tropomyosin, have been localized in the epididymal epithelium and are thought to be components of the contractile system of the distal excurrent duct (Yagi and Paranko, 1992). Furthermore, other genes such the prostaglandin receptor (PTGER3) and the ADRA1A are highly expressed in the human cauda epididymidis. The presence of these transcripts corroborates with the different studies that described the involvement of these gene products in the contractile system in the cauda and of the vas deferens in rodent species (Cosentino et al., 1984; Honner and Docherty, 1999; Chaturapanich et al., 2002; Mewe et al., 2006). The presence of these transcripts in the human cauda epididymidis transcriptome suggests that the distal segment of the human epididymis may be similar to the vas deferens in rodent species that focuses on muscular contraction. This correlates well with the modest sperm reservoir capacity of the human epididymis. Taken together, these observations suggest that the human cauda epididymidis is poorly differentiated compared with other mammalian species.
Exhaustive microarray analyses of the mouse and rat epididymides have been performed and allow interspecies comparison between of gene expression patterns along the epididymis (Jelinsky et al., 2006). While the epididymal transcriptome appears similar when comparing the two rodent species, major differences are found in human. Many genes expressed by the human epididymis have different patterns of expression along the excurrent duct when compared with mouse and rat. This is well illustrated by the protooncogene c-ros which is expressed at a high level along the human epididymis (not shown) (Legare and Sullivan, 2004), while it is exclusively expressed in the initial segment of murine epididymis. Comparison between rodent and human epididymides is complicated, as the human epididydimis presents distinctive morphological features when compared with other species (Bedford, 1994; Turner, 1995). Even though the human epididymis peculiarities, as in rodent, its pattern of gene expression is highly regulated and varies from one segment to the other. When analysed on the basis of individual transcript, the gene expression patterns along the epididymis can however show major differences when comparing human with rodent species.
Based on two decades of observation, we know that spermatozoa undergo sequential modifications during the epididymal transit that are driven by changes in gene expression pattern along the excurrent duct. The function of the human epididymis has been questioned especially by fertility recovery in men following vasoepididymostomy performed in the proximal regions of the epididymis. Among the 2274 qualifiers differentially expressed along the epididymis that display a segment-specific pattern of expression, many of them are involved in specific biological functions. This contrasts with recently published secretome and proteome analysis of HE fluids suggesting that there are only minor changes in the electrophoretic patterns of major proteins secreted along the human epididymis (Dacheux et al., 2006). The transcriptome of the human epididymis supports the concept that the excurrent duct interacts in a sequential and segment-specific manner with maturing spermatozoa.
In conclusion, our results provide a large view of the highly regionalized human gene expression pattern that differs from the transcriptome analysis performed using rodent species. This study highlights the potential role of novel genes or gene families expressed along the human epididymis. The HE transcriptome will contribute to a better understanding of sperm maturation and brings new insights to resolve some male infertility problems.
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This work was supported by Canadian Institutes for Health Research (CIHR) grant to R.S. Mrs. Christine Légaré and Julie Laflamme are acknowledged for their help in biological material dissection and processing. The collaboration of ‘Québec Transplant’ nurses and coordinator as well as of associated surgeons is also acknowledged.
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
Amann RP, Howards SS. Daily spermatozoal production and epididymal spermatozoal reserves of the human male. J Urol (1980) 124:211–215.[ISI][Medline]
Bedford JM. The status and the state of the human epididymis. Hum Reprod (1994) 9:2187–2199.
Benjamini Y, Drai D, Elmer G, Kafkafi N, Golani I. Controlling the false discovery rate in behavior genetics research. Behav Brain Res (2001) 125:279–284.[CrossRef][ISI][Medline]
Boue F, Berube B, De Lamirande E, Gagnon C, Sullivan R. Human sperm-zona pellucida interaction is inhibited by an antiserum against a hamster sperm protein. Biol Reprod (1994) 51:577–587.[Abstract]
Cao W, Haig-Ladewig L, Gerton GL, Moss SB. Adenylate kinases 1 and 2 are part of the accessory structures in the mouse sperm flagellum. Biol Reprod (2006) 75:492–500.
Castella S, Fouchecourt S, Teixeira-Gomes AP, Vinh J, Belghazi M, Dacheux F, Dacheux JL. Identification of a member of a new RNase a family specifically secreted by epididymal caput epithelium. Biol Reprod (2004) 70:319–328.
Chaturapanich G, Maythaarttaphong S, Verawatnapakul V, Pholpramool C. Mediation of contraction in rat cauda epididymidis by alpha-adrenoceptors. Reproduction (2002) 124:887–892.[Abstract]
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][ISI][Medline]
Cooper TG, Yeung CH. Sperm maturation in the human epididymis. In: The Sperm Cell, Production, Maturation, Fertilization, Regeneration.—De Jonge C, Barratt C, eds. (2006) Birmingham, UK: Cambridge University Press. 72–107.
Cornwall GA, Hann SR. Specialized gene expression in the epididymis. J Androl (1995) 16:379–383.
Cornwall GA, Lareyre JJ, Matusik RJ, Hinton BT, Orgebin-Crist MC. Gene expression and epididymal function. In: The Epididymis: from Molecules to Clinical Practice.—Robaire B, Hinton BT, eds. (2002) New York: Kluwer academic/Plenum Publishers. 169–199.
Cosentino MJ, Takihara H, Burhop JW, Cockett AT. Regulation of rat caput epididymidis contractility by prostaglandins. J Androl (1984) 5:216–222.
Dacheux JL, Belghazi M, Lanson Y, Dacheux F. Human epididymal secretome and proteome. Mol Cell Endocrinol (2006) 250:36–42.[CrossRef][ISI][Medline]
Dube E, Chan PT, Hermo L, Cyr DG. Gene expression profiling and its relevance to the blood-epididymal barrier in the human epididymis. Biol Reprod (2007).
Ellerman DA, Cohen DJ, Da Ros VG, Morgenfeld MM, Busso D, Cuasnicu PS. Sperm protein "DE" mediates gamete fusion through an evolutionarily conserved site of the CRISP family. Dev Biol (2006) 297:228–237.[CrossRef][ISI][Medline]
Gatti JL, Castella S, Dacheux F, Ecroyd H, Metayer S, Thimon V, Dacheux JL. Post-testicular sperm environment and fertility. Anim Reprod Sci (2004) 82–83:321–339.
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][ISI][Medline]
Hayashi M, Fujimoto S, Takano H, Ushiki T, Abe K, Ishikura H, Yoshida MC, Kirchhoff C, Ishibashi T, Kasahara M. Characterization of a human glycoprotein with a potential role in sperm-egg fusion: cDNA cloning, immunohistochemical localization, and chromosomal assignment of the gene (AEGL1). Genomics (1996) 32:367–374.[CrossRef][ISI][Medline]
Hess RA. Estrogen in the adult male reproductive tract: a review. Reprod Biol Endocrinol (2003) 1:52.[CrossRef][Medline]
Hess RA, Bunick D, Lee KH, Bahr J, Taylor JA, Korach KS, Lubahn DB. A role for oestrogens in the male reproductive system. Nature (1997) 390:509–512.[CrossRef][Medline]
Hess RA, Bunick D, Lubahn DB, Zhou Q, Bouma J. Morphologic changes in efferent ductules and epididymis in estrogen receptor-alpha knockout mice. J Androl (2000) 21:107–121.[Abstract]
Honner V, Docherty JR. Investigation of the subtypes of alpha1–adrenoceptor mediating contractions of rat vas deferens. Br J Pharmacol (1999) 128:1323–31.[CrossRef][ISI][Medline]
Hsia N, Cornwall GA. DNA microarray analysis of region-specific gene expression in the mouse epididymis. Biol Reprod (2004) 70:448–457.
Hu ZH, Liu Q, Shang Q, Zheng M, Yang J, Zhang YL. Identification and characterization of a new member of serpin family- HongrES1 in rat epididymis. Cell Res (2002) 12:407–410.[CrossRef][ISI][Medline]
Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed TP. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics (2003) 4:249–264.[Abstract]
Jalkanen J, Kotimaki M, Huhtaniemi I, Poutanen M. Novel epididymal protease inhibitors with Kazal or WAP family domain. Biochem Biophys Res Commun (2006) 349:245–254.[CrossRef][ISI][Medline]
Jelinsky SA, Turner TT, Bang HJ, Finger JN, Solarz MK, Wilson E, Brown EL, Kopf GS, Johnston DS. The rat epididymal transcriptome: comparison of segmental gene expression in the rat and mouse epididymides. Biol Reprod (2007) 76:561–570.
Jervis KM, Robaire B. Dynamic changes in gene expression along the rat epididymis. Biol Reprod (2001) 65:696–703.
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.
Kirchhoff C. Gene expression in the epididymis. Int Rev Cytol (1999) 188:133–202.[ISI][Medline]
Kirchhoff C. Speciffic gene expression in the human and non-human primate epididymis. In: The Epididymis.—Robaire B, Hinton BT, eds. (2002) New york: Plenum press. 201–218.
Kirchhoff C, Derr P, von Horsten H H, Käppler-Hanno K, Obermann H, Osterhoff, Samalecos A. Gene expression in the Human Epididymis. In: The Third International Conference on the Epididymis.—Hinton BT, Turner TT, eds. (2003) Charlottesville. 192–201.
Kirchhoff C, Habben I, Ivell R, Krull N. A major human epididymis-specific cDNA encodes a protein with sequence homology to extracellular proteinase inhibitors. Biol Reprod (1991) 45:350–357.[Abstract]
Kirchhoff C, Krull N, Pera I, Ivell R. A major mRNA of the human epididymal principal cells, HE5, encodes the leucocyte differentiation CDw52 antigen peptide backbone. Mol Reprod Dev (1993) 34:8–15.[CrossRef][ISI][Medline]
Kirchhoff C, Osterhoff C, Habben I, Ivell R. Cloning and analysis of mRNAs expressed specifically in the human epididymis. Int J Androl (1990) 13:155–167.[ISI][Medline]
Kirchhoff C, Osterhoff C, Pera I, Schroter S. Function of human epididymal proteins in sperm maturation. Andrologia (1998) 30:225–32.[ISI][Medline]
Kirchhoff C, Osterhoff C, Young L. Molecular cloning and characterization of HE1, a major secretory protein of the human epididymis. Biol Reprod (1996) 54:847–856.[Abstract]
Legare C, Gaudreault C, St-Jacques S, Sullivan R. P34H sperm protein is preferentially expressed by the human corpus epididymidis. Endocrinology (1999) 140:3318–3327.
Legare C, Sullivan R. Expression and localization of c-ros oncogene along the human excurrent duct. Mol Hum Reprod (2004) 10:697–703.
Legare C, Thabet M, Gatti JL, Sullivan R. HE1/NPC2 status in human reproductive tract and ejaculated spermatozoa: consequence of vasectomy. Mol Hum Reprod (2006) 12:461–468.
Lin YC, Sun GH, Lee YM, Guo YW, Liu HW. Cloning and characterization of a complementary DNA encoding a human epididymis-associated disintegrin and metalloprotease 7 protein. Biol Reprod (2001) 65:944–950.
Mewe M, Bauer CK, Schwarz JR, Middendorff R. Mechanisms regulating spontaneous contractions in the bovine epididymal duct. Biol Reprod (2006) 75:651–659.
Moritz A, Lilja H, Fink E. Molecular cloning and sequence analysis of the cDNA encoding the human acrosin-trypsin inhibitor (HUSI-II). FEBS Lett (1991) 278:127–130.[CrossRef][ISI][Medline]
Oliveira CA, Carnes K, Franca LR, Hermo L, Hess RA. Aquaporin-1 and -9 are differentially regulated by oestrogen in the efferent ductule epithelium and initial segment of the epididymis. Biol Cell (2005) 97:385–395.[ISI][Medline]
Pastor-Soler N, Bagnis C, Sabolic I, Tyszkowski R, McKee M, Van Hoek A, Breton S, Brown D. Aquaporin 9 expression along the male reproductive tract. Biol Reprod (2001) 65:384–393.
Richardson RT, Sivashanmugam P, Hall SH, Hamil KG, Moore PA, Ruben SM, French FS, O’Rand 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][ISI][Medline]
Robaire B, Hermo L. Efferent ducts, epididymis and vas deferens: structure, functions and their regulation. In: The Physiology of Reproduction.—Knobil E, Neill J, eds. (1988) New York: Raven Press. 999–1080.
Saalmann A, Munz S, Ellerbrock K, Ivell R, Kirchhoff C. Novel sperm-binding proteins of epididymal origin contain four fibronectin type II-modules. Mol Reprod Dev (2001) 58:88–100.[CrossRef][ISI][Medline]
Shayu D, Chennakesava CS, Rao AJ. Differential expression and antibacterial activity of WFDC10A in the monkey epididymis. Mol Cell Endocrinol (2006) 259:50–56.[CrossRef][ISI][Medline]
Sipila P, Pujianto DA, Shariatmadari R, Nikkila J, Lehtoranta M, Huhtaniemi IT, Poutanen 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.
Smyth GK. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol (2004) 3:3–12.
Sullivan R. Interaction between sperm and epidiydimal secretory proteins. In: The Male Gamete: from Basic to Clinical Applications.—Gagnon C, ed. (1999) Vienna, IL: Cache River Press. 130–136.
Thimon V, Metayer S, Belghazi M, Dacheux F, Dacheux JL, Gatti JL. Shedding of the germinal angiotensin I-converting enzyme (gACE) involves a serine protease and is activated by epididymal fluid. Biol Reprod (2005) 73:881–890.
Thompson RC, Ohlsson K. Isolation, properties, and complete amino acid sequence of human secretory leukocyte protease inhibitor, a potent inhibitor of leukocyte elastase. Proc Natl Acad Sci USA (1986) 83:6692–6696.
Tomee JF, Koeter GH, Hiemstra PS, Kauffman HF. Secretory leukoprotease inhibitor: a native antimicrobial protein presenting a new therapeutic option? Thorax (1998) 53:114–116.[Abstract]
Turner TT. On the epididymis and its role in the development of the fertile ejaculate. J Androl (1995) 16:292–298.
Wettenhall JM, Simpson KM, Satterley K, Smyth GK. AffylmGUI: a graphical user interface for linear modeling of single channel microarray data. Bioinformatics (2006) 22:897–899.
Yagi A, Paranko J. Localization of actin, alpha-actinin, and tropomyosin in bovine spermatozoa and epididymal epithelium. Anat Rec (1992) 233:61–74.[CrossRef][Medline]
Yamazaki K, Adachi T, Sato K, Yanagisawa Y, Fukata H, Seki N, Mori C, Komiyama M. Identification and characterization of novel and unknown mouse epididymis-specific genes by complementary DNA microarray technology. Biol Reprod (2006) 75:462–468.
Yeung CH, Cooper TG. Acquisistion and development of sperm motility upon maturation in the epididymis. In: The Epididymis: from Molecules to Clinical Practice.—Robaire B, Hinton BT, eds. (2002) New York: Kluwer Academic/Plenum Publishers. 417–434.
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][ISI][Medline]
Zhang B, Kirov S, Snoddy J. WebGestalt: an integrated system for exploring gene sets in various biological contexts. Nucleic Acids Res (2005) 33:W741–W748.
Submitted on May 4, 2007; resubmitted on June 27, 2007; accepted on July 10, 2007.
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