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BOR - Papers in Press, published online ahead of print April 26, 2006.
Biol Reprod 2006, 10.1095/biolreprod.105.047811
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BIOLOGY OF REPRODUCTION 75, 240–251 (2006)
DOI: 10.1095/biolreprod.105.047811
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Differential Endocrine Regulation of Genes Enriched in Initial Segment and Distal Caput of the Mouse Epididymis as Revealed by Genome-Wide Expression Profiling1

Petra Sipilä 3 4, Dwi Ari Pujianto 3 4, Ramin Shariatmadari 4, Janne Nikkilä 5, Mari Lehtoranta 4, Ilpo T. Huhtaniemi 6, and Matti Poutanen 2 4

Department of Physiology,4 Institute of Biomedicine, University of Turku, FIN-20520 Turku, Finland Genolyze Ltd.5 DataCity, 20521 Turku, Finland Institute of Reproductive and Developmental Biology,6 Faculty of Medicine, Imperial College, London, London W12 ONN, United Kingdom

ABSTRACT

We have performed genome-wide expression profiling of endocrine regulation of genes expressed in the mouse initial segment (IS) and distal caput of the epididymis by using Affymetrix microarrays. The data revealed that of the 15 020 genes expressed in the epididymis, 35% were enriched in one of the two regions studied, indicating that differential functions can be attributed to the IS and the more distal caput regions. The data, furthermore, showed that 27% of the genes expressed in the IS and/or distal caput epididymidis are under the regulation of testicular factors present in the duct fluid, while bloodborne androgens can regulate for 14% of them. This is in line with the high testis dependency of epididymal physiology. We then focused on genes with moderate or strong expression, showing strict segment enrichment and strong dependency on testicular factors. Analyses of the 59 genes, including upregulated and downregulated genes, fulfilling the criteria indicated that the expression of 18 (17 downregulated genes; 1 upregulated gene) of 19 gonadectomy-responsive genes enriched in the IS was not maintained by the androgen treatment, whereas the expression of all six downregulated genes enriched in the distal caput and the majority of those with no strict segment enrichment of expression (28 of 34; consisting of 23 downregulated and 5 upregulated genes) were maintained by androgens. Hence, it is evident that testicular factors other than androgens are important for the expression of IS-enriched genes, whereas the expression of distal caput-enriched genes is typically regulated by androgens. Identical data were obtained by independent clustering analyses performed for the expression data of 3626 epididymal genes. Several novel genes with putative involvement in epididymal sperm maturation, such as a disintegrin and metallopeptidase domain 28 (Adam28) and a solute carrier organic anion transporter family, member 4C1 (Slco4c1), were identified, indicating that this approach is successful for identifying novel epididymal genes.

epididymis, male reproductive tract, mechanisms of hormone action, sperm maturation, testosterone

INTRODUCTION

Epididymal gene expression is characterized by strong segment specificity [1]. Secretory proteins are synthesized and secreted in a highly regionalized manner, resulting in a unique luminal environment in each of the epididymal regions; this phenomenon is apparently essential for proper sperm maturation [2]. In addition to being region-specific, a number of epididymal genes are tissue-specific, expressed exclusively in the adult epididymis. Such genes include the initial segment (IS)-specific ros1 proto-oncogene (Ros1) [3] and lipocalin 8 (Lcn8) [4] and the distal caput-specific lipocalin 5 (Lcn5) [5]. In addition, there are numerous other genes that are expressed predominantly in the epididymis and only at lower levels in other tissues. IS-specific cystatin 8 (Cst8) [6, 7], caput-specific glutathione peroxidase 5 (Gpx5) [8], and corpus- and cauda-specific cysteine-rich secretory protein 1 (Crisp1) [9] fall into this category. In addition to genes with sharply restricted expression in certain epididymal regions [4], some exhibit more gradual changes of expression between the different regions, leading to characteristic checkerboard-type expression patterns for many epididymal genes [5, 10, 11]. Furthermore, there are several epithelial cell types present in the ductus epididymis, and several epididymal genes exhibit cell specificity, most of them being expressed only in the principal cells.

A variety of epididymal functions are regulated directly or indirectly by androgens. For example, epididymal histology, intermediary metabolism, ion transport, synthesis and secretion of a number of epididymal proteins, and activity of certain enzymes have been shown to be under the control of androgens [12]. Furthermore, transport, acquisition of fertilizing capacity, and storage of spermatozoa are dependent on androgens [12, 13]. Androgenic control is mainly mediated by 5{alpha}-dihydrotestosterone (DHT) and less clearly by testosterone [T] [13, 14], which is avidly converted to DHT by steroid 5{alpha}-reductase isoenzymes, type 1 and 2, in the epididymis. The expression patterns of the two steroid 5 alpha reductases (Srd5a) in the epididymis exhibit a gradient. While the IS expresses high levels of both isoforms, their expression decreases in the more distal segments [15, 16]. DHT acts via binding to the androgen receptor (AR), which is expressed in all epididymal regions and in most of the epididymal cell types [17].

Although the expression of many epididymal genes is regulated by androgens, only a few of them have been shown to contain androgen response elements (AREs) in their promoter region. However, the 5'-flanking region of murine Crisp1 contains several putative AREs [18]; also, the murine Gpx5 [19, 20], Lcn5 [21], and reproductive homeobox 5 (Rhox5) [22] promoter regions contain functional AREs.

Interestingly, certain epididymal genes with a wider tissue distributions, such as gamma-glutamyltransferase 1 (Ggt1), are androgen regulated only in the epididymis; furthermore, Ggt1 transcripts are regulated differentially by androgen in the different epididymal regions [23], which suggests the possibility that they play a role as AR coregulators in the regulation of Ggt1 expression. Examples of tissue-specific coregulators of AR are known from other tissues [24, 25], and the findings on Ggt1 expression after gonadectomy suggest that a tissue-, segment-, and cell-specific combination of transcription factors and coregulators mediates the androgen regulation of epididymal genes.

In addition to androgens, mostly unknown testicular factors present in efferent duct fluid have been shown to regulate the maintenance of the epithelial structure and gene expression in the IS [26, 27]. These genes include Cst8 [6], v-raf murine sarcoma 3611 viral oncogene homolog (Araf) [28], Gpx5 [8, 29], a disintegrin and metallopeptidase domain 7 (Adam7) [30], and Lcn8 [4]. Similar to androgens, these testicular factors can also act as inhibitors of transcription [31], and in addition, they have been shown to stabilize the mRNA of Ggt1 [32]. Several testicular factors have been suggested, such as the androgen-binding protein, which regulates the expression of Srd5a1 [33], the basic fibroblast growth factor that regulates the expression of Ggt1 [34], and the germ cells themselves, or germ cell-associated factors, that regulate the expression of preproenkephalin 1 (Penk1) [35].

In the present study, we performed genome-wide expression profiling of mRNAs expressed in the mouse IS and distal caput (excluding the IS) epididymidis and analyzed the differences in gene expression between these two epididymal regions. These regions were selected for analysis because they are more crucial for epididymal sperm maturation [36] than the other epididymal regions. We furthermore performed expression profiling of the proximal region of the epididymis in gonadectomized mice with and without androgen replacement therapy. The data provide a global expression profile for genes expressed in the two proximal regions of the mouse epididymis and novel data on the differential regulation of the epididymal gene expression in the IS and distal caput epididymidis by androgens and other regulatory factors originating from the testis.

MATERIALS AND METHODS

Experimental Animals and Tissue Collection

By using microarrays, we aimed to analyze the segment specificity of the gene expression in the mouse IS and distal caput and to study the regulation of gene expression in these segments by androgens and other testicular factors. For this purpose, the RNA samples were collected from the following epididymal regions in 8-wk-old male FVB/N mice (NL-5960 AD; Harlan, Horst, The Netherlands) by pooling the following samples from four mice: the IS, the whole caput region containing the IS (caput), and the distal caput excluding the IS (caput - IS). Furthermore, we collected RNA samples from the caput 1 and 3 days after gonadectomy and 3 days after gonadectomy while the mice underwent DHT treatment (2 mg of DHT in oil by daily s.c. injection). In addition, one pooled RNA sample was collected from the caput 8 h after gonadectomy. All the above samples were analyzed in triplicate by Affymetrix arrays (see below).

For quantitative (q) RT-PCR, we used samples similar to those described for microarrays. In addition, we collected RNA samples from the corpus and cauda epididymides of intact and gonadectomized mice. Additional caput samples were collected 8 h and 7 and 14 days after gonadectomy, in the presence and absence of a T treatment. For the T treatment, a 2-cm-long silastic tube, containing 2 mg of T (Fluka BioChemica, Buchs, Switzerland), was placed s.c. in the back of the animal at the time of gonadectomy. At the end of the treatment, the mice were anesthetized, blood samples were collected by cardiac puncture, and tissue samples were collected after cervical dislocation. The sera were stored at –70°C until assayed for T, using diethyl ether extraction and standard radioimmunoassay as described previously [37]. Hence, two androgen replacements were used: For array analyses, androgen was given by DHT injection, whereas for qRT-PCR analyses, it was given by T implantation.

RNA samples from 15 other tissues were also collected to analyze the tissue distribution of genes of interest. All mice were handled in accordance with the institutional animal care policies of the University of Turku (Turku, Finland); they were specific pathogen free, fed with complete pelleted chow and tap water ad libitum in a room with controlled light (12L:12D) and temperature (21 ± 1°C).

DNA Microarray Analysis

Microarray analyses were carried out in the Finnish DNA Microarray Center at the Turku Center for Biotechnology according to protocols provided by the manufacturer (Affymetrix Inc., Santa Clara, CA). In short, double-stranded cDNA was synthesized from 5 µg of total RNA, followed by a transcription reaction to produce biotin-labeled cRNA. The cRNA was fragmented and hybridized to a test 3 array for quality control and then to Murine Genome U74v.2 A, B, and C arrays at 45° C for 16 h. The only exceptions were the 3-day postgonadectomy samples, which were hybridized only to chip A. The arrays were washed and stained in an Affymetrix Fluidics Station 400 with streptavidin phycoerythrin as the stain and then scanned in an Affymetrix scanner.

Bioinformatics

The raw data from Affymetrix probe results (CEL files) were preprocessed and analyzed in two different ways: 1) with MAS5.0 and GeneSpring to collect the most salient differentially expressed genes and 2) with R/Bioconductor and clustering methods to find more subtle similarities among the genes. This offered two alternative perspectives to the data, which complement each other, since each preprocessing and analysis highlights different aspects of the data [38].

GeneSpring analysis GeneSpring analysis was used especially to identify genes with a markedly differential expression in the IS and distal caput (caput - IS) epididymidis. The default MAS5.0 preprocessing (with a target intensity scaling of 50) was performed, and the gene level data obtained were imported to GeneSpring 5.1 (Silicon Genetics, Redwood City, CA). To compare the data from the different arrays, the signal intensity value for each gene on the array was normalized by per chip and per gene normalization. For per chip normalization, scaling the median intensity of the chip to unity normalized the data for a given chip. For per gene normalization, scaling the median intensity of that gene to unity normalized the data for a given gene. Furthermore, genes categorized as absent by Affymetrix software in all the samples were filtered from further analysis. The normalized and filtered values were then subjected to logarithmic transformation. The logarithm values were used to analyze genes expressed in the mouse IS and distal caput (caput - IS) epididymides and to compare them with the postgonadectomized samples. For statistical analyses, the nonparametric Wilcoxon-Mann-Whitney test was used to calculate P values (considering P ≤ 0.05 significant). The criteria used for determining the segment-enriched genes in the nontreated IS and distal caput were as follows: 1) a difference of 5-fold or more, 2) statistically significant changes, and 3) a raw expression level ≥100. The criteria used for determining differential gene expression between control and postgonadectomy samples were as follows: 1) a difference of 3-fold or more, 2) statistically significant changes, and 3) a raw expression level ≥100. Genes were considered androgen regulated if their expression was maintained with DHT injections equivalent to at least at 50% of the original level. In addition, if gene expression was not maintained with DHT injections, it was considered regulated by other testicular factors.

R/Bioconductor and clustering analysis This approach was used especially to obtain the clustering analyses for IS- and distal caput (caput - IS)-expressed genes. The original Affymetrix probe results (CEL data) were processed with the Bioconductor R package Affy [39, 40]. Probe level data were normalized by quantile normalization [40]. In addition to the normalization, three MAS5.0-equivalent transformations were applied to the data [39]: 1) background correction, 2) perfect match/mismatch correction, and 3) summation of probe level data to give signal values for each gene. After preprocessing, the data were filtered on the basis of the present and absent calls. Triplicate samples were required to have at least two present calls and one marginal call. In addition, in the single 8-h sample, the expression was required to be categorized as present. After this, the genes from intact mice were defined to be IS enriched if they were present and significantly more highly expressed in the IS (according to a one-sample t-test) than the mean expression plus SEM of all the mouse genes in the intact IS but were not expressed in the distal caput according to the same criteria. The same definition was used to determine the distal caput-enriched genes.

The preprocessed data were then clustered and visualized with the self-organizing map [SOM] [41] protocol and hierarchical clustering. To focus on the most interesting genes, only the genes that were present at least in one of the time points analyzed and were statistically significantly (P < 0.05) differentially expressed between the nontreated sample and at least in one of the treatments were included in the analysis. Next, the data were converted to a four-dimensional data matrix by averaging the replicate values and dividing with the values of the untreated epididymis. Genes with abnormally large norms (cut-off value = 3.0) were removed from the SOM analysis and analyzed separately, with hierarchical clustering performed with the Bioconductor R package (the similarity criterion was the average distance). The rest of the genes were analyzed by the SOM. The SOM was visualized with the U-matrix [42] that calculates and visualizes the distances between the model vectors attached to each map unit of the SOM. The programs used to train and visualize the SOM were based on the publicly available SOM-PAK [43] package. The size of the map was chosen to be 20 x 30 nodes, which results in about five genes per node on average. The neighborhood function was Gaussian with an SEM of 1 node. Sammon mapping [44] was used to confirm a reasonable organization of the SOM in the data space.

Expression Analysis In Silico

Affymetrix gene expression data from the control IS and caput - IS epididymidis (only genes identified as present by Affymetrix software) were compared with the epididymal gene expression data at the Jackson Laboratory (MGI; http://www.informatics.jax.org). At MGI, the RT-PCR results from 524 genes expressed in the adult mouse epididymis were available at the time of analysis, and the expression of these genes was classified as strong, moderate, trace, and absent. This comparison was used to determine the cut-off raw expression level for genes that were further analyzed. In addition, the expressed sequence tag (EST) data of the UniGene data bank (http://www.ncbi.nlm.nih.gov/UniGene/library.cgi?ORG=Mm&LID=2606) were used to predict the tissue distribution of the selected genes.

Quantitative Real-Time RT-PCR Analysis

Quantitative real-time RT-PCR (qRT-PCR) measurements were performed by the DNA Engine Opticon system (MJ Research, Inc., Waltham, MA) with continuous fluorescence detection. One hundred nanograms of DNase I (Invitrogen, Life Technologies, Inc., Carlsbad, CA)-treated total RNA was used, and the reactions were performed by the QuantiTect SYRB Green RT-PCR Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. The samples and standard curves were run in triplicate. For controlling qRT-PCR reactions, melting profiles for each primer pair were analyzed, and products were run on agarose gel and sequenced. The relative standard curve method [45] was used to calculate relative gene expression. Actin beta (Actb) was included as the endogenous normalization control to adjust for unequal amounts of RNA. The primers and annealing temperatures used are presented in Table 1.


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  		TABLE 1. Primers used for quantitative RT-PCR

Statistical Analysis

SigmaStat for Windows was used to perform a one-way analysis of variance for the androgen dependency of mRNA expression, analyzed by qRT-PCR. The difference between means for each segment analyzed was subsequently assessed by the Holm-Sidak test. The level of statistical significance was set as P ≤ 0.001.

RESULTS

Epididymal Genes Regulated by Androgens

The expression profiling was performed especially to identify segment-enriched genes regulated either by androgens or other testicular factors (Fig. 1). We therefore first analyzed the genes expressed in the IS and distal caput (caput - IS) and then compared these expression profiles with those obtained in the whole caput epididymis of untreated, gonadectomized, and DHT-treated gonadectomized mice. In these analyses, 1993 (13%) genes were significantly downregulated 1 day after gonadectomy, and 343 (17.2%) of these genes maintained their expression level in gonadectomized mice treated with DHT. Almost the same number of genes (2126 [14%]) were significantly upregulated after gonadectomy, and 249 (11.7%) of them responded to DHT treatment with reduced expression (Fig. 2). This indicates that of the 15 020 genes expressed in the caput epididymis, 27.4% (n = 4119) are under positive or negative regulation by testicular factors and that androgen upregulates (n = 343) or downregulates (n = 249) 14.3% of them. All the genes identified and expressed in the present study in the IS and distal caput (caput - IS), as well as the genes differentially expressed after gonadectomy, with and without androgen replacement, are presented in the supplementary material available online at http://www.biolreprod.org (see supplements 1–3).


Figure 1
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  		FIG. 1. Experimental flowchart of the study performed using Affymetrix Murine Genome U74v.2 arrays. Expression profiling was performed for the mouse initial segment (IS) and distal caput epididymidis not including the IS (caput - IS). Furthermore, the regulation of the genes by androgens and other testicular factors was analyzed by expression profiling of the whole caput epididymidis in intact and gonadectomized mice with and without a 3-day DHT replacement therapy. DHT(+) = gene expression recovered in gonadectomized mice >50% by DHT treatment; DHT(-) = gene expression recovered in gonadectomized mice by DHT <50%


Figure 2
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  		FIG. 2. Response of genes expressed in the IS and/or distal caput to gonadectomy and DHT replacement. C, Number of genes expressed in the intact caput; DR, genes significantly downregulated 1 day after gonadectomy; DR + DHT, genes with restored expression in the gonadectomized mice after a 3-day DHT treatment; UR, genes significantly upregulated 1 day after gonadectomy; UP + DHT, genes with restored expression in the gonadectomized mice after a 3-day DHT treatment

We then focused on the genes with moderate-to-high levels of expression (a raw expression level ≥100). The rationale behind selecting the raw value of 100 as the exclusion level in the Affymetrix arrays was based on comparing the expression data obtained by the oligo arrays to those present at the MGI Web site of the Jackson Laboratory. At the time the analysis was carried out, of the genes with an Affymetrix raw signal ≥100, a total of 102 were also analyzed in the MGI database, and of these, the RT-PCR signals were considered strong or moderate for 97 genes. Thus, of the 102 Affymetrix-positive genes (signal ≥100), 95.1% were also found to be expressed in the epididymis by the MGI RT-PCR database. However, when all the genes with a statistically significant expression in the Affymetrix arrays were compared with the MGI data bank, expression could be confirmed for 38% of the genes. Hence, limiting the analyses to genes with a raw signal ≥100 in Affymetrix analysis is expected to markedly reduce the number of false-positive results in our study. The study was focused on genes that were most profoundly affected by gonadectomy (a statistically significant, ≥3-fold change up or down 1 day after gonadectomy). Furthermore, the segment specificity of these genes was resolved by using the data from the expression profiles obtained separately for the IS and caput - IS epididymides. After combining these two data sets, we found 25 gonadectomy-responsive (24 downregulated and 1 upregulated) genes with a 5-fold higher expression in one of the regions (indicating segment enrichment) and 34 gonadectomy-responsive genes (27 downregulated and 7 upregulated) whose expression in the two epididymal regions (IS and distal caput) differed by less than 5-fold (considered nonsegment enriched). These two groups of gonadectomy-responsive genes were then divided into those that maintained their expression during DHT treatment (expression changed <50% compared with nongonadectomized mice) and those that did not, possibly being regulated mainly by testicular factors other than androgens (expression changed >50% despite the DHT treatment). The data revealed that most of the gonadectomy-responsive genes enriched in the IS region were not maintained by DHT treatment by the above criteria: one downregulated gene responded to DHT, while 18 (17 downregulated and 1 upregulated) genes did not. In contrast, all six downregulated genes enriched in the distal caput (caput - IS) were maintained by DHT according to the criteria used. Furthermore, the expression of most of the genes (28 of 34; consisting of 23 downregulated and 5 upregulated genes) that did not show strict segment enrichment was also maintained by DHT, while only six (four downregulated and two upregulated) nonsegment-enriched genes were not maintained by DHT. The complete lists of the genes in these categories are presented in Tables 2–5.


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  		TABLE 2. Initial segment enriched, gonadectomy responsive genes (down-regulated) in the mouse epididymis


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  		TABLE 5. Genes up-regulated by gonadectomy

To obtain further information on the putatively differential regulation of the IS- and distal caput-enriched genes, clustering analyses were performed to generate groups of genes sharing the same expression pattern across the three different time points after gonadectomy (8 h, 1 day, and 3 days), as well as the DHT treatment of gonadectomized mice. Genes with abnormally large norms (cut-off value = 3148 genes) were analyzed with hierarchical clustering, resulting in two main clusters of genes (Fig. 3a). Interestingly, the genes expressed differentially between the IS and distal caput regions were clearly localized in separate clusters. IS-enriched genes (yellow dots) were localized mainly to cluster 2 (16 genes: two additional genes in cluster 1), whereas the eight distal caput (caput - IS)-enriched genes (blue dots) were localized only to cluster 1 (Fig. 3a). Boxplots revealed that the genes in cluster 1, including the caput - IS-enriched genes, were clearly DHT responsive, while the genes in cluster 2, including the IS-enriched genes, were not androgen responsive (Fig. 3b). SOM analysis performed with the rest of the 3478 genes revealed several gene clusters. In the present study, we directed our interest to identifying the localization of IS- (yellow dots) and distal caput (caput - IS; blue dots)-enriched genes in SOM analysis (Fig. 4a). The analysis revealed that the IS- and distal caput-enriched genes were differentially distributed in SOM analysis. We could identify one large cluster containing only nonsegment-enriched and distal caput-enriched genes (Fig. 4a, cluster 1), whereas no IS-enriched genes were present in this cluster. The boxplot from the cluster revealed that these genes were downregulated 3 days after gonadectomy and were maintained at this level of control (nongonadectomized mice), or were even increased, by DHT replacement (Fig. 4b, cluster 1). In the other cluster enriched with distal caput-enriched genes (Fig. 4b, cluster 2), the genes responded to gonadectomy after 8 h and were increased after DHT replacement, similar to those in cluster 1. These genes thus represent those most sensitive to androgen removal. Interestingly, two clusters enriched with the IS-enriched genes, not containing any distal caput-enriched genes, were also identified (Fig. 4b, clusters 3 and 4). The genes in these clusters were downregulated after gonadectomy but not maintained at this level of control with DHT replacement. Conspicuously, the genes in cluster 3 were downregulated at 8 h and 1 day but upregulated 3 days after gonadectomy. DHT replacement prevented this upregulation, and gene expression returned to the level observed 1 day after gonadectomy. In cluster 4, gene expression levels with DHT replacement were higher than 3 days after gonadectomy but still maintained the same level as 1 day after gonadectomy. Thus, clusters 3 and 4, enriched by the IS-enriched genes, represented genes mainly regulated by testicular factors other than androgens.


Figure 3
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  		FIG. 3. Hierarchical clustering of the genes with abnormally large norms. a) The dendrogram of the hierarchical clustering revealing clusters of IS-enriched and distal caput (caput - IS)-enriched genes. The caput IS-enriched genes are concentrated in cluster 1 (blue dots), and the IS-enriched genes are concentrated in cluster 2 (yellow dots). b) Boxplots of the two clusters from the dendrogram representing the behavior of the gene expression across different time points after gonadectomy (8 h, 1 day, and 3 days) and DHT replacement (+DHT). Boxes depict the two middle quartiles of the data, and the line in the box represents the median. The whiskers extend to cover the data between +1.5 times SEM, and the data outside them are shown with circles


Figure 4
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  		FIG. 4. Self-organizing map (SOM) clustering. a) The distribution of the IS-enriched genes (yellow dots) and caput-enriched genes (blue dots) is shown in the data space as visualized by the SOM. The SOM has been visualized by the U-matrix method that depicts the approximate density of the data: the light shade represents dense data (clusters), and the dark shade represents sparse data (the small dots are the map units of the SOM). Clusters encircled with black lines are the most salient areas where only IS- or caput-enriched genes are present. b) Boxplots of the clusters indicated in the SOM represent the gene expression at different time points after gonadectomy (8 h, 1 day, and 3 days) and after DHT replacement (+DHT). The boxplots for clusters 1 and 2 represent the data space where the IS-enriched genes are absent and the distal caput (caput - IS)-enriched genes are enriched. The boxplots indicate that DHT treatment can normalize the expression for genes expressed in the distal caput effectively. The boxplots for clusters 3 and 4 indicate that the expression of the IS-enriched genes cannot be restored with DHT. For a detailed description of the boxplots, see the legend of Figure 3

Quantitative RT-PCR Analysis of Segment Enrichment and Tissue Distribution of Selected Epididymal Genes

A set of genes identified by the oligo arrays was then analyzed further by qRT-PCR. This was done to further determine the gene expression in the IS, distal caput, and corpus and cauda regions and to analyze tissue distribution for selected genes of interest. To select the genes with putative expression only in a restricted number of other tissues, we used the EST data (Tables 2–5) available in the UniGene data bank. All the genes analyzed by qRT-PCR showed a unique expression pattern in the different epididymal regions and tissues (Table 6) in accordance with the array data. Tissue distribution analysis revealed that many genes were expressed in only a restricted number of other tissues, as also predicted by the EST data. Concerning the putative function of the genes, several interesting expression patterns were identified. Absolute epididymis specificity was observed for Adam28. The expression of the solute carrier organic anion transporter family, member 4C1 (Slco4c1), was highly specific for the epididymis and seminal vesicles. The RIKEN cDNA 1700018G05 gene was highly specific for the epididymis and testis, whereas the adaptor-related protein complex 3, beta 2 subunit (Ap3b2), and glutamate receptor, ionotropic (Gria2), were expressed especially in the epididymis, brain, and pituitary. An interesting expression pattern for the RIKEN cDNA 2310032F03 was identified, as the gene was highly expressed in the IS, corpus, and cauda but was much less expressed in the distal caput (caput - IS).


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  		TABLE 6. Tissue distribution of selected testis-dependent and segment enriched or nonsegment-enriched genes by qRT-PCR analysis.*

Quantitative RT-PCR Analysis of Androgen Dependency of Selected Genes Identified in the Array

Quantitative RT-PCR was also used to confirm the androgen dependency of the genes identified by expression profiling (Fig. 5). For this purpose, we selected eight genes, of which the array analysis demonstrated four genes with expressions that were maintained by DHT treatment (expression level ≥50% from the original level) and four genes with expressions that were not maintained by DHT treatment (expression level <50% from the original level). In the present study, we used T replacement administered by s.c. implants. To confirm the success of gonadectomy and androgen replacement, serum T levels were measured at all time points studied. In the intact male mice, the serum T concentration was 4.8 nmol/L, and after a 1-day gonadectomy, it was reduced to 0.3 nmol/L. With T pellets, serum T concentrations were 2.8-fold higher than in intact mice. The data obtained by qRT-PCR were in agreement with the array analyses obtained, all genes being responsive to gonadectomy. There were significant differences of gene expression levels in intact mice (wild type) compared to all gonadectomy groups (P ≤ 0.001). The genes with almost exclusive expression in the IS (Gria2, Adam28, and Slco4c1) were not maintained by T replacement, while the genes expressed almost exclusively in the caput - IS (Ap3b2, RIKEN cDNA 1700018G05 gene) and those expressed in both segments (lipase, endothelial; Lipg, and NADP-dependent steroid dehydrogenase-like; Nsdhl), responded to androgen replacement (P ≤ 0.001).


Figure 5
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  		FIG. 5. Quantitative RT-PCR analysis to confirm the androgen dependency of selected IS-enriched (Gria2, RIKEN cDNA 2310032F03, Adam28, Slco4c1), distal caput-enriched (Ap3b2, RIKEN cDNA 1700018G05), and nonsegment-enriched (Lipg, Nsdhl) genes. RNA samples were extracted from the whole caput (containing the IS; black bars), corpus (white bars), and cauda (gray bars) of intact mice (wild type) 1, 3, 7, and 14 days after gonadectomy and from gonadectomized mice treated with testosterone (T) for 7 or 14 days. Expression levels of all selected genes were significantly downregulated after gonadectomy (P ≤ 0.001), as analyzed from the region with highest level of expression. Testosterone replacement maintained the expression of Ap3b2, RIKEN cDNA 1700018G05, Lipg, and Nsdhl, with a level that is significantly higher than in gonadectomized mice (P ≤ 0.001), and the error bars indicate SEM

DISCUSSION

We performed genome-wide expression profiling of mRNAs expressed in the mouse IS and the caput segments distal to the IS, with special reference to the testis dependency of genes expressed in these two proximal epididymal regions. The high reliability of the array results is suggested by comparisons of the Affymetrix data with those present in the literature, the RT-PCR data available at MGI, and the results obtained by qRT-PCR. The array data showed that 35% of the 15 020 genes expressed in the IS and/or distal caput (caput - IS) epididymidis were enriched in either of these proximal regions. A good correlation was found between the array and the RT-PCR data at the MGI database, with a total of 4208 genes showing a raw signal >100 in the arrays. Hence, we could hypothesize that at least this set of genes was expressed in the IS and distal caput (caput - IS) epididymidis. Previously, the identification of 15 278 genes expressed in the IS and the remainder of the epididymis had been reported [46]. To our surprise, a largely different set of genes was identified in these two studies. The differences might be due, to a great extent, to a different set of genes studied as well as to the different epididymal regions analyzed. Hsia and Cornwall [46] compared the IS to the rest of the epididymis, whereas in the present study, the IS was compared to the rest of the caput region. In the previous study's list of genes preferentially expressed in the epididymal regions distal from the IS [46], there were only a few genes that appeared to be caput enriched on the basis of the present study. This would indicate that many genes that are reported to be enriched in the distal epididymal regions are highly expressed in the corpus and cauda regions but not in the caput. In contrast, the IS and distal caput expression data obtained in this study correlated well (data not shown) with the mouse epididymal transcriptome data reported recently [47]. The better correlation between these studies might be because of the use of arrays from the same manufacturer and the similar divisions of epididymal tissue for the arrays.

In the present study, a massive downregulation of gene expression was observed 3 days after gonadectomy, as 44% of the genes expressed in the intact epididymis were totally absent (data not shown). In the rat epididymis, apoptosis reaches maximum levels 2–3 days after gonadectomy [48], which suggests that the downregulation of several epididymal genes at this time point is due to transcriptional changes occurring upon apoptotic cell death, rather than through direct androgen deficiency. For this reason, we focused our data analysis on the results obtained 1 day after gonadectomy. However, we are aware that with the strict limits used in the present study, several testis-dependent genes were likely to be excluded. For example, two known androgen-regulated genes, Gpx5 and Lcn5 [5, 19], did not fulfill the inclusion criteria, although they also displayed a significant change 1 day after gonadectomy in the present study. Our genome-wide expression profiling data are in full agreement with previous reports of certain IS-specific genes that were not typically under androgen control but were regulated by other testicular factors [4, 6, 49] and genes expressed in the distal caput that were more tightly regulated by androgen [5, 19].

Similarly, the results from hierarchical and SOM clustering analysis showed that distal caput-enriched genes were typically maintained with DHT, whereas IS-enriched genes were not. Furthermore, the group of genes upregulated 3 days after gonadectomy may be involved in apoptosis or cell survival, since apoptosis reaches its maximum level in the epididymis 3 days after gonadectomy [48]. Recently, the androgen regulation of mouse epididymal genes expressed in the caput, corpus, and cauda regions was studied using the Affymetrix Murine Genome U74v.2 A array [50]. Surprisingly, from the androgen-regulated genes described in the previous study [50], only a few were common with the genes that fulfilled the strict criteria given in the present study. The majority of the androgen-regulated genes reported in that study [50] were less than 3-fold downregulated 1 day after gonadectomy in the present study. Furthermore, many of the genes reported were downregulated only after 3 days of gonadectomy in the present data and were thus, by our assumption, not directly regulated by androgens but rather related to apoptosis. Furthermore, in the previous study, the DHT treatment was started 6 days after gonadectomy [50], which might have contributed significantly to the different results of the two studies.

To our knowledge, there are only few previous reports on the upregulation of epididymal genes after gonadectomy [50–53], whereas the downregulation of gene expression after gonadectomy is a well-known epididymal response. Interestingly, many upregulated genes were totally absent in the control epididymides and appeared only after gonadectomy. For example, several members of the chemokine-signaling pathway belong to this category. Chemokines are involved in inflammatory reactions, immune response, and apoptosis. Since gonadectomy induces apoptotic cell death of epididymal principal cells [48], the upregulation of the chemokine signaling probably reflects transcriptional changes occurring in apoptotic cells.

On the basis of the array data and the in silico analysis, mRNA expressions for several genes were further studied by the qRT-PCR technique. One of the genes analyzed, Adam28, was especially interesting, being highly expressed in the lymphocytes and epididymis [54, 55], and the present data demonstrated that its more precise localization in the epididymis is in the IS. Furthermore, Adam28 is regulated by testicular factors other than androgens, is enzymatically active [56], and has an ortholog in the human [55]. Adams have been proposed to have roles in proteolysis, adhesion, fusion, and intracellular signaling, and they have wide tissue distribution [57]. Several Adam members are expressed in spermatogenic cells [58], and seven different Adam family members, Adam7, -8, -10, -28, Adamts1, -4, and -12, have been reported to be expressed in the epididymis [30, 46], which suggests important roles for the protein products of these genes in male reproduction. Furthermore, the infertility of a disintegrin and metallopeptidase domain 2 (Adam2) (previously known as beta fertilin) [59] and a disintegrin and metallopeptidase domain 3 (Adam3) (previously known as cyritestin) [60] in knockout mice emphasizes their importance for male fertility. Interestingly, data from the MGI gene expression data bank showed that Adam2 was also expressed in the epididymis. Thus, further studies are required to elucidate the importance of the mainly epididymal expression of Adam28 and Adam2 for male fertility.

Another interesting gene, expressed predominantly in the IS, was Slco4c1. It was dramatically downregulated 1 day after gonadectomy, but androgen replacement did not maintain its expression, indicating that this gene was mainly dependent on other testicular factors. Analysis showed that the gene was highly expressed in the IS and seminal vesicles, suggesting its role in secretory functions. The epithelial cells in seminal vesicles are assumed to secrete proteins involved in decapacitation by modifying sperm surface proteins to facilitate the acquisition of the ability to fertilize the egg. Mikkaichi et al. [61] reported that human and rat homologs for Slco4c1 are expressed in the kidney, as a transporter for various compounds, such as thyroid hormones, cAMP, and methotrexate. Mouse Slco4c1 may be involved in the epididymal sperm maturation by acting as an ion transporter, enabling directed movement of organic anions within or between cells. The exchange of various compounds, including ions in the epididymis, is also important to maintain the strict epididymal homeostasis necessary for sperm maturation.

In conclusion, the present study provides novel information on the differential regulation of epididymal genes expressed in the IS and distal caput (caput - IS) segments. The data bank generated in the present study provides a tool for the identification of novel epididymal genes and segment-specific regulatory networks involved in epididymal sperm maturation.


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  		TABLE 3. Distal caput (caput-IS) enriched, gonadectomy responsive genes (down-regulated) in the mouse epididymis


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  		TABLE 4. Nonsegment-enriched, gonadectomy responsive genes (down-regulated) in the mouse epididymis

ACKNOWLEDGMENTS

We thank N. Messner for animal handling and T. Laiho for T measurements.

FOOTNOTES

1 Supported by grants from the Rockefeller and Ernst-Schering foundations and the Academy of Finland (projects 53272 and 207028). Back

2 Correspondence: Matti Poutanen, Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland. FAX: 358 2 250 2610; matti.poutanen{at}utu.fi Back

3 These authors contributed equally to this work. Back

Received: 22 September 2005.

First decision: 23 November 2005.

Accepted: 21 April 2006.

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