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Male Reproductive Tract |
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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epididymis, male reproductive tract
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Thus far, a number of regionally expressed genes in the epididymis have been identified, including those encoding secretory proteins with putative roles as proteases and protease inhibitors, antioxidant enzymes, modifying enzymes, growth factors, neuropeptides, and transporters [6]. Several of these genes are unique to the epididymis, implying specific roles in epididymal function, whereas other genes are common to several tissues, suggesting broader functions. In addition, regionally expressed genes that encode intracellular proteins have been identified, including transcription factors, signaling molecules, receptors, kinases, and proteins with unknown functions [6]. Together, these studies have provided valuable information. However, gene discovery in the epididymis has been limited, because approaches to monitor global gene expression patterns have not been widely used.
The use of microarrays is a well-established and powerful approach that not only enables rapid gene discovery but also a systematic study of gene expression. To date, microarray technology has been used to profile genes that are expressed during malignancy and other disease states [7, 8], altered following injury [9, 10], associated with gene mutations [11], and up- or downregulated following exposure to exogenous stimuli [12]. Specifically, within the male reproductive tract, microarrays have been used to identify genes that are differentially expressed in testicular germ cell tumorigenesis [13], FSH-stimulated Sertoli cells [14], testicular development [15], segment-specific expression in the rat epididymis [16], and mRNAs in spermatozoa from fertile men [17]. In several of these reports, commercially available microarrays consisting of a subset of known genes were used [7, 16]; in other studies, specific arrays were generated that were composed of cDNAs representing a particular tissue or chromosomal region [15, 18]. The microarray analysis of region-specific expression in the rat epididymis examined a 1200-clone membrane consisting of known families of genes involved in cell signaling, structure, metabolism, cell cycle, and DNA binding [16]. These studies showed the region-specific expression of several oxidative stress-related genes as well as several cathepsins. In subsequent studies, a 474 rat cDNA microarray was used to identify genes regulated by orchiectomy both throughout the whole epididymis as well as in a region-dependent manner [19]. A transient upregulation of several genes expressed in the epididymis was noted, and previously uncharacterized androgen-regulated genes were identified.
The goal of the present study was to use microarray technology to identify genes explicitly expressed in the initial segment of the mouse epididymis. Our intent was to obtain a comprehensive survey of genes that may be involved in early sperm maturation events as well as those genes that may contribute to the unique regulatory characteristics of this region. Furthermore, we were interested in broadening our knowledge of gene expression in the epididymis in general and, thus, wanted to examine microarrays that did not exclusively contain known genes but that also consisted of a large number of unknown genes. Because of the lack of available arrays composed of epididymal-derived cDNAs, we opted to examine an array of 15 247 mouse genes derived from pre- and peri-implantation embryos, developing female gonads, and newborn ovarian cDNAs. These arrays were thus derived, in part, from reproductive tissues and also consisted of a large number of cDNAs, of which 78% represented novel sequences.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Retired breeder male CD-1 strain mice were obtained from Charles River (Wilmington, MA). Mice were housed under a constant 12L:12D photoperiod and were allowed free access to food and water. All animal studies were carried out in accordance with the principles and procedures outlined in the NIH Guidelines for Care and Use of Experimental Animals.
RNA Isolation
For preparation of RNA for the microarray studies, epididymides from six mice were dissected into two segments consisting of the initial segment (region 1) and the remaining regions from the midcaput to the cauda (regions 2–5) (Fig. 1) and total RNA isolated from each segment using Trizol reagent (Invitrogen, Grand Island, NY). After Trizol isolation, total RNA was further purified using an RNeasy column (Qiagen, Valencia, CA). The RNA isolations for each epididymal segment were performed in triplicate, with each isolation prepared from six mice. For preparation of RNA for Northern studies, mouse epididymides were dissected into five regions as previously described [20].
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Microarray Hybridization
The mouse microarray consisted of 15 247 expressed sequence tags (ESTs) divided between three slides. This approximately 15 000-clone set was developed at the National Institutes of Aging (NIA) and has been sequence verified by sequencing from both the 5'- and 3'-termini. Approximately 15 000 unique cDNA clones were derived from 52 374 ESTs from pre- and peri-implantation embryos, Embryonic Day 12.5 female gonad/mesonephros, and newborn ovary [21]. More details about this clone set as well as further gene annotation and informatics can be found at the NIA website (http://lgsun.grc.nia.nih.gov/cDNA/15k.html).
Preparation of the arrays and probes and hybridization of the microarrays were carried out at the Vanderbilt University School of Medicine Microarray Shared Resource (Nashville, TN) under the direction of Dr. Shawn Levy. Total RNA from mouse epididymal regions 1 and 2–5 were used to generate the two probes used for hybridization. The initial segment (region 1) RNA was labeled with Cy-Dye 5 (Cy5) and regions 2–5 RNA with Cy-Dye 3 (Cy3). The Cy5- and Cy3-labeled probes were hybridized simultaneously to the same array. Three independent RNA isolations, labeling, and microarray hybridizations were performed.
For reverse transcription, 30 µg of total RNA from each epididymal segment and 3 µg of oligo-dT were incubated for 5 min at 70°C and then at room temperature for 10 min. Under low-light conditions, reagents were added to achieve a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 120 µM Cy5-dCTP (region 1 probe) or Cy3-dCTP (regions 2–5 probe), and 200 µM unlabeled dNTPs (except for dCTP). Two microliters of Superscript reverse transcriptase (Invitrogen) was added, and the reactions were incubated at 42°C for 2 h. The 30-µl probe reactions were hydrolyzed for 10 min at 70°C with 15 µl of 0.1 M NaOH and subsequently neutralized with 15 µl of 0.1 M HCl. The hydrolyzed region 1 and regions 2–5 probes were then combined and cleaned using the Qiagen polymerase chain reaction purification kit according to the manufacturer's protocol, then dried in a speed-vac. The combined dried probes were resuspended in 25 µl of 3x SSC (single strength: 0.15 M sodium chloride and 0.015 M sodium citrate), 24 mM Hepes (pH 7), 0.6 µg/µl of polyA RNA, and 0.225% SDS and then incubated for 2 min at 100°C before hybridization.
Each of the three slides consisting of the 15 247 ESTs was hybridized with the combined region 1 and regions 2–5 probes. The slides were first prehybridized in 1% BSA, 5x SSC, and 0.1% SDS for 45 min at 65°C and then rinsed with MilliQ water and isopropanol. Hybridization was done overnight at 65°C for 14–16 h in a humidified hybridization chamber. The slides were washed with gentle agitation in 2x SSC and 0.1% SDS for 5 min, with 1x SSC for 5 min, and with 0.1x SSC for 5 min at 55°C. Slides were dried and immediately scanned using a dual-laser Genepix 4000B array scanner with the Genepix Pro 4.0 software (Axon Instruments, Union City, CA) to detect both the Cy5 (red) and Cy3 (green) fluorescent signals representing hybridization of the region 1 and regions 2–5 probes, respectively, to each individual EST. The ESTs that bound only the region 1 probe exhibited a Cy5 (red) signal, whereas ESTs that bound only the regions 2–5 probe exhibited a Cy3 (green) signal. The ESTs that bound both probes equally exhibited yellow fluorescence. Plant cDNAs on each chip served as negative controls and were used as a qualitative assessment of the hybridization.
All cDNA clones on the microarray are available from American Type Culture Collection (ATCC; Manassas, VA).
Data Analysis
The raw data obtained from the scanned array images were analyzed with Genespring 5.0 software (Silicon Genetics, Redwood City, CA) and normalized as follows: Local background for each clone was determined by measuring the Cy5 and Cy3 fluorescence in a defined pixel area surrounding each spot. Background fluorescence levels were subtracted from the Cy5 and Cy3 fluorescence values, resulting in a net Cy5 and Cy3 fluorescence intensity for each clone. For each clone, the net median fluorescence intensity for the Cy5 (region 1) signal was divided by that for the Cy3 (regions 2–5) signal. The ratio of Cy5 to Cy3 fluorescence indicated the relative abundance of a clone transcript in the initial segment and in the remaining epididymal regions, respectively. Background-subtracted median values that were less than zero were set to zero and subjected to intensity-dependent LOWESS (locally weighted scatterplot smoother) normalization, in which 20% of the data were used for smoothing [22, 23]. This technique was used to correct for dye-related artifacts resulting from nonlinear rates of dye incorporation and inconsistencies in the relative fluorescence intensity between the red and green dyes. The normalized ratio of the median values (median fluorescence of epididymal region 1/median fluorescence of epididymal regions 2–5) from three independent microarray hybridization experiments was averaged for each gene/EST, and genes exhibiting 2-fold or greater expression in region 1 and a probability t-test P value of 0.05 or less are reported. Similarly, the ratios of the median fluorescence of epididymal regions 2–5 to the median fluorescence of epididymal region 1 were averaged, and genes exhibiting 2-fold or greater expression in epididymal regions 2–5 and a P value of 0.05 or less are reported. The Genespring cross-gene error model (Silicon Genetics) was used to obtain a more accurate estimate of the measurement precision of a gene by combining measurement variation and between-sample variation. The nomenclature of selected genes was updated by searching GenBank databases using the nucleotide BLAST program. Several ESTs were represented multiple times on gene chips. Because the normalized expression intensities were similar, only one expression profile is presented in Figures 2 and 3. However, all EST replicates are present in our Supplemental Data (http://www.ttuhsc.edu/cbb/faculty/cornwall/default.asp). The replicates represent different regions of sequence of the same gene.
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Northern Blot Analysis
The RNA samples (10 µg each) were heated at 95°C for 2 min and then loaded onto 1% agarose/2% formaldehyde/1x borate gel and electrophoresed. The gels were washed extensively in water to remove formaldehyde before transfer to a nylon membrane (Nytran Supercharge; Schleicher & Schuell, Keene, NH) by vacuum blotting (Appligene, Illkirch, France) at 50 mbar for 3 h in the presence of 10x SSC. The membranes were then washed for 5 min in 5x SSC before ultraviolet cross-linking (Stratalinker; Stratagene, La Jolla, CA). The membranes were prehybridized for 1.5 h at 65°C in Church buffer containing 0.5 M NaPO4 buffer (pH 7.4), 7% SDS, and 1 mM EDTA, then hybridized overnight at 65°C in the presence of 3 x 105 cpm probe/ml hybridization buffer. The cDNA clones were purchased from ATCC and their identities verified by sequence analysis. The cDNA inserts were isolated from each vector, and the probes were prepared using a random prime labeling method (Prime-It II; Stratagene). After hybridization, the blots were washed twice in 1x SSC and 0.1% SDS at room temperature for 15 min and then twice at 65°C for 15 min before exposure to film. To verify equal transfer of RNA onto the blot, the cDNA probes were removed from each blot by stripping twice at 55°C for 30 min in 0.1x SSC and 1% SDS that had been preheated to 100°C. The stripped blots were then reprobed under the same conditions described with a cDNA probe to 18S rRNA.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The NIA mouse gene array used in the present study consists of approximately 15 000 genes (78% novel, 22% known) derived from pre- and peri-implantation embryos, Embryonic Day 12.5 female gonad/mesonephros, and newborn ovary [21]. The approximately 15 000-clone set contains cDNA inserts with an average size of 1.5 kilobase and has been sequenced from both 5'- and 3'-ends to determine gene identities. The majority of classifiable genes (
3300) encode matrix/structural proteins, signal transduction, protein synthesis/translational control, energy/metabolism, and transcription/chromatin associated.
The mouse array was hybridized with probes representing the initial segment (region 1) and the remainder of the epididymis (regions 2–5) to identify genes that are highly enriched in the initial segment region. Of the total 15 298 genes examined, 11 had no detectable signal, as evidenced by the negative intensity values with either of the two probes (data not shown). As shown in the scatterplot of Figure 1, the majority of the remaining genes exhibited a ratio of region 1 to regions 2–5 (epi 1:epi 2–5 ratio) of 1.0, suggesting that their expression levels were similar in all epididymal regions. However, we cannot rule out that these genes could be expressed in region 1 and at lower levels in the remaining regions, the sum of which is similar to the region 1 expression levels. Alternatively, an epi 1:epi 2–5 ratio of 1.0 could also result from expression in region 1 and similar levels of expression in one other epididymal region. In the present study, 123 genes exhibited 2-fold or higher levels of expression in epididymal region 1, as shown by their distribution above the central axis in the scatterplot, whereas 216 genes had 2-fold or higher levels of expression in epididymal regions 2–5, as evidenced by their distribution below the central axis (Fig. 1).
Identification of Genes Enriched in Regions 1 and 2–5
Because of our interest in region 1-specific genes, we first carried out a detailed analysis of the genes that showed enriched expression in this region. Because of space limitations, we narrowed the group of 123 region 1-enriched genes down to 53 by selecting those that exhibited 2.5-fold or higher levels of expression. Shown in Figure 2 are the actual fluorescence intensities (three replicates) for each gene falling into this category as well as annotation data, average ratio of the medians (epi 1:epi 2–5) of the normalized data, and P values. The genes are ranked from those with the greatest fold-difference between epididymal regions, with the Cres3 gene showing 146-fold higher levels of expression in region 1 compared to regions 2–5, to those with 2.5-fold differences between the two epididymal regions. Across varying levels of intensity, which ranged from abundantly expressed genes such as Cres3 (average E1 [region 1] intensity, 39 881) to low-expression genes such as mouse C2 membrane binding (average E1 intensity, 890), the actual fluorescence intensities for each gene were similar between replicate microarray experiments (Fig. 2). The high reproducibility between the arrays and the corresponding t-test P values allowed the identification of differentially expressed genes with 95% confidence (P 
0.05). The genes that fit into this category included several that were previously shown to exhibit epididymal region 1-enriched expression, including Cres3 [24], lipocalin 2 (mouse 24p3) [25], and connexin 43 [26], thus supporting the validity of the microarray experiments. The microarray data also revealed many new region 1-specific genes, including those with putative functions in transcription (erm, a new ets-related ;obPEA3 group;cb transcription factor, TBX1, PDZK1), signal transduction (RASA1, Ppfibp2, tyrosine phosphatases), cell-cell interactions (tetraspanins, CE34), components of the ubiquitin pathway (UCH-L1, EG:25E8.2), as well as several genes with unknown functions (KIAA1579, RIKEN clone AK020732).
We also examined in detail the expression of several genes that showed specific expression in epididymal regions 2–5. Because of the larger number of genes in this category (n = 216), we included only those that exhibited 3.5-fold or higher levels of expression in the distal epididymis. As shown in Figure 3, the genes were again ranked from those showing the greatest difference between regions, with glycine decarboxylase exhibiting 15-fold greater expression in epididymal regions 2–5 compared to region 1, to those with 3.5-fold difference between regions. Similar to our results with region 1-specific genes, the actual fluorescence intensities and P values for genes in this category indicated reproducibility among the microarray replicates, thus allowing the detection of genes expressed in epididymal regions 2–5 with high levels of confidence. The genes in this group included those encoding proteins with putative functions in metabolism (carbonic anhydrase, sulfotransferase), transcription (a gene similar to zinc-finger protein 91, the corepressor Sin3B, a gene similar to makorin, a ribonucleoprotein with zinc-finger motifs), signal transduction (Wnt-signaling frizzled-related protein, CD97 antigen), and many genes with unknown functions (membrane-associated protein 17, LOC226921).
Validation of Microarray Data
Northern blot analyses were carried out to confirm the ranking of gene expression levels determined by microarray analysis. Genes that were determined to be specific for region 1, specific for regions 2–5, and similar in all epididymal regions were randomly selected for analysis. As shown in Figure 4, the expression profiles by Northern blot analysis correlated well with those established by microarray. The Cres3, erm (etv5), milk fat globule factor, lipocalin 2, prostein, Alex3, a RIKEN clone, and synaptotagmin-like 4 genes all showed region 1-enriched expression, with the expression profiles paralleling the ratios determined by microarray. Zinc-finger 364 and ladinin 1 genes had average median ratios (epi 1:epi 2–5) close to 1.0, suggesting similar levels of expression in regions 1 and 2–5, as demonstrated by Northern blot analysis (Fig. 4). Finally, the elafin-like 1 and lactotransferrin genes exhibited epi 1:epi 2–5 ratios of 0.1 and epi 2–5:epi 1 ratios of 7.5 and 9.4, respectively. Thus, these genes were specific for regions 2–5 by microarray and were expressed only in regions 2–5 by Northern blot analysis. These results illustrate the validity of the microarray data.
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Identification of New Genes Expressed in the Epididymis
To broaden our knowledge of genes expressed in the epididymis, independent of region, we used the Genespring software to globally assess the expression of several groups of genes based on their molecular functions. The genes presented in Figure 5 represent a small proportion of the approximately 3000 genes on the microarray that have identifiable molecular functions. Several of the genes were categorized in specific groups but were not annotated; thus, these sequences were screened against GenBank using the BLAST program to determine putative identities. As shown in Figure 5, genes with proposed molecular functions in terms of proteases/protease inhibitors, DNA binding, signal transduction, and roles in cancer were identified in the epididymis. In addition, many of the genes exhibited regionalized expression, including those with enriched expression in the initial segment (epi 1:epi 2–5 ratio of 1.5–5) as well as those enriched in more distal epididymal regions (epi 1:epi 2–5 ratio of 0.8–0). In particular, several members of the ADAM and ADAMTS family of transmembrane-bound metalloprotease and disintegrin domain-containing proteins were identified, including ADAM8, ADAM10, and ADAMTS 1, 4, and 12. Other proteases expressed in the epididymis include the apoptotic cysteine protease caspase 3; serine proteases TMPRSS2, Prss15, and Prss25; members of the ubiquitin pathway, such as USPs, sentrin (ubiquitin-like), and fafx; 26S proteasomal ATPases PSMC 1, 3, and 5; and the proprotein-processing protease subtilisins/kexin isozyme SKI-1. Several serine protease inhibitors (Spi, Spint) were also detected in the epididymis.
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In addition to proteases and their inhibitors, the microarray analysis also identified the presence of a number of DNA-binding proteins. In addition to RNA polymerases I, II, and III, several transcriptional activators of the JAK/STAT pathway were present, as were CRSP2 (cofactor for Sp1 transactivation) and two new genes with moderate to weak sequence identity with CRSPs. Other DNA-binding proteins included zinc-finger protein (Zpy288), members of the ATF family of bZIP transcription factors, and other bHLH-ZIP transcription factors, such as TCF12, USF, LISCH7, and Lztfl1. Metal response element-binding transcription factor 1 (Mtf1), nuclear transcription factor X-binding 1 (Nfx1), pre-ß cell leukemia transcription factor 1 (pbx1), pou domain class 5 transcription factor 1 (pou5f1), prostate-specific ets transcription factor (pse), interferon-dependent positive-acting transcription factor 3
(isgf3g), and hepatocyte nuclear factor 1
(TCF1) were also detected in the epididymis.
Genes representing components of various signaling pathways also were expressed in the epididymis, some of which exhibited highly regionalized patterns of expression. Initial segment-enriched genes included milk fat globule-epidermal growth factor (EGF) as well as several components associated with tumor necrosis factor (TNF) stimulation, such as lipopolysaccharide-induced TNF factor (Litaf), TNF-induced protein (Tnfaip2), TNF-receptor superfamily member 23 (Tnfrsf2), and TNF-receptor and TNF receptor-associated factor protein (Ttrap). Growth factor-signaling molecules included insulin-like growth factor 1 (Igf1), fibroblast growth factor regulatory protein (Fgfrp), hepatocyte growth factor (Hrf), and growth factor receptor-bound protein 7 (Grb7). Several bone morphogenetic genes (Bmps) as well as constitutive photomorphogenic proteins (COPS) involved in the ubiquitin pathway were also detected.
Finally, genes associated with oncogenesis or tumor suppression were also detected in the epididymis. In addition to Jun, Fos, Kit, Myb, and Myc genes, a number of the Ras family of oncogenes were expressed (Rabs) as well as the tumor suppressor genes Tssc 3, 4, and 8.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Microarray analysis was used to examine gene expression in the mouse epididymis with the goal of identifying genes having enriched expression in the initial segment region. Not surprisingly, the majority of cDNAs on the microarray chips showed epi 1:epi 2–5 ratios of 1.0, suggesting similar levels of expression in all epididymal regions. However, of the remaining clones, 123 showed 2-fold or higher levels of expression in the initial segment region. Although only a small group of genes was examined by Northern blot analysis, the data showed that across all levels of gene expression (range of average E1 fluorescence intensities, 15–39 881), the expression patterns reflected that predicted by the ratio of the medians in the microarray studies. These data support the microarray data and suggest that the average ratios of the medians are adequate predictors of gene expression profiles.
New Genes and Putative Functions
Initial segment Because the microchips used cDNAs and ESTs derived from the developing mouse embryo and newborn ovary rather than cDNAs from a differentiated epididymis, the microarray analysis was biased toward identifying new subsets of genes. Specifically, these studies provide further insight regarding developmentally expressed genes that also function in the adult epididymis and, thus, may play important roles in sperm maturation. Given the vast amount of information acquired by microarray analysis, it is difficult to discuss in great detail the roles that these newly identified genes may play. Furthermore, many of the sequences are novel; thus, putative functions are not known. However, by examining groups of genes with associated biological functions, new insight is gained regarding signaling pathways and other regulatory mechanisms that may mediate region-specific functions. For example, the presence of the initial segment-enriched expression of synaptogamin-like 4, a member of a family of proteins that are important for membrane trafficking in the nerve terminal and modulate dense core vesicle exocytosis in endocrine cells [27], suggests parallel roles in the proximal epididymis. As demonstrated in PC12 cells, Slp4 negatively controls regulated secretion via specific interactions with the GDP-bound form of Rab27A [28], a small GTP-binding protein shown by our microarray studies to be expressed in the epididymis as well. It is postulated that the interactions of Slp4 with Rab27A are involved in late (postdocking) stages of granule exocytosis [28]. Also, Slp4 complexes with other Rab family members and may play a role in other aspects of cellular trafficking, such as transport of dense core vesicles from the trans-Golgi network to the cell membrane [27]. The region-specific expression of Slp4 in the epididymis implies that the mechanisms of cellular trafficking and secretion may differ between regions.
Other initial segment-enriched genes include erm (etv5), the second member of the ets family of transcription factors to show highly regionalized expression in the epididymis. Another ets family member, PEA3, exhibits highly restricted expression in the initial segment and the brain [29], is regulated by testis factors [4], and both binds and transactivates the promoters for the initial segment-expressed genes GGT [30] and glutathione peroxidase (gpx5) [31]. The recent observation suggesting that both erm and Pea3 are downstream targets of FGF8 signaling [32] supports the model that these transcription factors may be effectors of testis factor signaling, possibly via FGF8.
Also of interest is the initial segment-enriched milk fat globule factor (MFG-E8) that previously was shown to be present on the apical head of pig spermatozoa and that binds to zona pellucida glycoproteins [33, 34]. A soluble integrin-binding protein containing two Notch-like EGF domains and two discoidin domains, milk fat globule factor may mediate cell-cell interactions by binding to integrins via its RGD (Arg-Gly-Asp) motif; thus, one possible role may be during fertilization. In other studies, milk fat globule factor was shown to link apoptotic cells to phagocytes. Specifically, when secreted from activated macrophages, this factor bound to apoptotic cells by recognizing aminophospholipids and, as a result of this binding, was able to bind via its RGD motif to phagocytes [35]. These observations suggest that an alternative function for this protein may be in the removal of dead or damaged spermatozoa.
Finally, the initial segment-enriched expression of Alex3, a member of the armadillo repeat family of proteins related to the armadillo gene of Drosophila and having potential functions in tumorigenesis, embryonic development, and maintenance of tissue integrity, is intriguing from the standpoint of its possible role as a tumor suppressor in epithelial cells [36]. We have previously shown that B-Myc, also a putative tumor suppressor, is predominantly expressed in the initial segment as well as in several hormonally regulated tissues, such as the prostate, ovary, and mammary gland, and is regulated by testis factors and androgens [37, 38]. Given the anecdotal evidence that epididymal cells are difficult to transform and the rarity of cancer in the epididymis, the presence of several tumor suppressors may be, in part, the reason.
Distal epididymis The microarray studies also yielded important insight regarding new gene families with roles in the more distal epididymal regions, including the many members of the whey acidic proteins (WAPs). As shown by our microarray data and confirmed by Northern blot analysis, the elafin-like 1 (SWAM1) gene is primarily expressed in the corpus and cauda epididymis and encodes a protein with antibacterial activity [39]. Related WAP genes that are also expressed in the epididymis include secretory leukocyte protease inhibitor (SLPI) [40], HE4 [41], and eppin [42]. These genes as well as several other WAP motif proteins, such as C20orfl 70, LOC164237, and WFDC3, form a cluster on human chromosome 20, suggesting they may have evolved from the same ancestral gene [43]. Their biological functions are not known, but elafin and SLPI exhibit antibacterial and antiprotease activity and may function in host defense or the regulation of endogenous proteases [44, 45]. Similarly, our microarray studies also revealed cryptdin 17, an antimicrobial peptide of the defensin family in the distal epididymal regions. At least 17 cryptdin isoforms have been identified in the small intestine, with specific localization in the Paneth cells in small intestinal crypts [46, 47]. Recent studies suggest that in addition to antimicrobial functions, these proteins may regulate salt and water secretion [48].
The studies presented herein identify 15 287 sequences that are expressed in the epididymis, many of which exhibit region-specific patterns of expression and the majority of which have, to our knowledge, not been shown previously to be expressed in this organ. The enormity of the data precludes even mentioning most of these new sequences, but all 15 287 clones, GenBank accession numbers, average epi 1:epi 2–5 ratios, P values, clone descriptions, and other pertinent information are presented in an Excel file (as well as in the original Genepix files) that can be obtained from our Supplemental Data (http://www.ttuhsc.edu/cbb/faculty/cornwall/default.asp). From these data, other regionally expressed genes can be selected for future study. Ultimately, the examination and study of many of these genes will shed light on the intricacies of epididymal function.
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FOOTNOTES |
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2 Correspondence: Gail A. Cornwall, Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430. FAX: 806 743 2990; gail.cornwall{at}ttuhsc.edu
Received: 24 July 2003.
First decision: 14 August 2003.
Accepted: 14 October 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/granuphilin- regulates dense-core vesicle exocytosis in PC12 cells. J Biol Chem 2002 277:39673-39678/granuphilin- inhibits dense-core vesicle exocytosis through interaction with the GDP-bound form of Rab27A in PC12 cells. J Biol Chem 2003 278:15390-15396This article has been cited by other articles:
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  Z. Mou, A. R. Tapper, and P. D. Gardner The Armadillo Repeat-containing Protein, ARMCX3, Physically and Functionally Interacts with the Developmental Regulatory Factor Sox10 J. Biol. Chem., May 15, 2009; 284(20): 13629 - 13640. [Abstract] [Full Text] [PDF]
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|
 |
|
 G. A. Cornwall New insights into epididymal biology and function Hum. Reprod. Update, March 1, 2009; 15(2): 213 - 227. [Abstract] [Full Text] [PDF]
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 P Sipila, J Jalkanen, I T Huhtaniemi, and M Poutanen Novel epididymal proteins as targets for the development of post-testicular male contraception Reproduction, March 1, 2009; 137(3): 379 - 389. [Abstract] [Full Text] [PDF]
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 |
|
 V. Thimon, E. Calvo, O. Koukoui, C. Legare, and R. Sullivan Effects of Vasectomy on Gene Expression Profiling along the Human Epididymis Biol Reprod, August 1, 2008; 79(2): 262 - 273. [Abstract] [Full Text] [PDF]
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E. Dube, L. Hermo, P. T.K Chan, and D. G Cyr Alterations in Gene Expression in the Caput Epididymides of Nonobstructive Azoospermic Men Biol Reprod, February 1, 2008; 78(2): 342 - 351. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Russo, G. A. Balogh, I. H. Russo, and and the Fox Chase Cancer Center Hospital Network P Full-term Pregnancy Induces a Specific Genomic Signature in the Human Breast Cancer Epidemiol. Biomarkers Prev., January 1, 2008; 17(1): 51 - 66. [Abstract] [Full Text] [PDF]
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|
 |
|
 V. Thimon, O. Koukoui, E. Calvo, and R. Sullivan Region-specific gene expression profiling along the human epididymis Mol. Hum. Reprod., October 1, 2007; 13(10): 691 - 704. [Abstract] [Full Text] [PDF]
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E. Dube, P. T.K. Chan, L. Hermo, and D. G. Cyr Gene Expression Profiling and Its Relevance to the Blood-Epididymal Barrier in the Human Epididymis Biol Reprod, June 1, 2007; 76(6): 1034 - 1044. [Abstract] [Full Text] [PDF]
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K. Yamazaki, T. Adachi, K. Sato, Y. Yanagisawa, H. Fukata, N. Seki, C. Mori, and M. Komiyama Identification and Characterization of Novel and Unknown Mouse Epididymis-Specific Genes by Complementary DNA Microarray Technology Biol Reprod, September 1, 2006; 75(3): 462 - 468. [Abstract] [Full Text] [PDF]
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P. Sipila, D. A. Pujianto, R. Shariatmadari, J. Nikkila, M. Lehtoranta, I. T. Huhtaniemi, and M. Poutanen 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, August 1, 2006; 75(2): 240 - 251. [Abstract] [Full Text] [PDF]
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K. Zhang, R. Gao, H. Zhang, X. Cai, C. Shen, C. Wu, S. Zhao, and L. Yu Molecular Cloning and Characterization of Three Novel Lysozyme-Like Genes, Predominantly Expressed in the Male Reproductive System of Humans, Belonging to the C-Type Lysozyme/Alpha-Lactalbumin Family Biol Reprod, November 1, 2005; 73(5): 1064 - 1071. [Abstract] [Full Text] [PDF]
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D. S. Johnston, S. A. Jelinsky, H. J. Bang, P. DiCandeloro, E. Wilson, G. S. Kopf, and T. T. Turner The Mouse Epididymal Transcriptome: Transcriptional Profiling of Segmental Gene Expression in the Epididymis Biol Reprod, September 1, 2005; 73(3): 404 - 413. [Abstract] [Full Text] [PDF]
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T. B. Koo, H. Song, I. Moon, K. Han, C. Chen, K. Murphy, and H. Lim Differential expression of the PEA3 subfamily of ETS transcription factors in the mouse ovary and peri-implantation uterus Reproduction, May 1, 2005; 129(5): 651 - 657. [Abstract] [Full Text] [PDF]
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S. Yenugu, R. T. Richardson, P. Sivashanmugam, Z. Wang, M. G. O'Rand, F. S. French, and S. H. Hall Antimicrobial Activity of Human EPPIN, an Androgen-Regulated, Sperm-Bound Protein with a Whey Acidic Protein Motif Biol Reprod, November 1, 2004; 71(5): 1484 - 1490. [Abstract] [Full Text] [PDF]
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