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Research Article |
Departments of Bioenvironmental Medicine3
Functional Genomics,4
Environmental Health Science Project for Future Generations,5 Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan
Center of Environmental Health Science for Future Generations,6 Chiba 260-8670, Japan
Department of Genomic Drug Discovery Science,7 Graduate School of Pharmaceutical Sciences,
Kyoto University, Kyoto 606-8509, Japan
Core Research for Evolutional Science and Technology,8 Japan Science and Technology Agency, Kawaguchi 332-0012, Japan
Center for Environment, Health and Field Sciences,9 Chiba University, Kashiwa 277-0882, Japan
ABSTRACT
To examine epididymal function, we attempted to identify highly expressed genes in mouse epididymis using a cDNA microarray containing PCR products amplified from a mouse epididymal cDNA library. We isolated one novel and four known genes—lymphocyte cytosolic protein 1 (Lcp1), complement subcomponents C1r/C1s, Uegf protein, and bone morphogenetic protein and zona pellucida-like domains 1 (Cuzd1), transmembrane epididymal protein 1 (Teddm1), and whey acidic protein 4-disulfide core domain 16 (Wfdc16)—with unknown functions in the epididymis. The novel gene, designatedSerpina1f(serine peptidase inhibitor [SERPIN], clade A, member 1f), harbors an open reading frame of 1 233 bp encoding a putative protein of 411 amino acids, including a SERPIN domain. These five genes were predominantly expressed in the epididymis as compared to other organs. In situ hybridization analysis revealed their epididymal region-specific expression patterns. Real-time RT-PCR analysis revealed a significant increase in mRNA expression of these genes around puberty. Castration decreased their expression, except forLcp1. Testosterone (T) restored these reduced expressions, except forTeddm1; however, this restoration was not observed with 17 beta-estradiol (E2). Administration of T and E2 combination recovered theSerpina1fmRNA concentration; this recovery was also observed with T alone. However, the recovery ofCuzd1andWfdc16mRNA concentrations was inadequate. Neonatal diethylstilbestrol treatment suppressed theCuzd1,Wfdc16, andSerpina1fmRNA expression in the epididymis of 8-week-old mice; this was not observed with E2. These results suggest that our microarray system can provide a novel insight into the epididymal function on a molecular basis, and the five genes might play important roles in the epididymis.
estradiol, gene regulation, male reproductive tract, testosterone, toxicology
INTRODUCTION
The epididymis is a long, convoluted duct that links the efferent ducts to the vas deferens, and is a highly specialized tissue that functions in the maturation, transportation, and storage of spermatozoa. During their transit through the epididymis, spermatozoa undergo biochemical and morphological changes to acquire motility and the ability to fertilize an oocyte in vivo. The spermatozoa maturation process is believed to involve the interaction of spermatozoa with proteins that are synthesized and secreted by the epididymal epithelium. Thus, the luminal environment, which is composed of these epididymal proteins, plays an important role in this process. The epididymis comprises four regions—the initial segment, caput, corpus, and cauda epididymis—and has a highly region-specific gene expression pattern. The luminal environments differ between regions. However, limited information is available regarding the molecular mechanisms that form and maintain the luminal environment through the regulation of tissue- and region-specific gene expression, leading to proper spermatozoa maturation [1].
The use of microarray analysis may be a feasible method for addressing this issue, because it is a powerful and high-throughput methodology for monitoring the expression of thousands of genes simultaneously. Using the microarray methodology, several studies have reported that the expression of epididymal genes is affected by androgen, androgen withdrawal/castration, ligation of efferent ducts, and aging [2–4]. Some of these genes are important and have not been previously reported. On the other hand, most of the genes detected by the microarrays were not such genes as were specifically or predominantly expressed in the epididymis, because the microarrays used in these studies were commercially available and harbored a subset of known genes.
A number of regionally expressed genes in the epididymis have been identified, including those encoding secretory proteins with putative roles, such as proteases, protease inhibitors, antioxidant enzymes, modifying enzymes, growth factors, neuropeptides, and transporters [5, 6]. Several of these genes are unique to the epididymis, which indicates their specific roles in epididymal function [5]. Thus, the elucidation of the expression pattern of the epididymal genes, including these specific genes, would provide important information for understanding the molecular events of sperm maturation in the epididymis.
In the present study, we constructed a cDNA microarray system by using a set of 1 751 nonredundant cDNA clones derived from an oligo-capped mouse epididymal cDNA library. In the first trial, in order to investigate the epididymal function, we attempted to identify the genes that were highly expressed in the epididymis by using the microarray. In this article, we report the identification and characterization of one novel gene and four known genes—lymphocyte cytosolic protein 1 (Lcp1), complement subcomponents C1r/C1s, Uegf protein, and bone morphogenetic protein (CUB) and zona pellucida (ZP)-like domains 1 (Cuzd1), transmembrane epididymal protein 1 (Teddm1), and whey acidic protein (WAP) 4-disulfide core domain 16 (Wfdc16)—with unknown functions in the epididymis.
MATERIALS AND METHODS
Animals
The ICR strain mice used in the present study were purchased from Japan SLC, Inc. (Hamamatsu, Japan). Various organs, including the whole epididymis (initial segment, caput, corpus, and cauda regions, but not efferent ducts or vas deferens), were dissected from the mice at Postnatal Day 1 (1 day), and at 1, 2, 4, 8, and 22 weeks of age. Fetuses were removed from the pregnant mice at Embryonic Day 17.5 (E17.5).
The bilateral orchidectomy experiment was performed in 8-week-old mice. The vascular supplies to the testes were ligated without compromising the epididymal blood supply; this was accomplished via an abdominal incision. The testes were excised from the epididymides and the associated fat pads. The epididymides and fat pads were returned into the tunica, and the incision was sutured. Androgen and/or estrogen replacement was performed by subcutaneous injection of 3 mg testosterone propionate (T) and/or 17ß-estradiol (E2; Sigma-Aldrich Japan, Tokyo, Japan) dissolved in 100 µl of corn oil as a vehicle [7]. T and/or E2 were administered every day from the time of castration. The mice were killed 7 days postcastration, and the epididymides were collected. The control mice were sham operated and killed at the same time points as the animals that underwent castration.
In the experiment involving neonatal estrogen treatment, newborn male mice were each subcutaneously injected with 5 µg of a potent synthetic estrogen, diethylstilbestrol (DES; Sigma-Aldrich Japan) or an endogenous estrogen E2 dissolved in 25 µl sesame oil for the first 5 successive days of life [8]. The control mice were injected with 25 µl of the vehicle. The mice were killed at the age of 8 weeks and dissected to obtain the epididymides.
The tissues for cDNA microarray and RT-PCR analyses were frozen in liquid nitrogen and stored at –80°C until use for RNA extraction. The epididymides used for in situ hybridization analysis were fixed with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). All animal experiments were approved by the Laboratory Animal Care Committee and conducted in accordance with the Guideline for Animal Experimentation of Graduate School of Medicine, Chiba University.
Complementary DNA Microarray
An in-house cDNA microarray carrying the PCR products of 1 751 unique clones derived from an oligo-capped cDNA library of the whole epididymis (initial segment, caput, corpus, and cauda regions, but not efferent ducts or vas deferens) obtained from 8-week-old mice was constructed as described previously [9, 10]. Total RNA was extracted from the fetuses (E17.5) and the epididymides (8 weeks) by using Trizol (Invitrogen, Tokyo, Japan) in accordance with the manufacturer's instructions. Total RNA from the fetuses was pooled, and poly (A)+ RNA was purified from the mixture by using Oligotex-dT 30 (Takara Shuzo Co., Ltd., Kyoto, Japan). The purification of the epididymal poly (A)+ RNA was done by the same method. One microgram of the poly (A)+ RNA was reverse transcribed to incorporate Cy3-dUTP or Cy5-dUTP (Amersham-Pharmacia Biotech Japan, Tokyo, Japan) during cDNA synthesis by using Superscript II reverse transcriptase (Invitrogen) and oligo (dT) primer in accordance with the manufacturer's protocol. Hybridization and fluorescence detection were essentially performed as described previously [11]. An independent cDNA microarray experiment was performed once by using the following two combinations: 1) epididymis-Cy3 and fetus-Cy5; and 2) epididymis-Cy5 and fetus-Cy3. The data obtained from the scanned images of the microarray experiment were analyzed using GeneSpring 5.0 software (Silicon Genetics, Redwood City, CA). Normalization of the data was subjected to intensity-dependent LOWESS normalization. The genes whose intensities were less than the mean plus 2-fold SDs of background levels were excluded from further analyses [8]. The full-length cDNA sequencing of the cDNA clones identified by the microarray analysis was performed in the Hitachi Science Systems Corporation (Tokyo, Japan). The genes possessing a high homology to the clone sequences were determined using the basic local alignment search tool (BLAST) method (http://www.ncbi.nlm.nih.gov/BLAST.html).
Real-time RT-PCR
Total RNA was purified by DNase treatment using RNeasy (Qiagen K.K., Tokyo, Japan) in accordance with the manufacturer's instructions. The cDNA templates for real-time RT-PCR analysis were synthesized from the total RNA by the method described above, using dTTP instead of Cy3-dUTP or Cy5-dUTP. Real-time RT-PCR analysis was performed in a DNA Engine Opticon (MJ Research Inc.) by using SYBR Green PCR Master Mix Reagent (Applied Biosystems Japan, Tokyo, Japan) as a detector, in accordance with the manufacturer's instructions. The fold change was calculated based on differences of the threshold cycle (Ct) value [6, 8]. The internal control used was ß-actin [6, 8]. The primer pairs used in the real-time RT-PCR analysis are listed in Supplemental Table 1 (available online at http://www.biolreprod.org/).
Reverse Transcription and Polymerase Chain Reaction
The organs used were the brain, heart, lung, liver, kidney, spleen, intestine, pancreas, testis, skin, muscle, ovary, nonpregnant uterus, and the whole epididymis; these organs were obtained from intact mice at the age of 8 weeks. The cDNA templates for the RT-PCR analysis were synthesized from the genomic DNA-free total RNA by using the method described above. The aliquots were amplified with 1 U of AmpliTaq Gold (Applied Biosystems Japan) in a final volume of 25 µl containing 1x PCR buffer II, 2.5 mM MgCl2, 0.2 mM dNTP, and 0.5 µM of the specific primer. The reaction was initiated at 95°C for 10 min, followed by 25 cycles consisting of denaturation at 95°C for 15 sec, annealing of primers to the target sequence, and primer extension at 57.2°C–64°C for 1 min. The PCR products were resolved on 2% agarose gels containing 0.1 µg/ml ethidium bromide. The RT-PCR analysis was performed in a GeneAmp PCR System 9700 (Applied Biosystems Japan). The primer pairs used in this analysis were the same as those used in the real-time RT-PCR analysis (Supplemental Table 1, available online at http://www.biolreprod.org/).
In Situ Hybridization
The fixed epididymides were dehydrated and embedded in Tissue-Tek OCT compound (Sakuraya Finetechnical Co., Ltd., Tokyo, Japan). Using a cryostat, 7-µm-thick sections were cut and mounted onto slides coated with 3-aminopropyltriethoxy-silane (Matsunami Glass Inc., Ltd., Osaka, Japan). The PCR was performed using specific primer pairs for each gene, and the PCR products were subcloned into a pGEM-T Easy vector (Promega, Tokyo, Japan). To synthesize antisense and sense probes for the in situ hybridization analysis, the cDNA was transcribed in vitro by using T7- and SP6-RNA polymerase (Roche Diagnostics Co., Mannheim, Germany), respectively, and digoxygenin-11-UTP (Roche Diagnostics Co.) was incorporated in accordance with the manufacturer's protocol. The hybridization and coloring reaction were performed as described previously [12], and the sections were counterstained with methyl green for detailed observation of the mRNA signals detected in the stromal tissue of the epididymis. The slides were examined using an Olympus BX51 light microscope (Olympus Optical Co., Ltd., Tokyo, Japan). The primer pairs used for the in situ hybridization analysis are listed in Supplemental Table 1 (available online at http://www.biolreprod.org/).
Statistical Analysis
Statistical analysis among the groups was carried out by the Student t-test, or the Mann-Whitney U-test when the distribution was nonnormal [8]. In gene expression analysis of postnatal development and of castrated mice with/without steroid hormones, significance was determined by one-way ANOVA followed by the Scheffé test.
RESULTS
Identification of Epididymal Genes in Microarray and Sequencing Analyses of the Detected Genes
A cDNA microarray system was constructed using a set of 1 751 cDNA clones derived from an oligo-capped mouse epididymal cDNA library. Next, using the microarray, we carried out the screening of the genes that showed 4-fold higher expression levels in the epididymis at the age of 8 weeks than in the fetus at E17.5 [10] and identified 9 epididymal genes (Supplemental Table 2, available online at http://www.biolreprod.org/). To confirm the microarray data, the expression levels of these genes were investigated by using real-time RT-PCR. The expression levels of these genes in the epididymis were significantly higher than those in the fetus (Supplemental Table 2, available online at http://www.biolreprod.org/). Of the 9 genes, glutathione peroxidase 3 (Gpx3), Gpx5, cysteine-rich secretory protein 1 (Crisp1), and RIKEN cDNA 9230104L09 gene (9230104L09Rik) have been previously reported in the epididymis [7, 13–15], whereas the other 5 genes (Lcp1, Cuzd1, Teddm1, Wdfc16, and 1 novel gene) have not been reported thus far.
Subsequently, the full-length cDNA sequence of the novel gene was analyzed. The novel gene harbors an open reading frame that is 1 233 bp in length. Conceptual translation of the cDNA shows a protein composed of 411 amino acids. Using the GenBank protein data base as a reference, the gene was identified as a novel member of the serine peptidase inhibitor (SERPIN) family. The novel gene was named Serpina1f (SERPIN, clade A, member 1f). The nucleotide sequence and the predicted amino acid sequence of Serpina1f have been submitted to the GenBank with accession number AB233454.
Gene Expression Levels in the Epididymis
To examine the organ distribution of the expression of these 5 genes (Lcp1, Cuzd1, Teddm1, Wfdc16, and Serpina1f), expression levels in 14 different organs were analyzed by RT-PCR (Fig. 1A). The Lcp1 gene was expressed at high levels in the lung, intestine, and epididymis; at middle levels in the liver, pancreas, and skin; and at low levels in the heart, muscle, ovary, and nonpregnant uterus. The Cuzd1 gene was expressed in the pancreas and epididymis. The transcripts of the Teddm1, Wfdc16, and Serpina1f genes were predominantly expressed in the epididymis. Thus, these five genes were strongly or predominantly expressed in the epididymis.
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Furthermore, we investigated the expression levels of these five genes in the epididymis during the postnatal period (at Day 1 and/or 1, 2, 4, 8, and 22 weeks) by real-time RT-PCR. Transcripts of all these genes were present from Day 1 or Weeks 1–22, but the signal varied during epididymal development (Fig. 1B). Lcp1, Cuzd1, Teddm1, and Serpina1f genes displayed similar developmental expression patterns, whereas Wfdc16 showed a different pattern. The expressions of Lcp1, Cuzd1, Teddm1, and Serpina1f genes were gradually upregulated from Day 1 or Week 1 to Week 2; their expression significantly increased from Week 2 to Week 4, and remained high until Week 22 (Fig. 1B). The expression level of the Wfdc16 gene increased gradually from Week 1 to Week 2, and showed a significant upregulation from Week 2 to Week 4; the upregulation was steadily maintained up to Week 22 (Fig. 1B).
Localization of Gene Expression in the Epididymis
In situ hybridization was performed to localize the mRNAs of these 5 genes in the epididymis at the age of 8 weeks. The Lcp1 and Wfdc16 genes were uniformly expressed throughout the stromal tissue of the epididymis (Fig. 2, A and F). The signals of the Cuzd1 mRNA were observed in the epithelial cells of all the regions (Fig. 2, B–D, J, and M) and at high levels, particularly in the corpus epididymis (Fig. 2, C, J, and M). The Teddm1 mRNA signals were detected only in the epithelial cells of the initial segment of the epididymis (Fig. 2, E, K, and N). The expression of Serpina1f mRNA was present in the epithelial cells of the caput, corpus, and cauda epididymis (Fig. 2, G–I, L, and O); mRNAs were abundant in the caput and cauda epididymis (Fig. 2, G, I, L, and O).
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Effects of Castration, with and Without Steroid Hormones, on Gene Expression Levels in the Epididymis
The effects of castration on the expression levels of the five genes in the epididymis were studied by real-time RT-PCR. The expression levels of the Cuzd1, Teddm1, Wfdc16, and Serpina1f genes were lower in the castrated mice than in the sham-operated mice, whereas the expression level of the Lcp1 genes was similar between the 2 groups (Fig. 3A). Next, to determine whether the expression of Cuzd1, Teddm1, Wfdc16, and Serpina1f is regulated by steroid hormones, we administered T, E2, or their combination (T + E2) to the castrated mice. Administration of T alone restored Cuzd1, Wfdc16, and Serpina1f mRNA levels to similar levels as those observed in the sham-operated mice, whereas the administration of E2 alone did not (Fig. 3B). In the castrated mice administered T + E2, Serpina1f mRNA level was approximately consistent with those observed in the sham-operated mice and in the castrated mice administered T alone (Fig. 3B). Cuzd1 mRNA level was not significantly different from that in the other groups; however, it tended to be higher than that in the castrated mice administered E2 and lower than those in the sham-operated mice and castrated mice administered T alone (Fig. 3B). The accumulation of Wfdc16 mRNA was detected; however, the expression level was lower than that in the sham-operated mice (Fig. 3B). The expression level of Teddm1 gene in the castrated mice did not recover by administration of any of the hormones studied here (Fig. 3B).
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Effects of Neonatal Estrogen Treatment on Gene Expression Levels in the Epididymis
We examined the effects of neonatal DES or E2 treatment on the expression levels of the 5 genes at the age of 8 weeks by real-time RT-PCR. In the mice that were neonatally treated with DES, the expression levels of Cuzd1, Wfdc16, and Serpina1f genes were significantly lower than those in the control mice, whereas the expression levels of Lcp1 and Teddm1 genes were similar to those in the control mice (Supplemental Table 3, available online at http://www.biolreprod.org/). In mice that were neonatally treated with E2, the expression levels of all these genes were identical to those in the control animals (Supplemental Table 3, available online at http://www.biolreprod.org/).
DISCUSSION
In the present study, we detected nine genes with higher expression levels in the epididymis by using our in-house cDNA microarray, and the data were confirmed by real-time RT-PCR (Supplemental Table 2, available online at http://www.biolreprod.org/). It has been reported that the Gpx3, Gpx5, Crisp1, and 9230104L09Rik genes are predominantly expressed and play an important role in the epididymis [7, 13–15]. However, thus far, the characteristics of the other five genes (four known genes, Lcp1, Cuzd1, Teddm1, Wfdc16, and one novel gene, Serpina1f) have not been reported with respect to the epididymis. Thus, this is the first study that shows their predominant expression in the epididymis (Fig. 1A). Identification of the genes that are expressed specifically/predominantly in the epididymis, which indicates their specific functions in the epididymis, is crucial to understanding the molecular basis of sperm maturation [5]. These results suggest that our microarrays might be a useful tool for providing an insight into the molecular mechanisms of sperm maturation in the epididymis.
Next, we investigated the characteristics of the expression of the Lcp1, Cuzd1, Teddm1, Wfdc16, and Serpina1f genes in the epididymis. A significant increase in the mRNA expressions of these five genes was observed around puberty (Fig. 1B) when the plasma testosterone concentration increases [16]. Castration results in the removal of circulating androgens and estrogens emerging from the testis, as well as the cessation of luminal fluid coming from the testis. T and/or E can replace only the circulating component, and will influence only the functions dependent upon the circulating steroids. Thus, expression of the Cuzd1, Wfdc16, and Serpina1f genes declines after castration, but is restored after administering T alone or T + E2; however, administering E2 alone did not restore the gene expressions (Fig. 3), indicating their androgen-dependent expression. The inadequate recovery of Cuzd1 and Wfdc16 mRNA concentrations following T + E2 administration raises the possibility that E2 has an opposite effect to that of T [7]. However, as Teddm1 gene is expressed in the initial segment, which is maintained by the testicular fluid but not the androgen [17, 18], it is not surprising that its expression declines after castration and is not restored by androgen (Fig. 3). In this regard, it may be worthwhile to study the effects of ligation of the efferent ducts on Teddm1 gene expression, because androgens were maintained but luminal fluid was removed by this operation.
Based on the amino acid sequence, SERPINA1F was considered as a novel member of the SERPIN family with a putative function as a serine protease inhibitor. Members of the SERPIN family are expressed in many tissues, and they control many physiological processes, such as blood coagulation, inflammation, tumor cell metastasis, apoptosis, neurite extension, etc. In the male reproductive tissues, including the epididymis, several genes with the SERPIN domain have been shown to play special roles in sperm maturation and the fertility process [5, 14, 19–22]. Therefore, SERPINA1F might play an important role in epididymal function.
WFDC16 has one WAP domain that functions as a four-disulfide core protease-inhibitor. In mice, the Wfdc16 gene is mapped in chromosome 2 where genes with the WAP domain form a cluster [23]. Many of the known Wap genes in this cluster yield high transcript levels in the epididymis and testis [24]. One of them, an epididymal protease inhibitor, is dominantly expressed in the epididymis; it is bound to the ejaculated spermatozoa, and exhibits antimicrobial activities in humans and mice [23, 25, 26]. It has been reported that other WAP domain proteins in this cluster, such as secretory leukoprotease inhibitor and elastase-specific inhibitor, possess antimicrobial activity and are expressed in the male reproductive organs, including the epididymis [2, 27, 28]. When collected, WFDC16 might possess resistance against invading microorganisms and regulate the inhibition of a wide spectrum of microbial proteolytic enzymes in the epididymis.
CUZD1 (annotated as UO-44 in rats and humans; GenBank accession nos. AF022147 and AF305835, respectively) is composed of two domains: the CUB domain and the ZP domain [29]. The CUB domain is found in the developmentally regulated proteins that participate in embryogenesis and organogenesis. CUZD1 has been reported to play a role in the proliferation and differentiation of the female reproductive epithelial cells [30]. Furthermore, this protein has been shown to be involved in the interaction and signaling with extracellular components in uterine and ovarian cancer cells in vitro [31]. In reproductive tissues, including the epididymis, stromal-epithelial interactions play an important role in normal development [32]. The ZP domain proteins are involved in sperm-egg recognition, and are found in sperm receptors ZP2 and ZP3 [33]. Imamura et al. have proposed that CUZD1 may be involved in the activation of trypsinogen within the zymogen granules and contribute to zymogen assembly or stability [34]. Therefore, CUZD1 might play specific roles in the development, growth, and differentiation of the epididymal epithelium via interactions with the extracellular matrix and in the mediation of protein secretion, leading to the formation and maintenance of a proper luminal environment for sperm maturation.
The TEDDM1 protein contains one DUF716 domain. However, a well-defined function has not yet been assigned to this domain. The expression of this gene was restricted to the epithelium of the initial segment of the epididymis (Fig. 2, E, K, and N), which is essential for the sperm maturation process [35]. Polyomavirus enhancer activator 3 (PEA3) transcription factor is predominantly expressed in the initial segment, and its consensus sites (AGGAAA/G) are commonly found in the promoters of the genes expressed in this region [36, 37]. In fact, eight consensus sites are present in the promoter region of the Teddm1 gene. Lan et al. have hypothesized a unique signal pathway in the initial segment involving PEA3 [36]. Thus, TEDDM1 might be associated with the initial segment-specific signaling pathway that is involved in early sperm maturation events.
LCP1 (annotated as l-plastin; GenBank accession no. AK088202) is a member of the actin-binding protein family, and is specifically expressed in hematopoietic cells and cancer cells but not in nonhematopoietic cells [38]. It is notable that Lcp1 gene expression was found in the normal epididymis (Fig. 1A), thereby implying that the protein encoded by this gene plays a specific role in epididymal function. The epithelium and interstitium of the epididymis contain many lymphocytes [39]. It is believed that the halo cells involved in epididymal immune function are lymphocytes that have infiltrated the epithelium from the stromal tissue [40]. The interstitium, where the Lcp1 gene was expressed (Fig. 2A), has been thought to function as a reservoir of lymphocytes for the epithelial compartment [39]. The halo cells become visible around puberty [40]; Lcp1 gene expression also increased around that same time (Fig. 1B). Furthermore, the protein encoded by this gene is proposed to be involved in the control of cell adhesion and motility [41]. Based on our data and these findings, it can be concluded that lymphocytes are the sites of Lcp1 gene expression within the interstitium. It is possible that LCP1 is associated with the migration of lymphocytes into the epithelium. To demonstrate this association, more data on the relationship of the timing between emergence of the halo cells and the increase in LCP1 expression around puberty are required.
Next, we examined the effects of neonatal DES or E2 treatment on the expression levels of the 5 genes in the epididymis at the age of 8 weeks. DES suppressed Cuzd1, Wfdc16, and Serpina1f gene expression levels; however, it did not suppress the Lcp1 and Teddm1 gene expression levels (Supplemental Table 3, available online at http://www.biolreprod.org/). Recently, it has been reported that human fetuses and neonates are exposed to various exogenous chemicals, including the endocrine disruptor chemicals that possess the capacity to mimic estrogen action. In utero or neonatal exposure to exogenous estrogen compounds could result in adverse effects on the reproductive system, including the epididymis in humans, rodents, and other species [6, 8, 42–45]. Altogether, the Cuzd1, Wfdc16, and Serpina1f genes might be associated with the epididymal dysfunctions following neonatal exposure to exogenous estrogen compounds, such as DES. In contrast to these results obtained with DES, E2 did not show any effects (Supplemental Table 3, available online at http://www.biolreprod.org/). We have previously reported that DES might have a specific gene regulatory mechanism in the epididymis [8]. Thus, these genes would help us in understanding the epididymal dysfunction induced by neonatal exposure to exogenous estrogen compounds. These genes could be useful as biological indicators of dysfunction and/or signs that are otherwise overlooked; study of these genes could lead to the development of biomarkers that can predict and diagnose the adverse effects of exogenous estrogen compounds.
In conclusion, using our new microarray system, we detected one novel gene (Serpina1f, a novel member of the SERPIN family) and four known genes (Lcp1, Cuzd1, Teddm1, and Wfdc16) with high expression levels in mouse epididymis. These genes showed specific expression patterns with regard to development, localization, and regulation in the epididymis. Neonatal DES treatment suppressed the expression of Cuzd1, Wfdc16, and Serpina1f genes; however, E2 did not have this effect. Our microarray system can provide a novel insight into the epididymal function on a molecular basis. These five genes might play important roles in the epididymis. The Cuzd1, Wfdc16, and Serpina1f genes may help us to understand not only normal epididymal functions, but also the dysfunctions induced by neonatal exposure to exogenous estrogen compounds, such as DES. Furthermore, these three genes could contribute to the development of a new method for evaluating the effects of exposure to exogenous estrogen compounds.
ACKNOWLEDGMENTS
We thank Dr. Yushin Ono from Chiba University for his technical assistance in the construction and analysis of the cDNA microarray.
FOOTNOTES
1 Supported by Ministry of Education, Culture, Sports, Science and Technology of Japan. The nucleotide and conceptual amino acid sequence of serine peptidase inhibitor, clade A, member 1f (SERPINA1F) has been deposited in GenBank under the GenBank accession number AB233454. 
2 Correspondence: FAX: 81 43 226 2018; mkomi{at}faculty.chiba-u.jp
Received: 29 September 2005.
First decision: 1 November 2005.
Accepted: 16 May 2006.
REFERENCES
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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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5 mice). *P < 0.01 vs. Day 1 and/or 1 week and 2 weeks, 
P < 0.05 vs. 4 weeks
