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Male Reproductive Tract |
Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman, Washington 99164
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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androgen, epididymis, microarray
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-dihydrotestosterone (DHT) [4], secretion and resorption of many different molecules and proteins [2], and storage of spermatozoa [5]. The epididymis is made up of four different types of cells: the principal cells, basal cells, clear cells, and narrow cells. Principal cells are located throughout the epididymis and are the main secretory cells [3]. The principal cells in the caput, corpus, and cauda regions of the epididymis secrete different profiles of proteins that are thought to be important for maturation of spermatozoa [3]. Principal cells are also the main cell type in the epididymis that decrease secretion as androgen levels drop [6–8]. Immunohistochemistry reveals that a high level of androgen receptor is present in all three regions of the epididymis [9].
Testosterone is transported from the testis to the epididymis by androgen binding protein (ABP) [10–12] and is converted to DHT by 5-reductase [4]. DHT binds to androgen receptor (AR), a member of the steroid receptor superfamily, and interacts with the androgen response element in the promoters of genes regulated by androgens. The binding of DHT to the receptor aids in inducing or reducing transcription of the androgen-responsive genes.
Bilateral castration removes all circulating androgens as well as testicular factors that can also influence transcription. In 1926, Benoit reported that, after castration, the epididymis regressed and it was concluded that this regression occurred due to the loss of some unknown testicular substance or factor [8], which turned out to be testosterone. This regression is due to the absence of spermatozoa and the reduced amount of luminal secretions [6]. Many different genes have been identified as being androgen responsive in the epididymis. Some of these include the caput-specific gene glutathione peroxidase 5 (Gpx5) [13] and the corpus-specific genes carbonic anhydrase 2 (Car2) and carbonic anhydrase 4 (Car4) [14]. Other transcripts are regulated in the epididymis by both androgen and testicular factors. Expression of these genes is reduced after castration and, upon androgen replacement, their expression does not return to the original level. One example of this type of regulation is observed for the 
-glutamyl transpeptidase gene [15].
There is a high level of androgen receptor in the murine epididymis and, because of the importance of the organ in regulation of sperm maturation, identification of the transcripts that are regulated by androgens after a 48-h treatment may be important to help elucidate sperm maturation. Castrated mice, DHT supplementation, and a genomic approach to investigate which transcripts in the epididymis are androgen regulated are used in this study. The genomic approach utilizes the Affymetrix MGU74Av2 GeneChip oligonucleotide microarrays (Affymetrix, Santa Clara, CA), which contained probe sets for over 12 000 different transcripts. Transcripts were identified that were both upregulated and downregulated by androgens in the epididymis. Also identified are androgen-regulated transcripts that are expressed in other tissues but have an enriched expression profile in the caput, corpus, or cauda.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult B6129 male mice (4–6 mo old) were housed in an animal care accredited facility and had access to food and water ad libitum in accordance with the rules of the American Association for Accreditation of Laboratory Animal Care. All animal protocols were approved by the Washington State University Animal Care and Use Committee and were in accordance with the National Research Council standards established by the Guide for Care and Use of Laboratory Animals.
Mice were separated into three treatment groups: wild type (no castration), castrated + oil, and castrated + DHT. Bilateral castrations were done through the abdominal route. Anesthesia was performed with an i.p. injection of ketamine (100.0 mg/kg) and xylazine (10.0 mg/kg). In castrated mice, care was taken to leave the intact epididymis in the scrotal area. The efferent ducts and the testicular arteries and veins were ligated and the testes removed above the ligation point. After 7 days of recovery, all castrated mice were divided into two groups and treated. Control-castrated mice were treated with a 100 µl injection of 90% sesame oil and 10% ethanol (v/v). Treated-castrated mice were injected with 5 mg of DHT (Sigma-Aldrich, St. Louis, MO) dissolved in 90% sesame oil and 10% ethanol (v/v) once every 24 h, for a total treatment of 10 mg DHT over a 48-h period. All mice were killed 48 h postinjection by CO2 asphyxiation, and the epididymides were removed and divided into three sections (caput, corpus, and cauda) for RNA extraction and purification. Each treatment group contained multiple castrated mice (two for control group 1, three for control group 2, four for treatment group 1, five for treatment group 2) and all experiments were repeated two independent times.
RNA Extraction
Due to the small amount of tissue in the regressed epididymides, samples were pooled to have enough RNA for the Affymetrix chips and subsequent follow-up analysis with real-time PCR and Northern blots. RNA was isolated from the caput, corpus, and cauda by using the TRIzol method according to manufacturer's instructions (Invitrogen, San Diego, CA). RNA concentration and purity were obtained by spectrophotometry. RNA used for microarray analysis had a A260/A280 ratio greater than 1.85. Ten micrograms of total RNA were resolved on a 1.2% denaturing agarose gel to check for RNA integrity.
Affymetrix GeneChip Target Preparation
Ten micrograms of total RNA isolated from the caput, corpus, or cauda from mice that were castrated and treated with DHT, castrated and treated with oil, or untreated were collected in two independent experiments. These were used to synthesize biotinylated cRNA target, which was used for the MGU74Av2 GeneChip. RNA was reverse transcribed using Superscript II (Invitrogen) reverse transcriptase into double-stranded cDNA using oligo dT primers that contained a T7 promoter. The cDNA was extracted with phenol-chloroform and a phaseLock gel (Eppendorf, Hamburg, Germany) and precipitated with ethanol and ammonium acetate. Biotinylated cRNA was synthesized using the cDNA as a template in an in vitro transcription reaction using the MEGAscript high-yield transcription kit (Ambion, Austin, TX) as described by the manufacturer with the following modification: biotinylated CTP and UTP were added to the reaction mix. The resulting biotinylated target cRNA was purified using Rneasy columns (Qiagen, Valencia, CA), alcohol precipitated, and quantified using a spectrophotometer. The fragmented biotin-labeled cRNA (15 µg) was spiked with Eukaryotic Hybridization control and hybridized to the MGU74Av2 microarray overnight at 45°C. After the hybridization, the array was washed, stained with phycoerythrin-coupled streptavidin, and processed using the Affymetrix GeneChip Fluidics Workstation 400 as described for the Mini Euk 2v3 protocol. Microarrays were then scanned using a Hewlett-Packard Gene Array Scanner (Hewlett-Packard Co., Palo Alto, CA).
Microarray Analysis Using GeneSpring
Expression analysis of all replicate microarray experiments was performed with GeneSpring 5.1 (Silicon Genetics, Redwood City, CA) using the absolute analysis data generated by Microarray Suite version 5.0 software (MAS 5.0; Affymetrix). All experiments were scaled to a signal value of 125. Initially, genes that were expressed in the caput, corpus, and cauda regions of the epididymis and had a raw signal of greater than 150 in both replicate experiments were targeted for further analyses. These genes were further sorted to identify specific genes that were regulated by DHT when comparing the castrated + oil groups versus the castrated + DHT groups. Differential expression was defined as those transcripts that had a difference of 2-fold or greater with a raw signal value of greater than 150 in both replicates when comparing the DHT versus the control samples. Genes were considered enriched in a specific region of the epididymis if the expression was 2-fold or higher in that region when compared with the other regions. Statistical analyses were performed using GeneSpring software by using the Cross-gene-error-model in combination with a one-way ANOVA (significance level set at P < 0.05)
Real-Time PCR
Real-time PCR was used to confirm differences in transcription that were observed in the microarray analysis. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to generate cDNA from the total RNA isolated from the caput, corpus, and cauda regions of the epididymis from the wild-type, castrated + oil, and castrated + DHT animals. Total RNA (1 µg) was reverse transcribed into cDNA by an oligo(dT)12-18 primer using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's instructions and used as the template for the real-time PCR. Primer Express 2.0 software (Applied Biosystems, Foster City, CA) was used to generate specific primers for genes that were investigated (Table 1). Real-time PCR was performed in a 96-well plate using a 7000 ABI prism sequence detection system (Applied Biosystems). The template cDNA for each specific region of the epididymis was used in triplicate for each real-time PCR. The PCR contained between 5 and 10 ng of template cDNA, 1x SYBR green master mix (Applied Biosystems), and 600 nM of specific forward and reverse primers. The threshold cycle (CT), which indicates the relative abundance of a particular transcript, was calculated for each reaction by the 7000 ABI prism sequence detection system. Primers for the housekeeping gene, ribosomal protein S2, were used to normalize CT values for each sample. The difference in expression between the castrated + oil versus castrated + DHT was calculated using the formula 2-
CT as described in the SyBR green protocol,
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Northern Blots
Ten micrograms of total RNA were resolved on a 1.2% denaturing agarose gel, transferred onto a Nylon membrane (Hybond-N; Amersham, Piscataway, NJ), and cross-linked to the membrane by ultraviolet irradia tion. Membranes were prehybridized for 1 h at 65°C in Church and Gilbert solution (0.5 M sodium phosphate, 7% SDS, 1 mM EDTA, 1.0% BSA). The cDNA probes were radiolabeled with 50 µCi of [-32P] deoxyaden osine triphosphate at 37°C for 1 h using a random primer-labeling kit (Invitrogen). The reaction was terminated by adding 50 µl of 0.5 M EDTA, and free nucleotides were removed from the reaction with a Se phadex G50 spin column. Blots were hybridized with the labeled probe overnight at 65°C in Church and Gilbert solution. After hybridization, blots were washed one time in 2x SSC, 0.1% SDS at 26°C for 30 min, two times in 0.2x SSC, 0.1%SDS at 65°C for 30 min, and then exposed to a phosphor-imaging screen (Molecular Dynamics, Amersham Biosci ences, Little Chalfont, UK) overnight. Membranes were stripped with boil ing 1% SDS and each blot was hybridized with ribosomal S2 probe to ensure equal loading of RNA [16]. Levels of mRNA for each specific probe are expressed as a ratio of (specific probe)/ribosomal protein S2 using ImageQuant analysis software version 1.1 (Molecular Dynamics).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Absolute analysis using MAS 5.0 revealed that 2547 genes (20.5% of the total transcripts represented on the mi croarray) were present in the caput, 2525 genes (20.3%) were present in the corpus, and 2626 genes (21.2%) were present in the cauda. Genes that had a raw signal of greater than 150 in only one specific region were considered en riched in that particular segment of the epididymis. One hundred sixty-two genes were enriched in the caput, 55 genes were enriched in the corpus, and 133 genes were enriched in the cauda.
Confirmation of the Methodology to Identify Androgen-Regulated Genes in the Epididymis
The epididymal gene expression microarray data were filtered using GeneSpring 5.0 software to produce a list of genes that were regulated by androgens and enriched in each specific region: the caput, corpus, and cauda epidid ymis. Genes that were considered upregulated in the epi didymis by androgen met two criteria. First, the raw Af fymetrix signal had to be greater than 150 in both samples in the treated group, and second, the fold-change difference had to be two or greater versus the control, treated animals. A total of 24 genes and 5 expressed sequence tags (ESTs) met these criteria and their expression patterns shown in Figure 1. Both epididymal retinoic acid-binding protein and a disintegrin and metalloprotease 7 have been previously reported to be regulated by androgens in the epididymis [17, 18]. Northern blot analyses were performed on these two genes, Erabp and Adam7, to confirm that the castration and subsequent treatment with DHT was a valid model sys tem to study androgen regulation of genes in the epididymis of mice. The Erabp and Adam7 transcripts were upregu lated in all three regions of the epididymis in castrated mice treated with androgen (Fig. 2). Other genes previously shown to be regulated by androgens, including thiopurine methyltransferase (Tpmt) [19] and glutathione S-transferase mu (Gstm2) [20], were also present in the results (Fig. 1).
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Glutathione peroxidase 5 (Gpx5), a gene previously shown to be expressed primarily in the caput region [13], was also found to be highly expressed in the caput epididymis by microarray analysis (Fig. 3). This expression pattern was also confirmed using Northern blot analysis using the same RNA samples that were used for the microarray analysis. In the Northern blot, the message for Gpx5 was upregulated 2.2-fold, which agrees with the 2.1-fold increase observed in the array data (Fig. 4A). Car2, a gene that is expressed exclusively in the corpus, was also confirmed with Northern blot analysis using the same RNA that was used for target preparation for the microarray analysis (7.4-fold in the array data and 1.7-fold in the Northern blot data). This data demonstrates that the methodology used to look at regionally enriched genes with the Affymetrix oligonucleotide microarray GeneChip platform is valid.
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Regulation of Genes Upregulated by Castration
Castration resulted in a set of transcripts that had increased expression in all regions of the epididymis. This increase in steady-state mRNA levels could be repressed when the mice were treated with DHT. A list of these androgen-repressed genes is shown in Figure 7. To confirm that the repression actually occurred in the DHT-treated mice (Fig. 8), Northern blot analysis was performed on insulin growth factor binding protein 5 (Igfbp5) and secreted phosphoprotein-1 (Spp-1). The Northern blot analysis showed an upregulation of Igfbp5 in the caput of 2.5-fold, in the corpus of 1.4-fold, and in the cauda of 1.8-fold in the castrated mice. Real-time PCR was also used to verify the microarray results on the gene Spp-1. In the array data, Spp-1 was downregulated 9.5-fold in the caput, 1.4-fold in the corpus, and 11.6-fold in the cauda. Combining RNA from all three regions and using it as a template for real-time PCR, a 2.8-fold (±0.2) decrease in Spp-1 expression was observed, confirming the pattern observed in the micorarray data.
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Androgen Regulation of Genes in the Caput, Corpus, Cauda Epididymis
The main goal was to identify androgen-regulated genes that were enriched in the caput, corpus, or cauda. Using the lists created in GeneSpring, a set of 25 known genes and 8 ESTs that were regulated 2-fold or more in DHT-treated, castrated mice versus control, castrated mice was identified in the caput. Relative levels of transcripts for these genes are shown in Figure 3. Six genes from this list were previously reported to be regulated by androgens. These include mannosidase 1, alpha (Man1a) [21], angiotensin converting enzyme (Ace) [22], glutamine synthetase (Glul) [20], myo-inositol synthase A1 (Isyna1), aflatoxin B1 aldehyde reductase (Afar) [23], and Gpx5 [13]. Using real-time PCR, upregulation of several transcripts was confirmed. One of the transcripts confirmed was an EST (AI592789), which corresponds to amino acid transporter N2 (Aatn2) (26.2-fold ± 7.8; Fig. 4C). This gene was selected because it may play a roll in maintaining the environment of the caput epididymis. Another gene selected was alkaline phosphatase 2 (Akp2), which, according to the microarray experiment, was upregulated the greatest by androgen treatment (33.4-fold ± 2.5; Fig. 4D). Northern blot analysis confirmed that the previously reported androgen-regulated gene Gpx5 was upregulated in a similar manner when compared with the microarray data (Fig. 4A).
Six known genes and two ESTs were androgen regulated and enriched in the corpus epididymis (Fig. 5). Two of these genes, Car2 and Car4, were previously reported to be corpus specific and regulated by androgen. Three other genes from the corpus epididymis-enriched data set were also confirmed by real-time PCR. An EST corresponding to Glycine dehydrogenase (Gldc) was the transcript that was upregulated the greatest, according to the microarray data, and this was confirmed with real-time PCR (14.1-fold ± 1.2; Fig. 6C). Myomesin 2 (Myom2) was also selected for confirmation because it is novel in the fact that no androgen-regulated muscle protein has ever been reported in the epididymis and it was upregulated as well (7.9-fold ± 1.4; Fig. 6D). Protein tyrosine phosphatase receptor type 
(Ptpro), which encodes for PTP phi, a protein that is thought to modulate the focal adhesion protein paxillin, was also upregulated (16.1-fold ± 2.5; Fig. 6B). All three of these genes were upregulated and graphical representation of the real-time PCR data can be visualized in Figure 6.
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In the cauda region of the epididymis, nine genes that were enriched and regulated 2-fold or more by androgen were identified (Fig. 9). Serpine-2 and prostaglandin-endoperoxide synthase 2 (Ptgs2) were previously reported to be regulated by androgens [24, 25], and both transcripts were upregulated in the enriched-cauda set of genes. Serpine-2, a gene that plays a role in male reproductive development [26], was confirmed to be upregulated by real-time PCR using the same RNA that was used for microarray analysis (Fig. 10). According to the real-time PCR, a 20.6-fold (±5.5) upregulation of Serpine-2 was observed.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Car2 and Car4, which are corpus specific and androgen regulated, and the secretory protein, glutathione peroxidase 5, which is caput specific and androgen responsive, all exhibited the previously reported expression patterns in the microarray analysis [13, 14]. Analyses of the data indicate that, after castration, there is an overall decrease in transcription when compared with untreated mice. These results were not surprising because, recently, it was reported an overall decrease in gene expression in the epididymis occurs following castration in rats [20]. After the 48-h treatment with DHT, the majority of transcripts were upregulated in response to androgen treatment, with only a few downregulated in the treated animals. This indicates that androgen regulation in the epididymis has some repressive activities in addition to the well-documented ability of androgens to upregulate gene expression.
Within the three subregions of the epididymis, the greatest number of androgen-regulated genes were observed in the caput region when compared with regulation in either the corpus or cauda regions. This suggests that more androgen-responsive genes are active in the caput. The caput has higher levels of protein synthesis than the rest of the epididymis [30], and it follows that the high amount of protein synthesis may be linked to androgen-activated gene expression. The different regions of the epididymis have different functions that are thought to be important for sperm maturation. Each region is morphologically different and the luminal environment in each region is vastly different as well [31, 32]. The microenvironment of the caput is different than other regions of the epididymis and data presented from this experiment may identify genes that are important for keeping that environment unique. One of the transcripts identified as being androgen regulated and caput enriched was an EST (AI592789) that has 96% homology to the rat amino acid transporter SN2. AATN2 is a Na+-dependent transporter of neutral amino acids, such as alanine, serine, and glycine [33]. The concentration of many amino acids varies in the different regions of the epididymis [34] and the AATN2 protein may be a crucial regulator of this phenomenon.
The caput epididymis contains a high amount of the osmolyte myo-inositol, which is known to be important for maintaining the osmolarity in both the kidney [35] and the testis [36]. Previously, it was demonstrated that castrated rats had a decrease in the amount of myo-inositol present in the epididymal luminal fluid [37]. This decrease in myo-inositol could be reversed by treatment with androgen [37]. The authors postulated that myo-inositol 1-phosphate synthase A1 (Isyna1) was under the control of androgens. ISYNA1 is an isomerase that converts glucose-6-phosphate to myo-inositol-1-phosphate. In this microarray experiment, Isyna1 is regulated by androgen in the caput epididymis. This not only confirms the original hypothesis set forth in earlier literature that ISYNA1 is regulated by androgen [37] but also indicates that the microenvironment of the caput epididymis is controlled by androgens because both Aatn2 and Isyna1 are controlled by androgens. Both of these genes are probably very important for keeping the environment in the caput epididymis conducive for sperm maturation.
In the corpus region, there were only a few transcripts that were upregulated 2-fold or more by DHT treatment, when compared with the castrated controls. Two transcripts that were upregulated are the previously characterized Car2 and Car4 genes [14]. The remaining transcripts that fit the criteria of being upregulated 2-fold or more have not previously been identified in the epididymis or found to be regulated by androgens in any other tissues. One of these is the titin-associated protein myomesin 2. This protein is important for structure of the sarcomeric myofibrils and muscle contraction [38]. It is possible that androgen regulation of this gene in the epididymis is important for contractility of the corpus to move spermatozoa through the epididymis to the cauda region.
Androgen-regulated transcripts of the cauda include many of the kallikreins, which are serine proteases [39]. The proteolytic activity of proteases in the cauda region could be important for proper storage of spermatozoa. The serine proteases that were expressed in the cauda may be present to protect the spermatozoa from other proteins that may be detrimental to viability of the spermatozoa. The luminal fluid in the cauda may contain residual proteins that were important for sperm maturation in the caput or corpus but are no longer needed in the cauda and may be harmful to spermatozoa. These proteases may also be modifying surface proteins of the spermatozoa similar to other proteases found in the epididymis [40, 41]. According to the microarray data, Serpine-2 is regulated by androgens and this was confirmed by real-time PCR. Serpine-2 is a thrombin and urokinase inhibitor [42] and its function in the epididymis has not been investigated. Serpine-2 has been linked to the testis-determining pathway, as it is expressed specifically in male gonadal development in utero [26]. It has been speculated that Serpine-2 may regulate proteolysis, which could keep cells from migrating during development [43]. This inhibition of migration may play a role in keeping spermatids in the cauda epididymis until they are ready for release. Therefore, we postulate that Serpine-2 is important for the preservation of spermatids in the cauda epididymis.
Transcripts have been identified that were upregulated after castration, then repressed after treatment with DHT. One of these genes is secreted phosphoprotein-1 (Spp-1). SPP-1 is a phosphorylated glycoprotein and is hypothesized to be important for calcium regulation [44]. A shorter splice variant of Spp-1 is Osteopontin, recently reported to be regulated by androgens in the rat epididymis [45]. After rats were castrated, the amount of osteopontin decreased, but in rats treated with androgen, the level of osteopontin went back to normal [45].
In summary, this article has identified many different genes that were regulated by androgen in the epididymis. Many of these androgen-regulated transcripts have been identified as being region enriched. New genes have been identified that may be important for sperm maturation, and the data presented here opens the door to many new molecular-based research projects that can be performed on the epididymis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Michael D. Griswold, 531 Fulmer Hall, School of Molecular Biosciences, Washington State University, Pullman, WA 99164. FAX: 509 335 9688; griswold{at}mail.wsu.edu
Received: 5 December 2003.
First decision: 18 December 2003.
Accepted: 1 April 2004.
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REFERENCES |
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