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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 METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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epididymis, estradiol, gene regulation, male reproductive tract, testosterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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Each cystatin family is comprised of multiple members. The cystatin type 2 family contains the most members, of which at least six are predominantly expressed in the male reproductive tract. These are CST 8 (or CRES) [13], CST 9 (or Testatin) [14], CST T [15], human CST 11 [16], and CST SC and CST TE-1 [17]. The CRES is highly restricted to the proximal region of the epididymis and is present at low levels in the testis [13] and the anterior pituitary gonadotropes [18]. Testatin is expressed only in the pre-Sertoli cells of fetal testis, in Sertoli cells of the adult testis [14], and in (pro)spermatogonial cells in both fetal and adult testis [19]. The CST T is expressed exclusively in the testis [15] and mainly in spermatogenic cells (unpublished data). Human CST 11 is expressed only in the epididymis and is most abundant in the initial segment of epididymis. The CST SC transcript is expressed in Sertoli cells in a stage-dependent pattern, whereas the expression of CST TE-1 mRNA is highly restricted to the Sertoli cells in the testis and to the epithelial cells of the proximal region of the mouse epididymis [17]. All of these male reproductive tract cystatins have four conserved cysteine residues, but they lack some of the conserved motifs thought to be critical for binding to a cysteine proteinase of the papain family [20], such as the N-terminal glycine (Gly-9 in chicken egg-white cystatin) and Gln-X-Val-X-Gly in the first hairpin loop. Therefore, their biochemical or biological functions may differ from those of other type 2 cystatins.
In the present study, we report the identification and characterization of two novel cystatin members, termed mouse Cystatin E1 (mCST E1; GenBank accession no. AK020300; human CST E1 has recently been reported independently under the name CST 11 [16]) and mouse Cystatin E2 (mCST E2; GenBank accession no. AK020314; 
ystatin-related gene highly expressed in 
ididymis). These genes were identified in mice by searching the National Center for Biotechnology Information (NCBI) database for proteins containing homology to known type 2 cystatins. We describe the putative protein sequences and structures, the sequence comparison of similar cystatins, as well as the chromosomal locations and structures. To study the expression of these genes, we investigated the tissue distribution and localization of the corresponding mRNA and examined their regulation in the epididymis by the testosterone and/or 17ß-estradiol (E). The characteristics of these genes, such as the protein homology with known cystatins, structural motifs, chromosomal locations and structures, and tissue-specific expression, suggest that mCST E1 and mCST E2 are new members of the male reproductive tract subgroup of cystatin type 2 family.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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Mouse CST E1 and E2 were identified in mice by searching the protein sequences of known type 2 cystatins, especially the known members of the reproductive tract subgroup, against the NCBI database. Potential identity of the proteins was determined by a homology search using the protein-protein basic local alignment search tool (BLAST) method (http://www.ncbi.nlm.nih.gov/BLAST). According to the protein identity to known cystatins and the tissue where the gene was originally isolated, Mouse CST E1 and E2 were selected as new members of the reproductive tract subgroup within the cystatin 2 family for further study. Multiple protein sequences were aligned by the CLUSTAL W method. Signal peptide and its putative cleavage site were predicted according to the method of Nielsen et al. [21] using SignalP v2 (http://www.cbs.dtu.dk/services/SignalP). The prediction of N-glycosylation site was done using PROSITE, established by the Swiss Institute of Bioinformatics (http://ca.expasy.org/cgi-bin/scanprosite/scanprosite). The putative phylogenetic tree was constructed using GeneBee-NET (http://www.genebee.msn.su), established by the A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia [22, 23]. The chromosomal locations and gene structures were approved by searching the Mouse Genome Database (http://www.ncbi.nlm.nih.gov/genome/guide/mouse/).
Isolation of RNA and mRNA
Total RNA was isolated from adult 129/B6 mice organs using the Trizol method (Invitrogen Corp., Carlsbad, CA) following the manufacturer's recommendations. The polyadenylated [poly(A)+] RNA was prepared using a standard oligo-(deoxythymidine)-affinity chromatography method (Sigma-Aldrich, St. Louis, MO).
Reverse Transcriptase-Polymerase Chain Reaction
Five micrograms of DNase-treated total RNA from mouse epididymis was annealed with oligo(dT)12–18 at 45°C for 60 min in a solution with SuperScript II reverse transcriptase (Invitrogen) and RNasin (Promega, Madison, WI). Primers used to amplify mCST E1 from mouse epididymis cDNA were as follows: 5' primer, 5'-ccc tta agc ctg acc cag aga cac-3'; 3' primer, 5'-tga ggg ctt cta ttg att gca g-3'; Primers used to amplify mCST E2 from mouse epididymis cDNA were as follows: 5' primer, 5'-ggt aag ggc aaa gac agg aga ca-3'; 3' primer, 5'-cca gga cat gaa act gca gac a-3'. The polymerase chain reaction (PCR) was performed using the following thermal cycle profile: denaturation at 95°C for 2 min; by 30 cycles at 95°C for 30 sec, 59°C for 30 sec, 72°C for 1 min; and a final extension step of 72°C for 10 min. The reverse transcriptase (RT)-PCR products were purified and cloned into pGEM-T easy vectors (Promega).
Oligonucleotide Synthesis and DNA Sequencing
Both the primer synthesis and DNA sequencing analysis were performed in the Laboratory of Bioanalysis and Biotechnology at Washington State University, Pullman, Washington. Both T7 END (5'-TTGGACCCGACGTCGCA-3') and SP6 END (5'-AGCTATGCATCGAACGCGTT-3') primers were used for sequencing of the RT-PCR clones. The sequences of the cloned RT-PCR fragments were confirmed by the GenBank and European Molecular Biology Laboratory databases using BLAST.
Northern Blot Analysis and Presentation of Data
Two micrograms of mRNA isolated from adult mice organs such as brain, heart, kidney, liver, lung, skeletal muscle, spleen, pancreas, ovary, uterus, mammary gland, prostate, epididymis, and testis or 7µg of total RNA from intact epididymides and caput, corpus, and cauda of the epididymides were fractionated on a 1.0% agarose/formaldehyde gel. The RNA was transferred to Hybond N membranes (Amersham Pharmacia Biotech, Piscataway, NJ) and cross-linked to the membranes by exposure to 120 mJ of ultraviolet energy in a Stratalinker 1800 (Stratagene, La Jolla, CA).
The cDNA fragments obtained from RT-PCR (574 base pairs [bp] from mCST E1 and 589 bp from mCST E2) were radiolabeled with [
-32P]dATP using the Rad Prime DNA Labeling Kit (Invitrogen). The membranes were hybridized overnight at 65°C in a hybridization solution consisting of 1% BSA, 7% SDS, 1 mM EDTA, 0.5 M sodium phosphate (pH 7.2), and 1 x 106 cpm/ml of corresponding cDNA probe. After hybridization, the blots were washed with 2x SSC (single strength: 0.15 M sodium chloride and 0.015 M sodium citrate)/0.1% SDS for 5 min at room temperature, 2x SSC/0.1% SDS for 15 min at 65°C, and 0.2x SSC/0.1% SDS for 15 min at 65°C. The blots were exposed to a phosphor screen (Molecular Dynamics, Sunnyvale, CA) for 16–48 h. The signals were detected and analyzed using a Molecular Dynamics PhosphorImager 445 SI, ImageQuant software (Molecular Dynamics), and Microsoft Excel (Microsoft, Seattle, WA). Ribosomal protein S2 cDNA probe was used for normalizing the amount of loaded RNA as described previously by Mukherjee et al. [24]. In the figures, the bars indicate mean ± SEM for all hybridizations.
Surgical Procedures
All animal procedures were conducted according to the guidelines stated in the U.S. Public Health Service's Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Washington State University. Animals were housed in a standard animal facility with free access to food and water. Adult mice (25–30 g) were anesthetized with an i.p. injection of ketamine-xylazine mixture at a dose of 0.1 mg/kg (ketamine) and 0.05 mg/kg (xylazine). All ligations and sutures were done using nonabsorbable, braided 6-0 silk (Surgical Specialist Corp., Reading, PA).
Castration and Hormone Treatments
The testicular vascular supply was ligated without compromising the epididymal blood supply. The testis was dissected from the epididymis and associated fat pad and then excised. The epididymis and fat pad were returned, caput first, into the tunica vaginalis, and the incision was sutured. For sham operation, testis and epididymis were manipulated and returned into the tunica vaginalis, and the incision was sutured. Testosterone and/or estrogen replacements were done by s.c. injection of 3 mg of testosterone propionate (T; Sigma Chemical Co., St. Louis, MO) or E (Sigma Chemical) dissolved in sterile sesame-seed oil as a vehicle every day [25]. Mice were castrated at Day 0, and T and/or E were administered at the time of castration (T, E, or T + E maintenance). Control mice received 100 µl of vehicle every day postcastration. Experimental mice were killed on Days 7 and 14 postcastration (n = 3 per group). The epididymis from one side was put in Trizol for RNA extraction, whereas the contralateral epididymis was fixed in 4% paraformaldehyde (PFA)/0.25% glutaraldehyde (GA) for in situ hybridization.
Plasma T/E Determination
Blood was collected from each mouse via the retro-orbital plexus before any surgery or treatment. After the mouse was killed, cardiac puncture was used to obtain the blood from animals. Plasma T/E levels were determined by RIA at the Radioimmunoassay Core Laboratory through the Center of Reproductive Biology at Washington State University using kits obtained from Diagnostic Systems Laboratories, Inc. (Webster, TX). The kit of testosterone RIA has a sensitivity of 0.05–25 ng/ml and a cross-reactivity of <6.6%. The estradiol RIA kit has a sensitivity of 6.5 pg/ml and a cross-reactivity of <1%.
Unilateral Efferent Duct Ligation
Efferent ductules of the left testis were ligated at their junction with the extratesticular rete testes without compromising testicular or epididymal blood supply [26]. As a control, the right testis was manipulated similarly to the left testis, but its efferent ductules were not ligated. Each testis was returned into its tunica vaginalis, and the incisions were sutured. Ligated and control epididymides were removed on Days 1, 2, 3, 7, 10, and 30 postligation (n = 3 per group) and put in Trizol for RNA extraction or fixed in 4% PFA/0.25% GA for in situ hybridization.
In Situ Hybridization Analysis
In situ hybridization analysis was performed as previously described [27, 28] with slight modifications. Mouse positive strands (sense probe) or negative strands (antisense probe) of full-length mCST E1 and mCST E2 cDNAs were cloned into pGEM-T vector (Promega). The plasmids were used as the templates, and sequences of T7 (5'-AATACGACTCACTATAGGG-3') and Sp6 (5'-ATTTTAGGTGACACTATAG-3') promoter were used as primers to produce PCR products, which were used as templates for in vitro transcription.
Radiolabeled antisense or sense probes were generated from a reaction consisting of 10 µl of [-33P]UTP (catalog no. NEG-607H; NEN, Boston, MA), 20 U of RNase inhibitor (Promega), and 50 U of T7 RNA polymerase (Invitrogen). The cRNA probes were cleaved to approximately 200 bp with 40 mM NaHCO3/60 mM Na2HCO3 (pH 10.2) so that they had similar access to cells in each tissue section. Epididymides were fixed in freshly prepared, 4% (v/v) PFA/0.25% GA for 16 h at 4°C. Sections (thickness, 6 µm) were deparaffinized with xylene and treated with 10 µg/ml of proteinase K (Invitrogen) for 30 min at 37°C, then acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0). Slides were hybridized overnight at 55°C in hybridization mixture containing 1 x 106 cpm probe per 30 µl of hybridization solution, stringently washed the next day, dipped in Kodak emulsion NTB-2 (Eastman Kodak Co., Rochester, NY), and exposed for 4–7 days at room temperature in darkroom. The sections were counterstained lightly with hematoxylin and eosin-Y and mounted with GVA mounting solution (Zymed, South San Francisco, CA).
Sections were observed with an Olympus SZX12 microscope (Olympus America, Inc., Melville, NY), and photomicrographs were taken under bright-field and dark-field illumination with an Olympus OLY-200 digital camera (Olympus America) using Olympus MagnaFire Camera Imaging and Control (version 1.0; Olympus America). Digital images were captured and assembled using Adobe Photoshop 6.0 (Adobe Systems, San Jose, CA) and Microsoft PowerPoint (Microsoft, Redmond, WA)
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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The objective of the present study was to identify and characterize the expression of new gene members of the cystatin family 2 in male reproductive tissues. Two new members of cystatin family 2, named Cystatin E1 and E2, were identified in mice by searching the NCBI database for proteins containing homology to known type 2 cystatins. Both genes were isolated originally from mouse epididymis. The gene encoding the putative 139 amino acid protein was named mCST E1 (GenBank accession no. AK020300; the amino acid sequence has 53% identity to human CST 11), whereas the gene encoding the putative 133 amino acid protein was named CST E2 (GenBank accession no. AK020314).
According to the deduced amino acid sequences, mCST E1 and mCST E2 show similarities to family 2 cystatins. Both genes have four conserved cysteine residues at the C-terminal end in exact alignment with that of other members of the family (Fig. 1a). These transcripts also contain secretory signal sequences with putative cleavage sites between position 28 (R) and 29 (K) of mCST E1 and between position 21 (A) and 22 (W) of mCST E2. These cleavage sites are at similar positions to those of other cystatin family members (Fig. 1a). The mCST E1 contains a predicted C-terminal N-glycosylation site NCTD at N134, whereas no N-glycosylation site was found in mCST E2. A phylogenetic tree (Fig. 1b) shows the evolutionary relationship between mCST E1, mCST E2, and selected known cystatins. A close phylogenetic relationship exists between mCST E1 and mouse CRES and between mCST E2 and Testatin. The level of sequence homology between mCST E1 and mCST E2 is only 22%, and that between these two genes and other closely related cystatins is approximately 30–37%. Although mCST E1 and mCST E2 have characteristics of cystatins, they lack some of the short conserved amino acid regions thought to be important for the cysteine proteinase inhibitory function [20]. The mCST E1 protein possesses only the Proline-Tryptophan (PW) motif in the second hairpin loop. In addition, only the P residue is conserved in mCST E2, whereas other regions commonly present in cystatins are absent (Fig. 1a).
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The gene localization on mouse chromosomes and the gene structures were identified by searching the Mouse Genome Database. The mCST E1 corresponds to Sanger gene F005232 and mCST E2 to Sanger gene F005223. Both genes were determined to be located on mouse chromosome 2, and they both have three exons and two introns. Although the length of intron sequences is quite different between these two genes, the intron/exon junctions are in the same location within the coding sequence of the two genes (Fig. 1c).
Tissue-, Region-, and Cell-Specific Expression of mCST E1 and mCST E2 mRNA
To determine the tissue expression patterns of mCST E1 and mCST E2, Northern blot analyses were performed using mRNA isolated from cultured mouse Sertoli cells, Leydig cells, and a number of tissues from adult male and female animals. As shown in Figure 2, b and c, the mCST E1 and mCST E2 transcripts were highly expressed in the epididymis (and only in the caput of the epididymis). The high abundance transcripts migrate with an apparent size of approximately 0.7 kilobase. The mCST E1 gene was also present in the prostate. In addition, the mCST E2 transcript was also observed in the testis and prostate, but the signal was weak. The expression of these two genes was not detected in other organs that were analyzed. Transcripts of several sizes were observed on the blot probed with mCST E2 probe. This indicates either the existence of different RNA splicing forms or incompletely processed mRNAs, or it represents mCST E2 mRNAs that include regions beyond those we identified as components of this gene, possibly including an upstream fourth exon as described for CST 8 or an additional 3' exon [29].
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To localize the expression of mCST E1 and mCST E2 to specific cells in the epididymis, in situ hybridization was performed. Longitudinal sections of mouse adult epididymis were incubated with antisense and sense [-33P]UTP-labeled cRNA probes generated from the mCST E1 and mCST E2 cDNA. Both mCST E1 (Fig. 3, aA and aB) and mCST E2 (Fig. 3, bA and bB) mRNAs were highly regionalized and predominantly expressed in the caput epididymis, with no signal in the corpus or cauda epididymis. Higher magnification revealed that expression of mCST E1 (Fig. 3, aC and aD) and E2 (Fig. 3, bC and bD) was present only in the epithelial cells of the caput region. The mCST E1 mRNA was abundant in the initial segment (segment I) and in segment II, but the expression level was lower in segment III, of the caput epididymis. The mCST E2 transcripts were highly expressed only in segment I, and weak signal was detected in segment II. No mCST E2 mRNA signal was seen in segment III. No mCST E1 or mCST E2 mRNA was detected in the efferent duct, sperm, or conjunctive tissues (Fig. 3, aE, aF, bE, and bF). Thus, the expression of mCST E1 mRNA is highly restricted to the epithelial cells in segments I, II, and III, whereas mCST E2 mRNA is expressed only in the epithelial cells of segments I and II of the caput epididymis.
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Effect of Castration on mCST E1 and mCST E2 Expression
The effect of castration on mCST E1 and mCST E2 mRNA expression was studied by Northern blot analysis (Fig. 4). Total RNA extracted from the epididymides at 4 and 7 days postcastration was hybridized with the 32P-radiolabelled mCST E1 and mCST E2 cDNA probes. High levels of transcripts were detected in RNA from intact animals. Both mCST E1 and mCST E2 signals decreased by 65% on day 7 postcastration. Testosterone levels were 1.6–27 ng/ml on Day 0 and were undetectable (<0.05 ng/ml) by Day 7 postcastration.
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Effect of Efferent Duct Ligation on the mCST E1 and mCST E2 mRNA Expression
To test for possible contributions of testicular factors to the regulation of gene expression, unilateral efferent duct ligation was used to prevent the flow of testicular fluid into one epididymis. After ligation, mCST E1 and mCST E2 mRNA levels decreased dramatically. On Day 10 after ligation, the mCST E1 (Fig. 5a) expression showed an 85% reduction, and mCST E2 (Fig. 5b) showed a 95% reduction, compared to that of nonligated, intact epididymides. In situ hybridization showed similar results: The signal of mCST E1 (Fig. 5, cC and cD) decreased dramatically, and that of mCST E2 (Fig. 5, cG and cH) was almost undetectable 30 days after efferent duct ligation.
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Hormonal Regulation of the mCST E1 and mCST E2 mRNA Expression
To determine if exogenous testosterone can restore mCST E1 and E2 expression in the epididymides of castrated mice, T was administered to mice at the time of castration in a T-maintenance paradigm. The administration of T for 7 days did not result in any recovery of the expression of either gene. On the contrary, T treatment further repressed the expression of these genes in the epididymis following castration (Fig. 4). This suggests that hormones other than T or other testicular factors up-regulate the expression of these genes.
Estrogen receptors (ER), both ER and ERß, are expressed in the caput epididymides of mice [30], and it has been reported that estrogen plays an important role in spermatogenesis and in the epididymis [31]. Therefore, experiments were carried out to determine if the administration of exogenous E or E+T to castrated mice restored the gene expression in epididymis. Immediately after castration, E and/or T were administered. As shown in Figure 6, a and b, E treatment for 1 wk resulted in a partial recovery of mCST E1 mRNA. The E+T treatment for 1 wk failed to up-regulate the mCST E1 mRNA level. On the other hand, mCST E1 transcripts decreased to 50% of castrated levels and to 20% of intact levels after 7 days of E+T treatment in the castrated animals. However, no recovery of mCST E1 mRNA was detected after 14 days of E treatment in the castrated mice, and E+T administration for 14 days reduced the mCST E1 mRNA level to 50% of intact levels and 70% of castrated levels. Figure 6, c and d, shows the regulation of mCST E2 mRNA expression by E and E+T administration in castrated mice. The expression of mCST E2 mRNA was restored by E treatment for 7 and 14 days in castrated mice to the same levels as in sham-operated mice. The levels of mCST E2 transcripts decreased dramatically to less than 10% of that in the sham-operated animals after the administration of E+T for 1 and 2 wk. The RIA analysis showed that circulating levels of T and E were undetectable in castrated mice and approximately 10-fold higher and 15-fold higher, respectively, in treated mice than in sham-operated animals.
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Potentially, significant changes in the weight of epididymides and the abundance of mRNAs in the sham-operated, castrated, T-treated, E-treated, and E+T-treated epididymides may prevent Northern blot analysis from reflecting actual expression changes in the tissues. Therefore, in situ hybridization analysis was performed to confirm the results of Northern blot analysis. Longitudinal sections of mouse adult epididymides after 14 days of castration with or without hormone administration were incubated with antisense and sense cRNA probes generated from the mCST E1 and mCST E2 cDNA. All the sections were exposed to the NB-2 emulsion for 7 days, and all the digital images taken under bright-field and dark-field illumination were captured under the same conditions. The weight of the epididymis was decreased at 14 days postcastration (Figs. 7, C and D, and 8, C and D), and E administration did not prevent this reduction in weight compared to that of an epididymis from a castrated animal (Figs. 7, E and F, and 8, E and F). Both E+T and T maintenance did prevent the reduction in weight of the epididymis in castrated animals (Figs. 7, G, H, I, and J, and 8, G, H, I, and J). Similar to the results shown in Northern blot analysis (Fig. 6), the abundance of mCST E1 mRNA signals did not change in epididymides of sham-operated (Fig. 7, A and B), castrated (Fig. 7, C and D), or castrated with E treatment (Fig. 7, E and F) animals. The decrease of mCST E1 expression in epididymides of E+T-treated mice (Fig. 7, G and H) and of T-treated (Fig. 7, I and J) mice was noticeable. Figure 8 shows that mCST E2 mRNA expression decreased dramatically 14 days after castration (Fig. 8, C and D) compared with the sham-operated mice (Fig. 8, A and B). The mCST E2 signals were restored to intact levels after E treatment (Fig. 8, E and F). The mCST E2 mRNA in the epididymis of E+T-maintained mice was reduced compared to E-treated mice. However, the mCST E2 signal was still detectable in E+T-treated epididymis (Fig. 8, G and H). Administration of T decreased the mCST E2 level dramatically, and the mCST E2 expression was almost undetectable (Fig. 8, I and J). These results of in situ hybridization analyses are also consistent with the results of Northern blot analysis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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The results from the Mouse Genome Database search showed that both mCST E1 and mCST E2 are located on mouse chromosome 2, the same chromosome where CST C [33], CRES [29], and CST T [15] are located. In contrast, members of families 1 and 3 have been mapped to the long arm of chromosome 3 [34, 35]. In the human, several family 2 cystatins, including CST 11 [16] as well as CST C, CST D, S, SN, and SA [36], have been identified to cluster on human chromosome 20, which shows a region of linkage conservation with the distal region of mouse chromosome 2. In addition, both mCST E1 and mCST E2 contain three exons and two introns (Fig. 1c). The sizes of the coding exons are similar to those of the mouse CST C, and the exon/intron junctions are in identical locations as in CST C [33], CST SC, and CST TE-1 (unpublished data), and CRES, although CRES possesses an extra exon encoding 5' untranslated sequences [29]. These observations are consistent with mCST E1 and mCST E2 being members of the male reproductive tract subgroup within family 2 cystatins.
The mCST E1 and mCST E2 mRNA showed expression patterns similar to those of CRES [13] and CST TE-1 [17] in the epididymis. All transcripts are predominantly expressed in the caput epididymis, with no signal detected in the corpus or cauda epididymis. The expression of these genes is highly restricted to the epithelial cells of the caput region. However, mCST E1 and mCST E2 are detected in segments I and II of the caput epididymis, whereas CRES and CST TE-1 transcripts are restricted to the epithelial cells in the initial segment (segment I) of the epididymis. Both CRES and mCST E2 have very low expression in the testis, whereas CST TE-1 is highly expressed in both the testis and epididymis. The complicated expression pattern of these cystatins suggests that they might interact with different proteinases involved in male reproductive tract function.
Normal epididymal function depends on both circulating androgens and testicular factors [37–40]. These factors include a variety of molecules found in the seminiferous tubular fluid, such as luminal androgens [41], androgen-binding proteins [42], estrogen [31], growth factors [43], and probably unknown molecules as well. It was reported that human CST 11 transcription shows region-specific regulation by testosterone in the epididymis, and its expression requires multiple proteins in addition to androgens [16]. The regulation of mCST E1 and mCST E2 mRNA expression is distinct. Although the expression of both genes decreased after castration, which eliminates both circulating androgens and testicular factors, T administration failed to restore either of the messages. Moreover, T further repressed the transcription of both genes, especially that of CST E2 (Fig. 8, I and J). It is not surprising that these two genes were regulated by T, because androgen-response elements are found in both gene promoters. To determine whether testicular factors are important in regulation of mCST E1 and mCST E2 expression, efferent duct ligation, which prevents testicular fluid from entering the epididymis without altering the concentration of circulating androgens, was performed. Both mCST E1 and mCST E2 mRNA levels decreased to less than 15% of the intact levels after 10 days of ligation (Fig. 5, a and b), which is much lower than the expression level after 7 days postcastration (Fig. 4). This suggests that testicular factors are necessary for the expression of mCST E1 and mCST E2 mRNA and that circulating androgens down-regulate the expression of both genes. It has been reported that prostatic androgen-repressed message-1 (PARM-1) is reduced by 70% within 12 h of testosterone administration to castrated rats [44], and SGP-2 (known as Clusterin) mRNA is also repressed by testosterone in the prostate [45]. In addition, no region-specific regulation of mCST E1 or mCST E2 by androgen was detected by in situ hybridization. This indicates that the regulation pattern of mCST E1 and mCST E2 may be different from that of human genes, such as CST 11.
In males, estrogen is present at a low concentration in the blood stream but can be extraordinarily high in semen and as high as 250 pg/ml in rete testis fluid [46, 47], which is higher than the level of serum estradiol in the female [48]. Male reproductive tissues express ERs [49–52], and both the and ß forms of the ER are present in the caput epididymis of mice [30]. Both ER and ERß knockout mice displayed a phenotype of abnormal morphology of the epididymis [53, 54]. These studies suggest that estrogen plays a role in the regulation of epididymal function. To determine if estrogen regulates the expression of mCST E1 and mCST E2 mRNA, we treated the castrated mice with E or E+T for 1 and 2 wk. Our results showed that mCST E1 was only slightly up-regulated by E, whereas mCST E2 transcript was restored to near-normal levels by E administration postcastration. This indicates that mCST E1 is more dependent on other testicular factors than estrogen. Moreover, as shown by in situ hybridization (Fig. 8), the level of mCST E2 was much higher in the epididymides of E+T-treated animals than in those treated with T alone. This suggests that E up-regulates the mCST E2 mRNA expression, whereas T represses the expression. The observation that transcript level can be modulated by both androgens and estrogens is not completely unique. Turner et al. [55] have demonstrated that the androgen-dependent transcripts C3 and SGP-2 can also be modulated by estrogens in the prostate. Jaussi et al [56] have reported that the administration of estrogen, either alone or in combination with testosterone, to female mice could affect the expression of a variety of androgen-responsive genes within kidney. Furthermore, epidermal growth factor mRNA is partially repressed in kidney following testosterone treatment of mice, but simultaneous treatment with E appears to antagonize this effect [57]. Further experiments should be done to interpret the mechanisms involved in the modulation of mCST E1 and mCST E2 by testosterone and estrogen.
Mammalian spermatozoa undergo a complex process of maturation in the epididymis, resulting in their forward motility and fertilizing ability [58]. Epididymal spermatozoa are largely transcriptionally and translationally quiescent. This suggests that the proteins secreted by the epithelium of the epididymis are very important in sperm maturation [59]. A large number of proteinases and proteinase inhibitors have been found in the reproductive tract tissues. Several proteinase inhibitors have been described in the epididymal fluid or tissue, including acrosin and 2-macroglobulin [60], protein C inhibitor [61], matrix metalloproteinases (tissue inhibitors of metalloproteinases) [62], eppin [63], HE4 (an extracellular proteinase inhibitor) [47], human CST 11 [16], CST TE-1 [17], and CRES [64]. The CRES protein is exclusively synthesized by the proximal caput epithelium in the epididymis and then secreted into the lumen [65]. It is localized in sperm acrosomes, and it may play a role in the regulation of intra-acrosomal protein processing or be involved in fertilization [64]. It is also reported that the recombinant protein of human CST 11 has antimicrobial activity [16]. Because mCST E1 and mCST E2 mRNA have similar localization in the epididymis to that of CRES and hCST 11, these genes may interact with corresponding proteinases and play critical roles in sperm maturation or protect against invading pathogens by inhibiting microbial proteinases.
In summary, mCST E1 and mCST E2 are members of the reproductive tract subgroup within family 2 cystatins according to the gene sequences, gene structures, and gene localizations. In the mouse, mCST E1 and mCST E2 are highly expressed only in the epithelial cells of the caput epididymis. Both transcripts are repressed by androgen administration to castrated mice. However, mCST E1 mRNA expression is more dependent on testicular factors other than estrogen, whereas mCST E2 is more estrogen-responsive and its expression probably involves other testicular factors as well. So, although both mCST E1 and mCST E2 share significant homology with family 2 cystatins and have similar distribution in tissues and similar localization in epididymis, these genes may have different functions, because their regulations involve different hormones and testicular factors.
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ACKNOWLEDGMENTS |
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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: 10 December 2002.
First decision: 5 January 2003.
Accepted: 27 March 2003.
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expression in the rat epididymis. Biol Reprod 1996 54:1111-1119[Abstract] knockout mice. J Androl 2000 21:107-121[Abstract]This article has been cited by other articles:
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  E. Prabagaran, U. C. Hegde, S. B. Moodbidri, A. H. Bandivdekar, and V. P. Raghavan Postnatal Expression and Androgen Regulation of HOXBES2 Homeoprotein in Rat Epididymis J Androl, September 1, 2007; 28(5): 755 - 771. [Abstract] [Full Text] [PDF]
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