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Regular Article |
a Departments of Obstetrics and Gynecology,
b Biochemistry,
c Urologic Surgery, and
d Center for Reproductive Biology Research, Vanderbilt University Medical Center, Nashville, Tennessee 37232
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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epididymis, gene regulation, male reproductive tract, sperm maturation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Among the proteins present in the epididymis, four members of the lipocalin family have been identified in the mouse: protein 24p3 [7]; two proteins encoded by two duplicated genes, epididymal retinoic acid-binding protein (mE-RABP) [8, 9] and epididymal protein 17 kDa (mEP17) [10]; and prostaglandin D2 synthase (PGDS) [11, 12]. Lipocalins are small secretory proteins found in extracellular fluids (tears, nasal secretion, luminal uterine fluid, urine). These proteins possess a characteristic three-dimensional folding, which forms an internal hydrophobic ligand pocket for small lipophilic molecules such as retinoic acid [13, 14]. Rat and mouse E-RABP have this characteristic tertiary structure [15, 16]. Within the epididymis, expression of these lipocalins is regionalized. For example, mE-RABP is expressed in the distal caput [16], and the protein accumulates in the cauda [8], while mEP17 is expressed in the initial segment [10], and the protein is exclusively present in the proximal part (initial segment, caput) of the epididymis [17].
In the present study, we characterized PGDS (lipocalin-type glutathione independent) in the mouse epididymis, the only lipocalin reported to date, to our knowledge, with an enzymatic activity (EC 5.3.99.2) [18]. The enzymatic activity (prostaglandin H2 D-isomerase) is particularly well documented in the brain, where PGDS is considered to be a bifunctional protein: an enzyme producing prostaglandin D2, and a lipophilic ligand-binding protein considered to be a carrier with high affinity for several molecules such as retinaldehyde, retinoic acid, biliverdin, and bilirubin [19].
The PGDS is a major protein secreted by epididymal epithelial cells into the lumen of the tubule in several mammalian species, including the mouse [12], rat [20, 21], ram and stallion [22], bull [23], and human [24]. Moreover, PGDS is present in the milieu surrounding spermatozoa throughout the genital tract, from the testis to the semen, in most species. In the mouse epididymis, the protein was previously detected by immunohistochemistry in the epithelial cells of the efferent ducts and the distal caput and in the lumen, with an increasing staining gradient from distal caput to cauda [12]. Although PGDS mRNA was previously detected in mouse epididymis, its regionalization was not reported [11]. The aims of the present study were 1) to identify PGDS in mouse epididymis using a proteomic approach combining two-dimensional (2D) gel electrophoresis and mass spectrometry (MS), 2) to characterize its isoforms in 2D gel electrophoresis and quantify the protein in the caudal fluid, 3) to investigate its regionalization both at the mRNA and the protein level, and 4) to compare expression of PGDS in other species such as rat, hamster, and monkey. The proteomic approach we have used in this study allowed us to unambiguously identify murine PGDS in caudal epididymal fluid as a major protein and to demonstrate that matrix-assisted laser desorption ionization (MALDI) and nanoelectrospray ionization (nanoESI) MS are powerful techniques to identify an array of secreted proteins in the murine epididymal fluid.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Restriction enzymes and sequencing-grade trypsin were purchased from Promega (Madison, WI); reverse transcriptase, Concert Gel Extraction Systems, proteinase K, and RNase A from Gibco-BRL (Gaithersburg, MD); oligo(dT) primers, Sephadex G50, and enhanced chemiluminescence (ECL) Western blotting detection reagent from Amersham-Pharmacia Biotech, Inc. (Piscataway, NJ); Emulsion Ilford K5 from Polysciences, Inc. (Eppelheim, Germany); IPG strips, SDS, and Sypro Ruby from Bio-Rad (Hercules, CA); SuperFrost slides and Bouin fixative from VWR Scientific Products (Atlanta, GA); horseradish peroxidase-conjugated goat anti-rabbit IgG and diaminobenzidine substrate (DAB substrate-chromogen) from DAKO (Carpinteria, CA); DNA polymerase AmpliTaq Gold from Applied Biosystems (Foster City, CA); vector pCRII-Blunt-TOPO from Invitrogen Corporation (Carlsbad, CA); and D-19 developer from Eastman Kodak Company (Rochester, NY). All other products were purchased from Sigma (St. Louis, MO).
Animals and Organ Sampling
Investigations were conducted in accordance with the National Research Council publication Guide for Care and Use of Laboratory Animals. This study was performed on adult CD-1 mouse epididymis (Harlan Sprague-Dawley, Indianapolis, IN). Mice were killed under methoxyflurane (Mallinckrodt, Inc., Mundelein, IL) anesthesia, and the epididymis was then sampled. For protein extraction, the epididymis was dissected into four regions: initial segment, caput, corpus, and cauda (Fig. 1). For total RNA extraction, the epididymis was dissected into three regions only: caput, including initial segment; corpus; and cauda. The epididymis was kept intact to be fixed in Bouin fixative or 4% paraformaldehyde in PBS for immunodetection and in situ hybridization, respectively. Caudal epididymal fluid was obtained by retroflushing the vas deferens with PBS. The other species studied were adult rat (Wistar), hamster (Syrian Golden), and cynomolgus monkey (Macaca fascicularis). The epididymis was used fresh or after freezing at -80°C
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One-Dimensional and 2D Polyacrylamide Gel Electrophoresis
Proteins were extracted from initial segment, caput, corpus, and cauda epididymal tissues by homogenization in 50 mM Tris-HCl (pH 7.4), 0.5 mM EDTA (pH 8), and a 2% (v/v) protease-inhibitor cocktail (Sigma). The tissue homogenates were centrifuged twice at 15 000 x g for 30 min at 4°C, and the concentration of total proteins was estimated by the Bradford assay (Bio-Rad). The SDS-PAGE separation was carried out according to the method of Laemmli [25] on 12% (w/v) polyacrylamide gels. One-dimensional (1D) and 2D gel electrophoresis were performed with 15 and 180 µg of total protein, respectively. For 2D gel electrophoresis, isoelectric focusing was performed using pH 3–10 IPG strips (13 cm). Gels were stained with Sypro Ruby or transferred onto membrane (see below).
Detection of PGDS by Western Blot Analysis and Immunohistochemistry
Production of the specific antibody raised against pig PGDS and used in this study was previously described [22]. For Western blot analysis, the proteins were electrotransferred onto polyvinyldifluoride membrane (Millipore Corporation, Bedford, MA) in 25 mM Tris/190 mM Glycine/20% methanol buffer. After incubation in the blocking buffer (3% BSA in PBS), the membranes were incubated with the antibody diluted at 1:1000 (immune and preimmune serum). After washes, the blots were incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (Ig) G and then washed again. Immunoreactive proteins were detected by ECL according to the manufacturer's instructions.
For immunohistochemistry, tissues freshly dissected were postfixed in Bouin fixative for 48 h, washed intensively in 50% ethanol, and then dehydrated before embedding. Immunodetection was performed on 10-µm sections. Sections were boiled for 15 min in 1 M urea. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 30 min, and nonspecific-binding sites were blocked with 3% BSA. Tissue sections were exposed to the primary antibody (diluted at 1:200) overnight at 4°C. After incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG and washes, the detection was performed with diaminobenzidine substrate according to the manufacturer's instructions. The preimmune serum of the rabbit was used as negative control for Western blot analysis as well as for immunochemical staining.
Quantification
After 2D gel electrophoresis, quantification of PGDS was performed after staining with Sypro Ruby using an External Laser Molecular Imager FX scanner and the Quantity One software (Bio-Rad). The intensities of the five main spots of PGDS as well as of the spots corresponding to cysteine-rich secretory protein 1 (CRISP-1) and mE-RABP were measured.
N-Glycanase Digestion
N-glycanase digestion was performed using recombinant N-glycanase from Glyko, Inc. (Novato, CA), according to the manufacturer's instructions. Fifty micrograms of total caudal proteins were digested at 37°C for 18 and 36 h. After 18 and 36 h, an aliquot of 15 µl was removed and analyzed by Western blotting.
Sample Preparation for MS Analysis
After 2D gel electrophoresis and Sypro Ruby staining, spots revealed by the PGDS antibody were cut and washed in 30% methanol, then in 100 mM ammonium bicarbonate. After crushing the gel into small pieces, the proteins in the gel were reduced with 10 mM dithiothreitol (DTT) at 60°C and alkylated with 10 mM iodoacetamide at room temperature in the dark. The gel pieces were then washed in 50 µl of acetonitrile and subsequently dried under vacuum. In-gel trypsin digestion was performed overnight at 37°C in 25 µl of 50 mM ammonium bicarbonate with 0.02 µg/µl of trypsin.
MS Analysis
MALDI MS
Peptide mapping was performed by MALDI MS in the linear acquisition mode under optimized delayed-extraction conditions using 
-cyano-4-hydroxy-cinnamic acid as matrix (prepared at 10 mg/ml in 50/49/0.1 [v/v/v] acetonitrile/H2O/trifluoroacetic acid) with a Voyager MALDI DE-STR mass spectrometer (Applied Biosystems). After in-gel digestion, 1 µl of the supernatant was mixed on target with 1 µl of matrix. The mixture was allowed to dry and then analyzed.
NanoESI MS/MS Peptide sequence analyses were performed by nanoESI MS/MS on a QStar QqTOF mass spectrometer (MDS Sciex, Toronto, ON, Canada) equipped with a nanospray ion source (MDS Protana A/S, Odense, Denmark) [26]. After in-gel digestion, 1 µl of the supernatant was mixed with 4 µl of diluent composed of 60/39/1 (v/v/v) methanol/H2O/88% formic acid. The diluted sample was transferred to a nanospray glass tip and mass analyzed.
Reverse Transcriptase-Polymerase Chain Reaction and Cloning
Total RNA from liver, testis, and total epididymis as well as caput (including initial segment), corpus, and cauda epididymidis was extracted using the isothiocyanate guanidinium technique as described by Chomczynski and Sacchi [27]. Five hundred nanograms of total RNA were used for reverse transcriptase-polymerase chain reaction (RT-PCR). Reverse transcription was performed with Moloney murine leukemia virus RT and oligo(dT) primers. The specific reverse-transcript PGDS cDNA was then amplified by DNA polymerase (AmpliTaq Gold; Applied Biosystems) with a pair of 23-base primers: reverse primer, 5'-CTTTTATTTCTGAGTGACAGAGCAAAGGA-3'; and forward primer, 5'-ACCTGCTCTGCTCTGAGCAAATG-3'. The PCR was performed for 35 cycles (denaturation at 94°C for 45 sec, primer annealing at 50°C for 45 sec, and elongation at 62°C for 72 sec). The band obtained was extracted from the gel using the Concert Gel Extraction Systems according to the manufacturer's instructions, and the cDNA cloned in the vector pCRII-Blunt-TOPO. After sequence verification (dye-terminator technique), the cDNA was amplified and used for in situ hybridization.
In Situ Hybridization
The vector containing the PGDS cDNA was linearized using enzyme BamHI or EcoRV to produce antisense and sense riboprobes, respectively. After phenol/chloroform extraction and ethanol precipitation, 1 µg of each linearized plasmid was labeled with 35S-rUTP using the T7/SP6 Riboprobe in vitro transcription system (Promega) following the manufacturer's instructions. The BamHI digest was transcribed using the T7 polymerase for the synthesis of antisense riboprobe and the EcoRV digest using the SP6 polymerase for the synthesis of the sense riboprobe. Ribonucleotides were removed from the riboprobe using a G50 Sephadex column in buffer composed of 10 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% SDS, and 10 mM DTT and kept at -80°C until hybridization.
Each mouse epididymis was fixed in 4% paraformaldehyde-PBS at 4°C overnight, paraffin embedded, sectioned (7 µm) and mounted on SuperFrost slides. After deparaffinization in xylene and rehydration, sections were treated with 20 µg/ml of proteinase K for 30 min at 37°C, post-fixed in 4% paraformaldehyde-PBS, and acetylated in 0.1 M triethanolamine and 0.25% acetic acid. The hybridization was performed overnight at 60°C in a humidification chamber (300 000 cpm/section). Following hybridization, the slides were washed successively with the following buffers: 5x SSC (1x SSC: 150 mM NaCl and 165 mM sodium citrate)/20 mM ß-mercaptoethanol for 30 min at 50°C; 2x SSC/50% formamide for 30 min at 50°C; and 10 mM Tris-HCl (pH 8)/5 mM EDTA (pH 8)/0.5 M NaCl. After incubation with RNase A at 20 µg/ml, the slides were washed again in 2x SSC/50% formamide/20 mM ß-mercaptoethanol, 1x SSC, and 0.1x SSC (15 min at 37°C for each bath). After dehydration in ethanol (50%, 75%, 80%, 90%, and 100%), the sections were air-dried, dipped in emulsion, and exposed for 3 wk. The slides were developed with the D-19 developer (Eastman Kodak).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The proteomic map of epididymal caudal fluid obtained after 2D gel electrophoresis and Sypro Ruby staining revealed at least 15 major proteins, most of them composed of several isoforms of different pI values and molecular weights (Fig. 2A). To detect PGDS isoforms in mouse epididymal caudal fluid, we performed 2D Western blot analysis using a specific antibody raised against pig PGDS and characterized in a previous study [22]. In mouse caudal epididymal fluid, PGDS appeared as a protein composed of at least six isoforms (P1 to P6), with molecular weights ranging from 20 to 25 kDa and with pI values ranging from 6.5 to 8.8 (Fig. 2B).
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We performed N-glycanase digestion on caudal tissue extract. After 18 h of N-glycanase digestion, the mass of the band detected by the antibody decreased from 20–25 to 18.5 kDa (Fig. 3), which is in agreement with the PGDS theoretical molecular weight of 18 483.8, suggesting that the different mass isoforms observed in caudal fluid are essentially due to N-glycosylation of the primary sequence.
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To confirm PGDS identity, the spot P3 (pI, 7; 23 kDa) was digested by trypsin and analyzed by MALDI MS as described in Materials and Methods. Figure 4A presents the MALDI MS peptide map obtained after in-gel trypsin digestion of the P3 spot. Most of the observed peptides were in accordance with the PGDS theoretical peptide map (sequence obtained from the Swiss-Prot protein database [www.expasy.ch], accession no. O09114), covering more than 80% of the sequence (Fig. 5 and Table 1).
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To unambiguously identify PGDS, several of the tryptic peptides detected by MALDI MS were sequenced by nanoESI MS/MS (Fig. 5 and Table 1). Figure 4B shows the MS/MS spectrum obtained for the double-charge peptide ion at m/z 950.48, which corresponds to a monoisotopic peptide molecular weight of 1898.94. Interrogation of the Swiss-Prot database with the derived sequence information confirmed that this peptide had the sequence of the known PGDS tryptic fragment (145–161). The signal detected at m/z 1624.7 could not be readily assigned to PGDS tryptic peptide. The nanoESI MS/MS of the double-charge ion at m/z 812.84 (Fig. 4C) confirmed the peptide to be the N-terminal tryptic fragment of PGDS (1–14), with a substitution of pyroglutamic acid for the expected glutamic acid at the N-terminus. The database sequence for PGDS suggests two potential N-glycosylation sites on the asparagine residues 27 and 54 of the processed PDGS sequence (Table 1). The tryptic peptides incorporating these amino acids (19–32 and 43–61) were not detected by either MALDI or nanoESI MS, suggesting that both sites carry N-linked carbohydrates. Furthermore, in the peptide profile of Figure 4A, in the m/z range from 2300 to 3500, several signals separated by 162 mass units, representing the increment mass for an hexose sugar residue, were detected, suggesting that these signals arose from the different isoforms of the glycopeptides. Table 1 summarizes the different PGDS tryptic peptides detected and sequenced by MS.
The relative quantity of PGDS was evaluated as described in Materials and Methods. Among all the proteins detected in the caudal epididymal fluid after 2D gel electrophoretic separation and staining with Sypro Ruby, PGDS was estimated to represent 3% of the total protein content. From the different spots detected on the gel, several other proteins such as CRISP-1 (pI, 6.8; 26 kDa) and mE-RABP (pI, 5.8; 18.5 kDa) were identified by MS after trypsin digestion and peptide mapping (data not shown) and quantified. The CRISP-1 and mE-RABP represented 6% and 0.3% of the total protein content, respectively.
Immunolocalization of PGDS Protein in Mouse Epididymis
The presence of PGDS in the mouse epididymis was investigated by Western blot analysis after 1D gel electrophoresis using tissue extracts. The PGDS was almost undetectable in testis and initial segment. The protein was detected in the caput (24 kDa), corpus (20–24 kDa), and cauda (20–25 kDa), where a strong signal was obtained (Fig. 6). Using epididymal tissues fixed in Bouin solution and processed for antigen retrieval, PGDS was mostly detected in the corpus and caudal lumen (Fig. 7). Clear cells were strongly stained, whereas a faint staining was observed in the supranuclear region of the principal cells.
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Localization of PGDS mRNA by RT-PCR and In Situ Hybridization
The PGDS mRNA was detected by RT-PCR in the testis and in the caput, corpus, and cauda epididymidis using specific primers as described in Materials and Methods. In this experiment, liver RNA was used as negative control (Fig. 8). The cDNA obtained at 0.8 kilobases was extracted from the gel, cloned into the vector pCRII-Blunt-TOPO, and sequenced for verification after amplification. After linearization, antisense RNA was used to localize the mRNA by in situ hybridization (Fig. 9), and sense RNA was used as negative control. By in situ hybridization, PGDS mRNA was detected in the epididymal epithelium in all regions except the initial segment, with a strong signal in the proximal caput, middle caput, corpus, and cauda. No signal was obtained with the sense probe (data not shown).
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Detection of PGDS in Rat, Hamster, and Monkey Epididymis by Western Blot Analysis
Using Western blot analysis, PGDS was detected in epididymal tissue extracts of rat, hamster, and monkey after 1D gel electrophoresis (Fig. 10). In the rat epididymis, PGDS, the molecular weight of which was approximately 23 kDa, was present in increasing amounts from the initial segment to the cauda. After 2D gel electrophoresis, the isoforms of PGDS in the cauda presented acidic pI values (range, 3.8–5.6) (Fig. 11). In the hamster, the protein was detected in the testis and in increasing amounts from the initial segment (faint signal) to the cauda, where its mass was 20–27 kDa (Fig. 10). In the monkey, the amount of PGDS decreased from caput to cauda. The mass was polymorphic according to each region: 19 kDa in the caput, 22.5 kDa in the corpus, and 20 kDa in the cauda (Fig. 10).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Identification and Molecular Characterization of Murine Epididymal PGDS
Spectrometric instrumental techniques such as MALDI MS and nanoESI MS are powerful tools for protein identification and characterization. Tissue protein extracts are fractionated by 2D gel electrophoresis. Protein bands of interest are excised from the gel and proteolyzed, and the peptides are extracted. The peptide mixtures are mapped by MALDI MS [29, 30]. The resulting protein-specific patterns are either used to query protein databases for protein identification or compared to a computerized peptide map generated from the sequence of a known protein [31]. Protein identity is then confirmed by fragmenting one or several peptides by nanoESI MS/MS [32]. These techniques allow investigators to sequence peptides at or below the femtomole level, to measure such things as molecular weights of more than 200 kDa, and to identify postribosomal modifications and other types of structural parameters [33, 34].
In this study, MS enabled the unambiguous identification of PGDS according to its trypsin fragmentation pattern (peptide map) and the sequences of tryptic fragments by nanoESI MS/MS. Moreover, data revealed several structural elements of the protein, such as the presence of a pyroglutamate residue at the N-terminal of the protein, and suggested glycosylations with the presence of several signals separated by 162 mass units, representing the increment mass for a hexose sugar residue. It is generally admitted that N-pyroglutamate rearrangement prevents or minimizes N-terminal degradation by monohexopeptidases. The presence of a N-terminal pyroglutamate on a protein or a peptide may, in some instances, enhance or be responsible for its biologic function or activity, as shown for the tripeptide thyrotropin-releasing factor [35]. Two-dimensional gel electrophoresis showed that PGDS is a highly polymorphic protein distributed among several isoforms presenting different pI values in the mouse as well as in the rat cauda epididymidis (Figs. 2 and 11). Similar results were previously shown for sheep and horse [22]. Using N-glycanase digestion, the molecular weight decreased to the theoretical mass (after cleavage of the peptide signal, 18.5 kDa), suggesting that mouse epididymal PGDS, like human epididymal PGDS [24], is N-glycosylated. According to its primary sequence, mouse PGDS has two putative N-glycosylation sites. Further study would be needed to determine if one or both sites (as suggested by our MS data) are glycosylated. Glycosylations of primary structure are very common in the epididymis, because most epididymal proteins are resolved into several isoforms that are observed as a "train of spots" from 2D gel electrophoresis, as shown in the pig [36] and stallion [37].
PGDS Is One of the Major Proteins in the Fluid of Mouse Cauda Epididymidis
This study shows that PGDS is one the major proteins in the lumen of the mouse epididymal duct. Among the proteins in the caudal fluid and detected after 2D gel electrophoresis and Sypro-Ruby staining (as sensitive as silver staining), PGDS was estimated to represent 3% of the total protein content. In the horse, PGDS represents 20% of the proteins in the fluid of the proximal caput [22, 37]. Two other known major proteins were also identified using this strategy: CRISP-1 (6%), and mE-RABP (0.3%).
Regionalization of PGDS Protein and mRNA
Within the epididymis, many genes encoding secretory proteins exhibit highly regionalized expression [4, 5]. In situ hybridization showed that PGDS mRNA was not detected in the initial segment but was detected in the epithelium from proximal caput to cauda. Hoffmann et al. [11] had previously performed in situ hybridization in mouse epididymis and showed expression in the epithelium, but they did not report region specificity. Immunodetection performed in the present study gave a protein localization pattern similar to that of the mRNA, with the protein being detected at increasing amounts in the lumen from caput to cauda by Western blot analysis. This result suggests that the protein is secreted in increasing amounts from the proximal to the distal part of the epididymis, leading to an accumulation in the caudal lumen. Our study confirms the previous study of Gerena et al. [12], who, using immunohistochemistry and an antibody raised against mouse recombinant PGDS, reported an increasing gradient of PGDS from the caput to cauda in the mouse epididymal lumen. They also reported the presence of PGDS in the efferent ducts and caput epithelium, which we could not detect. This discrepancy may be explained by differences in sensitivity of the antibodies used and/or immunodetection techniques.
In contrast to PGDS, another major protein of the caudal fluid, the lipocalin mE-RABP, also possesses its highest concentration in the cauda, but the protein is secreted only in the distal caput and accumulates in the lumen of the distal epididymidis during the transit [8, 16]. Theses two proteins, which belong to the same lipocalin superfamily, both accumulate in the cauda epididymidis but display a different pattern of mRNA expression. This suggests the existence of several modes of regulation that control the expression, secretion, and accumulation of epididymal lipocalins.
PGDS Is Present in Rat, Hamster, and Monkey Epididymis
The sequence similarity of PGDS ranges from 70% to 90% among the following species: bovine, equine, murine, human, ovine, and porcine. The antibody used in this study was raised against the N-terminal peptide of the porcine sequence, which includes the conserved GXW-specific lipocalin sequence, explaining in part the cross-reactivity between species.
In the hamster and monkey, as in the mouse, PGDS shows polymorphic isoforms, at least at the molecular weight level, because different molecular weights are observed according to the region (Fig. 10). This change may originate from posttranslational modifications of the secreted protein in the lumen or from secretion under different isoforms according to the region. The latter is the case for clusterin in the stallion [37], which is secreted under isoforms of higher molecular weights in the distal caput than in the proximal caput. Similarly, in the monkey, PGDS presents a higher molecular weight in the corpus than in the caput.
The increasing gradient of PGDS concentration within the epididymis was also found by Western blot analysis in rat and hamster. To our knowledge, PGDS has not been reported previously in the hamster epididymis. In the rat, the mRNA was previously found throughout the organ [20], and the protein was characterized in 2D gel using protein extracts from the caput [21]. One notable difference between the three studied rodents is the presence of PGDS in the initial segment of the rat and hamster, whereas it is absent in the initial segment of the mouse. Another lipocalin, the newly discovered mEP17, is specifically secreted in both the mouse and rat initial segment [17]. Because the main function of the initial segment in rodents is the reabsorption of ions, proteins, and fluid, one would expect a similar pattern of protein secretory activity. Because PGDS is not expressed in the mouse initial segment, this may suggest that PGDS is not involved in the process of reabsorption.
In contrast to PGDS patterns in rodents, epididymal PGDS in domestic mammals such as sheep, horse [22], and bull [23] is more abundant in the proximal part of the organ than in the distal part. Although PGDS was previously detected in human epididymis, its specific regionalization was not reported [24]. In the cynomolgus monkey, PGDS is more abundant (Fig. 10) in the proximal part of the epididymis, and this result is also found in the baboon (data not shown). Because this species is close to human, it would be very interesting to see if PGDS is also more abundant in the proximal part than in the distal part of the human epididymis.
PGDS and Male Reproduction
It is interesting to compare the localization of PGDS in the epididymis and the acquisition of sperm fertilization ability. In domestic mammals such as sheep, horse, and pig, the motility and fertilization ability of spermatozoa (oocyte recognition and fertilization) first appear in the anterior region (distal caput) [38]. In rodents, several studies have shown that fertilization ability is acquired in a more distal part of the epididymis, when the sperm reach the proximal cauda [39]. In all species, the development of sperm fertilizing capacity occurs in a milieu rich in PGDS, which suggests this protein may be involved in the sperm maturation process. Targeted disruption of PGDS in the mouse [40] suppressed tactile pain, but the reproductive performance of the animals was not reported. Previous studies have suggested that PGDS might be a biochemical marker of sperm quality [23, 41], but the biochemical function of PGDS has not been completely elucidated. A recent review characterized the brain PGDS as a bifunctional protein, having both enzymatic and lipocalin (lipophilic ligand-binding) activities [19]. In the male genital tract, although PGDS enzyme activity is detected in seminal plasma, its function in vivo is unclear, because PGD2 is not found in seminal plasma [23]. The PGDS may function as a lipocalin. Tanaka et al. [42] showed that the human recombinant PGDS is able to bind, with high affinity, several molecules such as retinaldehyde, retinoic acid, biliverdin, and bilirubin. The presence of several lipocalins in the epididymal fluid [7, 9, 10, 12] suggests that these proteins have an important function in sperm maturation and/or storage. Furthermore, a protein such as PGDS that is conserved during evolution from rodents to primates is likely to have an essential function. Further studies will be needed to determine whether the biochemical (ligand-binding) and physiological functions of the different epididymal lipocalins are redundant.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Rockefeller/Ernst Shering Foundation and by NIH grants HD 36900 and GM 58008. 
2 Correspondence: Marie-Claire Orgebin-Crist, Center for Reproductive Biology Research, Vanderbilt University, School of Medicine, Medical Center North, Room C3306, Nashville, TN 37232-2633. FAX: 615 343 7797; m-c.orgebin-crist{at}mcmail.vanderbilt.edu
Accepted: September 25, 2001.
Received: May 1, 2001.
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