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a Station de Physiologie de la Reproduction des Mammifères Domestiques, URA INRA-CNRS 1291, 37380 Nouzilly, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Castration and efferent duct ligation in the ram led to a decrease in PGDS mRNA and secretion. PGDS mRNA was not detected in the stallion 1 mo after castration, and it was restored by testosterone supplementation.
This study showed that PGDS is present in the environment of spermatozoa throughout the male genital tract. Its function in the maturation and/or protection of spermatozoa is unknown.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Epididymal fluid proteins have two main origins. Most are synthesized by the epididymal cells and secreted in the lumen. Others originate from the testis and are carried either by the rete testis fluid or by germ cells. Few epididymal proteins have been found to have a blood origin.
It has previously been shown that major epididymal protein secretions are regionalized throughout the organ, and it has been supposed that this specialization of each part of the epididymis (caput, corpus, and cauda) has an important significance for the sequential acquisition of mature sperm properties.
In most species studied, spermatozoa found in the distal corpus are able to fertilize more than 50% of eggs [1], and thus the caput and corpus appear to be key regions for sperm maturation. It was therefore of interest to study their fluid composition in further detail and to elucidate the protein-spermatozoa interactions that occur in these regions. Furthermore, the anterior region has been shown to be the most active in protein secretory activity. In the boar this region secretes 80% of the total luminal proteins secreted by the organ [2].
One of the major proteins secreted by the anterior region of the ram and stallion epididymis was identified in this study as glutathione-independent prostaglandin D2 synthase (PGDS) (prostaglandin-H2 D-isomerase, EC 5.3.99.2), and its regulation was studied. This enzyme was previously described in the central nervous system and male genital tract in men and in rats, but no studies have to date been done on the presence of PGDS in the epididymal milieu surrounding spermatozoa.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dulbecco's Modified Eagle's medium without methionine and cysteine (DMEM-), x-ray films (Kodak X-OMAT-XAR5; Eastman Kodak, Rochester, NY), keyhole limpet hemocyanin (KLH), m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS), orthophenylenediamine dihydrochloride (OPD), 3-([3-cholamidopropyl]dimethylammonio])-1-propanesulfonate, and goat anti-rabbit IgG coupled to horseradish peroxidase were purchased from Sigma Chemical Co. (St. Louis, MO). [35S]Methionine and [35S]cysteine (35S protein-labeling mix, EXPRE35S35S) were purchased from NEN (Les Ulis, France); [
-32P]dCTP from Amersham (Les Ulis, France); acrylamide (30% acrylamide, 0.8% N,N-methylenebisacrylamide) from Millipore (St Quentin, France); ampholytes pH 2–11 (Servalytes) from Serva (Heidelberg, Germany); ampholytes pH 3–10 (Ampholytes), Coomassie brilliant blue (Phastgel Blue R), and electrophoresis calibration kit (standard proteins) from Pharmacia (Saclay, France); and Bradford assay from Bio-Rad (Paris, France). All other chemicals were of molecular biology grade and were purchased from Sigma.
Animals and Organ Sampling
Eight normal adult rams (Ile de France) and eight normal adult horses (Pony) were used in this study. The efferent ducts of one testis of one animal from each species were ligated with sterile braided silk (close to the extratesticular rete testis) for 30 days in the stallion and 45 days in the ram, and the ipsilateral epididymides of these animals were used for controls. For one ram the testis on each side was removed through a scrotal incision, and the epididymis was returned to the scrotum. After 1 mo the two epididymides were sampled. One horse was castrated for 1 mo and one for 15 days before the study period. The 15-day-castrated animal (250 kg) then received i.m. injections of 180 mg of testosterone (Interteston DC, Intervet, France) every 3 days for 15 days.
The epididymis and testis were obtained at castration in the stallions and after slaughter for the rams. The epididymis was subdivided into 10 regions (0–9) as previously described in the ram [3, 4] and in the stallion (Fig. 1). For Northern blot analysis, epididymal and testicular tissues were collected, frozen in liquid nitrogen, and kept at -70°C until analysis. For the protein analysis, the epididymal luminal fluids of the epididymis were obtained by perfusion with a PBS solution as previously described [5]. Rete testis fluids (RTF) were sampled in vitro from the gonad. Semen samples from the stallion and ram were collected via an artificial vagina.
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All samples were centrifuged (1500 x g for 15 min) to remove sperm. The supernatants were centrifuged (15 000 x g for 10 min) and kept at -20°C until use. For in vitro incubation, the samples were incubated as described below.
In Vitro Secretion of [35S]Methionine-Cysteine-Labeled Proteins from Tissue Samples and Isolated Tubules
In vitro secretion of [35S]methionine-cysteine-labeled proteins was estimated from testis and vasa efferentia samples (minced tissues) and all epididymal regions (isolated tubules) according to the methods described previously [5]. Briefly, samples of tissue and closed end-tubules were incubated in 0.5 ml DMEM- in the presence of 100 µCi 35S protein-labeling mix and under 95% O2:5% CO2 at 32°C. After 5 h of incubation, the incubation medium was collected after separation from the tissues, and the lumen fluid of each tubule was collected by perfusion with DMEM solution. The incubation medium and the luminal fluid were then centrifuged (16 000 x g for 10 min), and the supernatants were stored at -20°C until use.
Peptide Synthesis and Antibody Production
A 15-amino acid peptide from porcine PGDS (sequence no. X92979 in GenBank), corresponding to amino acids 27–41 (SLQPNFQEDKFLGRW), was synthesized with a cysteine residue added at the C-terminal (Eurogentec, Seraing, Belgium). This peptide was conjugated with KLH as protein carrier through the cysteine residue, as described by Green et al. [6]. Briefly, 10 mg KLH in 0.92 ml 10 mM sodium phosphate, pH 7.2, was incubated (30 min, room temperature) with 1.2 mg MBS in 0.18 ml dimethylformamide. Excess MBS was removed by gel filtration at 4°C on a Sephadex (Pharmacia and Upjohn, Kalamazoo, MI) G-25 column equilibrated with 50 mM sodium phosphate, pH 6. Peak fractions of KLH-MBS (1 ml total volume) were coupled with 5 mg peptide dissolved in 1 ml of 250 mM sodium phosphate, pH 7.2. Three female rabbits whose preimmune serum was negative against epididymal fluid proteins were immunized with the peptide-KLH conjugates. Four multisite intradermal antigen injections were made at 2-wk intervals. Proteins were diluted in complete Freund's adjuvant for the first injection and in incomplete Freund's adjuvant for the three others. A fifth i.v. injection was made in the ear vein after i.v. injection of 12.5 mg/kg promethazine (Phénergan, Theraplix Rhône-Poulenc, Paris, France). Titration of antibodies against PGDS was determined by ELISA assay with the peptide as antigen. Of the three immunized rabbits, one generated antibodies that recognized PGDS and glutathione peroxidase (GPX) in both the ram and the stallion. Only four contiguous amino acids (-PNFQ-) in the peptide sequence used for the immunization protocol were present both in porcine PGDS and porcine GPX. The antisera of the two other rabbits that specifically recognized PGDS were used in the present study.
Evaluation of Relative Amounts of PGDS by ELISA
Amounts of PGDS in epididymal fluids from different regions were compared in both species by an ELISA method. Proteins from various epididymal fluids (from 200 µg/ml to 100 ng/ml in 50 mM carbonate buffer, pH 9) were coated on ELISA plates. The binding sites of the well were then saturated by incubation with 10% goat serum in TBS (0.05% Tween 20, 50 mM Tris, 200 mM NaCl, pH 7.5). The antiserum (1:10 dilution in TBS) was incubated for 1 h at 37°C. The second antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG) was used at 1:5000 dilution and incubated under the same conditions. The color was revealed by OPD. Epididymal fluids of three animals of each species were compared for a nonsaturated total amount of protein (200 ng); the values of the various epididymal samples were expressed as percentages in relation to the epididymal sample that had the maximum absorbance in each ELISA test.
Gel Electrophoresis
SDS-PAGE separation was carried out for all samples according to the method of Laemmli [7] on gels (14 x 16 cm or 6 x 8 cm) formed with 6–16% linear concentrations of polyacrylamide. The quantities of protein were similar with the electrophoresis separation techniques (one-dimensional [1D] SDS-PAGE: 50 µg; two-dimensional [2D]: 150 µg).
Isoelectric focusing was performed using the O'Farrell technique [8] modified as described previously [9]. The pH gradient was obtained with 1% pH 3–10 ampholytes and 1% pH 2–11 servalytes. Isoelectric focusing was run at 20 mA, 0.1 W/tube, and 700 V for a total of 10 000 V/h followed by 20 mA, 0.1 W/tube, and 3000 V and a total of 2000 V/h. The 2D separation was performed on a 6–16% acrylamide gel (1.5 mm thick) running at 40 mA.
For detection of radioactive proteins, the gels were impregnated with a fluorography enhancer (Amplify; Amersham) after silver staining [10] and then dried and exposed on preflashed x-ray film for several days at -80°C or on a PhosphoImaging screen (Storm; Molecular Dynamics, Paris, France). Quantification of the radioactivity on the gels was assessed by the software program ImageQuant (Molecular Dynamics).
For protein quantification in the fluid, the gels were scanned after Coomassie brilliant blue R250 staining with a Bio-Rad model Gel Doc 1000 imaging densitometer and analyzed with ImageQuant.
Immunodetection
For immunodetection, the proteins from acrylamide gels were electrotransferred (0.8 mA/cm2 for 2 h) by a semi-dry technique on 0.2-µm nitrocellulose membrane (Schleicher et Schuell, Dassel, Germany). The transferred nitrocellulose membranes were stained with Ponceau Red S (Sigma) and incubated overnight with the same blocking solution as used for ELISA at 4°C.
The primary antibody was diluted at 1:2000 in 5% goat serum in TBS and incubated for 1 h at 37°C. Blots were then washed with the same buffer and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG diluted at 1:10 000 in 5% goat serum in TBS for 30 min at 37°C. After several washings, immunoreactive proteins were detected by chemiluminescent substrate (Western Blot Chemiluminescence Reagent Plus; NEN, Boston, MA) according to the manufacturer's instructions.
N-Terminal Amino Acid Sequence Analysis
After the transfer of the proteins to a polyvinylidene difluoride membrane (Hyperbond; Porton Instruments, Tarzana, CA), the membranes were lightly stained with Coomassie Blue R-250 and the protein spots excised. The N-terminal amino acid sequence of the proteins was determined on a Porton sequencer (Model LF3000; Beckman, Palo Alto, CA) using the reagents and methods recommended by the manufacturer. Some spots were submitted to trypsin digestion on the membrane, and the protein fragments were sequenced after separation by HPLC. Similarities between the amino acid sequences obtained and those of other known proteins were sought in GenBank, PIR, EMBL, Genpept, PDB, and SwissProt using BLAST or FASTA software [11].
RNA Extraction
Total RNA was prepared from 200 mg frozen tissue according to the isothiocyanate guanidinium technique described by Chomczynski and Sacchi [12]. RNA was extracted from the testes, efferent ducts, and various epididymal regions. For each total RNA sample, 10 µg was separated by electrophoresis in 1% agarose formaldehyde gel in the presence of RNA markers (Promega, Charbonnières, France). RNA was then transferred to a nylon membrane (Hybond N+; Amersham) by capillary blotting in 10-strength SSC (1.5 M NaCl, 0.15 M trisodium citrate, pH 7) and fixed for 2 h at 80°C. The membrane was stored at -20°C until prehybridization.
DNA Probe and Northern Blot Hybridization
We obtained specific DNA probes for PGDS by reverse transcription-polymerase chain reaction (RT-PCR) using 5 µg total RNA extracted from region 3 (distal caput [13]) of a boar epididymis (adult Large White) as described above. RT was performed by Moloney murine leukemia virus reverse transcriptase (Gibco-BRL, Gaithersburg, MD) with oligo(dT) primers (Pharmacia). The specific reverse-transcript PGDS cDNA was then amplified by DNA polymerase (Goldstar; Eurogentec) with a pair of 23-base primers (reverse primer: 5'-GTC TCA GGT CTC GGG GTG TTG GA-3'; forward primer: 5'-CTG GAG AAG AAG AAG GTG CTG TC-3') corresponding to 484 bases (bases 211–695) of porcine PGDS cDNA. PCR was performed for 30 cycles (94°C, 30 sec; 55°C, 30 sec; 72°C, 30 sec). The DNA obtained was sequenced to verify its identity with PGDS and was then labeled with [-32P]dCTP using the Prime-a-Gene Labeling System from Promega. Hybridization was performed using 32P-labeled probes as described by Thomas [14]. The membrane was prehybridized in 6-strength SSC, 5-strength Denhardt's solution, 0.5% SDS, 100 µg/ml herring sperm DNA, and 50% formamide for 2 h at 42°C. Overnight hybridization to the labeled probe was performed in the same solution but without Denhardt's solution for one night at 42°C. The membrane was washed once in double-strength SSC, 0.1% SDS and twice in 0.10-strength SSC, 0.1% SDS. The PGDS transcript was visualized by exposure on preflashed x-ray film.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A major polymorphic monomeric protein of 27 kDa, pI 4.5–6.5, was present in the middle caput epididymidis (region 2) fluid of the ram. It was composed of at least 8 visible isoforms in silver-stained 2D electrophoresis and was identified as PGDS by N-terminal amino sequencing of two of its spots (Fig. 2A, spots 1 and 2) and by sequencing of several peptides obtained by trypsin digestion of a third spot (Fig. 2A, spot 3). All the sequences were highly homologous to all the PGDS from various species already sequenced; the highest percentage of similarity was found for bovine PGDS [15] (Chart 1). The 8 isoforms of the protein found in the anterior region were recognized by the polyclonal antibody obtained against a PGDS peptide deduced from the porcine sequence (Fig. 2C). The protein was detected by Western blotting in testicular fluid and throughout the epididymis (Fig. 3A). Molecular isoforms of PGDS were characterized by immunoblots from 2D gel electrophoresis (Fig. 2, B–E). PGDS was represented by 5 main acidic isoforms in the RTF, with a molecular mass of 30 kDa and a range of pI 4.2–5.5 (Fig. 2B). Eight spots were detected by Western blotting in the caput epididymal fluid, with a molecular mass of 27 kDa and a pI range of 4.5–6.5 (Fig. 2C); in contrast, in the cauda (Fig. 2D) and in the ejaculate (Fig. 2E), only six spots were found, with a pI range of 5–7 and a molecular mass decrease of about 5 kDa. The highest concentration of PGDS was observed in the anterior part of the epididymis as judged by protein staining intensity on electrophoretic gels (Fig. 4A). PGDS was 4 and 2.5 times lower by ELISA assay (Fig. 3B) in, respectively, rete testis and cauda fluids than in the caput fluid for the same amount of total luminal proteins. In the proximal epididymal caput (regions 0–1), this protein represented 15% of the total proteins present in the lumen.
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In the stallion, PGDS was immunologically detected in fluids from all parts of the epididymis (Fig. 5A and Fig. 6, A, C, and D), from the rete testis (Fig. 6B), and in seminal plasma (Fig. 6E). The N-terminal amino acid could not be directly sequenced (Fig. 6A, spot 1) and was probably blocked for the Edman reaction. After trypsin digestion of one of its spots (Fig. 6A, spot 2), the amino acid sequence obtained from peptides of the protein confirmed a high degree of homology and similarity to bovine PGDS (Chart 1). In the RTF, PGDS was represented by three acidic spots with a molecular mass of 30 kDa and a pI range of 5–5.8 (Fig. 6B), whereas the luminal epididymal protein comprised seven main spots in the proximal caput, with the same molecular mass and a range of pI 4.5–7.2 (Fig. 6, A and C). A reduction in molecular mass of about 5 kDa appeared for the most basic isoforms of the protein during epididymal transit (Fig. 6D). PGDS was represented by two spots of 27 kDa and pI of 5.8–6.2 in the ejaculate (Fig. 6E).
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The PGDS concentration was highest in the fluid from the anterior region of the epididymis (Fig. 7A) and decreased gradually to the cauda epididymidis, the concentration of PGDS in the caput being 5 times that in the cauda epididymidis (Fig. 5B). In regions 1–2, PGDS represented 8% of the total proteins found in the lumen.
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Secretion and mRNA Expression of PGDS in Testis, Vasa Efferentia, and Epididymis
The proteins secreted by isolated epididymal tubules, as well as PGDS mRNA expression, were analyzed to determine the origin of PGDS in epididymal fluids. In both the ram and stallion, PGDS was secreted only in the proximal caput epididymidis (Fig. 4B for the ram and Fig. 7B for the stallion). PGDS represented 25% of the total amount of proteins secreted in the proximal caput in the ram and 15% in the stallion.
PGDS isoforms secreted by the caput epididymal epithelium in the ram (Fig. 8A) were identical to the PGDS detected in the luminal fluid by silver staining (Figs. 2A and 8B). In the stallion, only the most basic forms were intensively secreted by the vasa efferentia epithelium (Fig. 9B) and proximal caput epithelium (Fig. 9C); the most acidic forms of PGDS found in the caput fluid (Fig. 9D) were secreted intensively only in the testis (Fig. 9A).
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A 0.8-kilobase mRNA was detected only in the proximal caput epididymidis of both the ram (regions 0–1) (Fig. 4C) and the stallion (regions 0–2) (Fig. 7C). Testicular PGDS transcripts were found in stallions (not shown), but we were not able to detect signals in any rams. PGDS mRNA could be detected in the efferent ducts of all the horses studied, whereas the transcript was detected in only half the rams.
Regulation of the Expression and Synthesis of PGDS in the Vasa Efferentia and Epididymis
Regulation of PGDS expression by testicular factors and testosterone was studied with ipsilateral-ligated efferent ducts and with castrated and castrated-testosterone-supplemented animals.
Ligation for 45 days and castration for 30 days induced a decrease in mRNA expression in the ram (Fig. 10). The protein secreted and detectable in the fluid of the proximal caput presented only the most basic isoforms found in the normal ram (Fig. 8, E and F).
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In the stallion, ligation induced the disappearance of mRNA in the efferent ducts but did not affect the level of PGDS mRNA in the proximal caput (Fig. 11, A and B) or its secretion (not shown). After 1 mo of castration, PGDS mRNA expression disappeared from the epididymal proximal caput (Fig. 11C), and the protein was no longer detected by Western blotting in epididymal fluids (not shown). Messenger RNA expression (Fig. 11D) and protein secretion (not shown) reappeared in the caput epididymidis after 15 days of injection of testosterone.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,11-epidioxy-15-hydroxyprosta-5,13-dienoate-D-isomerase, EC 5.3.99.2). We identified this compound by microsequencing the protein and by using polyclonal antisera obtained from a peptide deduced from the cDNA sequence of pig PGDS. This enzyme, which was previously identified in human cerebrospinal fluid as ß-trace protein [16], catalyses the isomerization of prostaglandin (PG) H2 to PGD2, a major primary prostaglandin present in most tissues [17]. This compound has also been detected immunologically in human seminal plasma as ß-trace [18], and it was recently confirmed to be PGDS [19]. This protein has been also detected immunologically in the cauda epididymal fluid and semen of the bull [15].
In the present study, the protein was detected by Western blotting in rams and stallions throughout the epididymal tubule. PGDS appeared as a polymorphic and monomeric 22- to 30-kDa molecule with a variable pI range according to the fluid origin and species. The origin of PGDS in the epididymal lumen was mostly related to synthesis and secretion processes by the epithelium of the tubule. The secretory activity was highly regionalized and was localized only in the initial segment (regions 0–1) of the ram epididymis and regions 0–2 (proximal and distal caput) of the stallion epididymis. Expression of mRNA for PGDS in epididymal tissues was detected only in the caput epididymal region where the protein was secreted. The secretion of PGDS in both the ram and horse was among the highest in this region (around 20%), and PGDS was a major protein of the epididymis, accounting for nearly 8% of all proteins secreted by the epididymis. In both species, the highest amount of PGDS was found in the fluid of the anterior region (around 10%). This protein could be considered as a good marker of the secretory activity of the anterior region, as also proposed for GPX [2].
The amount of PGDS progressively decreased during epididymal transit and reached a value 5-fold lower in the caudal fluid compared to the caput. This decrease could be linked in part to resorption of this protein by the epithelium, as has been described for other proteins such as clusterin and GPX [2]. The decrease could also be the result of partial degradation of the molecule in view of the progressive loss of its mass. Furthermore, the pI of PGDS isoforms in the ram became more basic in the cauda than in the caput. These modifications are probably linked to changes in the carbohydrate part of this glycoprotein that possesses two potential N-glycosylation sites [20–22].
The presence of PGDS in ram and stallion epididymal fluids was related not only to epididymal activity but also to testicular activity, as it was detected in the RTF. This protein has also been found in the RTF of the bull [15]. However, testicular PGDS has more acidic isoforms than the epididymal protein. As only one glutathione-independent PGDS gene has been observed to date [23], this polymorphism is probably due to differential degrees of glycosylation of the protein between tissues. Variations in the degree of glycosylation of PGDS have been found between blood and brain PGDS (ß-trace protein) in humans [24,25]. Such variations in protein glycosylation between the testis and the epididymis have also been found for other proteins secreted by the two organs, such as clusterin. As for PGDS, the isoforms of testicular clusterin are more acidic than the epididymal isoforms [2, 26]. In the stallion, the acidic isoforms of PGDS found in the first part of the epididymis were not found to be secreted by the epididymal epithelium, and they corresponded to the acidic testicular PGDS secretion that was not reabsorbed. Indeed, these isoforms were not observed in the caput epididymal fluid when the testicular fluid was prevented from entering the epididymis by efferent duct ligation. In the ram, the acidic testicular form was not found in the anterior part of the epididymis; it was probably all resorbed. Expression of PGDS mRNA (ß-trace mRNA) in the testis has been detected in Leydig cells in the mouse [27] and in cells within the seminiferous tubules in men [28]. A germ cell origin of PGDS is highly improbable, since PGDS is still found in the RTF of azoospermic rams (unpublished results).
The origin of PGDS in the seminal plasma of the ram was the epididymis, since no PGDS mRNA was detected in the seminal vesicle tissue and the protein was not detectable in seminal vesicle secretions or in the seminal plasma of vas deferens-ligated rams (data not shown).
The regulation of expression and synthesis of PGDS in the anterior part of the epididymis is not dependent on testicular factors, since efferent duct ligation did not abolish mRNA expression or protein synthesis in the caput epididymidis in either species studied. The reduction in PGDS expression and synthesis observed in the anterior caput in the ligated ram, and its disappearance from the efferent ducts of the ligated stallion, were probably a consequence of the general reduction in epithelial activities due to the suppression of testicular fluid.
The sensitivity to androgen is species dependent, since PGDS expression was still detectable in the castrated ram but not in the castrated stallion, where androgen administration completely restored PGDS secretion. However, the synthesis of PGDS was reduced and abnormal in castrated rams, since only the most basic isoforms were secreted. Furthermore, no modification of the protein was observed during epididymal transit.
The role of PGDS in all the fluids of the genital tract is uncertain. No information is available in vivo about enzyme activity of the secreted luminal PGDS. However, PGD2 can be formed in vitro by glutathione-independent PGDS in rat epididymal tissue homogenates [17], and thus it is probable that PGDS is potentially active in the epididymis. Bull testicular, epididymal, and seminal isoforms have been found to be capable of transforming PGH2 to PGD2 in the fluids after dilution [15]. However, the authors of this work suggested that an unknown inhibitor of PGDS may exist in vivo, at least in bull seminal plasma. Furthermore, the structural modifications observed in the ram and stallion when the protein passes through the epididymis (loss of about 5 kDa in both species and an increase in pI in the ram) may also alter the properties of this enzyme, as has been shown for other epididymal enzymes such as gamma-glutamyl transpeptidase during transit [29].
The substrate of PGDS, PGH2, is produced from arachidonic acid by cyclooxygenases, also known as prostaglandin G/H synthases, that have been described in at least two isoforms, termed COX-1 and COX-2. No information is available about the presence or concentration of PGH2 in either the RTF or the epididymal fluid. However, prostaglandin G/H synthase has been localized in the epithelium of the anterior part of the mouse epididymis [30], suggesting that PGH2 should be formed by the epididymal epithelium in the region where PGDS is secreted. Another potential source of PGH2 may be the spermatozoa themselves, since cyclooxygenases have also been localized in the male gamete [31–33]. The hypothesis of a sperm PGH2 origin is reinforced by the fact that arachidonic acid (the substrate of COX activity) can be released from sperm membranes by phospholipase A2 during the loss of phospholipids as the gametes transit the epididymis [34]. These hypotheses have to be investigated.
PGDS is the only one of the lipocalins that is associated with enzyme activity. The lipocalin family consists of various small secretory proteins that share a common feature of binding and transport of lipophilic molecules [35]. PGDS may act as a transporter of lipophilic substances, including prostaglandins. Moreover, PGDS was suggested to be a retinoid transporter with an affinity for retinoic acid as great as that of retinoic acid-binding protein [36]. However, in the epididymis this function is more probably performed by the epididymal retinoic acid-binding protein that is secreted from the corpus to the cauda epididymidis in mammals [2, 37, 38].
Another role in the maturation and maintenance of blood-tissue barriers has been proposed for PGDS, which is highly and specifically expressed in fluids protected by a blood-tissue barrier such as cerebrospinal fluid and male genital excurrent duct fluids [27].
The relationship between PGDS and the physiology of spermatozoa is unknown, but a relationship between this protein and the fertility of animals has recently been described [15]. This protein does not seem to bind to sperm membranes with high affinity, since it has not been detected in sperm membrane extracts obtained after sperm washing (personal results). However, this lack of sperm binding does not exclude potentially low-affinity interactions that could occur in the presence of the high concentration of PGDS in the caput epididymal fluid.
In fact, the wide distribution of this enzyme in the genital tract more probably has a continuous effect on the gamete, or on its surroundings, than a regionalized action on the sperm, as appears to be the case for epididymal sperm maturation processes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 33 02 47 42 77 43; jdacheux{at}tours.inra.fr
Accepted: October 5, 1998.
Received: July 22, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1-acid glycoprotein and serum retinol-binding protein and its relatives. FASEB J 1987; 1:209–214.[Abstract]-2-microglobulin superfamily. Biochem Int J 1987; 14:235–240.This article has been cited by other articles:
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  A. A. Moura, D. A. Chapman, H. Koc, and G. J. Killian Proteins of the Cauda Epididymal Fluid Associated With Fertility of Mature Dairy Bulls J Androl, July 1, 2006; 27(4): 534 - 541. [Abstract] [Full Text] [PDF]
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 J.-L. Gatti, S. Metayer, M. Belghazi, F. Dacheux, and J.-L. Dacheux Identification, Proteomic Profiling, and Origin of Ram Epididymal Fluid Exosome-Like Vesicles Biol Reprod, June 1, 2005; 72(6): 1452 - 1465. [Abstract] [Full Text] [PDF]
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S. Castella, S. Fouchecourt, A. P. Teixeira-Gomes, J. Vinh, M. Belghazi, F. Dacheux, and J.-L. Dacheux Identification of a Member of a New RNase A Family Specifically Secreted by Epididymal Caput Epithelium Biol Reprod, February 1, 2004; 70(2): 319 - 328. [Abstract] [Full Text] [PDF]
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S. Fouchecourt, S. Castella, F. Dacheux, and J.-L. Dacheux Prostaglandin D2 Synthase Secreted in the Caput Epididymidis Displays Spatial and Temporal Delay Between Messenger RNA and Protein Expression During Postnatal Development Biol Reprod, January 1, 2003; 68(1): 174 - 179. [Abstract] [Full Text] [PDF]
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S. Metayer, F. Dacheux, J.-L. Dacheux, and J.-L. Gatti Comparison, Characterization, and Identification of Proteases and Protease Inhibitors in Epididymal Fluids of Domestic Mammals. Matrix Metalloproteinases Are Major Fluid Gelatinases Biol Reprod, May 1, 2002; 66(5): 1219 - 1229. [Abstract] [Full Text]
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S. Fouchecourt, G. Charpigny, P. Reinaud, P. Dumont, and J.-L. Dacheux Mammalian Lipocalin-Type Prostaglandin D2 Synthase in the Fluids of the Male Genital Tract: Putative Biochemical and Physiological Functions Biol Reprod, February 1, 2002; 66(2): 458 - 467. [Abstract] [Full Text] [PDF]
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S. Fouchecourt, P. Chaurand, B. B. DaGue, J.-J. Lareyre, R. J. Matusik, R. M. Caprioli, and M.-C. Orgebin-Crist Epididymal Lipocalin-Type Prostaglandin D2 Synthase: Identification Using Mass Spectrometry, Messenger RNA Localization, and Immunodetection in Mouse, Rat, Hamster, and Monkey Biol Reprod, February 1, 2002; 66(2): 524 - 533. [Abstract] [Full Text] [PDF]
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S. Fouchécourt, S. Métayer, A. Locatelli, F. Dacheux, and J.-L. Dacheux Stallion Epididymal Fluid Proteome: Qualitative and Quantitative Characterization; Secretion and Dynamic Changes of Major Proteins Biol Reprod, June 1, 2000; 62(6): 1790 - 1803. [Abstract] [Full Text]
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R.L. Gerena, D. Irikura, N. Eguchi, Y. Urade, and G.J. Killian Immunocytochemical Localization of Lipocalin-Type Prostaglandin D Synthase in the Bull Testis and Epididymis and on Ejaculated Sperm Biol Reprod, March 1, 2000; 62(3): 547 - 556. [Abstract] [Full Text]
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