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Biology of Reproduction 59, 843-853 (1998)
©Copyright 1998 Society for the Study of Reproduction, Inc.

Rat Prostaglandin D2 Synthetase: Its Tissue Distribution, Changes during Maturation, and Regulation in the Testis and Epididymis1

Claudio Sorrentinoa, Bruno Silvestrinib, Laura Braghirolia, Sanny S.W. Chunga, Sabrina Giacomellib, Maria-Grazia Leoneb, Yan-bo Xiea, Ya-ping Suia, Meng-yun Moa, , and C. Yan Cheng2,a

a The Population Council, New York, New York 10021 b Institute of Pharmacology and Pharmacognosy, University of Rome "La Sapienza", Rome, Italy


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The changes in glutathione-independent prostaglandin D2 synthetase (PGD-S) during maturation in the rat were determined in selected organs by an RIA using PGD-S purified from rat cerebrospinal fluid and a monospecific anti-rat PGD-S polyclonal antibody. In a survey of its tissue distribution in various organ extracts and biological fluids, it was found that the concentration of PGD-S was highest in the epididymis—about 6- and 80-fold greater than that in the brain and testis, respectively. During maturation, PGD-S concentration increased steadily in the testis and epididymis; this is in contrast to the pattern of changes in the brain and liver, which showed a general trend of decline. Reverse transcription-polymerase chain reaction and Southern blotting were used to demonstrate the presence of PGD-S mRNA transcript in the testis and in Sertoli and germ cells. In the epididymis, the steady-state PGD-S mRNA level was highest in the caput, followed by the cauda and corpus. Orchiectomy induced a drastic reduction of PGD-S concentration in all three epididymal compartments. Administration of dihydrotestosterone (DHT) failed to restore the reduced epididymal PGD-S level except in the caput epididymis, where 4 days after DHT treatment the level of PGD-S was restored to about 50% of the pre-orchiectomized level; this suggests that the epididymal PGD-S level is not entirely regulated by androgen and that another yet to be identified testicular factor(s) is likely to be involved in its regulation. Germ cell-conditioned medium was also shown to stimulate PGD-S expression in the Sertoli cell. These results illustrate that PGD-S is an important molecule in testicular and epididymal function and that it is likely involved in spermatogenesis and sperm maturation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glutathione-independent prostaglandin (PG) D2 synthetase (prostaglandin H2 D-isomerase, EC 5.3.99.2; PGD-S) is the enzyme that catalyzes the conversion of the cyclooxygenase-derived intermediate PGH2 to PGD2 in the presence of sulfhydryl compounds. However, under different conditions, PGH2 can also be converted to PGD2 by glutathione-dependent PGD synthetase, glutathione-S-transferase, and even albumin (for review, see [1]). Besides being investigated under its own name, PGD-S was also independently studied as cerberin 30 and ß-trace protein before their identification as PGD-S [2–5]. PGD2 is known to be involved in diversified physiological functions such as thermoregulation, sleep induction and sedation, release of pituitary gonadotropins, and smooth muscle contraction and relaxation (for reviews, see [6–8]). Furthermore, PGD2 is the major PG in the brain, where it acts both as a neuromodulator and as a tropic factor. PGD-S, or its enzymatic activity, has been detected in many mammalian organs and biological fluids (for reviews, see [6–8]). In particular, the mRNA encoding for PGD-S is localized mainly in epithelia or near the blood-tissue barriers [9]. Using in situ hybridization, a specific temporal expression pattern of PGD-S mRNA has been detected in mice during pre- and postnatal development at sites suggestive of its potential role in the transport and/or maturation and maintenance of the blood-organ barriers [9]. Similar to other PG synthetases, PGD-S is found in the rough endoplasmic reticulum and in the outer nuclear membrane; however, PGD-S is also detected in the cerebrospinal fluid (CSF), interphotoreceptor matrix, seminal fluid, aqueous humor, amniotic fluid, and other biological fluids (for reviews, see [6, 7]). More importantly, PGD-S constitutes more than 10% of the total protein in the CSF and is the secretory product of astrocytes, illustrating that it also exists in a nonmembrane-bound soluble form [10].

When the primary sequence of PGD-S was determined, it was shown to be a member of the lipocalin family, which is composed of secretory proteins that bind and transport small lipophilic molecules [11, 12]. There is growing evidence suggesting that lipocalins may also be involved in other functions such as regulation of immune response and mediation of cellular homeostasis (for review, see [11]). As such, PGD-S may exert other physiological function(s) in addition to its enzymatic activity. In fact, PGH2, its highly reactive substrate, is very unstable; and if PGD-S indeed solely acts as an enzyme, it would be expected to be localized exclusively in the proximity of cyclooxygenase, close to the production site of PGH2. On the contrary, as mentioned above, PGD-S is found in a soluble form at high concentrations in other compartments, such as the CSF [4, 5, 10], where a constant supply of PGH2 is unlikely to be available. PGD-S has recently been shown to be a bifunctional molecule [12]. In addition to its enzymatic activity, PGD-S binds retinal and retinoic acid in vitro with affinities similar to those of other retinoid transporters, such as retinoic acid-binding protein; and the binding of retinoic acid exerts a noncompetitive inhibition of PGD-S enzymatic activity, revealing the presence of two biologically active sites on PGD-S [12]. An immunofluorometric assay has also been established quantifying PGD-S in various human tissue extracts and fluids [13]. Nonetheless, the precise PGD-S concentration, as well as its expression and regulation in the male reproductive system, is currently unknown. Thus we thought it pertinent to 1) establish a specific RIA to quantify PGD-S in selected reproductive organs in the rat and its changes during postnatal development, 2) produce recombinant human (rh) PGD-S to allow its possible use for other physiological and pharmacological studies, 3) assess the cellular distribution of PGD-S in the seminiferous epithelium and its level in various compartments of the epididymis, and 4) partially characterize the regulation of PGD-S in the testis and epididymis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents

PMSF, 3-[N-morpholino]propanesulfonic acid (MOPS), dihydrotestosterone (DHT), reduced glutathione, Ham's F-12 nutrient mixture/Dulbecco's Modified Eagle's medium (F12/DMEM; 1:1, v:v), salmon testis DNA, and tRNA were obtained from Sigma Chemical Company (St. Louis, MO). Grace's insect medium was purchased from Invitrogen (Carlsbad, CA). Metofane was purchased from Mallinckrodt Veterinary (Mundelein, IL). Formalin-fixed Staphylococcus aureus (Sa) cells were from Gibco BRL Life Technologies (Gaithersburg, MD). Restriction enzymes such as BamHI, EcoRI, Nco I, and Not I were from Boehringer-Mannheim (Indianapolis, IN). Thrombin was from Calbiochem (San Diego, CA); RNA STAT 60, from Tel-Test "B", Inc. (Friendswood, TX). All reagents used for reverse transcription-polymerase chain reaction (RT-PCR), 5' end-labeling, and nick translation were obtained from Promega (Madison, WI). [{alpha}-32P]dCTP (specific activity, 3000 Ci/mmol) and [{gamma}-32P]ATP (specific activity, 6000 Ci/mmol) were from Amersham (Arlington Heights, IL).

Animals

The use of animals for the experiments described in this paper was approved by the Rockefeller University Institutional Animal Care and Use Committee with protocol numbers 94132 and 97117. Male Sprague-Dawley rats between 10 and 120 days of age or adult rats (250–300 g BW) were obtained from Charles River (Kingston, MA). Blood was collected from animals by cardiac puncture under light Metofane anesthesia and allowed to clot overnight at 4°C, and serum was obtained by centrifugation at 2500 x g for 10 min at 4°C. Rats were killed by CO2 asphyxiation, and CSF was immediately collected as previously described [2, 3] by cannulation of the cisterna magna using a 25-gauge, 5/8-inch hypodermic needle attached to a 1-ml syringe. Routinely, 50–100 µl of CSF was collected from each adult rat (250–300 g BW). CSF were centrifuged at 15 000 x g for 5 min to remove cellular debris and stored at -20°C until use. Organs were then promptly removed and used for cytosol preparation. Brain, liver, kidney, testes, epididymides, ventral prostate, and seminal vesicles were homogenized by a Brinkman polytron homogenizer (Brinkman Instruments Co., Westbury, NY) in TG buffer (10 mM Tris, pH 7.4, at 22°C, containing 10% glycerol v:v, 1 mM EDTA, and 1 mM PMSF) with an organ-to-buffer ratio of 1:3 for brain, liver, and kidney and 1:10 for testis, epididymis, seminal vesicle, and ventral prostate. The homogenates were centrifuged at 45 000 x g for 1 h at 4°C. The clear supernatants were used as cytosols and stored at -20°C until use. In some experiments the epididymis was dissected into three anatomical compartments, namely, caput, corpus, and cauda [14, 15], and homogenized separately. To determine the effect of castration and androgen replacement on PGD-S production, adult male rats were orchiectomized as previously described [16]; on Day 3 after orchiectomy, some rats were injected s.c. with 5 mg DHT per rat daily for 5 consecutive days. DHT was suspended in corn oil. Control rats received the same volume of corn oil alone. Rats from each group were killed daily, and selected organs were removed for cytosol extraction.

Purification of PGD-S from Rat CSF

PGD-S was purified from rat CSF by HPLC as previously described [2, 3] with minor modifications. Briefly, after an initial Vydac C8 (4.6 x 250 mm, i.d.; Separations Group, Hesperia, CA) HPLC step, fractions containing PGD-S were pooled, lyophilized, resuspended in 500 µl of solvent A (5% acetonitrile/95% H2O, containing 0.1% trifluoroacetic acid v:v), and further purified by a Vydac C4 (4.6 x 250 mm, i.d.). Fractions from this Vydac C4 step containing PGD-S were pooled, lyophilized, resuspended in PBS (10 mM sodium phosphate, 150 mM NaCl, pH 7.4 at 22°C), and concentrated to about 100 µl using an Amicon (Danvers, MA) Microcon-10 ultrafiltration unit. PGD-S in this sample was then purified to apparent homogeneity using a Pharmacia Superose 12 gel-permeation HPLC column (3.2 x 300 mm, i.d.; Pharmacia Biotech, Piscataway, NJ). Proteins were eluted under isocratic conditions with PBS at a flow rate of 40 µl/min. The purity of the protein was assessed by SDS-PAGE [17] and silver staining [18].

Calibration of Anti-Rat PGD-S Antiserum for RIA

To assess the optimal dilution of antiserum to be used for RIA, serial dilutions of antiserum (from 1:100 to 1:5000) in RIA buffer (PBS containing 0.5% BSA, w:v) to a final volume of 100 µl were prepared. Each of these samples was incubated with a constant amount of tracer (about 15 000 cpm of [125I]PGD-S in 100 µl) and 300 µl RIA buffer, in a final volume of 500 µl/assay tube for 36 h at 4°C. Thereafter, 25 µl of a suspension of Sa cells, previously washed three times with Sa cell buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.4 at 22°C, containing 0.5% Nonidet P-40 and 0.2% BSA w:v), was added to each tube and incubated for an additional 1 h at 4°C. Samples were then centrifuged at 2500 x g for 20 min; supernatants were aspirated, and radioactivity in pellets was determined by spectrophotometry in a Packard (Meriden, CT) Cobra II gamma counter. A working dilution of 1:300 was selected, which yielded a 30% binding.

Standards for RIA

Because of the limited availability of purified PGD-S, a pool of rat CSF (CSF001STD) was used for calibration of the standard curve. Each standard was run in triplicate at serial dilutions ranging from 0.001 to 15 µl. When calibrated against a batch of highly purified rat PGD-S in which the protein concentration was estimated by Coomassie blue dye-binding assay [19]), 1 µl of CSF001STD was found to contain 50.4 ng PGD-S. In that report, it was shown that the recombinant PGD-S could be used for calibration of standard curves to substitute for crude CSF.

PGD-S RIA

To quantify PGD-S in unknown sample, each assay tube contained 200 µl of RIA buffer, 100 µl standard or sample diluted in RIA buffer, 100 µl of antiserum diluted 1:300, and 100 µl [125I]PGD-S (about 15 000 cpm) to give a final volume of 500 µl. The assay mixture was then vortexed and incubated for 36 h at 4°C. Thereafter, 25 µl of the Sa cell suspension was added to each test tube, incubated for 1 h at 4°C, and then centrifuged at 2500 x g for 20 min at 4°C. The supernatant was aspirated, and radioactivity in the pellet was determined. Each standard or sample was run in triplicate. The intraassay and interassay variations were 10% and 12%, respectively. The minimal detectable dose was 0.05 ng PGD-S, and 50% displacement was at 3.5 ng. All data were analyzed using a computer program adapted from Rodbard et al. [20].

Production of rhPGD-S

Recombinant human PGD-S was produced in a baculovirus-based in vitro expression system using a BaculoGold Transfection kit from Pharmingen (San Diego, CA). Briefly, two specific primers of human PGD-S [21] were prepared as follows: 5'-CCGCTCGGATCCAGGAGAATGGCTACTCAT-3' (sense primer, nucleotides 57–86, with a mutated BamHI site underlined) and 5'-CCCTGGGAATTCCTATTGTTCCGTCATGCA (antisense primer, nucleotides 630–659, with a mutated EcoRI site underlined). These primers were used to synthesize a full-length human PGD-S cDNA by PCR (30 cycles: denaturation at 94°C, 1 min; annealing at 58°C, 2 min; and extension at 72°C, 3 min) from a human fetal brain cDNA expression library (Clontech, Palo Alto, CA), using procedures detailed elsewhere [22, 23]. This PGD-S clone contained a BamHI and an EcoRI site in the 5' and the 3' end, respectively, for cloning into the pAcG2T baculovirus transfer vector (Pharmingen). The PGD-S cDNA was in frame with the pAcG2T to ensure a correct GST fusion product. This construct was cotransfected with the BaculoGold DNA (Pharmingen) into Spodoptera furgiperda (Sf)-9 cells. After 3 days, supernatant was collected and the virus containing the rhPGD-S clone was purified by dot-blot hybridization as described previously [24]. After amplification of the selected recombinant virus, titer was determined by end-point dilution assay. The 50% tissue culture infectious dose was calculated by Reed and Muench method [25] and converted to plaque-forming units/ml. To produce the rhPGD-S/GST fusion protein, Sf-9 cells (5 x 106) were cultured with 10 ml of Grace's insect medium containing 10% fetal bovine serum and 5% gentamicin sulfate (w:v) onto T 75 flasks at 27°C. When cells were about 80% confluent, medium was changed and an aliquot of the recombinant virus stock was added to each flask at a multiplicity of infection = 1. After 3–4 days, according to signs of infection, culture media and cells were harvested and separated by centrifugation at 800 x g for 5 min. Supernatant was concentrated by ultrafiltration, using an Amicon 8050 unit, to about 5 ml, and rhPGD-S conjugated with GST was purified by affinity chromatography by Glutathione Sepharose-4B (Pharmacia Biotech). Briefly, concentrated supernatant derived from Sf-9 cells was loaded onto the Glutathione Sepharose-4B column (15 x 65 mm, i.d.); the column was washed with 100 ml of PBS, and rhPGD-S/GST was then eluted with a buffer of 50 mM Tris containing 0.1% SDS (w:v), 0.1% Triton X-100 (v:v), and 20 mM reduced glutathione (pH 8.0 at 22°C) under gravity. Sf-9 cell supernatant (starting material), nonbound fractions (washout), and eluted rhPGD-S/GST were analyzed by SDS-PAGE on 12.5% T SDS-polyacrylamide gel under reducing conditions, and proteins were visualized by Coomassie blue staining and immunoblot. To remove the GST tag from rhPGD-S, 50-µl aliquots of the eluted protein (about 0.3 mg protein) were incubated with 0.01, 0.05, and 0.1 mg thrombin at 37°C for 8 h in 50 mM sodium citrate, 200 mM NaCl (pH 6.5) at 22°C (cleavage buffer) to a final volume of 100 µl. The cleaved PGD-S was then analyzed by immunoblot.

Preparation of Germ Cell Primary Cultures and Germ Cell-Conditioned Medium (GCCM)

Germ cells were isolated from adult Sprague-Dawley rats (250–300 g BW) by a mechanical procedure [26]. Total germ cells were cultured at 22.5 x 106 cells/9 ml in 100-mm Petri dishes in F12/DMEM containing gentamycin (20 µg/ml), sodium lactate (6 mM), sodium pyruvate (2 mM), and bacitracin (10 µg/ml). Cells were then incubated in a humidified atmosphere of 95% air:5% CO2 (v:v) at 35°C. To obtain GCCM, media from germ cell cultures were collected after 18 h of culture and centrifuged at 300 x g for 60 min at 4°C. GCCM was then concentrated in a Millipore (Bedford, MA) tangential ultrafiltration unit equipped with eight Minitan plates with a molecular weight cutoff at10 000.

Preparation of Sertoli Cell Primary Cultures and Sertoli Cell-Conditioned Medium

Primary Sertoli cell-enriched cultures and Sertoli cell-conditioned medium were prepared from testes of 20-day-old Sprague-Dawley rats as described previously [27, 28]. Sertoli cells at 4.5 x 106 cells/9 ml were plated in 100-mm Petri dishes in F12/DMEM containing sodium bicarbonate (1.2 g/L), 15 mM Hepes, 20 µg/ml gentamicin, 10 µg/ml insulin, 5 µg/ml transferrin, 5 µg/ml bacitracin, and 2.5 ng/ml epidermal growth factor. Cells were then cultured at 35°C in a humidified atmosphere of 95% air:5% CO2. After 36 h, cultures were hypotonically treated with 20 mM Tris, pH 7.5, at 22°C for 2.5 min to lyse contaminating germ cells [29]. In some experiments, Sertoli cells were cultured for an additional 18 h in the absence (control) or presence of GCCM using 0.05, 0.5, and 1.75 mg total protein per dish.

Sequential RT-PCR and Southern and Northern Blot Analysis

The presence of PGD-S mRNA in selected organs and cell types was assessed by RT-PCR and Southern blots as previously described [23, 28, 30]. Briefly, total RNAs were extracted from selected organs and primary cell cultures by the acid guanidinium thiocyanate-phenol-chloroform extraction method [31] using RNA STAT 60. Total RNA concentration in the samples was quantified by spectrophotometry at 260 nm. RNAs were reverse transcribed to cDNAs by avian myeloblastosis virus-reverse transcriptase. These cDNAs were then used as templates for PCR and amplified with two rat PGD-S primers [32]: 5'-CATGACACAGTGCAGCCC-3' (sense primer, nucleotides 113–130) and 5'-GTCGAACAGGAACGCGTA-3' (antisense primer, nucleotides 413–430). The identity of brain, epididymal, and testicular PGD-S cDNAs obtained by RT-PCR was confirmed by direct nucleotide sequencing using Sequenase (Amersham) as previously described [33]. In some experiments, the authenticity of the RT-PCR products was verified by Southern blots. Briefly, an internal oligonucleotide of 5'-GTGGTAGCTCCCTCCACAGAA-3' (sense primer, nucleotides 236–256) was 5' end-labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase and used as a probe for hybridization. Northern blots were performed [23, 30] with a gel-purified PGD-S cDNA probe of 318 base pairs (bp), which was obtained as described above and radiolabeled with [{alpha}-32P]dCTP by nick translation.

RT-"Hot-Nested" PCR

To study the regulation of the Sertoli cell PGD-S expression by germ cells, RT-"hot-nested" PCR was performed using total RNA extracted from Sertoli cells cultured alone (control) or cultured in the presence of different concentrations of GCCM. Samples were reverse transcribed and used as templates for PCR. ß-Actin was coamplified with PGD-S and used as an internal control. In all experiments, amplifications of both PGD-S and ß-actin cDNAs were in the linear range as determined by removing aliquots of samples at different amplification cycles between 15 and 35. To enhance the sensitivity and to yield results for densitometric scanning analysis, PCR was performed in which the sense primer was 5' end-labeled with [{gamma}-32P]ATP by T4 polynucleotide kinase. The two primers used for amplifying the ß-actin mRNA [34] were 5'-TCACCGAGGCCCCTCTGAACCCTA-3' (sense primer, nucleotides 314–337) and 5'-GGCAGTAATCTCCTTCTGCATCCT-3' (antisense primer, nucleotides 931–954). PCR was performed as detailed elsewhere [22, 23]. Briefly, the cycling parameters were performed as follows: 94°C for 1 min, annealing at 59°C for 2 min, and extension at 72°C for 15 min. A total of 20–25 cycles were performed. An aliquot of 10 µl from each sample tube was resolved onto a 5% T polyacrylamide gel, and the 318-bp PGD-S cDNA and the 641-bp ß-actin cDNA were visualized by autoradiography using x-ray films. Autoradiographs were densitometrically scanned at 600 nm, and the results were normalized against ß-actin. Statistical analysis was performed by Student's t-test using GB-STAT Statistical Analysis Software Package (Version 3.0) from Dynamic Microsystems, Inc. (Silver Spring, MD).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Purification of PGD-S from Rat CSF and Its Radioiodination

PGD-S was purified from rat CSF by sequential HPLC as described in Materials and Methods. In the final gel-permeation HPLC step, a total of 9 protein peaks were observed when the eluent was monitored by UV absorbance at 280 nm (Fig. 1A). The purity of protein used for radiolabeling was assessed by SDS-PAGE under reducing conditions. Aliquots of selected HPLC fractions were resolved onto a 12.5% T SDS-polyacrylamide gel. Silver staining showed that a protein with an electrophoretic mobility of PGD-S, about 30 kDa, was present in fractions 13–15 (Fig. 1B) corresponding to fractions under protein peak 4 (Fig. 1A) whose identity was confirmed by direct protein microsequencing and immunoblot (data not shown). Figure 1C shows the autoradiogram of 125I-labeled PGD-S, where about 15 000 cpm of the labeled protein was resolved by SDS-PAGE under reducing conditions onto a 12.5% T SDS-polyacrylamide gel. A single radioactive band with an apparent molecular weight corresponding to that of PGD-S was noted (Fig. 1C). These analyses confirmed the purity of PGD-S and the radiolabeled tracer.



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  		FIG. 1. Purification of PGD-S from rat CSF. Rat PGD-S was purified from CSF by sequential HPLC as described in Materials and Methods. A) The chromatogram of the last purification step on a Superose 12 micropore HPLC column (3.2 x 300 mm, i.d.). A total of 9 protein peaks were noted when the eluent was monitored by UV absorbance at 280 nm. The solid bar indicates where PGD-S was eluted. The arrow indicates where the sample was injected into the column. Aliquots of 10 µl from selected fractions were resolved onto 12.5% T SDS-polyacrylamide gel under reducing conditions and silver stained (B). The presence of PGD-S was detected in fractions 13–15 under protein peak 4. Lane M, molecular weight markers, consisting of about 0.2 µg protein each of corresponding proteins. D, dye front. Purified rat PGD-S (from fraction 14) was labeled with 125I by Iodo-Gen (Pierce Chemical Co., Rockford, IL). An aliquot of the labeled protein (about 15 000 cpm) was resolved onto a 12.5% T SDS-polyacrylamide gel under reducing conditions, the gel was dried, and the labeled protein was visualized by autoradiography as shown in C. A single radioactive band with an electrophoretic mobility corresponding to that of PGD-S was noted, confirming the purity of protein used for the RIA.

Production and Purification of rhPGD-S

To circumvent the difficulty of obtaining highly purified PGD-S for immunoassays and other pertinent physiological study in the long run, we have prepared rhPGD-S to assess whether it can generate a complete and parallel displacement curve, since early studies revealed that antibody prepared against human PGD-S cross-reacted across species [10]. The rhPGD-S/GST fusion protein prepared as described in Materials and Methods was purified from the Sf-9 spent media by affinity chromatography. The purity of the recombinant protein was assessed by SDS-PAGE under reducing conditions. Figure 2A is a Coomassie blue-stained polyacrylamide gel showing the purity of the affinity-purified rhPGD-S conjugated with a GST tag (lane 5 vs. lanes 1, 3, and 4). The identity of PGD-S was confirmed by immunoblot (Fig. 2B). When the rhPGD-S conjugated with GST was treated with thrombin, which enzymatically cleaved the GST tag from the fusion protein, the apparent molecular weight reduced from 43 kDa to about 20 kDa, which is slightly lower than the native PGD-S of 30 kDa (result not shown), apparently as a result of reduced glycosylation since it is known that PGD-S is a glycoprotein [2]. The identity of the rhPGD-S was also confirmed by direct protein microsequencing from its N-terminus (data not shown).



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  		FIG. 2. Purification of rhPGD-S by affinity chromatography. The rhPGD-S/GST was purified by affinity chromatography using a Glutathione Sepharose-4B column (15 x 65 mm, i.d.) from the spent media of Sf-9 cells transfected with baculovirus containing the full-length PGD-S cDNA. A shows the Coomassie blue-stained SDS-polyacrylamide gel (12.5% T) under reducing conditions. Lane 1, starting material: crude spent media (about 100 µg protein) of Sf-9 cells transfected with baculovirus containing the full-length human PGD-S cDNA. Lane 2, BRL prestained molecular weight markers. Lanes 3 and 4, washout of nonbound proteins from the affinity column in aliquots of 20 µl (about 30 µg protein) and 10 µl (about 15 µg protein), respectively. Lane 5, glutathione-eluted rhPGD-S conjugated with GST (about 5 µg protein). B is an immunoblot confirming the immunoreactivity of the rhPGD-S/GST in the spent medium of transfected Sf-9 cells before (sample 1) and after (sample 2) affinity chromatography.

RIA Specificity

The specificity of the RIA for PGD-S was assessed through comparison of displacement curves generated using increasing amounts of rat CSF, purified rat PGD-S, rat albumin, rat transferrin, and rhPGD-S to compete for the binding of 125I-rat PGD-S onto its antibody (Fig. 3). Both rat CSF and purified rat PGD-S induced complete and parallel displacement curves. In contrast, rat albumin and transferrin up to a dose of 50 µg/assay tube failed to displace the binding of the 125I-PGD-S onto its antibody, illustrating the specificity of the assay. The rhPGD-S/GST also partially cross-reacted with the anti-rat PGD-S antibody, confirming previous observations that anti-PGD-S antibody against human PGD-S cross-reacted with that of other species [10].



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  		FIG. 3. Characterization of the RIA for rat PGD-S. Increasing doses of purified rat PGD-S, rat CSF, rhPGD-S, rat albumin, or rat transferrin were used to compete for the binding of 125I-rat PGD-S onto the anti-rat PGD-S antibody. The batch of purified PGD-S used in this experiment was found to contain 17.8 ng protein/µl when estimated by Coomassie blue dye-binding assay [19]. The pool of rat CSF (CSF001STD, protein concentration: 204 µg/ml when estimated by Coomassie blue dye-binding assay) was found to contain 50.4 ng PGD-S/µl when titrated against the purified rat PGD-S. The abscissa is the dose in log scale expressed as µg protein per assay tube. The ordinate is value expressed as percentage of B/B0, where B and B0 are bound counts in the presence (B) and absence (B0) of competitor.

Tissue Distribution of Immunoreactive PGD-S during Postnatal Development

The changes in concentration (µg/g tissue) of PGD-S during maturation were assessed by RIA in brain, testis, epididymis, liver, and kidney of adult male rats at ages ranging between 10 and 120 days as shown in Figure 4. In the testis (Fig. 4A) and the epididymis (Fig. 4B), the concentration of PGD-S increased progressively during postnatal development; these patterns are in contrast to that of the brain and liver, for which the PGD-S concentration shows a general trend of reduction during maturation (Fig. 4, A and B vs. C and D). In the epididymis of adult rats at ages ranging between 60 and 120 days, the concentration of PGD-S was by far the highest among values in any organ examined in any age group (Fig. 4B). In the testis, the concentration of PGD-S reached its highest level (4.6 ± 0.8 µg/g tissue) at 45 days of age, and was maintained at around 3 µg/g tissue at 60–120 days of age (Fig. 4A). In the brain, PGD-S concentration showed a modest increase until Day 35, after which it rapidly declined (Fig. 4C). In the liver, the concentration of PGD-S declined steadily with maturation (Fig. 4D). In the kidney, the concentration of PGD-S remained relatively unchanged during maturation (Fig. 4E). These results seemingly suggest that PGD-S is related to spermatogenesis and sperm maturation in the reproductive system in view of its high level in the testis and epididymis.



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  		FIG. 4. Age-dependent changes of PGD-S concentration in various organs of the male rat. The concentration (µg PGD-S/g tissue) of immunoreactive PGD-S in extracts of testis (A), epididymis (B), brain (C), liver (D), and kidney (E) was quantified by RIA. Data are expressed as mean ± SD with 4–6 animals per age group. Each sample was assayed in triplicate. All samples from a given organ were assayed simultaneously to avoid interassay variation.

Tissue and Cellular Distribution of PGD-S mRNA Transcript

Using RT-PCR in conjunction with Southern blot analysis, PGD-S mRNA transcript was found in the brain, testis, epididymis, liver, and kidney (Fig. 5). Negative controls in which PCR was performed in the absence of either RT product or PGD-S primer pair showed no positive signal (data not shown), suggesting the specificity of this analysis. Within the seminiferous epithelium, both Sertoli and germ cells were shown to express PGD-S (Fig. 5). Furthermore, in the epididymis, PGD-S was expressed in all three compartments, namely, caput, corpus, and cauda (Fig. 5). Northern blot analysis confirmed the expression of a 0.76-kilobase (kb) PGD-S mRNA transcript in the testis, brain, and Sertoli cells (Fig. 6, A and B). The apparent absence of PGD-S expression in germ cells as revealed by Northerns (Fig. 6, A and B) is possibly the result of lower sensitivity of this technique versus the RT-PCR visualized by Southern blot shown in Figure 5. Using the mechanical procedure of isolating germ cells, it has been shown that somatic cell contamination is virtually negligible, since RT-PCR failed to detect observable testin, which is a known somatic cell product in the testis [26]. Interestingly, results shown in Figure 6, C and D, reveal that the steady-state PGD-S mRNA level is the highest in the caput; this is also consistent with results of the RIA data illustrating that the PGD-S protein level is the highest in the caput as compared to the corpus and cauda epididymis (Fig. 7, A–C).



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  		FIG. 5. Tissue and cellular distribution of PGD-S mRNA transcript in adult male rats. Total RNAs were extracted from brain, testis, germ cells, Sertoli cells, epididymis, and the corresponding compartments and used for RT-PCR. An aliquot (10 µl) of the PCR product from each sample was resolved onto a 5% T polyacrylamide gel, transferred to Nytran membrane, and hybridized with a {gamma}-32P-labeled internal PGD-S oligonucleotide primer as described in Materials and Methods. The Southern blot was then exposed to an x-ray film.



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  		FIG. 6. Distribution of PGD-S mRNA transcript in selected organs of adult rats and testicular cell types. Northern blot analysis of total RNAs (about 30 µg RNA per lane) extracted from the brain, testis, germ cells, and Sertoli cells (A) and from the epididymis as well as the various epididymal compartments (C). The PGD-S mRNA (0.76 kb) was visualized by hybridizing the blot with an {alpha}-32P-labeled PGD-S 318-bp cDNA probe. B and D are the ethidium bromide staining of the corresponding Northerns shown in A and C, demonstrating the integrity of the 28S and 18S ribosomal RNA subunits and showing that similar amount of total RNA was loaded in each lane. Results of two separate experiments using different batches of samples show virtually identical observations.



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  		FIG. 7. Effects of castration and androgen replacement on the concentration of PGD-S in selected organs and serum of adult male rats. The concentration of immunoreactive rat PGD-S (µg/g tissue or µg/ml) and its changes after orchiectomy and DHT replacement were measured in the caput (A), corpus (B), and cauda (C) epididymis, liver (D), ventral prostate (E), seminal vesicle (F), and serum (G) by RIA. Between Days 3 and 7 after orchiectomy, each rat received a daily s.c. injection of DHT (5 mg/rat at 250 g BW). Each data point represents mean ± SD of 4 rats. Each sample was assayed in triplicate. All samples were assayed simultaneously to avoid interassay variation.

Effects of Orchiectomy and DHT Replacement on the Concentration of Immunoreactive PGD-S in Selected Organs

Since the PGD-S expression was highest in the caput epididymis, we sought to quantify the levels of PGD-S in the three compartments of the epididymis and to examine whether the production of PGD-S in the epididymis and selected reproductive organs is under the control of androgen, by performing orchiectomy. PGD-S concentration was determined by RIA in cytosols of caput, corpus, and cauda epididymis, liver, prostate, and seminal vesicle and in the serum of adult male rats, 1 through 7 days after orchiectomy (Fig. 7). Day 0 animals were control rats that did not undergo orchiectomy. Consistent with the Northern blot data, the PGD-S concentration at Day 0 was highest in the caput (335 ± 93 µg/g tissue), followed by the cauda (217 ± 87 µg/g tissue) and the corpus (147 ± 44 µg/g tissue) (Fig. 7, A–C). In all three compartments of the epididymis, PGD-S levels dramatically decreased after orchiectomy, declining to a level of 26 ± 15 µg/g tissue, 10 ± 0.6 µg/g tissue, and 46 ± 3 µg/g tissue in the caput, corpus, and cauda epididymis, respectively. In the caput epididymis, DHT replacement beginning on Day 3 after orchiectomy, followed by a daily s.c. administration of DHT at 5 mg/rat, induced a partial recovery (Fig. 7A). In the corpus and cauda epididymis, DHT administration had relatively little affect in restoring the reduced PGD-S levels (Fig. 7, B and C). These results suggest that other testis-derived factor(s) may be involved in the regulation of epididymal PGD-S in addition to androgens. In all other organs examined, which included liver (Fig. 7D), ventral prostate (Fig. 7E), and seminal vesicles (Fig. 7F), as well as in serum (Fig. 7G), neither orchiectomy nor DHT administration had any apparent effect on the basal PGD-S level, suggesting that androgen and testis do not play a role in regulating PGD-S in these organs.

Regulation of Sertoli Cell PGD-S Steady-State mRNA Level by Germ Cells

To partially characterize the regulation of PGD-S in the testis, we examined the effect of germ cell-derived factors on Sertoli cell PGD-S steady-state mRNA level by culturing Sertoli cells in the absence or presence of increasing doses of GCCM (0.05, 0.5, and 1.75 mg total protein per 4.5 x 106 Sertoli cells). RT-PCR was performed using a PGD-S primer pair, and coamplification was carried out with ß-actin using RNA extracted from these Sertoli cell cultures. It was shown that GCCM induced a dose-dependent stimulation of PGD-S expression in Sertoli cells (Fig. 8A) with a maximal stimulation of about 2.4-fold at the dose of 0.5 mg protein per 4.5 x 106 cells when the autoradiograms were normalized against ß-actin (Fig. 8B).



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  		FIG. 8. Regulation of Sertoli cell PGD-S steady-state mRNA level by GCCM. Sertoli cells were cultured in the absence (control) or presence of increasing amount of GCCM (0.05, 0.5, and 1.75 mg protein per dish). Total RNA was extracted for RT-PCR after an 18-h incubation. ß-Actin was coamplified with PGD-S in the PCR as an internal control. Samples were than resolved onto a 5% T polyacrylamide gel; the gels were dried and exposed to x-ray films. A shows the autoradiogram of an RT-PCR experiment. B is the densitometrically scanned data of three blots from different experiments using various batches of GCCM such as the one shown in A at 600 nm and normalized against ß-actin. Results are mean ± SD of three different experiments. ns, Not significantly different from control without GCCM; *p < 0.05.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A sensitive and specific RIA for PGD-S was established and was used to quantify the concentration of PGD-S in tissue extracts of adult rats during maturation. Even though the presence of PGD2 [35] and the level of ß-trace (PGD-S) in the epididymis [36] have been described, the relative expression and concentration of PDG-S in different compartments of the epididymis and changes during epididymal maturation were not known. The present report illustrates that the concentration and organ content of PGD-S increased sharply in the testis and epididymis during maturation. Moreover, the level of PGD-S in the epididymis is the highest in relation to all the organs examined and is predominantly accumulated and expressed in the caput epididymis, suggesting its likely involvement in sperm maturation. In contrast, the concentration and organ content of PGD-S in the brain and liver rose slightly after birth, peaked at 30–40 days of age, and declined rapidly thereafter. This trend of changes in PGD-S level during maturation in the brain is consistent with previous observations when the binding sites on the synaptic membrane for PGD2, as well as the enzymatic activity responsible for the production of PGD2, were quantified during aging [37]. Using in situ hybridization, a high level of PGD-S expression was also detected in developing mouse testes at 14.5 days postconception [9]. Interestingly, the expression of PGD-S in the mouse testis is turned off in 1- and 3-wk-old mice in which expression is turned on again at the onset of puberty [9], suggesting its likely involvement in spermatogenesis. Consistent with the observation by Hoffmann et al. [9], a low level of PGD-S was detected in the rat testis, about 1–2 µg/g tissue, which increased by about 2-fold to 4.5 µg/g tissue at 45 days of age and remained at this level thereafter. These results collectively, in conjunction with earlier observations demonstrating a correlation between PGD-S level in seminal plasma and the number of spermatozoa in humans [38], as well as the positive correlation between the level of PGD-S in the seminal plasma and higher fertility in the bull [39, 40], strongly suggest that PGD-S plays an important role in the development and maintenance of male reproductive function. Furthermore, it was reported that intratesticular administration of PGD2 in the rat induced a significant decrease in testicular testosterone level [41]; hence a possible role of PGD2 in the regulation of testicular biosynthesis of androgen, and maintenance of the necessary level of testosterone essential for sperm maturation, was proposed [42].

The fact that there is a differential expression of PGD-S and its protein level in the three compartments of the epididymis was not entirely surprising. Differences in PG concentration along the epididymis have already been reported [43, 44]. For instance, the amounts of PGE and PGF2{alpha} are greater in the cauda than in the caput epididymis [43, 45]. Since the PGD-S steady-state mRNA level and its protein concentration were found to be higher in the caput than in the cauda epididymis, this may suggest a different role of PGD2 in the epididymis in comparison to PGE and PGF2{alpha}, or a function of PGD-S on its own other than its enzymatic activity. Both PGE and PGF2{alpha} appear to be important regulators of sperm transport in the epididymis, acting on both sperm motility and caput epididymis contractility [43, 44]. PGs have also been proposed to have a functional role in sperm maturation (for review, see [44]). It is also known that castration induces a reduction of PG concentration in the epididymis (for review, see [44]) and that the synthesis of PGs in the epididymis is, at least in part, under the control of androgens [46]. In the present study, we have demonstrated that orchiectomy caused a drastic reduction of PGD-S level in all three compartments of the epididymis. This may indicate the presence of a testis-derived factor(s) regulating PGD-S synthesis and secretion. However, DHT replacement is unable to restore the reduced PGD-S concentration in either epididymal compartment, illustrating that one or more factors other than androgen are involved in the epididymal PGD-S regulation.

The discovery of PGD-S as a bifunctional protein that also acts as a retinoid transporter, binding retinoids with an affinity comparable to that of retinoic acid-binding proteins [12], opens a new perspective in examining the possible physiological role of PGD-S in the male reproductive system. In fact, retinoic acid is a metabolite of vitamin A, which is necessary for the maintenance of spermatogenesis [47–49] and is important for the differentiation of spermatogonia [50, 51]. Earlier studies have shown that the levels of retinoic acid and its binding proteins in the epididymis are unusually high in comparison to other parts of the male reproductive tract [52, 53]. As such, the known retinoic acid binding proteins together with PGD-S may both participate in the process of maintaining a high level of retinoic acid in the epididymis. In addition, the drastic reduction of PGD-S levels in the three compartments of the epididymis after orchiectomy was very similar to that of rat epididymal retinoic acid-binding protein (EP-RABP) under similar conditions, as well as the partial recovery observed only in the caput epididymis after androgen replacement [53], illustrating that EP-RABP and PGD-S may share similar physiological roles in the epididymis. Both PGD-S and EP-RABP are lipocalins, and the partial failure of DHT in restoring their levels in the epididymis after castration is in agreement with the complex multihormonal regulation reported for other members of the same protein family [54–56]. The predicted tertiary structure of PGD-S has a hydrophobic pocket [57] that contains a Cys-65 residue shown to be essential for the enzymatic activity [58]. Retinoic acid was found to noncompetitively inhibit PGD-S enzymatic activity by binding to a site different from PGH2 but near the same cavity [12], suggesting that this is a bifunctional protein having two active sites. In supporting the role of PGD-S in spermatogenesis, it is noteworthy that rats exposed to sodium selenite, an inhibitor of PGD-S [59], developed remarkable changes in testicular function and morphology, such as degenerative lesions involving the more advanced stages of spermatogenesis, resembling vitamin A deficiency [60].

In the present study, the PGD-S mRNA transcript was found to be expressed in both Sertoli and germ cells. These results are consistent with the work of Blodorn et al. [61], who localized transcripts for PGD-S mRNA in the human seminiferous epithelium by in situ hybridization and proposed its presence in the Sertoli cells as well as in the spermatogonia and spermatocytes. These results, however, are in contrast with those of Hoffmann et al. [9], who also localized PGD-S mRNA in the mouse testis by in situ hybridization but found it limited to the Leydig cell. The immediate explanation for this discrepancy is not known but could relate to the use of different species by these investigators.

In summary, a sensitive and specific RIA for PGD-S has been established, making it possible to quantitatively assess the differential tissue distribution of this lipocalin during maturation and other pathophysiological conditions. It is apparent that PGD-S will likely become a usual marker for quantifying epididymal function.


   FOOTNOTES
 
1 Supported in part by grants from the Sovena Foundation, the Noopolis Foundation, the Rockefeller Foundation (PS9601, PS9721), the CONRAD Program (CIG-96–05), and NIH (HD-13541). Back

2 Correspondence: C. Yan Cheng, The Population Council, 1230 York Avenue, New York, NY 10021. FAX: (212) 327–7678; yan{at}popcbr.rockefeller.edu Back

Accepted: May 20, 1998.

Received: March 11, 1998.


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