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Endocrinology Vol. 141, No. 2 710-721
Copyright © 2000 by The Endocrine Society

ARTICLES

Sertoli Cell Prostaglandin D2 Synthetase Is a Multifunctional Molecule: Its Expression and Regulation1

Eileen T. Samy, Jonathan C. H. Li, Josephine Grima, Will M. Lee, Bruno Silvestrini and C. Yan Cheng

The Population Council (E.T.S., J.C.H.L., J.G., C.Y.C.), New York, New York 10021; the Department of Zoology, University of Hong Kong (E.T.S., J.C.H.L., W.M.L.), Hong Kong, People’s Republic of China; and the Department of Pharmacology of Natural Substances and General Physiology, University of Rome La Sapienza (B.S.), Rome 00185, Italy

Address all correspondence and requests for reprints to: C. Yan Cheng, Ph.D., The Population Council, 1230 York Avenue, New York, New York 10021. E-mail: yan{at}popcbr.rockefeller.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PGD2 synthetase (PGD-S; PGH2 D-isomerase; EC 5.3.99.2) is a bifunctional protein first identified in the mammalian brain. It acts as a PGD2-producing enzyme and a retinoid transporter. PGD-S is present in the testis, where its protein and messenger RNA levels are similar to those in the brain. In view of its diversified regulatory functions, we investigated its regulation using primary cultures of Sertoli cells in vitro to assess its role in the testis. When Sertoli cells were cultured in serum-free medium to allow the formation of specialized junctions, it was found that PGD-S expression increased steadily with time, coinciding with the formation of inter-Sertoli junctions in vitro. However, neither germ cells (using a Sertoli/germ cell ratio between 1:1 and 1:30 when Sertoli cells were cultured at a density of 5 x 104 cells/cm2) nor germ cell-conditioned medium affected the expression of Sertoli cell PGD-S in vitro. These results thus unequivocally demonstrated that germ cells do not play a role in regulating testicular PGD-S expression. Although FSH, dihydrotestosterone, and testosterone had no apparent effect on Sertoli cell PGD-S expression, the addition of progesterone (1 x 10-11 to 1 x 10-9 M) and T3 (1 x 10-11 to 1 x 10-9 M) to Sertoli cell cultures elicited a significant increase in PGD-S expression by as much as 4.5- and 2.5 fold, respectively. As PGD-S is a known retinoid transporter, the effects of all-trans-retinoic acid and all-trans-retinal on Sertoli cell PGD-S expression were also assessed. Both compounds were found to induce Sertoli cell PGD-S expression. In summary, PGD-S is a putative Sertoli cell product whose expression is regulated by progesterone, metabolites of vitamin A, and T3. In view of its dual biological properties, a study of its regulation and physiology will yield new insights into understanding its role in the testis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PGD2 SYNTHETASE (PGD-S) is a 30-kDa peptide with dual biological activities. It is the enzyme (1) that catalyzes the conversion of cyclooxygenase-derived intermediate PGH2 to PGD2 (for review, see Ref. 2). In addition, PGD-S has high affinity for metabolites of vitamin A whose Kd are comparable to those of retinoic acid-binding proteins, making it a genuine vitamin A carrier (3). PGD-S was formerly known as ß-trace protein (4) and cerebrin 30 (5). It constitutes almost 10% of the total protein in the cerebrospinal fluid (CSF) and aqueous humor (5, 6). PGD-S is expressed in a wide range of tissues and organelles, such as brain (7), retina (8, 9), cochlea (10), epididymis (11, 12, 13), testis (11, 12, 13), prostate (12), rough surface endoplasmic reticulum, and outer nuclear membrane of oligodendrocytes in adult rats (14). They are also found in biological fluids such as CSF, seminal plasma, cauda epididymal fluid, rete testes fluid (15), and amniotic fluid (16, 17). In view of its high expression in blood-brain, blood-retina, blood-aqueous humor, and blood-testis barriers, as demonstrated by in situ hybridization during devel-opment, it was postulated that it plays a role in the maturation and maintenance of blood-tissue barriers (11).

PGD-S is a member of the lipocalin family (18, 19, 20), which binds and transports small lipophilic ligands (21, 22) such as retinol, progesterone, lectin, yellow-brown retinoid, biliverdins, and some odorants, including pheromones (21, 22). As retinoids play an important role in regulating a variety of biological processes, including differentiation, morphogenesis, and cell proliferation, the fact that PGD-S is a transporter of retinoids seemingly suggests that it may be involved in diversified biological functions. Among all-trans-retinoids, PGD-S specifically binds to retinoic acid and retinal, but not retinol (3). It has been known for decades that retinoids are crucial for male fertility (23, 24). In view of the localization pattern of PGD-S in the testis and its affinity for metabolites of vitamin A, PGD-S may play a crucial role in spermatogenesis. In this report we investigated the expression of PGD-S in Sertoli cells in vitro at the time when specialized junctions were being formed and in the presence of various compounds and hormones.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male Sprague Dawley rats at 1–90 days of age and adult rats at 250–300 g BW were obtained from Charles River Laboratories, Inc. (Kingston, MA). Rats were killed by CO2 asphyxiation, and testes were removed immediately for the isolation of testicular cells or processed for RNA extraction. The use of animals for this study was approved by the Rockefeller University animal care and use committee (Protocols 97117, 95129-R, and 97101).

Preparation of testicular cell cultures
Sertoli cells cultures. Sertoli cells were isolated from 20-day-old Sprague Dawley rats as previously described (25, 26). For low cell density cultures, Sertoli cells were plated at 5 x 104 cells/cm2 in 100-mm Petri dishes with 9 ml serum-free Ham’s F-12 nutrient mixture and DMEM (F12/DMEM; 1:1, vol/vol) to allow the formation of monolayer cultures (25) without specialized tight junctions (TJ) when assessed by transepithelial electrical resistance measurement (26). It is anticipated that both anchorage (AJ) and communicating gap (GJ) junctions are formed between Sertoli cells under these conditions (27). Cells were cultured in F12/DMEM, supplemented with 15 mM HEPES, 1.2 g/liter sodium bicarbonate, 10 µg/ml bovine insulin, 5 µg/ml human transferrin, 2.5 ng/ml epidermal growth factor, 20 mg/liter gentamicin, and 10 µg/ml bacitracin. For high cell density cultures, Sertoli cells were plated on Matrigel (Collaborative Biochemical Products, Bedford, MA)-coated (diluted 1:7 with F12/DMEM, vol/vol) 12-well dishes at a density of 0.5 x 106 cells/cm2 as previously described (26) to allow the formation of specialized junctions, including TJ, AJ, and GJ, where these cells mimic many of the morphological and functional features of the Sertoli cell in vivo. These cultures were incubated in a humidified atmosphere of 95% air and 5% CO2 (vol/vol) at 35 C. After 48 h of incubation, the cultures were hypotonically treated with 20 mM Tris (pH 7.4) for 2.5 min to lyse the residual germ cells, followed by two successive washes with F12/DMEM (28). These cells were allowed to recover for 20 h and were designated cultures at time zero. Thereafter, these cells were cultured alone, cocultured with germ cells, incubated with germ cell-conditioned medium (GCCM), or incubated with other biochemicals and hormones. Sertoli cell-conditioned medium (SCCM) was obtained essentially as previously described (29). Briefly, Sertoli cells were cultured at 5 x 104 cells/cm2 in 100-mm Petri dishes and incubated in a humidified atmosphere of 95% air and 5% CO2 (vol/vol) at 35 C for 4 days. Spent media were collected, centrifuged to remove cellular debris, concentrated by a Millipore Corp. Minitan tangential ultrafiltration unit equipped with eight Minitan plates (Mr cut-off at 10 kDa), and filtered through a 0.2-µm pore size filter unit. Protein estimation was performed by Coomassie blue dye binding assay (30) using BSA as standard.

Trypsinization of cultured Sertoli cells. We also assessed the effects of trypsinization on the expression of PGD-S in cultured Sertoli cells (5 x 104 cells/cm2 in 100-mm Petri dishes) when the inter-Sertoli junctions were disrupted by trypsin. Sertoli cells were prepared as described above, and media were removed on day 3 after hypotonic treatment, which was performed on day 1. Freshly prepared PBS containing 0.1% trypsin (wt/vol) and 0.02% EDTA was added to the culture and incubated for about 2 min to disrupt cell-cell and cell-substratum junctions. The action of trypsin was then stopped by the addition of F12/DMEM containing 5% horse serum and 2.5% FBS. The cells were then rinsed and washed three times by centrifugation (800 x g, 2 min), transferred to other culture dishes, and terminated at specific time points by RNA STAT-60 (Tel-Test B, Inc., Friendswood, TX).

Germ cells. Total germ cells were isolated from adult Sprague Dawley rats (300 g BW) by a mechanical procedure without any enzymatic treatments as previously described (31). Sequential filtrations were performed to remove cellular debris, spermatozoa, and elongated spermatids. When the final preparation was analyzed by DNA flow cytometry as previously described (31, 32), it consisted largely of spermatogonia, spermatocytes, and round spermatids with relative percentages of 17%, 18%, and 65%, respectively. Germ cells with a purity of greater than 90% when judged microscopically and by other criteria (31, 32) were reconstituted in F12/DMEM, supplemented with 6 mM sodium lactate, 2 mM sodium pyruvate, 20 mg/liter gentamicin, and 10 µg/ml bacitracin and were used within 1 h for coculture experiments. For preparation of crude GCCM, germ cells isolated as described above were plated at a density of 0.3 x 106 cells/cm2 in 100-mm Petri dishes and incubated in a humidified atmosphere of 95% air and 5% CO2 (vol/vol) at 35 C for 18 h as previously described (33). Spent media were collected and processed similarly to those of SCCM as described above. Protein estimation was performed by Coomassie blue dye binding assay (30) using BSA as standard.

Sertoli-germ cells cocultures. The cell numbers of freshly isolated germ cells, as described above, were determined by a hemocytometer. They were then reconstituted in F12/DMEM to give the desired cell density. Germ cells were cocultured with Sertoli cells (5 x 104 cells/cm2) in different ratios of Sertoli/germ cells at 1:1, 1:3, 1:5, 1:10, and 1:30, and cultures were terminated at specific time points by RNA STAT-60.

Sertoli cells cultured with GCCM, steroids, metabolites of vitamin A, and T3. Sertoli cell cultures (5 x 104 cells/cm2) prepared as described above were incubated with 1) 900 µg protein of crude GCCM; 2) various concentrations (1 x 10-5 to 1 x 10-11 M) of 17ß-hydroxy-5{alpha}-androstan-3-one (DHT), testosterone, or progesterone and FSH (10–300 ng/dish); or 3) various concentrations of all-trans-retinal and all-trans-retinoic acid (1 x 10-5 to 1 x 10-11 M) and T3 (1 x 10-5 to 1 x 10-13 M) at time zero. These cultures were terminated after 10 h of incubation by RNA STAT-60 for RNA extraction.

Preparation of membrane and cytosol extracts
Sertoli and germ cells freshly isolated from 20- and 90-day-old rats, respectively, were briefly rinsed with 3 times their packed cell volume with a lysis buffer (20 mM Tris, pH 7.4, containing 1 mM EDTA and 2 mM phenylmethylsulfonylfluoride). The cells were then resuspended in the lysis buffer with 5 times their packed cell volume and incubated at 4 C for 1 h. They were centrifuged at 15,000 x g for 30 min at 4 C. The supernatant was saved as cytosol extracts, and the pellets were resuspended in a solubilization buffer (20 mM Tris containing 1 mM EDTA, 2 mM phenylmethylsulfonylfluoride, 1% SDS, and 0.1% Triton X-100) six times their packed volume at 4 C for 1 h. The solubilized pellet was centrifuged at 40,000 x g for 90 min at 4 C, and the supernatant was collected and designated membrane extract. Protein estimation was performed by Coomassie blue dye binding assay (30) using BSA as standard.

Relative distribution of PGD-S among Sertoli and germ cells and their cellular extracts
Fifty micrograms of protein from the membrane extract and cytosol from Sertoli and germ cells, GCCM, and SCCM were analyzed in 10% SDS-PAGE (34) and with silver staining (35). The presence of PGD-S protein was detected by immunoblots using antirat PGD-S antiserum as previously described (6).

Glycerol- and lonidamine-induced disruption of cell junctions in the testis
For glycerol treatment, groups of adult male Sprague Dawley rats of 250–300 g BW (n = 4–6 rats/time point) were anesthetized with metofane. They received either an injection of 200 µl PBS (10 mM sodium phosphate, pH 7.4, containing 0.15 M sodium chloride) or 20% glycerol solution in PBS (vol/vol) through the polar axis of each testis as previously described (26, 36). Lonidamine, 1-(2,4-dichlorobenzyl)-1H- indazole-3-carboxylic acid, was suspended in methylcellulose (Sigma, St. Louis, MO; 0.25%, wt/vol). Adult rats (n = 4–6 rats/time point) were fed a single dose of lonidamine at 50 mg/kg BW (37, 38). Testicular RNA extracted at specified time points were used for Northern blots.

RNA extraction
Total RNA was isolated from tissues or cells by RNA STAT-60 (Tel-Test B, Inc.) according to the manufacturer’s protocols. The concentration of RNA was quantified by spectrophotometry at 260 nm. In some cases, the integrity of the RNA preparations was assessed by agarose gel electrophoresis.

Semiquantitative RT-PCR
Semiquantitative RT-PCR was performed essentially as previously described (13, 26, 27, 39). Briefly, about 2 µg total RNA were reversetranscribed into complementary DNAs (cDNAs) with 1 µg oligo- (deoxythymidine)15 using a mouse mammary leukemia virus RT kit (Promega Corp., Madison, WI) in a final reaction volume of 25 µl. To quantify and compare the expression of PGD-S messenger RNA (mRNA) between samples in a given treatment, PGD-S was coamplified with S16 using hot PCR. PCR products were examined over a range of 20–28 amplification cycles to ensure linearity in preliminary experiments to estimate the concentrations of primers and RT products for PCR. PCR was routinely performed by combining 3 µl of the RT product with 0.4 µg each of PGD-S primers (40) (sense primer, 5'-GTGGTAGCTCCCTCCACA-3', nucleotides 236–253; and antisense primer, 5'-GCTGAACAGGAACGCGTA-3', nucleotides 413–430), 80 ng each of rat ribosomal S16 primers (32) (sense primer, 5'-TCCGCTGCAGTCCGTTC-AAGTCTT-3', nucleotides 15–38; and antisense primer, 5'-GCCAAAC-TTCTTGGATTCGCAGCG-3', nucleotides 376–399), 5 µl 10 x PCR buffer, 3 µl MgCl2 (6 mM), 8 µl deoxy (d)-NTPs (200 µM each of dATP, dGTP, dCTP, and dTTP), 2.5 U Taq DNA polymerase, and sterile double distilled water to a final volume of 50 µl. The cycling parameters for PCR were as follows: denaturation at 94 C for 1 min, annealing at 58 C for 2 min, and extension at 72 C for 3 min for a total of 25 cycles, followed by a 15-min extension at 72 C in a Perkin-Elmer Corp. thermal cycler (Norwalk, CT). To enhance the detection limit and to yield data for semiquantitative analysis after densitometric scanning, PCR was performed by the inclusion of a trace amount of {gamma}-32P-labeled primers. Briefly, the sense primers of PGD-S and S16 were labeled at the 5'-end with [{gamma}-32P]dATP (SA, 6000 Ci/mmol; Amersham Pharmacia Biotech, Arlington Heights, IL) using T4 polynucleotide kinase (Promega Corp., Madison, WI). Approximately, 1 x 106 cpm were used per PCR reaction. To ensure the linearity of both PGD-S and S16, 10-µl aliquots of PCR product at 21, 23, 25, and 27 cycles were withdrawn and resolved onto 5% T [total acrylamide concentration (g/100 ml) = acrylamide + methylenebisacrylamide] polyacrylamide gels, using 0.5 x TBE (44.5 mM Tris-borate and 1 mM EDTA, pH 8.0) as a running buffer. The PCR products were visualized by ethidium bromide staining and autoradiography using Kodak X-Omat AR film (Eastman Kodak Co., Rochester, NY). The resultant autoradiograms were densitometrically scanned at 600 nm to estimate the relative concentrations of PGD-S and S-16 mRNA between samples.

Northern blots
Approximately 8–20 µg total RNA were denatured at 65 C for 15 min in RNA loading buffer containing 6% formaldehyde (vol/vol), 50% deionized formamide (vol/vol), and 1 x MOPS buffer (40 mM MOPS, 10 mM sodium acetate, and 1 mM EDTA, pH 7.0). The integrity of the RNA was assessed by 1% agarose-formaldehyde gel electrophoresis in 0.5 x MOPS buffer followed by ethidium bromide staining. RNA was then electroblotted onto a Nytran Plus membrane (Schleicher & Schuell, Keene, NH) and immobilized by UV cross-linking. Prehybridization was performed at 42 C for 1 h in 50% deionized formamide (vol/vol), 10% dextran sulfate (wt/vol), 1% SDS (wt/vol), 1 M sodium chloride, 0.5 µg/ml yeast transfer RNA, and 100 µg/ml denatured salmon sperm DNA. An {alpha}-32P-labeled PGD-S cDNA probe (195 bp) prepared by nick translation was used for hybridization at 42 C for about 18 h. After several high stringency washes (13), the radiolabeled PGD-S band was visualized by autoradiography as described above. To normalize the RNA loading in each lane, all blots were rehybridized with an {alpha}-32P-labeled S16 cDNA probe, and autoradiograms were densitometrically scanned at 600 nm.

Statistical analysis
Autoradiograms were densitometrically scanned at 600 nm using an UltroScan XL enhanced laser densitometer (Amersham Pharmacia Biotech). The radiolabeled PGD-S was normalized against S16, and results were plotted as the mean ± SD. Results were analyzed by Student’s t test and two-way ANOVA followed by Duncan’s new multiple-range test using the GB Statistical Analysis Package (version 3.0, Dynamic Microsystems, Inc., Silver Spring, MD).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Changes in PGD-S steady-state mRNA level in rat testis during maturation
To investigate whether there is any correlation between the expression of PGD-S in the testis and development, testes were removed from 1-, 5-, 10-, 20-, 30-, 45-, 60-, and 90-day-old rats, and RNAs were extracted for Northern analysis. It was found that the expression of PGD-S was rarely detectable in rats at 1–10 days of age, but its mRNA level rose steadily during maturation beginning at 20 days of age (Fig. 1Go, A and B). An age-dependent increase in PGD-S expression was observed regardless of the change in testicular weight (Fig. 1Go, A–C), and as much as a 40-fold increase in testicular PGD-S steady-state mRNA level was found in adult rats compared with that in immature rats at 20 days of age (Fig. 1CGo). When the increase in testicular weight during maturation was taken into account, a 50-fold increase in PGD-S mRNA levels per organ pair was noted (Fig. 1CGo). The results of this study illustrate that the surge of PGD-S expression in the testis correlates with the onset of the first wave of production of mature spermatozoa when fully developed germ cells begin to be found in the tubular lumen at 45–60 days of age.



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  		Figure 1. A–C, Correlation of PGD-S expression and testicular maturation. The PGD-S steady-state mRNA level was quantified in testes isolated from rats at 1, 5, 10, 20, 30, 45, 60, and 90 days of age, which illustrates an age-dependent increase in expression. A, Total RNA extracted from rat testis were quantified by spectrophotometry at 260 nm. Approximately 20 µg total RNA were loaded per lane and the Northern blot was hybridized with an {alpha}-32P-labeled 195-bp PGD-S cDNA probe as described in Materials and Methods. B, The same blot as that shown in A was rehybridized with an {alpha}-32P-labeled rat ribosomal S16 probe. C, Densitometric scanning analysis of the blots such as those shown in A and B. The relative expression of PGD-S per pair of testes is also shown to reflect the increase in organ weight during maturation. Each bar represents the mean ± SD of at least three rats normalized against S16.

 
Relative expression of PGD-S in Sertoli and germ cells
Although the expression of PGD-S in the testis is high compared to that in other organs, the relative contributions of Sertoli and germ cells to the overall testicular PGD-S steady state mRNA level are not known. When several batches (n = 4) of primary cultures of Sertoli and germ cells were used for analysis, it was shown that germ cells expressed only about one third of the PGD-S found in Sertoli cells when analyzed by semiquantitative RT-PCR (Fig. 2Go, A and B). The purity of the Sertoli and germ cells used in these experiments was greater than 95%, and they have been well characterized using various criteria as detailed previously, including microscopy, RT-PCR using primers coding for genes specific to Sertoli or germ cells, and DNA flow cytometry (25, 32, 41, 42). When the relative distribution of the PGD-S gene product was assessed by immunoblots using cytosol and membrane extracts of Sertoli and germ cells as well as GCCM and SCCM with a monospecific antirat PGD-S antibody (6), PGD-S was detected in the cytosols of Sertoli and germ cells as well as in SCCM (Fig. 2Go, C and D).



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  		Figure 2. A–D, Differential expression and localization of PGD-S in Sertoli and germ cells. A, Sertoli and germ cells were isolated from 20-day-old rat testes. Total RNA was extracted, and RT-PCR was performed as described in Materials and Methods. This is a typical autoradiogram showing the relative expression of PGD-S by Sertoli and germ cells. B, Densitometrically scanned results using autoradiograms such as that shown in A. Each bar represents the mean ± SD of at least three experiments normalized against S16. C, The localization of PGD-S protein in different subcellular fractions and media was identified by SDS-PAGE and immunoblots. This is a silver-stained SDS-polyacrylamide gel (12.5% T) running under reducing conditions with 50 µg protein from each sample in each lane. Lane 1, Protein marker; lane 2, Sertoli cell membrane extracts; lane 3, Sertoli cell cytosol; lane 4, germ cell membrane extracts; lane 5, germ cell cytosol; lane 6, SCCM; lane 7, GCCM; lane 8, rat CSF. D, Corresponding immunoblot of the same gel as that shown in C. The presence of PGD-S protein was detected using 1% antirat PGD-S antibody. Immunoreactive PGD-S was detected in both Sertoli and germ cell cytosols as well as in SCCM. Rat CSF served as a positive control.

 
Expression of PGD-S in cultured Sertoli cells when specialized junctions were formed
When Sertoli cells were cultured at low cell density, at either 2.5 x 104 cells/cm2 (data not shown) or 5 x 104 cells/cm2 (Fig. 3Go, A and B), and at a high cell density (0.5 x 106 cells/cm2) on Matrigel-coated dishes in serum-free F12/DMEM to allow the formation of specialized junctions, which included AJ, GJ at low cell density, and AJ, GJ, and TJ at high cell density (27), it was found that the expression of PGD-S increased steadily with time in culture. Its level of expression became significantly higher at 2 days of culture and stayed high thereafter at both cell densities (Fig. 3Go, A–D), seemingly suggesting that its expression in Sertoli cells may correlate with the formation of specialized inter-Sertoli cell junctions. However, when the Sertoli cell steady-state PGD-S mRNA level was assessed at the time when cells were freshly isolated from tubules and also when cells were subjected to hypotonic treatment, it was found that the expression of PGD-S actually declined when cells were being dispersed from the intact tubules and throughout various steps of isolation, including hypotonic treatment (Fig. 4Go). PGD-S expression did not increase until cells were allowed to recover and plated on dishes apparently at the time when cell junction was formed (Fig. 4Go, A–C). These results in conjunction with the developmental pattern of PGD-S, as shown in Fig. 1Go, seem to suggest that the increase in PGD-S expression coincides with the establishment of the blood-testis barrier.



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  		Figure 3. A–C, Changes in the steady-state PGD-S mRNA level when specialized inter-Sertoli cell junctions were formed in vitro. Sertoli cells prepared from 20-day-old rats were cultured at two different cell densities, low cell density (5 x 104 cells/cm2; A) and high cell density (0.5 x 106 cells/cm2; C), as described in Materials and Methods. Cultures were terminated at specific time points by RNA STAT-60. Hot RT-PCR was performed to assess the changes in PGD-S steady-state mRNA levels under these culture conditions. A and C are autoradiograms showing the expression of PGD-S in Sertoli cells cultured at low and high cell densities, respectively. B and D are the corresponding densitometric scanned results using autoradiograms such as those shown in A and C. Each bar represents the mean ± SD of at least three experiments normalized against S16. ns, Not significantly different from the control; *, significantly different from the control (P < 0.01). H, Hours; D, days.

 


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  		Figure 4. A–C, Changes in the Sertoli cell PGD-S steady-state mRNA level at the time when cells were isolated from the seminiferous tubules, subjected to hypotonic treatment, and cultured at different time points. A, Northern blot showing the changes in expression of Sertoli cell PGD-S in a different time frame. Sertoli cells prepared from 20-day-old rats were cultured at low cell density (5 x 104 cells/cm2) as described in Materials and Methods. Cultures were terminated at specific time points by RNA STAT-60. Total RNA extracted was quantified by spectrophotometry at 260 nm, and approximately 15 µg total RNA were used for Northern analysis using an {alpha}-32P-labeled PGD-S cDNA probe as described in Materials and Methods. B, The same blot as that shown in A, but rehybridized with an {alpha}-32P-labeled rat ribosomal S16 probe. C, The densitometric scanning analysis of the blots such as those shown in A and B. Each bar represents the mean ± SD of at least three experiments normalized against S16. ns, Not significantly different from the control at time zero; *, significantly different from the control at time zero (P < 0.01).

 
Effect of trypsinization on the expression of PGD-S in cultured Sertoli cells
In the above study it was noted that the Sertoli cell PGD-S steady-state mRNA level was reduced drastically within 24 h after cells were isolated from the intact seminiferous tubules and subjected to various treatments and throughout the period of hypotonic treatment and subsequent recovery (Fig. 4Go). It was not until Sertoli cells were plated and formed a monolayer with AJs and GJs that the level of PGD-S expression bounced back to the basal level found in cells immediately after their isolation from the tubules (Fig. 4Go, A–C). These changes may be attributed to the culture shock to which the Sertoli cells were exposed. To investigate this possibility, we examined the effect of trypsinization on the expression of PGD-S in cultured Sertoli cells when junctions were artificially disrupted. Sertoli cells were cultured at low density (5 x 104 cells/cm2), hypotonic treatment was performed 48 h after cell isolation, and cells were allowed to recover for 24 h and treated as cultures at time zero (Fig. 5Go). On day 3, cells were trypsinized as described in Materials and Methods. When PGD-S steady-state mRNA levels of the trypsinized Sertoli cells cultures were compared with those in the normal untreated Sertoli cells cultures (Fig. 5Go), it was found that trypsinization can reduce the PGD-S mRNA level by as much as 2.5-fold within 24 h, and this level of reduction persisted until day 6 (Fig. 5Go vs. Fig. 3Go, A and B).



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  		Figure 5. Effects of trypsinization on Sertoli cell PGD-S expression. Sertoli cells prepared from 20-day-old rats were cultured at low cell density (5 x 104 cells/cm2) and were trypsinized on day 3 as described in Materials and Methods. Cultures were terminated at specific time points by RNA STAT-60. Semiquantitative RT-PCR was performed to assess the changes in mRNA level of PGD-S expression after trypsinization. This graph shows the results of the densitometrically scanned data where the expression of PGD-S was normalized against S16. Each bar represents the mean ± SD of at least three experiments normalized against S16. ns, Not significantly different from the control cultures without trypsin treatment; *, significantly different from the control cultures (P < 0.01).

 
Glycerol- and lonidamine-induced disruption of specific junctions in the testis
The in vitro studies described above have illustrated that the expression of PGD-S apparently relates to the inter- Sertoli junction formation and disruption. Therefore, two in vivo models were used to further assess this correlation. Lonidamine is an antispermatogenic agent known to disrupt the junctional complexes between Sertoli and germ cells (39, 43, 44). Glycerol treatment is also known to induce irreversible disruption of inter-Sertoli tight junctions within 1–2 weeks after its administration (36). It was found that lonidamine treatment caused a decrease in PGD-S expression in the testis during the first 2 days after treatment at the time when the seminiferous epithelium appeared to be normal when examined by light microscopy following hematoxylin staining (39) and that germ cells remained attached to the epithelium (Fig. 6AGo), although the Sertoli-germ cell junctions were in the process of being damaged (43). However, when germ cells, in particular elongated and round spermatids, were depleted from the epithelium at 6–15 days after lonidamine treatment, the testicular PGD-S level had already bounced back to its basal level (Fig. 6AGo), suggesting that germ cells per se indeed had no effect on the expression of PGD-S. Rather, it is the disruption of the Sertoli-germ cell junctions that causes the reduction. These results also illustrate that PGD-S may be a sensitive marker to reflect a disruption of the Sertoli-germ cell junctions. Glycerol treatment also caused a decrease in PGD-S mRNA levels throughout the study (Fig. 6BGo) when the inter-Sertoli tight junctions were irreversibly disrupted, which took place at around 2 weeks postglycerol treatment (36). Again, germ cells remained attached to the epithelium at 15–24 h when examined by light microscopy (26). It is noteworthy that lonidamine is not cytotoxic to Sertoli cell in vitro with a dose of up to 1 µg/ml in vitro (39). In both experimental models, large numbers of germ cells were still found in the epithelium without major morphological changes by light microscopy when there was a significant change in PGD-S expression, illustrating that it is a sensitive marker to detect the early events of germ cell loss from the testis.



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  		Figure 6. A and B, A study to assess the relationship between the disruption of cell junctions in the testis and PGD-S expression in lonidamine-treated (A) and glycerol-treated (B) rats. A, Graph showing results of the Northern analysis of PGD-S mRNA using 20 µg total RNA isolated from rat testes, when adult rats (250–300 g BW) received a single oral dose of lonidamine at 50 mg/kg BW. Thereafter, testes were removed at 1, 2, 6, and 15 days and compared with control (Ctrl) rats at time zero before drug administration. Data were normalized against S16. B, Graph showing results of the Northern analysis using 20 µg total RNA from the testes of control rats (Ctrl; no treatment) and 15 h, 24 h, and 8 wk after an intratesticular injection of 20% glycerol solution. Data were normalized against S16. Densitometric scanning was performed as described in Materials and Methods. Results are expressed as the mean ± SD of at least three experiments. ns, Not significantly different from the control; *, significantly different from the control (P < 0.01).

 
Effects of germ cells on the expression of Sertoli cell PGD-S steady-state mRNA levels
In view of the observation that there is a drastic increase in PGD-S expression during maturation in the testis and the fact that the ratio of Sertoli cells/germ cells increases drastically during aging to as much as 1:50 (45, 46), we sought to examine whether germ cells can up-regulate the expression of Sertoli cell PGD-S steady-state mRNA levels. Neither germ cells (at a Sertoli/germ cell ratio of 1:1; Sertoli cells at 5 x 104 cells/cm2; Fig. 7Go, A and B) nor GCCM (Fig. 7Go, C and D) appeared to affect the expression of Sertoli cell PGD-S in vitro, as the increase in the PGD-S levels shown in Fig. 7Go, A–D, is simply due to the endogenous increase in Sertoli cell PGD-S when cells were cultured alone (Fig. 7Go vs. Fig. 3Go). This conclusion was reached when increasing numbers of germ cells were cocultured with Sertoli cells using germ cell/Sertoli cell ratios of 1:1, 3:1, 5:1, 10:1, and 30:1 (Sertoli cells at 5 x 104 cells/cm2) for 20 h in vitro, and no significant changes in the expression of PGD-S were detected (Fig. 8Go, A–C). The rationale of choosing a 20-h incubation period for this set of experiments is that there is no endogenous change in Sertoli cell PGD-S expression, as shown in Fig. 3Go, A–D. If germ cells indeed have a positive effect on Sertoli cell PGD-S expression, a dose-dependent effect of germ cells on Sertoli cell PGD-S expression is anticipated. These results thus unequivocally demonstrated that germ cells per se do not play a role in regulating testicular PGD-S expression in vitro.



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  		Figure 7. A–D, Effects of germ cells (GC) and GCCM on Sertoli cell (SC) PGD-S steady-state mRNA level. A, Sertoli cells prepared from 20-day-old rats were cocultured with germ cells (SC:GC ratio at 1:1; SC at 5 x 104 cells/cm2) as described in Materials and Methods. Cultures were terminated at specific time points by RNA STAT-60. Hot RT-PCR was performed to assess the PGD-S mRNA level, and autoradiograms such as that shown in A were densitometrically scanned to provide a semiquantitative analysis (B). C, GCCM protein (900 µg) was incubated with Sertoli cells (5 x 104 cells/cm2) as described in Materials and Methods. Cultures were terminated at specific time points for RNA extraction. B and D, are the corresponding densitometrically scanned results using autoradiograms such as those shown in A and C. Each bar represents the mean ± SD of at least three experiments normalized against S16. ns, Not significantly different from control; *, significantly different from control (P < 0.01).

 


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  		Figure 8. A–C, Effects of increasing numbers of germ cell on Sertoli cell PGD-S steady-state mRNA level expression. A, Different numbers of germ cells were cocultured with Sertoli cells (5 x 104 cells/cm2) for 20 h (H) using Sertoli/germ cell ratios of 1:1, 1:3, 1:5, 1:10, and 1:30. Total RNA was extracted, and the steady-state PGD-S mRNA level was assessed by hot RT-PCR. B, The control experiment that was performed similarly to that shown in A, except that the cocultures were terminated at time zero (at the time when germ cells were added to Sertoli cells). C, Corresponding densitometrically scanned results using autoradiograms such as those shown in A and B. Each bar represents the mean ± SD of at least three experiments of the Sertoli-germ cell cocultures after 20-h incubation as shown in A, normalized against S16 and the basal PGD-S expression at time zero as shown in B. This was done to take into account the change in relative RNA level contributed by Sertoli cells in the samples being analyzed due to an increase in GC numbers. ns, Not significantly different from control.

 
Effects of steroids, FSH, metabolites of vitamin A, and T3 on PGD-S steady-state mRNA level in Sertoli cell cultures
Effects of steroids and FSH. The effects of DHT, testosterone, progesterone, and FSH on Sertoli cell PGD-S expression were assessed because it is known that Sertoli cells have the machinery to metabolize C19 steroids under the influence of FSH (47, 48, 49, 50, 51). Recent studies also illustrate that germ cells are capable of producing steroids in the testis (52, 53, 54). Although FSH, DHT, and testosterone had no apparent effect on SC PGD-S expression (data not shown), the addition of progesterone at 1 x 10-11 to 1 x 10-9 M elicited a significant increase in PGD-S expression (Fig. 9Go, A and B); however, progesterone at higher concentrations between 1 x 10-7 and 1 x 10-5 M failed to stimulate Sertoli cell PGD-S expression (Fig. 9Go, A and B), illustrating a biphasic effect on Sertoli cell PGD-S expression.



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  		Figure 9. A–E, Effects of steroids, metabolites of vitamin A, and T3 on Sertoli cell PGD-S expression. Sertoli cells (5 x 104 cells/cm2) were cultured in the presence of progesterone (A and B), all-trans-retinal (C), all-trans-retinoic acid (D), and T3 (E) at various concentrations. Cultures were terminated after 10 h of incubation by RNA STAT-60. Semiquantitative RT-PCR was performed to assess the mRNA level of PGD-S, and autoradiograms such as that shown in A were densitometrically scanned to provide a semiquantitative analysis, as shown in B–E. Results are expressed as the mean ± SD of three experiments normalized against S16. ns, Not significantly different from the control; *, significantly different from the control (P < 0.01).

 
Effects of metabolites of vitamin A. All-trans-retinoic acid and all-trans-retinal were shown to induce a significant increase in PGD-S expression, except that retinal displayed a biphasic effect, as it relinquished its stimulating effect at 10-5 M (Fig. 9Go, C and D).

Effects of T3. T3 at 1 x 10-11 to 1 x 10-9 M stimulated the Sertoli cell PGD-S steady-state mRNA level by as much as 2.5-fold (Fig. 9EGo) after 10 h of incubation, but at higher concentration (between 1 x 10-4 and 1 x 10-5 M), T3 failed to stimulate Sertoli cell PGD-S expression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Studies using Northern blots have shown that PGD-S is expressed in a variety of mammalian tissues. High levels of PGD-S are found in male reproductive organs such as the testis, prostate, and epididymis (7, 12, 13, 55). It is also found in the fluids of the male reproductive tract, such as seminal plasma, cauda epididymal fluid, and rete testis fluid (12, 13, 15), suggesting that PGD-S may be a secretory product of testicular cells. Recent studies have shown that PGD-S is a fertility-associated protein in the bull, as its concentration in the seminal plasma of bulls with above average fertility is 3.5-fold greater than that in bulls of average and below average fertility (15). PGD-S is also associated with fertility in humans. The seminal plasma concentration of lipocalin-type PGD-S is lower in oligozoospermic than in normozoospermic samples, but no significant correlation can be detected between the PGD-S concentration and other parameters of spermatogenesis (12, 16).

Both Sertoli and germ cells are found to contribute to the overall pool of PGD-S in the seminiferous epithelium behind the blood-testis barrier, and Leydig cells have been shown to express PGD-S (11). As such, PGD-S is produced by all three cell types in the testis, namely Sertoli, germ, and Leydig cells. As a maturation-dependent increase in PGD-S is detected in the rat testis, it is our belief that PGD-S is involved in male sexual functions and possibly maturation. Using in situ hybridization, PGD-S mRNA was first detected in mouse embryos at 14.5 days postconception in mesenchymal cells destined to become leptomeninges and in developing testis; however, it was not found in testis in 1- and 3-week-old mice whose expression was then up-regulated at the onset of puberty (11). PGD-S mRNA was also detected during differentiation of the eye (11). Thus, it is likely that PGD-S is involved in the differentiation and development of multiple organs, including the testis.

It has been postulated that PGD-S may play a role in the formation and possibly the maintenance of blood-tissue barriers in view of its specific expression pattern at the blood-brain, blood-retina, blood-aqueous humor, and the blood-testis barriers, as illustrated by in situ hybridization (11). The findings described herein have shown that the expression of PGD-S increases significantly at the onset of puberty, coinciding with the time when the blood-testis barrier is formed, further strengthening this hypothesis. In this study we have used several approaches to assess whether the expression of PGD-S indeed relates to the assembly and disassembly of cell junctions. First, an in vitro Sertoli cell culture model was used to study PGD-S expression at the time when specialized junctions were being formed. Sertoli cells were cultured at low density and were allowed to form specialized junctions, such as AJ and GJ. It was found that the expression of PGD-S appeared to correlate with the establishment of inter-Sertoli junctions. In some experiments Sertoli cell cultures were trypsinized after junctions were formed to disrupt the already assembled junctions, and it was found that the PGD-S mRNA level of the trypsinized cells declined significantly vs. that of the untreated cells, suggesting PGD-S may indeed play a role in junction formation. Secondly, an in vivo model involving drug-induced disruption of Sertoli-germ specialized junctions or inter-Sertoli tight junctions was used to assess whether there is any change in PGD-S expression. Glycerol treatment is known to have a long term irreversible effect on Sertoli-Sertoli tight junctions without destroying Sertoli cells and seminiferous tubules (36). The disruption of the blood-testis barrier after an intratesticular injection of glycerol may be due to an alteration in the cytoskeleton elements involved in the tight junctional complexes (36). Lonidamine is an antispermatogenic agent known to disrupt the junctional complexes between Sertoli and germ cells (43), possibly by rearranging the subcellular cytoskeleton microfilaments (44), thereby inducing massive depletion of germ cells from the seminiferous epithelium (39). Our results have shown that injection of glycerol into rat testes caused a significant decrease in testicular PGD-S expression between 15 h and 8 weeks after glycerol treatment at the time when the inter-Sertoli tight junctions were disrupted, as demonstrated by [125I]BSA and [3H]inulin diffusion studies (36). However, in the lonidamine-treated rats, it was found that the PGD-S level decreased only during the first 2 days after lonidamine treatment; thereafter, PGD-S returned to its normal level. Early studies have shown that 24 h after a single dose of lonidamine (50 mg/kg BW) in vivo, Sertoli cells displayed extensive vacuolation and retraction of the apical cytoplasm, with consequent release of the immature spermatids, focal swelling of the smooth endoplasmic reticulum, and enlargement of the intercellular germ-Sertoli cell spaces located above the junctional complex (43). By 48 h after treatment, the cytoplasmic matrix of Sertoli cells became remarkably electron dense, and the cisternae of the smooth endoplasmic reticulum appeared swollen and developed large vacuoles (43). By 10–15 days after lonidamine treatment, Sertoli cells became normal in appearance (43). As such, the early reduction in PGD-S expression in lonidamine-treated rats that coincides with the early phase of disruption of Sertoli-germ cell junctions seemingly suggests that the expression of Sertoli cells PGD-S relates to the assembly and reassembly of junctions.

In the present study it is noted that the steady-state PGD-S mRNA level was drastically reduced when Sertoli cells were isolated from the tubules and dispersed onto tissue culture plates; its level then bounced back to a level similar to that when cells were freshly isolated. This suggests that PGD-S may be a culture shock protein. However, these changes in expression can also be interpreted as follows. When freshly isolated Sertoli cells, which appear as cell clumps of 10–20 cells, are seeded onto culture dishes, cells slowly disperse and become monolayer cultures within 2 days; it is conceivable that extensive junction disassembly and reassembly take place at this time. As such, the decrease in PGD-S expression may be due to the disruption of cell junctions when cells disperse from the cell clumps to form monolayer cultures, which is consistent with the observations of the lonidamine- and glycerol-treated rats, illustrating that a decline in PGD-S expression correlates with junction disruption. When junctions are established in these cultures, the level returns to normal. If this postulation is correct, PGD-S may be a novel marker to monitor the integrity of junctions in a way just opposite that of testin, as testin expression increases drastically when Sertoli-germ cell junctions are disrupted both in vitro and in vivo (26, 39). However, the mechanistic role of PGD-S or its enzymatic products, such as PGD2, in junction formation is not known. For instance, it is not known whether Sertoli cells express receptors for PGs or when they produce PGD2 during development. Also, it is possible that PGD-S may act as an autocrine and/or paracrine factor in the testis. Work is now in progress to address some of these questions.

PGD-S is a member of the lipocalin family that binds small lipophilic proteins (18, 19, 20) in addition to its enzymatic activity. Structurally, PGD-S is composed of an {alpha}-helix and eight-stranded ß-sheets that construct an antiparallel ß- barrel structure containing a hydrophobic pocket (2). Cys65 is crucial for the enzyme activity (56), but not for retinoid binding, as substitution of Cys65 by Ala did not have any effect on retinoid binding (3). However, when Cys65 was modified with glutathione-S-transferase, the retinoid binding was abolished, possibly due to steric hindrance by glutathione-S-transferase (3). Binding of retinoic acid onto PGD-S interferes with the interaction between PGH2 and Cys65, which explained why retinoic acid could inhibit PGD-S enzyme activity (3). The fact that PGD-S has high affinity for retinoic acids may explain how cellular retinoic acid-binding protein (CRABP) and CRABP-II knockout mice remain essentially normal (57, 58) physiologically, as other molecules, such as PGD-S, can assume one of the functions of CRABP and CRABP-II. In our study to assess the effects of two vitamin A metabolites on the expression of Sertoli cell PGD-S, both all-trans-retinoic acid and all-trans-retinal can induce an increase in Sertoli cell PGD-S expression. The mechanism by which these biomolecules, such as retinal and retinoic acid, trigger activation of the PGD-S gene is not known. It is also not known whether the increase in the PGD-S level relates to an increase in the ability of Sertoli cells to transport small molecules within and also between cells through direct contacts.

Previous reports illustrated that receptors of T3 are found in high concentrations in Sertoli cells, being maximally expressed in the fetus and from 1–5 days postnatally and decreasing significantly at 15–20 days (59). Also, it is known that T3 is important to spermatogenesis (for review, see Ref. 60). Lepperdinger et al. (61) reported that the homolog of PGD-S in Xenopus binds to retinal, retinoic acid, and T3 in vitro. From our studies, it was found that T3, retinal, retinoic acid, and progesterone can all stimulate Sertoli cell PGD-S expression in vitro. These results suggest that these hormones or biomolecules somehow regulate the transport function of Sertoli cells via PGD-S.

In summary, PGD-S is likely to act as a PGD2-producing enzyme and a carrier protein in Sertoli cells to transport molecules that are important for spermatogenesis, such as retinoic acid, retinal, and T3. Moreover, its expression increases when junctions are formed, suggesting its possible involvement in vesicular trafficking.


   Footnotes
 
1 This work was supported in part by grants from the CONRAD Program (CIG-96–05), the NIH (HD-13541), and the Noopolis Foundation. Back

Received July 12, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Urade Y, Fujimoto N, Hayaishi O 1985 Purification and characterization of rat brain prostaglandin D synthetase. J Biol Chem 260:12410–12415[Abstract/Free Full Text]
  2. Urade Y, Watanabe K, Hayaishi O 1995 Prostaglandin D, E, F synthases. J Lipid Mediators Cell Signaling 12:257–273[CrossRef][Medline]
  3. Tanaka T, Urade Y, Kimura H, Eguchi N, Nishikawa A, Hayaishi O 1997 Lipocalin-type prostaglandin D synthase (ß-trace) is a newly recognized type of retinoid transporter. J Biol Chem 272:15789–15795[Abstract/Free Full Text]
  4. Clausen J 1961 Proteins in normal cerebrospinal fluid not found in serum. Proc Soc Exp Biol Med 107:170–172
  5. Leone MG, Saso L, Del Vecchio A, Mo MY, Silvestrini B, Cheng CY 1993 Micropurification of cerebrospinal fluid proteins by high performance electrophoresis chromatography (HPEC). J Neurochem 61:533–540[Medline]
  6. Giacomelli S, Leone MG, Silvestrini B, Cheng CY 1996 Astrocytes synthesize and secrete prostaglandin D synthetase in vitro. Biochim Biophys Acta 1310:269–276[Medline]
  7. Ujihara M, Urade Y, Eguchi N, Hayashi H, Ikai K, Hayaishi O 1988 Prostaglandin D2 formation and characterization of its synthetases in various tissues of adult rats. Arch Biochem Biophys 260:521–531[CrossRef][Medline]
  8. Goh H, Urade Y, Fujimoto N, Hayaishi O 1987 Content and formation of prostaglandins and distribution of prostaglandin related enzyme activities in the rat ocular system. Biochim Biophys Acta 921:302–311[Medline]
  9. Beuckmann CT, Gordon WC, Kanaoka Y, Eguchi N, Marcheselli VL, Gerashchenko DY, Urade Y, Hayaishi O, Bazan NG 1996 Lipocalin-type prostaglandin D synthase (ß-trace) is located in pigmented epithelial cells of rat retina and accumulates within interphotoreceptor matrix. J Neurosci 16:6119–6124[Abstract/Free Full Text]
  10. Tachibana M, Fex J, Urade Y, Hayaishi O 1987 Brain-type prostaglandin D synthetase occurs in the rat cochlea. Proc Natl Acad Sci USA 84:7677–7680[Abstract/Free Full Text]
  11. Hoffmann A, Bachner D, Betat N, Lauber J, Cross G 1996 Developmental expression of murine ß-trace in embryos and adult animals suggests a function in maturation and maintenance of blood-tissue barriers. Dev Dynamics 207:332–343[CrossRef][Medline]
  12. Tokugawa Y, Kunishige I, Kubota Y, Shimoya K, Nobunaga T, Kimura T, Saji F, Murata Y, Eguchi N, Oda H, Urade Y, Hayaishi O 1998 Lipocalin-type prostaglandin D synthase in human male reproductive organs and seminal plasma. Biol Reprod 58:600–607[Abstract/Free Full Text]
  13. Sorrentino C, Silvestrini B, Braghiroli L, Chung S, Giacomelli S, Leone MG, Xie YB, Sui YP, Mo MY, Cheng CY 1998 Rat prostaglandin D2 synthetase: its tissue distribution, changes during maturation, and regulation in the testis and epididymis. Biol Reprod 59:843–853[Abstract/Free Full Text]
  14. Urade Y, Fujimoto N, Kaneko T, Konishi A, Mizuno N, Hayaishi O 1987 Postnatal changes in the localization of prostaglandin D synthetase from neurons to oligodendrocytes in the rat brain. J Biol Chem 262:15132–15136[Abstract/Free Full Text]
  15. Gerena RL, Irikura D, Urade Y, Eguchi N, Chapman DA, Killian GJ 1998 Identification of a fertility-associated protein in bull seminal plasma as lipocalin-type prostaglandin D synthase. Biol Reprod 58:826–833[Abstract/Free Full Text]
  16. Melegos DN, Diamandis EP, Oda H, Urade Y, Hayaishi O 1996 Immunofluorometric assay of prostaglandin D synthase in human tissue extracts and fluids. Clin Chem 42:1984–1991[Abstract/Free Full Text]
  17. Yu H, Diamandis EP 1995 Prostate-specific antigen immunoreactivity in amniotic fluid. Clin Chem 41:204–210[Abstract/Free Full Text]
  18. Nagata A, Suzuki Y, Igarashi M, Eguchi N, Toh H, Urade Y, Hayaishi O 1991 Human brain prostaglandin D synthase has been evolutionarily differentiated from lipophilic ligand-carrier proteins. Proc Natl Acad Sci USA 88:4020–4024[Abstract/Free Full Text]
  19. Peitsch MC, Boguski MS 1991 The first lipocalin with enzymatic activity. Trends Biochem Sci 16:363[CrossRef][Medline]
  20. Igarashi M, Nagata A, Toh H, Urade Y, Hayaishi O 1992 Structural organization of the gene for prostaglandin D synthase in the rat brain. Proc Natl Acad Sci USA 89:5376–5380[Abstract/Free Full Text]
  21. Pervaiz S, Brew K 1987 Homology and structure-function correlations between {alpha}1-acid glycoprotein and serum retinol-binding protein and its relatives. FASEB J 1:209–214[Abstract]
  22. Ali S, Clark AJ 1988 Characterization of the gene encoding ovine ß-lactoglobulin. Similarity to the genes for retinol binding protein and other secretory proteins. J Mol Biol 199:415–426[CrossRef][Medline]
  23. Mitranond V, Sobhon P, Tosukhowong P, Chindaduangrat W 1979 Cytological changes in the testes of vitamin A-deficient rats. I. Quantitation of germinal cells in the seminiferous tubules. Acta Anat 103:159–168[Medline]
  24. Van Pelt AM, de Rooij DG 1990 The origin of the synchronization of the seminiferous epithelium in vitamin A-deficient rats after vitamin A replacement. Biol Reprod 42:677–682[Abstract]
  25. Grima J, Pineau C, Bardin CW, Cheng CY 1992 Rat Sertoli cell clusterin, {alpha}2-macroglobulin, and testins: biosynthesis and differential regulation by germs cells. Mol Cell Endocrinol 89:127–140[CrossRef][Medline]
  26. Grima J, Wong CCS, Zhu LJ, Zong SD, Cheng CY 1998 Testin secreted by Sertoli cells is associated with the cell surface, and its expression correlates with the disruption of Sertoli-germ cell junctions but not the inter-Sertoli tight junction. J Biol Chem 273:21040–21053[Abstract/Free Full Text]
  27. Chung SSW, Lee WM, Cheng CY 1999 A study on the formation of specialized inter-Sertoli cell junction in vitro. J Cell Physiol 181:258–272[CrossRef][Medline]
  28. Galdieri M, Ziparo E, Palombi F, Russo MA, Stefanini M 1981 Pure Sertoli cell cultures: a new model for the study of somatic-germ cell interactions. J Androl 5:249–259
  29. Cheng CY, Mather JP, Byer AL, Bardin CW 1986 Identification of hormonally responsive proteins in primary Sertoli cell culture medium by anion-exchange high performance liquid chromatography. Endocrinology 118:480–488[Abstract]
  30. Bradford MM 1976 A rapid and senisitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  31. Aravindan GR, Mruk D, Lee WM, Cheng CY 1997 Identification, isolation, and characterization of a 41-kilodalton protein from rat germ cell-conditioned medium exhibiting concentration-dependent dual biological activities. Endocrinology 138:3259–3268[Abstract/Free Full Text]
  32. Aravindan GR, Pineau CP, Bardin CW, Cheng CY 1996 Ability of trypsin in mimicking germ cell factors that affect Sertoli cell secretory function. J Cell Physiol 168:123–133[CrossRef][Medline]
  33. Pineau C, Syed V, Bardin CW, Jegou B, Cheng CY 1993 Germ cell-conditioned medium contains multiple factors that modulate the secretion of testins, clusterin, and transferring by Sertoli cells. J Androl 14:87–98[Abstract/Free Full Text]
  34. Cheng CY, Musto NA, Gunsalus GL, Bardin CW 1983 Demonstration of heavy and light protomers of human testosterone-estradiol-binding globulin. J Steroid Biochem 19:1379–1389[CrossRef][Medline]
  35. Wray WT, Boulikas T, Wray VP, Hancock R 1981 Silver staining of protein in polyacrylamide gels. Anal Biochem 118:197–203[CrossRef][Medline]
  36. Eng F, Wiebe JP, Alima LH 1994 Long-term alterations in the permeability of the blood-testis barrier following a single intratesticular injection of dilute aqueous glycerol. J Androl 15:311–317[Abstract/Free Full Text]
  37. Corsi G, Palazzo G, Germani C, Barcellona PS, Silvestrini B 1976 1-Halobenzyl-1H-indazole-3-carboxylic acids. A new class of antispermatogenic agents. J Med Chem 19:778–783[CrossRef][Medline]
  38. Silvestrini B, Palazzo G, De Gregorio M 1984 Lonidamine and related compounds. Prog Med Chem 21:110–135[Medline]
  39. Grima J, Zhu LJ, Cheng CY 1997 Testin is tightly associated with testicular cell membrane upon its secretion by Sertoli cells whose steady-state mRNA level in the testis correlates with the turnover and integrity of inter-testicular cell junctions. J Biol Chem 272:6499–6509[Abstract/Free Full Text]
  40. Urade Y, Nagata A, Suzuki Y, Fujii Y, Hayaishi O 1989 Primary structure of rat brain prostaglandin D synthetase deduced from cDNA sequence. J Biol Chem 264:1041–1045[Abstract/Free Full Text]
  41. Chung SSW, Zhu LJ, Mo MY, Silvestrini B, Lee WM, Cheng CY 1998 Evidence for cross-talk between Sertoli and germ cells using selected cathepsins as markers. J Androl 19:686–703[Abstract/Free Full Text]
  42. Mruk D, Zhu LJ, Silvestrini B, Lee WM, Cheng CY 1997 Interactions of proteases and protease inhibitors in Sertoli-germ cell cocultures preceding the formation of specialized Sertoli-germ cell junctions in vitro. J Androl 18:612–622[Abstract/Free Full Text]
  43. De Martino C, Malcorni W, Bellocci M, Floridi A, Marcante ML 1981 Effects of AF 1312 TS and lonidamine on mammalian testis. A morphological study. Chemotherapy [Suppl 2] 27:27–42
  44. Malorni W, Meschini S, Matarrese P, Arancia G 1992 The cytoskeleton as a subcellular target of the antineoplastic drug lonidamine. Anticancer Res 12:2037–2045[Medline]
  45. Wong V, Russell LD 1983 Three-dimensional reconstruction of a rat stage V Sertoli cell. I. Methods, basic configuration, and dimensions. Am J Anat 167:143–161[CrossRef][Medline]
  46. Weber JE, Russell LD, Wong V, Peterson RN 1983 Three-dimensional reconstruction of a rat stage V Sertoli cell. II. Morphometry of Sertoli-Sertoli and Sertoli-germ-cell relationships. Am J Anat 167:163–179[CrossRef][Medline]
  47. Hall P 1984 Cellular organization for steroidogenesis. Int Rev Cytol 86:53–95[Medline]
  48. Bardin CW, Cheng CY, Musta NA, Gunsalus GL 1988 The Sertoli cell. In: Knobil E, Neill JD, Ewing LL, Greenwald GS, Market CL, Pfaff DW (eds) The Physiology of Reproduction. Raven Press, New York, vol 1:933–974
  49. Sanborn BM, Caston LA, Buzek SW, Ussuf KK 1987 Hormonal regulation of Sertoli cell function. Adv Exp Med Biol 219:561–588[Medline]
  50. Tilbe KS, Wiebe JP 1981 Sertoli cell capacity to metabolize progesterone: variation with age and the effect of follicle-stimulating. Endocrinology 108:597–604[Medline]
  51. Welsh MJ, Wiebe JP 1978 Sertoli cell capacity to metabolize C19 steroids: variation with age and the effect of follicle-stimulating hormone. Endocrinology 103:838–844[Medline]
  52. Levallet J, Bilinska B, Mittre H, Genissel C, Fresnel J, Carreau S 1998 Expression and immunolocalization of functional cytochrome P450 aromatase in mature rat testicular cells. Biol Reprod 58:919–926[Abstract/Free Full Text]
  53. Nitta H, Bunick D, Hess RA, Janulis L, Newton SC, Millette CF, Osawa Y, Shizuta Y, Toda K, Bahr JM 1993 Germ cells of the mouse testis express P450 aromatase. Endocrinology 132:1396–1401[Abstract]
  54. Hess RA, Bunick D, Bahr JM 1995 Sperm, a source of estrogen. Environ Health Perspect [Suppl 7] 103:59–62
  55. Hochwald GM, Pepe AJ, Thorbecke GJ 1967 Trace protein in biological fluids. IV. Physicochemical properties and sites of formation of {chi}-trace and ß-trace proteins. Proc Soc Exp Biol Med 124:961–966[Medline]
  56. Urade Y, Tanaka T, Eguchi N, Kikuchi M, Kimura H, Toh H, Hayaishi O 1995 Structural and functional significance of cysteine residues of glutathione-independent prostaglandin D synthase. Identification of Cys65 as an essential thiol. J Biol Chem 270:1422–1428[Abstract/Free Full Text]
  57. Gorry P, Lufkin T, Dierich A, Rochette-Egly C, Decimo D, Dolle P, Mark M, Durand B, Chambon P 1994 The cellular retinoic acid binding protein I (CRABPI) is dispensable. Proc Natl Acad Sci USA 90:932–936
  58. Lampron C, Rochette-Egly C, Gorry P, Dolle P, Mark M, Lufkin T, LeMeur M, Chambon P