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Monash Institute of Medical Research,4 and Department of Physiology,5 Monash University, Clayton, Victoria 3168, Australia
ABSTRACT
Prostaglandins (PGs), particularly PGE2, have been implicated in the control of testicular steroidogenesis, spermatogenesis, and local immunity. However, virtually nothing is known about the expression or activity of the prostaglandin-endoperoxide synthases (PTGSs; also referred to as the cyclooxygenases), the specific rate-limiting enzymes responsible for PG production, in the adult testis. This activity was investigated in rats under normal conditions and during lipopolysaccharide-induced inflammation using quantitative real-time PCR, in situ hybridization, Western blotting, and PGE2 measurements by ELISA. The mRNA for both the "constitutive" Ptgs1 and the "inducible" Ptgs2 forms was detected in multiple testicular cell types. Testicular Ptgs2 expression was substantially higher than that of Ptgs1, and testicular production of PGE2 in vitro was found to be suppressed by a specific PTGS2 inhibitor (NS-398), but not by an inhibitor of PTGS1. Further investigation indicated that 1) PGE2 production in the adult testis is attributable to constitutive expression of PTGS2 by somatic (Leydig cells and Sertoli cells) and spermatogenic cells; 2) testicular macrophages constitutively produce relatively low levels of PTGS2 and PGE2 but are the only cell type to respond significantly to an inflammatory stimulus by increasing production of PGE2; and 3) testicular PTGS2 expression and intratesticular PGE2 levels are only marginally affected by acute inflammation. These data point toward a previously unanticipated maintenance role for the "inducible" PTGS2 enzyme in normal testicular function, as well as an anomalous response of testicular PTGS2 to inflammatory stimuli. Both observations are consistent with the reduced capacity of the testis to initiate and support inflammatory reactions.
immunology, Leydig cells, male reproductive tract, Sertoli cells, testis
The prostaglandins (PGs) are a family of lipid-soluble metabolites of arachidonic acid that act as autocrine and paracrine signaling molecules throughout the body. These molecules are particularly important in the regulation of vascular responses, apoptosis, and inflammation [1]. They also regulate several key reproductive processes in the female, including ovulation, fertilization, and parturition [2], but their roles in the male reproductive system, especially in the adult, are very poorly defined. Studies have shown that PGs are present in the testes of humans and other species [3, 4]. In the adult rat testis, PGE2 and PGF2
predominate, with PGD2, PGE1, and PGF1 detected at much lower levels [5, 6].
Numerous studies have examined the effects of nonsteroidal anti-inflammatory drugs (NSAIDS), such as aspirin and indomethacin, which are inhibitors of PG production, or the effects of PGs themselves on male fertility and spermatogenesis [7–9]. These studies have produced conflicting results so that both stimulatory and inhibitory effects of PGs overall on spermatogenesis have been proposed, highlighting the likely complexity of roles for these molecules in controlling various aspects of testicular function. Prostaglandins, particularly PGE2 and PGF2, have been directly and indirectly implicated in controlling Leydig cell development in the immature testis, production of proinflammatory cytokines such as interleukin 1 (IL1) and IL6 by the Leydig and Sertoli cells, autoregulation of steroidogenesis in the adult, and the decline in Leydig cell function that occurs during aging [10–15]. In addition, PGE2 and PGF2 are produced by mature spermatozoa and play a role in the acrosome reaction [16].
Published data suggest that PGs do not play a critical role in the regulation of the testicular vasculature [17, 18], but PG production by testicular macrophages may be involved in modulating local immune and inflammatory responses. Several studies have shown that the macrophages of the testis possess reduced inflammatory capacity and may actually play an active role in protecting the developing germ cells by locally suppressing immune responses [19–21]. In 1995, Kern and colleagues reported that rat testicular macrophages constitutively produce PGE2 and PGF2 in vitro [22], and that the nondiscriminatory NSAID indomethacin blocked the unique ability of cultured rat testicular macrophages to suppress lymphocyte activation and increased their capacity to produce IL1 and IL6 [22, 23]. Since PGE2 has been shown to inhibit the proinflammatory activity and modulate the immune properties of monocyte-derived cells (macrophages and dendritic cells) in other tissues [24–26], it was suggested that endogenous production of PGE2 by these cells was responsible for maintaining a noninflammatory, potentially immunosuppressive state within the testis.
Prostaglandin-endoperoxide synthases (PTGSs) catalyze the conversion of arachidonic acid into the prostaglandin precursor PGH2, which is the essential rate-limiting step in the production of all PGs, prostacyclins, and thromboxanes [27]. There are two forms of the PTGS enzyme, which have the same activity but are produced from different genes. In general, PTGS1 (also called cyclooxygenase 1) is constitutively expressed and has numerous housekeeping functions, such as maintenance of gastric pH levels. Surprisingly, the Ptgs1 knockout mouse shows relatively few defects, characterized by delayed parturition, low gastric pH, and minor deficits in inflammatory responses [28]. In contrast to PTGS1, PTGS2 (also called cyclooxygenase 2) has some housekeeping functions, but its expression can be greatly induced during inflammation. This inducible PTGS2 expression and its subsequent PG production both promote and eventually resolve the inflammation cascade [29]. Knockout mouse studies have demonstrated essential roles for PTGS2 in normal physiology, inflammation, and immunoregulation. Female Ptgs2 knockout mice suffer from reduced fertility at multiple levels, although male fertility appears to be unaffected [30–32]. Double deletions of the PTGS isoforms are lethal [33].
Among the tissues of the male reproductive tract, PTGS2 expression is most prominent by far in the vas deferens [34]. Both PTGS1 and PTGS2 are detectable in the epididymis [35] and testis [36, 37] under normal conditions. Within the testis, Ptgs2 mRNA has been detected in Sertoli cells [14] and in both progenitor and adult Leydig cells of the rat [10, 13] and hamster [38]. However, little is known concerning either the respective contributions of the two PTGS isoforms to testicular PG production or the capacity of different testicular cells for PG synthesis. Furthermore, the response of PTGS isoform expression and PG production in the testis to an inflammatory stimulus should provide important information necessary for understanding the unique immunologic environment of this organ. In the following study, these fundamental issues were investigated, providing evidence that PTGS2 plays the more important role in normal testicular function and displays an anomalous pattern of expression during inflammation that is consistent with a reduced capacity for supporting inflammatory responses in the testis.
Adult (80–100 days old) and immature (20 days old) male Sprague-Dawley rats from the Monash University Central Animal House were maintained under standardized conditions of lighting (12L:12D) and nutrition (food and water ad libitum) throughout the experimental period. Studies were performed in accordance with the Australian National Health and Medical Research Council Guidelines on Ethics in Animal Experimentation and were approved by the Monash Medical Centre Animal Experimental Ethics Committee. All reagents were obtained from the Sigma-Aldrich Chemical Co. (St. Louis, MO) unless otherwise stated.
Collection of Tissues and Interstitial Fluid
For tissue distribution studies, adult male rats were killed by CO2 overdose, and samples of testis, epididymis (caput and cauda), anterior prostate, vas deferens, liver, lung, cortical kidney, adrenal gland, and cortical brain were removed. Tissues were taken and immediately snap frozen in 1 ml TRIzol (Invitrogen, Carlsbad, CA) using liquid nitrogen and were stored at –80°C for subsequent RNA extraction. For the lipopolysaccharide (LPS) stimulation time course experiments, adult rats were injected (i.p.) with either 0.1 mg/kg body wt or 5 mg/kg body wt LPS (Escherichia coli strain 0127:B8) in saline or with pyrogen-free saline alone as control. Testis and liver were collected after 0, 3, 6, 12, 18, 24, or 72 h for RNA extraction. Interstitial fluid was obtained from the testes of adult rats injected (i.p.) with pyrogen-free saline or LPS (0.1 mg/kg) by drip collection (16 h, 4°C) in a preweighed 15-ml tube containing 50 µl of 1% (w/v) methoxyamine hydrochloride/8.2% (w/v) sodium acetate/10% ethanol, in a modification of the method described by Hedger and Hettiarachchi [39]. The volume of the recovered fluid sample was determined and was stored at –20°C in the methoxyamine solution prior to assay for PGE2.
In order to obtain testis sections for in situ hybridization studies, adult rats were injected with either saline or LPS (5 mg/kg) 3 h prior to collection of tissue, and the testes were fixed by perfusion with Bouin fixative (15 min) via the descending aorta under ether anesthetic, as previously described [20, 21]. Testes were removed, cut transversely into three equal pieces, postfixed in Bouin fixative for 5 h, and processed for embedding into paraffin. Sections of 5-µm thickness were collected onto Superfrost Plus slides (Menzel-Glaser, Braunschweig, Germany).
Cell and Tissue Fragment Isolation and Culture
Sertoli cells were isolated from immature rat testes, as previously described [40]. Briefly, isolated seminiferous tubules were subjected to serial collagenase/hyaluronidase digestion, resulting in Sertoli cell aggregates that were cultured in Dulbecco modified Eagle medium (DMEM) containing 0.1% BSA for 24 h at 32°C to allow attachment, followed by a change of medium to deplete contaminating germ cells. The purity of the resulting Sertoli cells was routinely 85%–90%, with the major contamination (8%) from peritubular cells and residual germ cells.
Leydig cells were isolated from adult rat testes by collagenase dispersal and centrifugal elutriation, as previously described [41]. The resulting Leydig cells were routinely >90% pure, as assessed by esterase and 3β-hydroxysteroid dehydrogenase staining [42].
Testicular macrophages were purified from saline-perfused adult rat testes, as previously described [43]. Peritoneal macrophages were isolated by peritoneal lavage of adult rats, followed by 30 min of culture in GIBCO Macrophage-SFM medium (Invitrogen) at 37°C to allow the macrophages to attach to the culture dish, and four washes to remove nonadherent contaminating cells. Immunolocalization of the macrophage-specific markers ED1 and ED2 was used to determine that testicular and peritoneal macrophage cultures were routinely >90% pure [44].
Pachytene spermatocytes and round spermatids were isolated from adult rat testes using centrifugal elutriation, as previously described [45]. The purity of the germ cells was routinely >85% for each cell type.
Whole-testis fragments of approximately 5 mm in each linear dimension were manually dissected using a scalpel blade from decapsulated adult rat testes that had been perfused with saline prior to collection [39]. Seminiferous tubule fragments were obtained from decapsulated, saline-perfused adult rat testes [46]. The seminiferous tubules were manually separated from the interstitial tissue in a Petri dish containing DMEM, and the dissociated testes were resuspended in DMEM to a volume of 50 ml in a centrifuge tube. The tubules were allowed to settle for 5 min at room temperature and were collected by removal of the supernatant containing the majority of the interstitial tissue. Tubules then were cut into approximately 2-mm lengths using a scalpel blade under a dissecting microscope.
The isolated testicular cells (approximately 1 x 106 cells/well), seminiferous tubules (50 x 2 mm fragments/well), and whole-testis fragments (5 mm x 5 mm x 5 mm fragments/well) were cultured at 37°C (testicular and peritoneal macrophages) or 32°C (all other cultures) with or without 10 µg/ml LPS for 3 h (for RNA and protein isolations), or for 24 h (for measurement of PGE2), at a final volume of 3 ml in a 9.6-cm2 well plate. Testicular macrophages isolated from each individual testis were cultured in a single well at a final volume of 3 ml. Peritoneal macrophages from a single rat produced duplicate wells. Leydig cells, whole-testis and tubule fragments, and germ cells were cultured in DMEM; Sertoli cells were cultured in DMEM/F12; and macrophages were cultured in GIBCO Macrophage-SFM medium. Fetal calf serum (Invitrogen) at a final concentration of 10% was added to the 24-h cultures only. Cells were harvested by lysis in 1 ml TRIzol for RNA extraction, or lysis in RIPA buffer (150 mM NaCl/1% [v/v] Nonidet P-40 [BDH-Merck, Darmstadt, Germany]/0.5% [v/v] Tween 20/0.1% [w/v] SDS/1 mM EDTA) containing 1% (v/v) Protease Inhibitor Cocktail III (Calbiochem, San Diego, CA) for protein extraction.
Specific Inhibition of PTGS1 and PTGS2 In Vitro
The PTGS1 inhibitor valeroyl salicylate (Cayman Chemical, Ann Arbor, MI), was dissolved in ethanol at a stock concentration of 195 mM. The PTGS2 inhibitor NS-398 (Cayman Chemical) was dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 50 mM. Whole-testis fragments (5 mm x 5 mm x 5 mm fragments/well) were cultured in GIBCO Macrophage-SFM medium/10% fetal calf serum in the absence or presence of 10 µg/ml LPS in triplicate 9.6-cm2 wells for 24 h at 32°C together with valeroyl salicylate (0.005–5 mM in a maximum volume of 12 µl/ml ethanol) and NS-398 (0.001–10 µM in a maximum of 1 µl/ml DMSO). At the end of the incubation, medium was collected for measurement of PGE2. Control cultures received the ethanol or DMSO vehicle solutions at the same maximum concentration as the experimental cultures.
RNA Extractions and cDNA Synthesis
Tissues and primary cell cultures from which RNA was to be extracted were snap frozen using liquid nitrogen and were stored at –80°C. Total RNA was purified by lysis in TRIzol and scraping of cells from tissue culture wells. This was followed by chloroform extraction using 0.2 ml chloroform/isoamyl alcohol (24:1). Extractions were centrifuged at 12 000 x g for 15 min at 4°C. The aqueous layer was precipitated by addition of 0.5 ml isopropanol, followed by incubation at room temperature for 10 min and centrifugation at 12 000 x g for 10 min at 4°C. RNA pellets were washed with 1 ml of 70% ethanol followed by centrifugation for 7 min at 7500 x g and dissolution in 20 µl diethylpyrocarbonate (DEPC)-treated water. RNA solutions were treated with DNAse to remove genomic DNA using the DNAfree kit (Ambion Inc., Austin, TX) according to the manufacturer's instructions. RNA (1–2 µg) was used in each reaction, except in the case of peritoneal macrophage cDNA synthesis, which was optimized to 400 ng RNA for each reaction. Oligo-dT-primed cDNA was synthesized using the Superscript kit (Invitrogen) according to the manufacturer's instructions. For each cDNA, a non-reverse transcribed control was produced in which primers and RNA were subject to the same synthesis conditions minus reverse transcriptase enzyme.
Quantitative real-time PCR analysis was performed using the Roche LightCycler (Roche, Mannheim, Germany) and the FastStart DNA Master SYBR-Green system (Roche). Rat Ptgs1 cDNA was amplified using the specific primers 5'-GCCTCGAACCACTACCAATGT-3' and 5'-GTGGTGGGTGAAGTGTTGTG-3'; rat Ptgs2 using 5'-GATTGACAGCCCACCAACTT-3' and 5'-ACGTGGGGAGGGTAGATCAT-3'; rat IL1β using 5'-CCAGGATGAGGACCCAAGCA-3' and 5'-CCCGACCATTGCTGTTTCC-3'; and rat β-actin using 5'-GATATCGCTGCGCTCGTC-3' and 5'-TGGGGTACTTCAGGGTCAGG-3'. Amplification conditions for each primer set consisted of 10 min of denaturation at 95°C, followed by 46 repeats of 15 sec of 95°C, 5 sec of 63°C, and 18 sec of 72°C in the presence of 3 mM Mg2+. Sample cDNAs were diluted 1:10 or 1:20 immediately prior to PCR reactions. The LightCycler software was used to calculate crossing point (CP) data points using the second derivative maximum method. Melting curve data acquisition was conducted from 68°C to 97°C to confirm product purity. For all real-time PCR reactions, standard curves were generated using purified PCR products in sequential 10-fold dilutions. In the LPS time course experiment in which all samples were derived from the same tissue type (testis or liver), quantification was performed using routine normalization to the measurement of the housekeeping gene β-actin cDNA for each sample to correct for minor differences in input RNA quantity and efficiency of cDNA synthesis, as previously described. [21]. In experiments where different tissues or cell types were compared, it was found that levels of β-actin per gram tissue or per cell, as well as those of other housekeeping genes (GAPDH and L32), differed significantly between each tissue or cell type, preventing normalization by this method. Consequently, the quantitative real-time PCR data was normalized to the total amount of RNA used in each cDNA reaction, which was calculated from spectrophotometric quantification of total RNA measured at the 260-nm wavelength (Biophotometer; Eppendorf, Hamburg, Germany). This value is directly proportional to the number of cells used to prepare the RNA [47]. All data in each experiment were determined from RNA extracted at the same time, and the resulting samples were run in parallel to minimize variation in extraction efficiency and cDNA synthesis. All experiments contained replicate samples, and samples were subjected to repeated measurements in order to obtain an indication of the variability arising from this method. With the exception of the tissue distribution data, which are presented as nanogram cDNA per milligram RNA, mRNA levels were standardized against the value for an appropriate reference group within the same quantitative real-time PCR run, which was assigned the arbitrary value of 1.0. In initial experiments, PCR product sizes were verified by agarose gel electrophoresis and sequenced, using the above primers to confirm identity. The absence of contaminating genomic DNA in cDNA samples was confirmed using non-reverse transcribed controls. PCR product purity was routinely monitored by LightCycler determination of the PCR product melting temperature.
Samples to be analyzed by Western blotting were lysed in RIPA buffer containing 1% (v/v) Protease Inhibitor Cocktail III (Calbiochem) at 4°C with homogenization using a Janke & Kunkel Ultraturrax T25 homogenizer (IKA Labortechnik, Staufen, Germany) as required. Lysates were incubated on ice for 10 min, followed by 10 min of rotatation at 4°C and 10 min of centrifugation at 18 000 x g to recover soluble fraction. Total protein concentration was determined using the DC protein assay kit (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. Protein (150 µg) for each sample was diluted 4 in 1 with sample buffer (0.5 M Tris, 16% [w/v] SDS, 50% [v/v] glycerol, 20% [v/v] β-mercaptoethanol, and 0.08% [w/v] bromophenol blue, pH 6.8) and was incubated at 65°C for 5 min, followed by electrophoresis in a 10% polyacrylamide gel and transfer onto an Immobilon P membrane (Millipore, Bedford, MA) in 20 mM Tris/150 mM glycine/10% (v/v) methanol. Membranes were blocked in Tris-buffered saline, pH 7.4, containing 5% (w/v) nonfat dry milk powder and 0.3% (v/v) Tween 20 (TBS-TM) for 2 h at room temperature, then probed overnight with 1:1000 rabbit anti-mouse PTGS2 polyclonal antibody (catalog no. 160106; Cayman Chemical) in TBS-TM, followed by three 10-min washes in TBS-TM. The anti-mouse PTGS2 sera cross-reacts with the rat PTGS2, since it was raised against a peptide from the C-terminus region identical in mouse and rat PTGS2 proteins. Membranes then were probed with 1:10 000 biotin-conjugated anti-rabbit IgG (Silenus, Melbourne, Australia) in TBS-TM for 1 h, washed, and probed with 1:7500 streptavidin-conjugated horseradish peroxidase (Silenus) diluted in TBS-TM for 1 h, followed by detection with the Enhanced Chemiluminescence system (Amersham) according to the manufacturer's instructions.
Localization of Ptgs2 in the testis by in situ hybridization was performed as previously described [48]. A rat Ptgs2 3' UTR PCR product was amplified from testis macrophage cDNA using the primers 5'-GCAGATACCGGCAACTGTCT-3' (forward) and 5'-GCCAGCAATCTGACGTACAA-3' (reverse). These PCR products were ligated into pGEMTEasy (Promega Corp., Madison, WI) and transformed into cells of the DH5 host strain. Positive colonies were identified via PCR, verified by sequencing, and the plasmids used as PCR templates to derive products using "Bluescript" forward and reverse primers with the Ptgs2 probe region flanked by the T7 and Sp6 promoter sequences. These PCR products were used as the template for PCR-based production of DIG-cRNAs from the T7 and Sp6 promoters. Hybridization and washing both were performed at 50°C. Both antisense and sense (negative control) RNAs were used on each sample. Sections were counterstained with Mayer hematoxylin.
PGE2 in media samples was subjected to methyloximation by dilution of 1:1 with 0.12 M methyloxyamine hydrochloride (Sigma Chemical) in sodium acetate (1 M, pH 5.6) containing 10% ethanol and by incubation overnight at room temperature. PGE2 content was determined by a previously described radioimmunoassay [49] using methyloximated PGE2 standard and methyloximated 3H-PGE2 tracer. The antiserum was raised against PGE2 methyloxime and displayed cross-reactivies of 270% with PGE1, 0.3% with 15-keto-PGE2, and less than 0.1% with other related cyclooxygenase products. The sensitivity of the assay was 0.36 nmol/l, and the intraassay and interassay coefficients of variation were 8% and 12%, respectively, for five assays.
Data were analyzed by 1) Student t-test, 2) one-way or two-way ANOVA following suitable transformation to normalize the data and to equalize variance to compare results between groups in conjunction with either a Tukey or Student-Neumann-Kuels multiple comparisons test, or 3) Kruskal-Wallis one-way ANOVA on ranks in conjunction with Dunn multiple comparisons with a control group, as appropriate. All statistical analyses were performed using the GraphPad Prism version 2 software program (GraphPad Software Inc., San Diego, CA).
Ptgs1 and Ptgs2 Expression in the Testis and Other Tissues
Ptgs1 and Ptgs2 transcripts were present in samples of testis, liver, lung, kidney, prostate, adrenal glands, brain cortex, epididymis, and vas deferens as measured by quantitative real-time PCR (Fig. 1). Overall Ptgs1 expression was highest in the lung, followed by the vas deferens, with the remaining tissues showing variable but comparatively lower levels of expression (Fig. 1A). Ptgs1 expression levels were consistently very low in the testis samples relative to all other tissues. Testis Ptgs2 mRNA expression was similar to that of the epididymis, kidney, lung, and adrenal gland, but was lower than either the brain cortex or vas deferens (which was at least 10-fold greater than any other tissue). Ptgs2 expression was significantly lower in the normal prostate and liver than in all other tissues measured. Comparison of the relative levels of expression of the two enzymes in the testis indicated that Ptgs2 levels were 8.1 ± 3.9 times higher (mean ± SEM, n = 6 animals) than that of Ptgs1 based on nanograms of cDNA produced per milligram of total RNA extracted.
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Ptgs1 and Ptgs2 mRNA Responses to LPS in the Testis and Liver
Ptgs1 and Ptgs2 mRNA levels were measured in samples of testis and liver from rats injected with 0.1 mg/kg LPS from 0–72 h prior to collection. In the testis, Ptgs1 and Ptgs2 mRNA levels were not significantly different from control (0 h) following LPS treatment for up to at least the first 6 h, but they fell progressively to be significantly lower than control values at 24 and 72 h (Fig. 2, A and B). In contrast, a typical tissue response to LPS was observed in the liver: Ptgs2 expression was significantly increased, peaking at 3 h after LPS stimulation, whereas Ptgs1 was unaffected (Fig. 2D). Increasing the dose of LPS 50-fold (5 mg/kg) did not stimulate either Ptgs1 or Ptgs2 expression in the testis within the acute (3 h) period, and a similar long-term decline in expression was observed (data not shown).
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Ptgs1 and Ptgs2 Expression and Regulation in Specific Cell Populations of the Testis
Ptgs1 mRNA expression was detected in cultured (3 h) testicular macrophages, Sertoli cells, Leydig cells, round spermatids, and pachytene spermatocytes, as well as whole-testis and seminiferous tubule fragment cultures (Fig. 3A). In each cell population, Ptgs1 mRNA was expressed at similar levels and was unaffected by stimulation with 10 µg/ml LPS. Basal expression of Ptgs2 in every cell type was at least as high as that found in resting testicular macrophages, and round spermatids actually contained approximately 20-fold higher levels than the resting macrophages (Fig. 3B). Treatment with 10 µg/ml LPS stimulated Ptgs2 mRNA levels in testicular macrophages an average of 120-fold (over 3 h), but its effects on the other testicular cell types were not significant. Under basal conditions, testicular macrophage Ptgs2 mRNA levels were much lower than those found in peritoneal macrophages cultured under the same conditions, but LPS stimulation produced a similar upregulation of Ptgs2 mRNA after 3 h in both cell types (Fig. 4A). The high levels of Ptgs2 expression in the unstimulated peritoneal macrophages was not due to cryptic activation of the cells, as basal production of several inflammatory cytokines, including IL1β (Fig. 4B), by these cells was not elevated. Consistent with previous observations [23, 43], isolated testicular macrophages showed a relatively reduced capacity to produce IL1β mRNA upon LPS stimulation.
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In testes from both normal and LPS-treated animals, expression of Ptgs2 mRNA was confirmed within both the seminiferous tubules and interstitial tissue compartments by in situ hybridization (Fig. 5). Within the tubules, strongest labeling appeared to be associated with the cytoplasm of spermatogonia/spermatocytes and Sertoli cells, with more diffuse labeling in round and elongating spermatids. In the interstitium, labeling was associated with the Leydig cells and macrophages, with macrophages in the LPS-treated testes showing the highest level of expression, as expected.
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Western blotting detected a protein band of approximately Mr 72 x 10–3 in lysates of whole testis, testicular macrophages, and round spermatids (Fig. 6), corresponding to the expected size of the PTGS2 protein. The protein was not detectable in unstimulated testicular macrophages, but only the testicular macrophages showed a substantial upregulation of protein levels in LPS-stimulated cultures.
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PGE2 Production in Rat Testicular Interstitial Fluid and Specific Cell Populations of the Testis
Interstitial fluid collected from saline-injected rat testes contained levels of PGE2 ranging from 9.2–30.6 ng/ml, with a mean of 18 ng/ml PGE2. Concentrations of PGE2 in testicular interstitial fluid from LPS-treated rats varied from 14.9–66.7 ng/ml, but overall were not significantly elevated relative to the saline-injected controls (Fig. 7A).
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PGE2 was detectable in the medium from all cultures except that of pachytene spermatocytes and unstimulated macrophages. In contrast to the Ptgs2 mRNA expression pattern, the PGE2 produced by cultured round spermatids appeared to be little different from that produced by the other testicular cell types. Following LPS stimulation, macrophages responded by producing significantly more (over 200-fold) PGE2 than basal levels (Fig. 7B). Consistent with the Ptgs2 expression data, however, there was little or no significant change in PGE2 production by the other cell types following LPS treatment.
Relative Contributions of PTGS1 and PTGS2 to Testis PGE2 Production In Vitro
Although expression levels indicated a considerably higher production of Ptgs2 versus Ptgs1 in the normal testis, there was the possibility that this relative expression was influenced by differences between the mRNA in stability, PCR efficiency or, possibly, in extraction. Consequently, to determine the actual contribution of the two PTGS isoforms to PGE2 production by the testis, cultured testis fragments were assessed for production of PGE2 in the presence of specific inhibitors of PTGS1 or PTGS2. PGE2 production by the cultured testis was only significantly reduced in the presence of excessively high levels (>500 mM) of the selective PTGS1 inhibitor, valeroyl salicylate (Fig. 8A). At this concentration, valeroyl salicylate also is able to inhibit PTGS2 [50]. Most importantly, PGE2 production was completely inhibited in the presence of a relatively low concentration (<0.1 µM) of the selective PTGS2 inhibitor, NS-398 (Fig. 8B). At this concentration, NS-398 is a highly specific inhibitor of PTGS2, but not of PTGS1 [51]. Similar results were obtained from cultures of testis fragments incubated with 10 µg/ml LPS (data not shown). These data indicate that most, if not all, of the PGE2 produced by the adult rat testis is due to the activity of PTGS2 rather than of PTGS1.
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Based on relative mRNA expression levels and the response to PTGS-specific inhibitors, the inducible form of PTGS2 was found to be much more important than PTGS1 for local PG production in the adult rat testis under basal or inflamed conditions. Even more unexpected was the fact that PTGS2 was constitutively expressed not only by somatic (Sertoli and Leydig) cells of the testis, as previously reported [13, 14], but also by spermatogenic cells in the normal testis. The presence of Ptgs2 mRNA and protein in the postmeiotic germ cells in particular is entirely consistent with observations that mature spermatozoa, which are transcriptionally inactive, are able to produce PGs [52–54]. However, the presence of this enzyme in the germ cells throughout spermatogenesis also suggests a specific physiologic role for germ cell-derived PG production within the seminiferous epithelium itself. In contrast to an earlier report [22], freshly isolated testicular macrophages expressed relatively low levels of PTGS2 and did not produce PGE2 unless stimulated with LPS. All other testicular cells studied appeared to be much less sensitive, or even insensitive, to stimulation by LPS, at least as far as PG production was concerned. Together, these data indicate an endogenous role for PGs produced by the action of PTGS2 from a number of different cell types in both normal testicular functions and during inflammation. This indicates that more detailed studies on the specific regulation of PTGS2 gene expression within the adult testis would clearly be very worthwhile.
Previous studies on the expression of PTGS forms in male reproductive tissues have produced quite variable results, with several reports of very high levels of PTGS2 in the vas deferens and lower levels in the epididymis, but not in the testis of several species, including the rat [34, 38, 55]. On the other hand, several studies support our observation that Ptgs2 mRNA is expressed in the rat testis [36, 37]. Although Ptgs2 expression in the adult rat testis was much lower than that found in the vas deferens in the present study, it was nonetheless comparable to levels found in many other tissues, including the epididymis. The presence of PTGS2 in the normal testis is consistent with numerous studies showing that PGs are produced constitutively in the testis, with PGE2 and PGF2 present at the highest concentrations [3–5]. Previously, basal and stimulated PG production have been reported in cultured rat Sertoli cells [14, 56], Leydig cells [11], and testicular macrophages [20, 22]. In a novel observation, however, our data establish that germ cells also contain PTGS2, that both premeiotic and postmeiotic cells express Ptgs2 mRNA, and that round spermatids at least can produce PGE2 under basal conditions.
Curiously, there was an apparent difference in the relative level of expression of Ptgs2 in the round spermatids as measured by quantitative real-time PCR and in situ hybridization: although isolated round spermatids displayed significantly higher levels of mRNA than any other cell type when measured by quantitative real-time PCR, they appeared to express lower levels compared with either the Sertoli cells or spermatocytes, particularly when measured by in situ hybridization. Moreover, PTGS2 protein levels in round spermatids as measured by Western blot, and the production of PGE2 by these cells did not appear to indicate a relatively higher level of enzyme expression in these cells. At this stage we have no obvious explanation for these apparent anomalies. Certainly, there is evidence that mRNA in round spermatids is stabilized and transcriptionally inactivated [57], and it is possible that the relatively higher value measured by quantitative real-time PCR simply reflects the fact that stability or recovery of mRNA from round spermatids during isolation is more efficient than for the other cell types. Moreover, round spermatids are considerably smaller than most other cell types in the testis, and hence expressing mRNA levels for a functionally regulated gene such as Ptgs2 against either total RNA or a constitutive housekeeping gene might lead to its overestimation. The observation that PGE2 production by the isolated germ cell populations in culture was low relative to either Sertoli cells or Leydig cells suggests that the production and/or activity of the enzyme itself may be inhibited to some degree in the germ cells. Alternatively, as germ cells are normally dependent upon Sertoli cell support for their normal function, it is possible that synthesis in vitro does not reflect the relative amounts of PGE2 normally capable of being produced by this cell type in vivo.
While speculating about the reasons for the apparent relative differences obtained using each of the four detection methods, particularly with respect to the round spermatids, it is worth noting that a study by Frungieri and colleagues [38] recently reported that PTGS2 was absent from the testes of several species, including the rat, based on immunohistochemical data. This suggests difficulties with the detection of the enzyme by this method in the testis, which would be consistent with our own immunohistochemical observations in adult rat testis using two different polyclonal antibodies (Winnall and Hedger, unpublished data). In the larger context, however, given both the overall contradictions in the literature regarding PTGS2 expression in the testis [34, 36, 37, 55] and the obvious quantitative differences observed using several different assay approaches in the present study, it would be best to adopt a cautious approach to the interpretation of any relative expression data in different tissues and cell types based on any single method of detection for this enzyme, as for any gene or protein.
The data in the present study and that of a previous study using highly purified resident testicular macrophages [20] indicate that although there was detectable Ptgs2 mRNA in unstimulated testicular macrophages, PTGS2 production and PGE2 synthesis are not constitutively elevated in these cells under normal conditions. An explanation for the difference between these results and those of Kern and Maddocks [22] may be that macrophages in the earlier study had become activated in culture either by endogenous endotoxin in the culture medium or serum or by the isolation procedure itself. For example, collagenase, which was used to isolate testicular macrophages in the earlier study but not in the present study, has been shown to activate testicular macrophages [58]. Another interesting finding was the fact that peritoneal macrophages displayed a relatively high level of Ptgs2 mRNA expression under basal conditions, which could not be attributed to activation in vitro, since inflammatory cytokine production was not similarly elevated. It should be noted that while constitutive Ptgs2 mRNA expression by rat peritoneal macrophages has not been reported previously, several studies have reported significant basal PGE2 production by these cells [59].
These experiments represent the first published study on the effect of LPS stimulation on PG production in the testis. Significantly, with the possible exception of the Sertoli cells, the principal somatic and germ cells of the testis showed little or no significant response to LPS in culture as far as Ptgs2 expression or PGE2 production was concerned. In fact, the data suggest that expression of PTGS2 in these cells undergoes a gradual decline for at least 72 h following an inflammatory event in vivo. On the other hand, the testicular macrophages showed a very robust increase in Ptgs2 mRNA and PGE2 production following LPS stimulation, which was in direct contrast with the relatively poor response of IL1β to LPS in these cells. This observation confirms that the capacity of the testicular macrophages to respond to LPS is intact, even though production of several key inflammatory cytokines by these cells is inhibited [23, 43]. Nonetheless, testicular inflammation did not significantly increase total PGE2 or Ptgs2 expression levels in the whole testis during the acute phase (3–6 h) of the response. One possible explanation for this lack of increase could be that the testicular macrophages comprise less than 1% of the total cells in the rat testis. By comparison, the spermatogenic cells comprise about 80% of all cells in the rat testis, whereas the Leydig and Sertoli cells represent about 9% of the total [60–62]. Constitutive, or possibly even declining, production of PGE2 by the somatic and germ cells could obscure even large changes in production by the testicular macrophages after LPS treatment, with the net result that inflammation has relatively little effect on overall PGE2 levels in the testis in the short term. While the data in the present study are not consistent with the hypothesis that the reduced inflammatory phenotype of the testicular macrophages is due to endogenous and constitutive production of PGE2 by these cells, as suggested by Kern and colleagues [22], it remains entirely feasible that constitutive PG production by other testicular cells may be responsible instead. This hypothesis requires further examination. Moreover, although most attention has been addressed to PGs of the E and F series, it should also be recognized that PTGS2 is in the pathway to PGs of the D and J series, which also have profound anti-inflammatory actions [63].
Apart from the immunoregulatory actions of the PGs, expression of PTGS2 and production of PGs by numerous cell types in the testis suggest an overall, ongoing role in controlling the development, steroidogenic activity, and inflammatory functions of the Sertoli and Leydig cells [10, 13–15, 64–66]. Since PG production regulates changes in vascular permeability and blood flow in other tissues, most notably during inflammation [1], endogenous PG production by the testis also may be involved in maintaining fluid balance and vascular function. Balanced against this, the limited data so far available have not provided any direct evidence for this possibility [17, 18]. Finally, while the expression of PTGS2 and nascent PGE2 production by the developing germ cells may simply prefigure the production of PGs by the mature spermatozoa, this expression pattern of PTGS2 also suggests a potential role for PGs in communication between the spermatogenic cells and the Sertoli cells within the seminiferous epithelium. Studies with PG inhibitors have indicated both negative [7, 9] and positive [8] roles for PGs in spermatogenesis, but given the widespread sites of production and multiple sites of action within the testis it cannot be assumed that such effects necessarily involve direct actions at the level of the seminiferous epithelium. Obviously, much further investigation will be necessary to establish the precise role of PGs in controlling spermatogenesis itself.
ACKNOWLEDGMENTS
Thanks are due to Dr. Kazutaka Saito and Ms. Nicole Dellios for expert technical assistance.
FOOTNOTES
3Current address: School of Biomedical Sciences, University of Newcastle, Callaghan, NSW 2308, Australia. 
1Supported by a Program Grant from the National Health and Medical Research Council (NHMRC grant no. 143786), an NHMRC Project Grant (no. 194423), and by NHMRC Fellowships awarded to M.P.H. (no. 143788) and M.K.O. (no. 143781).
Correspondence: 2Wendy R. Winnall, Centre for Reproduction and Development, Monash Institute of Medical Research, 27-31 Wright St., Clayton, VIC 3168, Australia. FAX: 61 3 9594 7111; e-mail: wendy.winnall{at}med.monash.edu.au
Received: 17 April 2006.
First decision: 8 May 2006.
Accepted: 11 January 2007.
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 T. M. Onorato, P. W. Brown, and P. L. Morris Mono-(2-ethylhexyl) Phthalate Increases Spermatocyte Mitochondrial Peroxiredoxin 3 and Cyclooxygenase 2 J Androl, May 1, 2008; 29(3): 293 - 303. [Abstract] [Full Text] [PDF]
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