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Species-specific expression of microsomal prostaglandin E synthase-1 and cyclooxygenases in male monkey reproductive organs
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Prostaglandins, Leukotrienes and Essential Fatty Acids 71 (2004) 233–240
Species-specific expression of microsomal prostaglandin E synthase-1 and cyclooxygenases in male monkey reproductive organs$ M. Lazarusa,d, N. Eguchia, S. Matsumotoa, N. Nagataa, T. Yanob, G.J. Killianc, Y. Uradea,* a
Department of Molecular Behavioral Biology, Osaka Bioscience Institute, Suita, 6-2-4 Furuedai Suita, Osaka 565-0874, Japan b Nagahama Institute for Biochemical Science, Oriental Yeast Co., Nagahama, Shiga 526-0804, Japan c Department of Dairy and Animal Science, J.O. Almquist Research Center, Pennsylvania State University, University Park, PA 16802, USA d Department of Neurology, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA Received 21 January 2004; accepted 29 March 2004
Abstract We investigated the tissue distribution and cellular localization of microsomal PGE synthase-1 (mPGES-1) and cyclooxygenase (COX)-1 and -2 in male monkey reproductive organs. Western blotting revealed that monkey mPGES-1 was expressed most intensely in the seminal vesicles, moderately in the testis, and weakly in the epididymis and vas deferens. The tissue distribution profile was quite different from those profiles for rats, rabbits, and pigs, e.g., rat mPGES-1 was the most abundant in the vas deferens, and the rabbit and pig enzymes, in the testis. Immunohistochemical staining with mouse monoclonal anti-human mPGES1 antibody revealed that monkey mPGES-1 was localized in spermatogonia, Sertoli cells, and primary spermatocytes of testis and in epithelial cells of the epididymis, vas deferens, and seminal vesicles. In monkeys, COX-1 was localized in epithelial cells of the epididymis and vas deferens, whereas COX-2 was dominantly found in epithelial cells of the seminal vesicles. r 2004 Elsevier Ltd. All rights reserved. Keywords: Microsomal prostaglandin E synthase-1; Cyclooxygenase; Male genital organs; Immunohistochemistry; Monoclonal antibody
1. Introduction Prostaglandin (PG) E2 was originally discovered as a vasodilatory substance in human seminal fluid [1]. PGE2 has been implicated in a wide variety of reproductive activities, e.g., male fertility, tubule contractility, nonpregnant and pregnant uterine contractility, the sperm acrosome reaction, and fertilization [2–4]. $ This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Grant 12558078 to Y.U. and Grant 13557016 to N.E.) and by grants from the Takeda Science Foundation (to M.L. and Y.U.), Science and Technology Agency (Grant 199041 to M.L.), the US Department of Agriculture (Grant 97-35203-9806 to G.J.K.), and Osaka City. *Corresponding author. Department of Molecular Behavioral Biology, Osaka Bioscience Institute, Suita, 6-2-4 Furuedai Suita, Osaka 565-0874, Japan. Tel.: +81-6-6872-4851; fax: +81-6-68722841. E-mail address: [email protected] (Y. Urade).
0952-3278/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2004.03.018
Glutathione-dependent, membrane-bound PGE synthase-1 (mPGES-1) catalyzes the isomerization of PGH2, a common precursor of various prostanoids, to PGE2 [5–7]. PGH2 is produced from arachidonic acid by cyclooxygenase (COX)-1 or COX-2 [8]. Coordinate upregulation of mPGES-1 and COX-2 was reported to occur in a human lung cancer cell line, A549, after treatment of the cells with interleukin-1b (IL-1b [9], and in rat osteoblasts [10] and rat and mouse peritoneal macrophages [10,11] after stimulation of them with IL1b, tumor necrosis factor-a, or lipopolysaccharide. Moreover, the induction of mPGES-1 was demonstrated to take place in granulosa cells of bovine ovarian follicles after in vivo administration of gonadotropins [12]. However, little is known about the cellular localization of PGE2-producing enzymes in mammalian genital organs. Recently, we reported that mPGES-1 was involved in PGE2 production in male mouse genital organs, where it was coordinately expressed with COX-1 and COX-2 [13].
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In the present study, we determined the tissue distribution and cellular localization of mPGES-1 and COXs in male monkey genital organs to gain insight into the nature of PGE2-producing cells of primates. We report here the highly species-specific distribution of mPGES-1 in monkey, rat, rabbit, and pig male genital organs and show that monkey mPGES-1 is colocalized with COX-1 in the testis, epididymis, and vas deferens and with COX-2 in seminal vesicles.
munohistochemistry of sections of the monkey caput epididymis and seminal vesicles, as described below. Positive clones were subcloned twice by the limiting dilution method. Ascites tumors were induced by injection of 1 107 hybridoma cells into the peritoneal cavity of pristane-primed BALB/C mice, and ascites fluids were collected after approx. 2 weeks. In order to purify mouse immunoglobulin (Ig) G from ascites and culture medium, we used a Proteus-A antibody purification kit (ProChem, Acton, MA) according to the manufacturer’s instructions.
2. Materials and methods 2.3. Western blot detection 2.1. Animals and materials Monkey (Macaca fascicularis) tissues were donated by R-Tech Ueno (Sanda, Japan), and rabbit tissues, by Kitayama Labes (Nagano, Japan). Pig tissues were obtained from a local slaughterhouse. Inbred C57BL/6 and BALB/C mice and Sprague–Dawley rats were purchased from Shizuoka Laboratory Animal Center (Shizuoka, Japan). All procedures used in handling animals were approved by the Animal Research Committee of Osaka Bioscience Institute. All commercially available chemicals were of analytical grade. Oligonucleotides were purchased from Amersham Biosciences Corp (New Jersey, USA). 2.2. Production of monoclonal antibody against human mPGES-1 Human mPGES-1 was recombinantly expressed in E. coli, purified as previously described [11], and used for immunization of mice. Monoclonal antibodies were . produced by the method of Kohler and Milstein [14]. In brief, an emulsion of human mPGES-1 (40 mg) and complete Freund’s adjuvant was injected intraperitoneally into 3 BALB/C mice. The mice were boosted every 2 weeks with the same amount of antigen mixed with an equal volume of Freund’s incomplete adjuvant until an adequate response was achieved, as judged by the results of enzyme-linked immunosorbent assay. An intravenous boost with 20 mg of the antigen in 0.2 ml of saline was carried out, and the mice were then euthanized 3 days after the injection. Subsequently, 5 108 spleen cells were fused with 2.5 108 P3U1 myeloma cells by the addition of 50% (w/v) polyethylene glycol 4000. The resulting cell suspension of hybrids was inoculated into three 96-well microtiter plates containing selective RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 10% fetal calf serum, antibiotics, 2mercaptoethanol, hypoxanthine, aminopterin, thymidine, and l-glutamine. The supernatants of cultures showing hybrid growth were screened for production of anti-human mPGES-1 antibody by Western blotting of microsomes of monkey seminal vesicles and by im-
Tissues were homogenized with an Ultra Turrax blender (Janke & Kunkel, Staufen, Germany) in phosphate-buffered saline (PBS; 1 ml/100 mg material) in the presence of protease-inhibitor complete (Roche Diagnostics, Mannheim, Germany). Cellular debris was removed by centrifugation at 7000 g and 4 C for 20 min. The microsomal fraction was then obtained by centrifugation at 100,000 g and 4 C for 1 h. Microsomal proteins were resolved sodium dodecyl sulfate polyacrylamide gel electrophoresis with 14% or 6% precast gels (Tefco, Tokyo, Japan) and electrophoretically transferred onto Immobilon polyvinyl difluoride (PVDF) membranes (Millipore, Bedford, MA) at 200 mA for 1 h or stained with Coomassie brilliant blue to confirm equal loading of proteins. After blockage of non-specific binding sites with BlockAce (Dainippon Seiyaku, Osaka, Japan) for 1 h at 25 C, the membranes were incubated at 4 C overnight with guinea pig polyclonal anti-mouse mPGES-1 IgG (2 mg/ml; [11]), mouse monoclonal anti-human mPGES-1 antibody (1 mg/ml), rabbit polyclonal anti-COX-1 antibody (dil. 1:1000; Oxford Biomedical Research, Oxford, MI), or mouse monoclonal anti-rat COX-2 antibody (1 mg/ml; BD Biosciences, Franklin Lakes, NJ) as appropriate, applied in 10% BlockAce in PBS containing 0.2% (v/v) Tween-20. As secondary antibody, horseradish peroxidase-coupled donkey anti-guinea pig, anti-mouse or anti-rabbit IgG (Jackson Laboratories, West Grove, PA) was added at a concentration of 10 mg/ml, and incubation was carried out 1 h at room temperature. Detection was performed with an enhanced chemiluminescence kit (Amersham Biosciences Corp.) according to the manufacturer’s instructions. 2.4. Immunoperoxidase staining After deep anesthesia with ketamine (intramuscular) and pentobarbital (intravenous), M. fascicularis monkeys were perfused with PBS via the left ventricle of the heart. The genital organs were removed and soaked in Bouin’s fixative [15]. After deep anesthesia with pentobarbital (intraperitoneal), rats were perfused via the left
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ventricle of the heart with 20 mM sodium phosphate (pH 7.4) containing 4% paraformaldehyde and 4% sucrose, and then with Bouin’s solution. Genital organs were removed and soaked overnight at 4 C in Bouin’s fixative. After embedment in paraffin, the monkey and rat tissues were sectioned (10 mm) and mounted on slides. The sections were then deparaffinized in xylene and rehydrated in ethanol with increasing concentrations of water. Endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide in PBS for 30 min at 25 C. The tissues were then treated with 0.3% pepsin in 0.01 M hydrochloric acid for 5 and 30 min for COXs and mPGES-1 immunostaining, respectively, at room temperature. Thereafter, non-specific binding sites were blocked for 1 h with 10% sheep serum (Life Technologies, Tokyo, Japan) in PBS that contained 0.1% Triton X-100 and 0.1% sodium azide. Primary and secondary antibodies were diluted in PBS containing 1% sheep serum and 0.1% Triton X-100. Monkey tissue sections were exposed to anti-mouse and antihuman mPGES-1 antibodies (4 and 2 mg/ml, respectively) for 2 days as well as to anti-COX-1 (dil. 1:500) and anti-rat COX-2 (2 mg/ml) antibodies for 1 day at 4 C; rat tissue sections were incubated with guinea pig
polyclonal mPGES-1 antibody (4 mg/ml) for 2 days at 4 C. After 1 h of incubation at 4 C with biotinylated anti-guinea pig, anti-mouse, or anti-rabbit IgG (7.5 mg/ ml; Vector Laboratories, Burlingame, CA), tissues were rinsed 3 times in PBS containing 0.1% Triton X-100. A Vectastain Elite avidin–biotin–peroxidase kit (Vector Laboratories) with diaminobenzidine substrate was used according to the manufacturer’s protocol. Tissues were counterstained with hematoxylin. Finally, sections were dehydrated, rinsed in xylene, and mounted with MountQuick (Daido Sangyo, Tokyo, Japan).
3. Results 3.1. Expression of mPGES-1, COX-1, and COX-2 in monkey, rat, rabbit, and pig male genital organs We previously generated a guinea pig polyclonal antimouse mPGES-1 antibody [11]. This polyclonal antibody not only reacted with mouse mPGES-1 but also cross-reacted with the rat, rabbit, pig, and monkey enzymes (Fig. 1A) as well as with the human enzyme (data not shown). We used this polyclonal antibody for (C)
(A) Monkey
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Lane 1 = mouse vas deferens Prostate
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Lane 2 = monkey seminal vesicles
Fig. 1. Western blotting analysis of mPGES-1 in male genital organs. (A) and (B) Expression of mPGES-1, COX-1, and COX-2 immunoreactive proteins in monkey, rat, rabbit, and pig male genital organs. Microsomal fractions were obtained from the testis (lane 1), caput (lane 2) and cauda (lane 3) epididymis, vas deferens (lane 4), seminal vesicles (lane 5), and prostate (lane 6) of the monkey, rat, rabbit, and pig. Microsomes of the genital organs (15 mg of protein) were electrophoresed and subsequently transferred onto a PVDF membrane. The membranes were immunostained with guinea pig polyclonal anti-mouse mPGES-1 antibody (A) or anti-COX-1 and COX-2 antibodies (B). Arrows in ‘‘B’’ indicate the positions of COX-1 and COX-2 on the right. (C) Specificity of monoclonal anti-human mPGES-1 antibody. Microsomes (15 mg of protein) of mouse vas deferens (lane 1) and monkey seminal vesicles (lane 2) were electrophoresed and subsequently transferred onto a PVDF membrane (left and right panels) or stained with Coomassie brilliant blue (middle panel). The membranes were then immunostained with polyclonal anti-mouse (left panel) or monoclonal antihuman (right panel) mPGES-1 antibodies. Molecular weight markers (Mr 103) are shown on the right, and the arrow on the left indicates the position of mPGES-1.
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Western blotting of the microsomal fraction to investigate the tissue distribution profiles of mPGES-1 in monkey, rat, rabbit, and pig male genital organs (Fig. 1A). Western blotting experiments were performed on specimens from several animals of each species and the same results were obtained in each case. The level of mPGES-1-immunoreactive protein was moderate in the microsomal fraction of monkey testis (lane 1), weak in that of the caput (lane 2) and cauda (lane 3) epididymis and vas deferens (lane 4), and strong in that of the seminal vesicles (lane 5). No immunoreactivity was detectable in the membrane fraction of the prostate (lane 6) or in the cytosolic fractions of any of the tissues of male monkey genital organs (data not shown). In the male rat reproductive tract (Fig. 1A), the mPGES-1 immunoreactivity indicated the expression of the enzyme to be weak in the testis (lane 1) and caput and cauda epididymis (lanes 2 and 3, respectively), moderate in the seminal vesicles (lane 5), very strong in the vas deferens (lane 4), and barely detectable in the prostate (lane 6). In the case of the male rabbit genital organs, the immunoreactive signals were the most intense in the testis (lane 1) and were much weaker in the other genital organs (lanes 2–6). For the pig organs, the immunoreactivity was the most intense in the testis (lane 1), next strongest in the prostate (lane 6), weak in the caput and cauda epididymis, vas deferens (lanes 2– 4), and almost undetectable in the seminal vesicles (lane 5). Western blot analyses with the anti-COX-1 antibody (Fig. 1B, upper panel) and anti-rat COX-2 antibody (Fig. 1B, lower panel) revealed that the immunoreactive signal for COX-1 was weak in the monkey caput and cauda epididymis (lanes 2 and 3, respectively) and prostate (lane 6), moderate in the vas deferens (lane 4), but not detectable in the testis (lane 1) or seminal vesicles (lane 5). In contrast, the immunoreactive band for COX-2 was detected almost exclusively in the
microsomal sample of the seminal vesicles (lane 5). The anti-rat COX-2 antibody partially cross-reacted with monkey COX-1 (lanes 2–4), with the positive band being at a position indicating a slightly lower molecular weight than that of COX-2. 3.2. Production of anti-human mPGES-1 monoclonal antibody We raised a monoclonal anti-human mPGES-1 antibody by immunization of mice with human mPGES-1 protein recombinantly expressed in E. coli [11]. Western blot analysis of the microsome fraction prepared from monkey seminal vesicles (Fig. 1C, middle panel, lane 2) showed that the monoclonal antibody highly specifically reacted with mPGES-1 protein of monkey seminal vesicles (Fig. 1C, right panel, lane 2), giving a single band at a position of Mr ¼ 17; 000 identical in size with mPGES-1 (arrow in Fig. 1C). However, the monoclonal antibody showed no cross-reactivity with mouse mPGES-1 (lane 1), although the secondary anti-mouse IgG antibody stained 2 protein bands, heavy and light chains of IgG, in the microsomal fraction of mouse vas deferens (Fig. 1C, middle panel, lane 1). 3.3. Immunohistochemical localization of mPGES-1 in monkey and rat testis We determined the cellular localization of mPGES-1 in monkey testis by immunoperoxidase staining with the mouse monoclonal anti-human mPGES-1 antibody (Fig. 2). Immunohistochemical experiments were performed on specimens from several animals. In each case the same results were obtained. Positive immunostaining was observed in the basal compartment of the seminiferous tubules of the testis but not in the interstitium (Figs. 2A and B). In the basal compartment, Sertoli cells and primary spermatocytes were positively stained. Moreover, spermatogonia and round spermatids were
Fig. 2. Immunolocalization of mPGES-1 in monkey and rat testis. (A, B) Low- and high-magnification micrographs of sections of the monkey testis showing reaction with anti-human mPGES-1 monoclonal antibody (A and B, bars, 100 and 15 mm, respectively). (C) High-magnification micrograph of rat testis showing reaction with anti-mouse mPGES-1 polyclonal antibody (bar, 20 mm). The sections were counterstained with hematoxylin. The arrows in ‘‘B’’ indicate positive staining for mPGES-1 in spermatogonia cells (Spg), Sertoli cells (Ser), primary spermatocytes (Spc), and round spermatids (rS) of the monkey testis. The arrow in ‘‘C’’ indicates positive staining for mPGES-1 in Sertoli cells (Ser) of the rat testis.
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lightly stained. Identical positive signals were detected in adjacent sections stained with polyclonal anti-mouse mPGES-1 antibody, whereas no positive signals were detected in the sections stained with mouse IgG or the polyclonal antibody preabsorbed with an excess amount of mPGES-1 (data not shown). When we immunostained the sections of monkey testis with anti-COX-1 antibody, the immunoreactivity was barely detectable in the basal cells of the seminiferous tubule (data not shown). Localization of mPGES-1 in the monkey testis was remarkably different from that in the mouse testis, in which mPGES-1 was localized in the Leydig cells of the interstitium [13]. When localization of mPGES-1 in the rat testis was investigated with the polyclonal antibody, the immunoreactivity for mPGES-1 was also observed in the Leydig cells (data not shown). Moreover, we
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observed a weak immunostaining for mPGES-1 in Sertoli cells of the seminiferous tubules of the rat testis (Fig. 2C). In the adjacent sections stained with polyclonal anti-mouse mPGES-1 antibody preabsorbed with an excess amount of recombinant mPGES-1, no positive signals were detected (data not shown). These results further indicate that the cellular localization of mPGES-1 in the testis varies among mammalian species. 3.4. Immunohistochemical localization of mPGES-1 and COX-1 in monkey epididymis and vas deferens In the monkey epididymis, mPGES-1 immunoreactivity was localized in tubular epithelial cells of the caput (Figs. 3A and B) and cauda (Figs. 3D and E) portions but was not detected in those cells of the corpus portion. The epithelium of the caput epididymis was intensely
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Fig. 3. Immunolocalization of mPGES-1 and COX-1 in monkey epididymis and vas deferens. Light micrographs of sections of the caput (A–C) and cauda (D–F) epididymis and of the distal vas deferens, i.e., ampulla (G–I) were reacted with anti-human mPGES-1 monoclonal antibody (left and middle photos; bars, 100 and 15 mm, respectively) or anti-COX-1 antibody (right photos; bar, 15 mm). The sections were counterstained with hematoxylin.
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and uniformly positive for mPGES-1, with a clear enrichment of the immunoreactivity in the nucleus. In the cauda epididymis, the immunoreactivity was also intense in the nuclei and in a zone between the nuclei and the basal surface of principal cells (Fig. 3E). No positive signals were detected in adjacent sections stained with mouse IgG or the polyclonal antimPGES-1 antibodies preabsorbed with an excess amount of recombinant mPGES-1 (data not shown). The COX-1 immunoreactivity was also detected in the basal compartment of the epithelium in the caput and cauda epididymis (Figs. 3C and F), but not in the corpus epididymis, with a significant enrichment in the nucleus, similar to the case of mPGES-1. In adjacent sections stained with the anti-rat COX-2 antibody, we also observed weak positive signals in the epithelium (data not shown), probably due to the cross-reactivity of this antibody with COX-1. In the vas deferens, strong immunoreactivities for mPGES-1 and COX-1 were detected in the epithelium from the proximal segment to the distal portion of the vas deferens and ampulla (Figs. 3G–I). No positive signals for mPGES-1 or COX-1 were detected in the smooth muscle surrounding the epithelium of the vas deferens (Figs. 3G–I). The cellular localization of mPGES-1 and COX-1 in rat epididymis and vas deferens was essentially the same as that of these enzymes in the monkey tissues described above and of the mouse ones [13], suggesting that the physiological function of PGE2 in these tissues is common among these species. 3.5. Cellular localization of mPGES-1 and COX-2 in monkey and rat accessory glands The immunoreactivity for mPGES-1 was strong in the epithelium of the monkey seminal vesicular gland (Figs. 4A and B) but was not detected in the monkey prostate (data not shown). In adjacent sections of the
seminal vesicles stained with mouse IgG or guinea pig polyclonal anti-mPGES-1 antibody in the presence of an excess amount of recombinant mPGES-1, no positive signals were detected. Monkey seminal vesicle epithelial cells were also positively stained with anti-COX-2 antibody (Fig. 4C) but not with anti-COX-1 antibody (data not shown). In rats, mPGES-1 immunoreactivity was detected in the epithelium of the seminal vesicles (data not shown) and prostate (Fig 4D). No positive staining was observed when we used the anti-mouse mPGES-1 antibody preabsorbed with recombinant mPGES-1.
4. Discussion In this study, we determined the tissue distribution of mPGES-1 in male monkey genital organs to get insight into the production site of PGE2 in primates. Western blotting with polyclonal anti-mPGES-1 antibody revealed that mPGES-1 was widely expressed in male monkey genital organs (Fig. 1). However, the tissue distribution profile of mPGES-1 in monkeys was quite different from that for rat, rabbit, or pig (Fig. 1). In male rat genital organs, mPGES-1 was expressed the most intensely in the vas deferens, with significantly lower expression in the different segments of the epididymis. This profile of mPGES-1 expression is in good agreement with previous findings on PGE2 production in the genital organs of rats, in which PGE2 was detected in a high concentration in the vas deferens and at much lower levels in the testis and epididymis [16–19]. Also, this profile is very similar to that of male mouse genital organs previously reported [13]. In contrast, in the rabbit and pig male reproductive tracts, mPGES-1 was the most intensely expressed in the testis. These results clearly indicate that mPGES-1 is expressed in a highly species-specific manner in the male genital organs of various species.
mPGES-1
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Rat prostate (D)
Fig. 4. Immunolocalization of mPGES-1 and COX-2 in monkey seminal vesicles and of mPGES-1 in rat prostate gland shown are light micrographs at low (A, bar, 100 mm) and high (B and C, bars, 15 mm) magnification of monkey seminal vesicles reacted with anti-human mPGES-1 antibody (A and B) or anti-rat COX-2 antibody (C). The section in ‘‘A’’ was not counterstained with hematoxylin. A micrograph (bar, 40 mm) of a section of rat prostate showing the epithelium reactive with anti-mouse mPGES-1 antibody is also presented (D).
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A remarkable difference between monkeys and rodents was also found in the cellular localization of mPGES-1. The mPGES-1 immunoreactivity was localized in various types of cells in the basal compartment of the seminiferous epithelium of the monkey testis (Fig. 2), whereas it was found only in Sertoli cells of the seminiferous tubule and in Leydig cells of the interstitium in the rat testis, and exclusively in Leydig cells of the mouse testis [13]. Taken together, these results suggest that different types of cells, i.e., Leydig, Sertoli cells or other epithelial cells, are responsible for the PGE2 production in the testis of rodents and primates to modulate the contractions of the testicular capsule through direct action on the peritubular myoid cells [13,20]. PGE2 also modulates the transport of sperm in the excurrent ducts of the epididymis and its subsequent expulsion at coitus from the vas deferens because of its smooth muscle relaxant activity [21–23]. We previously hypothesized that PGE2 produced by mPGES-1 in epithelial cells of the mouse epididymis and vas deferens is secreted toward the basal surface of the epithelium and acts on the surrounding smooth muscle layer [13]. This idea is supported by the cellular localization of mPGES-1 in epithelial cells of the caput and cauda epididymis and of the vas deferens in monkey genital organs (Fig. 3). PGE2 may also be, in part, released into the semen of monkeys from the epididymis or vas deferens, although it is believed that the seminal vesicles are the main source of PGE2 in the semen [24,25]. A species-specific difference in mPGES-1 expression was also observed in the seminal vesicles. In the male monkey reproductive tract, mPGES-1 expression was the strongest in the epithelial cells of the seminal vesicles; whereas mPGES-1 was only weakly expressed in seminal vesicles of various other species, i.e., mouse, rat, rabbit, and pig, as compared with its level in other organs of the reproductive tract (Fig. 4; [13]). The high expression of mPGES-1 in monkey seminal vesicles may explain why the 19-hydroxy derivative of PGE2 (19-OH PGE2) is remarkably abundant in the semen of primates [26–28] and acts as a potent immunosuppressor in the female tract following insemination [29–31]. Other reports also address the ability of both PGE2 and 19OH PGE2 supplied by the seminal plasma to facilitate sperm transport by regulating uterine contractility in the female reproductive tract [32] or to contribute to the acrosome reaction of the sperm [33]. In 1934, von Euler discovered a new group of hormones in the human seminal fluid and named these compounds PGs based on the assumption that they were produced in the prostate gland [1]. Later studies have indicated that the seminal vesicles are the preferential site of production [24,25] rather than the prostate gland. Presently, we showed by Western blotting and immunohistochemistry the absence of mPGES-1 in the
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monkey prostate, and thus, this finding supports the idea that the prostate of primates is not capable of producing PGE2. On the other hand, mPGES-1 was expressed in the epithelium of the rat prostate (Fig. 4), which is in good agreement with studies that have previously shown that PGE2 can be synthesized in the rat prostate gland [18]. These results suggest that the physiological function of the prostate is different between rodents and primates. Our Western blotting and immunohistochemical results revealed that mPGES-1 was expressed coordinately with COX-1 in the epithelial cells of the monkey caput and cauda epididymis and vas deferens (Fig. 3), but with COX-2 in epithelial cells of the monkey seminal vesicles (Fig. 4). Previously, the expression of COX-2 was detected immunohistochemically in epithelial cells of the human ejaculatory duct and seminal vesicles, by the use of monoclonal COX-2 antibody [34]; although, in monkeys, this antibody cross-reacted with both COX1 and COX-2 (Fig. 1B). COX-2 has long been recognized as the inducible COX isozyme [35]. However, COX-2 is constitutively expressed in the monkey seminal vesicles (Figs. 1 and 4), similarly as in the vas deferens of rodents [13,36] and the leptomeningeal cells in the rat brain [37], suggesting that COX-2 may contribute to the production of PGs in these tissues and cells. Recently, PGH2 19-hydroxylase (CYP4F8), catalyzing the conversion of PGH2 to 19-OH PGH2, was identified in human seminal vesicles, and COX-2, CYP4F8, and mPGES-1 were proposed to be closely linked for the production of 19-OH PGE2 [28]. Therefore, constitutive COX-2 may also be functionally coupled to other downstream enzymes, such as CYP4F8, rather than to mPGES-1 in the arachidonic acid cascade. In conclusion, the results of our immunohistochemical study on mPGES-1 and COXs in male monkey reproductive organs suggest that mPGES-1 is coordinately expressed with COX-1 and COX-2 to produce PGE2 and 19-OH PGE2, respectively, in the male reproductive system of primates. We also found highly species-specific expression patterns of mPGES-1 and COXs among monkeys, rodents (mice and rats), rabbits, and pigs, suggesting the need for critical consideration of animal models to study the physiological functions of COXs and mPGES-1.
Acknowledgements We thank Dr. Osamu Hayaishi for his encouragement of and during this study. We also acknowledge Mr. T. Deguchi, Mr. T. Hirado, and Mr. H. Osama for technical assistance; and Ms. S. Sakae, Ms. M. Yamaguchi, and Ms. T. Nishimoto for their secretarial assistance.
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References . [1] U.S. von Euler, Uber die spezifische blutdrucksenkende Substanz des menschlichen Prostata- und Samenblasensekretes, Klin. Wochenschr. 14 (1935) 1182–1183. [2] M. Bygdeman, A. Gillespie, Prostaglandins in human reproduction, in: J.B. Lee (Ed.), Prostaglandins, Elsevier, New York, 1982, pp. 215–249. [3] C.L. Joyce, N.A. Nuzzo, L. Wilson Jr., L.J. Zaneveld, Evidence for a role of cyclooxygenase (prostaglandin synthetase) and prostaglandins in the sperm acrosome reaction and fertilization, J. Androl. 8 (1987) 74–82. [4] S. Narumiya, Y. Sugimoto, F. Ushikubi, Prostanoid receptors: structures, properties, and functions, Physiol. Rev. 79 (1999) 1193–1226. [5] T. Miyamoto, S. Yamamoto, O. Hayaishi, Prostaglandin synthetase system-resolution into oxygenase and isomerase components, Proc. Natl. Acad. Sci. USA 71 (1974) 3645–3648. [6] N. Ogino, T. Miyamoto, S. Yamamoto, O. Hayaishi, Prostaglandin endoperoxide E isomerase from bovine vesicular gland microsomes, a glutathione-requiring enzyme, J. Biol. Chem. 252 (1977) 890–895. [7] P.J. Jakobsson, S. Thoren, R. Morgenstern, B. Samuelsson, Identification of human prostaglandin E synthase: a microsomal, glutathione-dependent, inducible enzyme, constituting a potential novel drug target, Proc. Natl. Acad. Sci. USA 96 (1999) 7220–7225. [8] W.L. Smith, D.L. Dewitt, Prostaglandin endoperoxide H synthases-1 and -2, Adv. Immunol. 62 (1996) 167–215. [9] S. Thoren, P.J. Jakobsson, Coordinate up- and down-regulation of glutathione-dependent prostaglandin E synthase and cyclooxygenase-2 in A549 cells. Inhibition by NS-398 and leukotriene C4, Eur. J. Biochem. 267 (2000) 6428–6434. [10] M. Murakami, H. Naraba, T. Tanioka, et al., Regulation of prostaglandin E2 biosynthesis by inducible membrane-associated prostaglandin E2 synthase that acts in concert with cyclooxygenase-2, J. Biol. Chem. 275 (2000) 32783–32792. [11] M. Lazarus, B.K. Kubata, N. Eguchi, Y. Fujitani, Y. Urade, O. Hayaishi, Biochemical characterization of mouse microsomal prostaglandin E synthase-1 and its colocalization with cyclooxygenase-2 in peritoneal macrophages, Arch. Biochem. Biophys. 397 (2002) 336–341. [12] F. Filion, N. Bouchard, A.K. Goff, J.G. Lussier, J. Sirois, Molecular cloning and induction of bovine prostaglandin E synthase by gonadotropins in ovarian follicles prior to ovulation in vivo, J. Biol. Chem. 276 (2001) 34323–34330. [13] M. Lazarus, C.J. Munday, N. Eguchi, et al., Immunohistochemical localization of microsomal PGE synthase-1 and cyclooxygenases in male mouse reproductive organs, Endocrinology 143 (2002) 2410–2419. . [14] G. Kohler, C. Milstein, Continuous cultures of fused cells secreting antibody of predefined specificity, Nature 256 (1975) 495–497. [15] R.L. Gerena, N. Eguchi, Y. Urade, G.J. Killian, Stage and region-specific localization of lipocalin-type prostaglandin D synthase in the adult murine testis and epididymis, J. Androl. 21 (2000) 848–854. [16] K. Gerozissis, F. Dray, Selective and age-dependent changes of prostagladin E2 in the epididymis and vas deferens of the rat, J. Reprod. Fertil. 50 (1977) 113–115. [17] K. Gerozissis, F. Dray, In-vitro prostanoid production by the rat vas deferens, J. Reprod. Fertil. 67 (1983) 389–394. [18] L.A. Klein, J.S. Stoff, Prostaglandin synthesis is independent of androgen levels in rat male genitalia, J. Lab. Clin. Med. 109 (1987) 402–408.
[19] F. Dray, K. Gerozissis, Interrelationships between androgens and prostaglandins in the vas deferens of prepubertal rats, Arch. Androl. 2 (1979) 189–196. [20] A. Tripiciano, A. Filippini, F. Ballarini, F. Palombi, Contractile response of peritubular myoid cells to prostaglandin F2a, Mol. Cell. Endocrinol. 138 (1998) 143–150. [21] P. Hedqvist, U.S. von Euler, Prostaglandin-induced neurotransmission failure in the field-stimulated, isolated vas deferens, Neuropharmacology 11 (1972) 177–187. [22] M.J. Cosentino, H. Takihara, J.W. Burhop, A.T. Cockett, Regulation of rat caput epididymidis contractility by prostaglandins, J. Androl. 5 (1984) 216–222. [23] P.B. Patra, R.M. Wadsworth, D.W. Hay, I.J. Zeitlin, The effect of inhibitors of prostaglandin formation on contraction of the rat, rabbit and human vas deferens, J. Auton. Pharmacol. 10 (1990) 55–63. [24] K. Gerozissis, P. Jouannet, J.C. Soufir, F. Dray, Origin of prostaglandins in human semen, J. Reprod. Fertil. 65 (1982) 401–404. [25] E. Bendvold, K. Svanborg, M. Bygdeman, S. Noren, On the origin of prostaglandins in human seminal fluid, Int. J. Androl. 8 (1985) 37–43. [26] H.T. Jonsson, B.S. Middleditch, D.M. Desiderio, Prostaglandins in human seminal fluid: two novel compounds, Science 187 (1975) 1093–1094. [27] R.W. Kelly, P.L. Taylor, J.P. Hearn, R.V. Short, D.E. Martin, J.H. Marston, 19-Hydroxyprostaglandin E1 as a major component of the semen of primates, Nature 260 (1976) 544–545. [28] J. Bylund, M. Hidestrand, M. Ingelman-Sundberg, E.H. Oliw, Identification of CYP4F8 in human seminal vesicles as a prominent 19-Hydroxylase of prostaglandin endoperoxides, J. Biol. Chem. 275 (2000) 21844–21849. [29] T.H. Tarter, S. Cunningham-Rundles, S.S. Koide, Suppression of natural killer cell activity by human seminal plasma in vitro: identification of 19-OH-PGE as the suppressor factor, J. Immunol. 136 (1986) 2862–2867. [30] R.W. Kelly, Immunomodulators in human seminal plasma: a vital protection for spermatozoa in the presence of infection, Int. J. Androl. 22 (1999) 2–12. [31] F.C. Denison, V.E. Grant, A.A. Calder, R.W. Kelly, Seminal plasma components stimulate interleukin-8 and interleukin-10 release, Mol. Hum. Reprod. 5 (1999) 220–226. [32] D.F. Woodward, C.E. Protzman, A.H. Krauss, L.S. Williams, Identification of 19 (R)-OH prostaglandin E2 as a selective prostanoid EP2-receptor agonist, Prostaglandins 46 (1993) 371–383. [33] M. Schaefer, T. Hofmann, G. Schultz, T. Gudermann, Anew prostaglandin E receptor mediates calcium influx and acrosome reaction in human spermatozoa, Proc. Natl. Acad. Sci. USA 95 (1998) 3008–3013. [34] A. Kirschenbaum, D.R. Liotta, S. Yao, et al., Immunohistochemical localization of cyclooxygenase-1 and cyclooxygenase-2 in the human fetal and adult male reproductive tracts, J. Clin. Endocrinol. Metab. 85 (2000) 3436–3441. [35] M.K. O’Banion, Cyclooxygenase-2: molecular biology, pharmacology, and neurobiology, Crit. Rev. Neurobiol. 13 (1999) 45–82. [36] J.A. McKanna, M.Z. Zhang, J.L. Wang, H. Cheng, R.C. Harris, Constitutive expression of cyclooxygenase-2 in rat vas deferens, Am. J. Physiol. 275 (1998) R227–233. [37] C.T. Beuckmann, M. Lazarus, D. Gerashchenko, et al., Cellular localization of lipocalin-type prostaglandin D synthase (b-trace) in the central nervous system of the adult rat, J. Comp. Neurol. 428 (2000) 62–78.
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Species-specific expression of microsomal prostaglandin E synthase-1 and cyclooxygenases in male monkey reproductive organs$ M. Lazarusa,d, N. Eguchia, S. Matsumotoa, N. Nagataa, T. Yanob, G.J. Killianc, Y. Uradea,* a
Department of Molecular Behavioral Biology, Osaka Bioscience Institute, Suita, 6-2-4 Furuedai Suita, Osaka 565-0874, Japan b Nagahama Institute for Biochemical Science, Oriental Yeast Co., Nagahama, Shiga 526-0804, Japan c Department of Dairy and Animal Science, J.O. Almquist Research Center, Pennsylvania State University, University Park, PA 16802, USA d Department of Neurology, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA Received 21 January 2004; accepted 29 March 2004
Abstract We investigated the tissue distribution and cellular localization of microsomal PGE synthase-1 (mPGES-1) and cyclooxygenase (COX)-1 and -2 in male monkey reproductive organs. Western blotting revealed that monkey mPGES-1 was expressed most intensely in the seminal vesicles, moderately in the testis, and weakly in the epididymis and vas deferens. The tissue distribution profile was quite different from those profiles for rats, rabbits, and pigs, e.g., rat mPGES-1 was the most abundant in the vas deferens, and the rabbit and pig enzymes, in the testis. Immunohistochemical staining with mouse monoclonal anti-human mPGES1 antibody revealed that monkey mPGES-1 was localized in spermatogonia, Sertoli cells, and primary spermatocytes of testis and in epithelial cells of the epididymis, vas deferens, and seminal vesicles. In monkeys, COX-1 was localized in epithelial cells of the epididymis and vas deferens, whereas COX-2 was dominantly found in epithelial cells of the seminal vesicles. r 2004 Elsevier Ltd. All rights reserved. Keywords: Microsomal prostaglandin E synthase-1; Cyclooxygenase; Male genital organs; Immunohistochemistry; Monoclonal antibody
1. Introduction Prostaglandin (PG) E2 was originally discovered as a vasodilatory substance in human seminal fluid [1]. PGE2 has been implicated in a wide variety of reproductive activities, e.g., male fertility, tubule contractility, nonpregnant and pregnant uterine contractility, the sperm acrosome reaction, and fertilization [2–4]. $ This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Grant 12558078 to Y.U. and Grant 13557016 to N.E.) and by grants from the Takeda Science Foundation (to M.L. and Y.U.), Science and Technology Agency (Grant 199041 to M.L.), the US Department of Agriculture (Grant 97-35203-9806 to G.J.K.), and Osaka City. *Corresponding author. Department of Molecular Behavioral Biology, Osaka Bioscience Institute, Suita, 6-2-4 Furuedai Suita, Osaka 565-0874, Japan. Tel.: +81-6-6872-4851; fax: +81-6-68722841. E-mail address: [email protected] (Y. Urade).
0952-3278/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2004.03.018
Glutathione-dependent, membrane-bound PGE synthase-1 (mPGES-1) catalyzes the isomerization of PGH2, a common precursor of various prostanoids, to PGE2 [5–7]. PGH2 is produced from arachidonic acid by cyclooxygenase (COX)-1 or COX-2 [8]. Coordinate upregulation of mPGES-1 and COX-2 was reported to occur in a human lung cancer cell line, A549, after treatment of the cells with interleukin-1b (IL-1b [9], and in rat osteoblasts [10] and rat and mouse peritoneal macrophages [10,11] after stimulation of them with IL1b, tumor necrosis factor-a, or lipopolysaccharide. Moreover, the induction of mPGES-1 was demonstrated to take place in granulosa cells of bovine ovarian follicles after in vivo administration of gonadotropins [12]. However, little is known about the cellular localization of PGE2-producing enzymes in mammalian genital organs. Recently, we reported that mPGES-1 was involved in PGE2 production in male mouse genital organs, where it was coordinately expressed with COX-1 and COX-2 [13].
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In the present study, we determined the tissue distribution and cellular localization of mPGES-1 and COXs in male monkey genital organs to gain insight into the nature of PGE2-producing cells of primates. We report here the highly species-specific distribution of mPGES-1 in monkey, rat, rabbit, and pig male genital organs and show that monkey mPGES-1 is colocalized with COX-1 in the testis, epididymis, and vas deferens and with COX-2 in seminal vesicles.
munohistochemistry of sections of the monkey caput epididymis and seminal vesicles, as described below. Positive clones were subcloned twice by the limiting dilution method. Ascites tumors were induced by injection of 1 107 hybridoma cells into the peritoneal cavity of pristane-primed BALB/C mice, and ascites fluids were collected after approx. 2 weeks. In order to purify mouse immunoglobulin (Ig) G from ascites and culture medium, we used a Proteus-A antibody purification kit (ProChem, Acton, MA) according to the manufacturer’s instructions.
2. Materials and methods 2.3. Western blot detection 2.1. Animals and materials Monkey (Macaca fascicularis) tissues were donated by R-Tech Ueno (Sanda, Japan), and rabbit tissues, by Kitayama Labes (Nagano, Japan). Pig tissues were obtained from a local slaughterhouse. Inbred C57BL/6 and BALB/C mice and Sprague–Dawley rats were purchased from Shizuoka Laboratory Animal Center (Shizuoka, Japan). All procedures used in handling animals were approved by the Animal Research Committee of Osaka Bioscience Institute. All commercially available chemicals were of analytical grade. Oligonucleotides were purchased from Amersham Biosciences Corp (New Jersey, USA). 2.2. Production of monoclonal antibody against human mPGES-1 Human mPGES-1 was recombinantly expressed in E. coli, purified as previously described [11], and used for immunization of mice. Monoclonal antibodies were . produced by the method of Kohler and Milstein [14]. In brief, an emulsion of human mPGES-1 (40 mg) and complete Freund’s adjuvant was injected intraperitoneally into 3 BALB/C mice. The mice were boosted every 2 weeks with the same amount of antigen mixed with an equal volume of Freund’s incomplete adjuvant until an adequate response was achieved, as judged by the results of enzyme-linked immunosorbent assay. An intravenous boost with 20 mg of the antigen in 0.2 ml of saline was carried out, and the mice were then euthanized 3 days after the injection. Subsequently, 5 108 spleen cells were fused with 2.5 108 P3U1 myeloma cells by the addition of 50% (w/v) polyethylene glycol 4000. The resulting cell suspension of hybrids was inoculated into three 96-well microtiter plates containing selective RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 10% fetal calf serum, antibiotics, 2mercaptoethanol, hypoxanthine, aminopterin, thymidine, and l-glutamine. The supernatants of cultures showing hybrid growth were screened for production of anti-human mPGES-1 antibody by Western blotting of microsomes of monkey seminal vesicles and by im-
Tissues were homogenized with an Ultra Turrax blender (Janke & Kunkel, Staufen, Germany) in phosphate-buffered saline (PBS; 1 ml/100 mg material) in the presence of protease-inhibitor complete (Roche Diagnostics, Mannheim, Germany). Cellular debris was removed by centrifugation at 7000 g and 4 C for 20 min. The microsomal fraction was then obtained by centrifugation at 100,000 g and 4 C for 1 h. Microsomal proteins were resolved sodium dodecyl sulfate polyacrylamide gel electrophoresis with 14% or 6% precast gels (Tefco, Tokyo, Japan) and electrophoretically transferred onto Immobilon polyvinyl difluoride (PVDF) membranes (Millipore, Bedford, MA) at 200 mA for 1 h or stained with Coomassie brilliant blue to confirm equal loading of proteins. After blockage of non-specific binding sites with BlockAce (Dainippon Seiyaku, Osaka, Japan) for 1 h at 25 C, the membranes were incubated at 4 C overnight with guinea pig polyclonal anti-mouse mPGES-1 IgG (2 mg/ml; [11]), mouse monoclonal anti-human mPGES-1 antibody (1 mg/ml), rabbit polyclonal anti-COX-1 antibody (dil. 1:1000; Oxford Biomedical Research, Oxford, MI), or mouse monoclonal anti-rat COX-2 antibody (1 mg/ml; BD Biosciences, Franklin Lakes, NJ) as appropriate, applied in 10% BlockAce in PBS containing 0.2% (v/v) Tween-20. As secondary antibody, horseradish peroxidase-coupled donkey anti-guinea pig, anti-mouse or anti-rabbit IgG (Jackson Laboratories, West Grove, PA) was added at a concentration of 10 mg/ml, and incubation was carried out 1 h at room temperature. Detection was performed with an enhanced chemiluminescence kit (Amersham Biosciences Corp.) according to the manufacturer’s instructions. 2.4. Immunoperoxidase staining After deep anesthesia with ketamine (intramuscular) and pentobarbital (intravenous), M. fascicularis monkeys were perfused with PBS via the left ventricle of the heart. The genital organs were removed and soaked in Bouin’s fixative [15]. After deep anesthesia with pentobarbital (intraperitoneal), rats were perfused via the left
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ventricle of the heart with 20 mM sodium phosphate (pH 7.4) containing 4% paraformaldehyde and 4% sucrose, and then with Bouin’s solution. Genital organs were removed and soaked overnight at 4 C in Bouin’s fixative. After embedment in paraffin, the monkey and rat tissues were sectioned (10 mm) and mounted on slides. The sections were then deparaffinized in xylene and rehydrated in ethanol with increasing concentrations of water. Endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide in PBS for 30 min at 25 C. The tissues were then treated with 0.3% pepsin in 0.01 M hydrochloric acid for 5 and 30 min for COXs and mPGES-1 immunostaining, respectively, at room temperature. Thereafter, non-specific binding sites were blocked for 1 h with 10% sheep serum (Life Technologies, Tokyo, Japan) in PBS that contained 0.1% Triton X-100 and 0.1% sodium azide. Primary and secondary antibodies were diluted in PBS containing 1% sheep serum and 0.1% Triton X-100. Monkey tissue sections were exposed to anti-mouse and antihuman mPGES-1 antibodies (4 and 2 mg/ml, respectively) for 2 days as well as to anti-COX-1 (dil. 1:500) and anti-rat COX-2 (2 mg/ml) antibodies for 1 day at 4 C; rat tissue sections were incubated with guinea pig
polyclonal mPGES-1 antibody (4 mg/ml) for 2 days at 4 C. After 1 h of incubation at 4 C with biotinylated anti-guinea pig, anti-mouse, or anti-rabbit IgG (7.5 mg/ ml; Vector Laboratories, Burlingame, CA), tissues were rinsed 3 times in PBS containing 0.1% Triton X-100. A Vectastain Elite avidin–biotin–peroxidase kit (Vector Laboratories) with diaminobenzidine substrate was used according to the manufacturer’s protocol. Tissues were counterstained with hematoxylin. Finally, sections were dehydrated, rinsed in xylene, and mounted with MountQuick (Daido Sangyo, Tokyo, Japan).
3. Results 3.1. Expression of mPGES-1, COX-1, and COX-2 in monkey, rat, rabbit, and pig male genital organs We previously generated a guinea pig polyclonal antimouse mPGES-1 antibody [11]. This polyclonal antibody not only reacted with mouse mPGES-1 but also cross-reacted with the rat, rabbit, pig, and monkey enzymes (Fig. 1A) as well as with the human enzyme (data not shown). We used this polyclonal antibody for (C)
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Fig. 1. Western blotting analysis of mPGES-1 in male genital organs. (A) and (B) Expression of mPGES-1, COX-1, and COX-2 immunoreactive proteins in monkey, rat, rabbit, and pig male genital organs. Microsomal fractions were obtained from the testis (lane 1), caput (lane 2) and cauda (lane 3) epididymis, vas deferens (lane 4), seminal vesicles (lane 5), and prostate (lane 6) of the monkey, rat, rabbit, and pig. Microsomes of the genital organs (15 mg of protein) were electrophoresed and subsequently transferred onto a PVDF membrane. The membranes were immunostained with guinea pig polyclonal anti-mouse mPGES-1 antibody (A) or anti-COX-1 and COX-2 antibodies (B). Arrows in ‘‘B’’ indicate the positions of COX-1 and COX-2 on the right. (C) Specificity of monoclonal anti-human mPGES-1 antibody. Microsomes (15 mg of protein) of mouse vas deferens (lane 1) and monkey seminal vesicles (lane 2) were electrophoresed and subsequently transferred onto a PVDF membrane (left and right panels) or stained with Coomassie brilliant blue (middle panel). The membranes were then immunostained with polyclonal anti-mouse (left panel) or monoclonal antihuman (right panel) mPGES-1 antibodies. Molecular weight markers (Mr 103) are shown on the right, and the arrow on the left indicates the position of mPGES-1.
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Western blotting of the microsomal fraction to investigate the tissue distribution profiles of mPGES-1 in monkey, rat, rabbit, and pig male genital organs (Fig. 1A). Western blotting experiments were performed on specimens from several animals of each species and the same results were obtained in each case. The level of mPGES-1-immunoreactive protein was moderate in the microsomal fraction of monkey testis (lane 1), weak in that of the caput (lane 2) and cauda (lane 3) epididymis and vas deferens (lane 4), and strong in that of the seminal vesicles (lane 5). No immunoreactivity was detectable in the membrane fraction of the prostate (lane 6) or in the cytosolic fractions of any of the tissues of male monkey genital organs (data not shown). In the male rat reproductive tract (Fig. 1A), the mPGES-1 immunoreactivity indicated the expression of the enzyme to be weak in the testis (lane 1) and caput and cauda epididymis (lanes 2 and 3, respectively), moderate in the seminal vesicles (lane 5), very strong in the vas deferens (lane 4), and barely detectable in the prostate (lane 6). In the case of the male rabbit genital organs, the immunoreactive signals were the most intense in the testis (lane 1) and were much weaker in the other genital organs (lanes 2–6). For the pig organs, the immunoreactivity was the most intense in the testis (lane 1), next strongest in the prostate (lane 6), weak in the caput and cauda epididymis, vas deferens (lanes 2– 4), and almost undetectable in the seminal vesicles (lane 5). Western blot analyses with the anti-COX-1 antibody (Fig. 1B, upper panel) and anti-rat COX-2 antibody (Fig. 1B, lower panel) revealed that the immunoreactive signal for COX-1 was weak in the monkey caput and cauda epididymis (lanes 2 and 3, respectively) and prostate (lane 6), moderate in the vas deferens (lane 4), but not detectable in the testis (lane 1) or seminal vesicles (lane 5). In contrast, the immunoreactive band for COX-2 was detected almost exclusively in the
microsomal sample of the seminal vesicles (lane 5). The anti-rat COX-2 antibody partially cross-reacted with monkey COX-1 (lanes 2–4), with the positive band being at a position indicating a slightly lower molecular weight than that of COX-2. 3.2. Production of anti-human mPGES-1 monoclonal antibody We raised a monoclonal anti-human mPGES-1 antibody by immunization of mice with human mPGES-1 protein recombinantly expressed in E. coli [11]. Western blot analysis of the microsome fraction prepared from monkey seminal vesicles (Fig. 1C, middle panel, lane 2) showed that the monoclonal antibody highly specifically reacted with mPGES-1 protein of monkey seminal vesicles (Fig. 1C, right panel, lane 2), giving a single band at a position of Mr ¼ 17; 000 identical in size with mPGES-1 (arrow in Fig. 1C). However, the monoclonal antibody showed no cross-reactivity with mouse mPGES-1 (lane 1), although the secondary anti-mouse IgG antibody stained 2 protein bands, heavy and light chains of IgG, in the microsomal fraction of mouse vas deferens (Fig. 1C, middle panel, lane 1). 3.3. Immunohistochemical localization of mPGES-1 in monkey and rat testis We determined the cellular localization of mPGES-1 in monkey testis by immunoperoxidase staining with the mouse monoclonal anti-human mPGES-1 antibody (Fig. 2). Immunohistochemical experiments were performed on specimens from several animals. In each case the same results were obtained. Positive immunostaining was observed in the basal compartment of the seminiferous tubules of the testis but not in the interstitium (Figs. 2A and B). In the basal compartment, Sertoli cells and primary spermatocytes were positively stained. Moreover, spermatogonia and round spermatids were
Fig. 2. Immunolocalization of mPGES-1 in monkey and rat testis. (A, B) Low- and high-magnification micrographs of sections of the monkey testis showing reaction with anti-human mPGES-1 monoclonal antibody (A and B, bars, 100 and 15 mm, respectively). (C) High-magnification micrograph of rat testis showing reaction with anti-mouse mPGES-1 polyclonal antibody (bar, 20 mm). The sections were counterstained with hematoxylin. The arrows in ‘‘B’’ indicate positive staining for mPGES-1 in spermatogonia cells (Spg), Sertoli cells (Ser), primary spermatocytes (Spc), and round spermatids (rS) of the monkey testis. The arrow in ‘‘C’’ indicates positive staining for mPGES-1 in Sertoli cells (Ser) of the rat testis.
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lightly stained. Identical positive signals were detected in adjacent sections stained with polyclonal anti-mouse mPGES-1 antibody, whereas no positive signals were detected in the sections stained with mouse IgG or the polyclonal antibody preabsorbed with an excess amount of mPGES-1 (data not shown). When we immunostained the sections of monkey testis with anti-COX-1 antibody, the immunoreactivity was barely detectable in the basal cells of the seminiferous tubule (data not shown). Localization of mPGES-1 in the monkey testis was remarkably different from that in the mouse testis, in which mPGES-1 was localized in the Leydig cells of the interstitium [13]. When localization of mPGES-1 in the rat testis was investigated with the polyclonal antibody, the immunoreactivity for mPGES-1 was also observed in the Leydig cells (data not shown). Moreover, we
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observed a weak immunostaining for mPGES-1 in Sertoli cells of the seminiferous tubules of the rat testis (Fig. 2C). In the adjacent sections stained with polyclonal anti-mouse mPGES-1 antibody preabsorbed with an excess amount of recombinant mPGES-1, no positive signals were detected (data not shown). These results further indicate that the cellular localization of mPGES-1 in the testis varies among mammalian species. 3.4. Immunohistochemical localization of mPGES-1 and COX-1 in monkey epididymis and vas deferens In the monkey epididymis, mPGES-1 immunoreactivity was localized in tubular epithelial cells of the caput (Figs. 3A and B) and cauda (Figs. 3D and E) portions but was not detected in those cells of the corpus portion. The epithelium of the caput epididymis was intensely
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Fig. 3. Immunolocalization of mPGES-1 and COX-1 in monkey epididymis and vas deferens. Light micrographs of sections of the caput (A–C) and cauda (D–F) epididymis and of the distal vas deferens, i.e., ampulla (G–I) were reacted with anti-human mPGES-1 monoclonal antibody (left and middle photos; bars, 100 and 15 mm, respectively) or anti-COX-1 antibody (right photos; bar, 15 mm). The sections were counterstained with hematoxylin.
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and uniformly positive for mPGES-1, with a clear enrichment of the immunoreactivity in the nucleus. In the cauda epididymis, the immunoreactivity was also intense in the nuclei and in a zone between the nuclei and the basal surface of principal cells (Fig. 3E). No positive signals were detected in adjacent sections stained with mouse IgG or the polyclonal antimPGES-1 antibodies preabsorbed with an excess amount of recombinant mPGES-1 (data not shown). The COX-1 immunoreactivity was also detected in the basal compartment of the epithelium in the caput and cauda epididymis (Figs. 3C and F), but not in the corpus epididymis, with a significant enrichment in the nucleus, similar to the case of mPGES-1. In adjacent sections stained with the anti-rat COX-2 antibody, we also observed weak positive signals in the epithelium (data not shown), probably due to the cross-reactivity of this antibody with COX-1. In the vas deferens, strong immunoreactivities for mPGES-1 and COX-1 were detected in the epithelium from the proximal segment to the distal portion of the vas deferens and ampulla (Figs. 3G–I). No positive signals for mPGES-1 or COX-1 were detected in the smooth muscle surrounding the epithelium of the vas deferens (Figs. 3G–I). The cellular localization of mPGES-1 and COX-1 in rat epididymis and vas deferens was essentially the same as that of these enzymes in the monkey tissues described above and of the mouse ones [13], suggesting that the physiological function of PGE2 in these tissues is common among these species. 3.5. Cellular localization of mPGES-1 and COX-2 in monkey and rat accessory glands The immunoreactivity for mPGES-1 was strong in the epithelium of the monkey seminal vesicular gland (Figs. 4A and B) but was not detected in the monkey prostate (data not shown). In adjacent sections of the
seminal vesicles stained with mouse IgG or guinea pig polyclonal anti-mPGES-1 antibody in the presence of an excess amount of recombinant mPGES-1, no positive signals were detected. Monkey seminal vesicle epithelial cells were also positively stained with anti-COX-2 antibody (Fig. 4C) but not with anti-COX-1 antibody (data not shown). In rats, mPGES-1 immunoreactivity was detected in the epithelium of the seminal vesicles (data not shown) and prostate (Fig 4D). No positive staining was observed when we used the anti-mouse mPGES-1 antibody preabsorbed with recombinant mPGES-1.
4. Discussion In this study, we determined the tissue distribution of mPGES-1 in male monkey genital organs to get insight into the production site of PGE2 in primates. Western blotting with polyclonal anti-mPGES-1 antibody revealed that mPGES-1 was widely expressed in male monkey genital organs (Fig. 1). However, the tissue distribution profile of mPGES-1 in monkeys was quite different from that for rat, rabbit, or pig (Fig. 1). In male rat genital organs, mPGES-1 was expressed the most intensely in the vas deferens, with significantly lower expression in the different segments of the epididymis. This profile of mPGES-1 expression is in good agreement with previous findings on PGE2 production in the genital organs of rats, in which PGE2 was detected in a high concentration in the vas deferens and at much lower levels in the testis and epididymis [16–19]. Also, this profile is very similar to that of male mouse genital organs previously reported [13]. In contrast, in the rabbit and pig male reproductive tracts, mPGES-1 was the most intensely expressed in the testis. These results clearly indicate that mPGES-1 is expressed in a highly species-specific manner in the male genital organs of various species.
mPGES-1
COX-2
Monkey seminal vesicles (A)
(B)
(C)
Rat prostate (D)
Fig. 4. Immunolocalization of mPGES-1 and COX-2 in monkey seminal vesicles and of mPGES-1 in rat prostate gland shown are light micrographs at low (A, bar, 100 mm) and high (B and C, bars, 15 mm) magnification of monkey seminal vesicles reacted with anti-human mPGES-1 antibody (A and B) or anti-rat COX-2 antibody (C). The section in ‘‘A’’ was not counterstained with hematoxylin. A micrograph (bar, 40 mm) of a section of rat prostate showing the epithelium reactive with anti-mouse mPGES-1 antibody is also presented (D).
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A remarkable difference between monkeys and rodents was also found in the cellular localization of mPGES-1. The mPGES-1 immunoreactivity was localized in various types of cells in the basal compartment of the seminiferous epithelium of the monkey testis (Fig. 2), whereas it was found only in Sertoli cells of the seminiferous tubule and in Leydig cells of the interstitium in the rat testis, and exclusively in Leydig cells of the mouse testis [13]. Taken together, these results suggest that different types of cells, i.e., Leydig, Sertoli cells or other epithelial cells, are responsible for the PGE2 production in the testis of rodents and primates to modulate the contractions of the testicular capsule through direct action on the peritubular myoid cells [13,20]. PGE2 also modulates the transport of sperm in the excurrent ducts of the epididymis and its subsequent expulsion at coitus from the vas deferens because of its smooth muscle relaxant activity [21–23]. We previously hypothesized that PGE2 produced by mPGES-1 in epithelial cells of the mouse epididymis and vas deferens is secreted toward the basal surface of the epithelium and acts on the surrounding smooth muscle layer [13]. This idea is supported by the cellular localization of mPGES-1 in epithelial cells of the caput and cauda epididymis and of the vas deferens in monkey genital organs (Fig. 3). PGE2 may also be, in part, released into the semen of monkeys from the epididymis or vas deferens, although it is believed that the seminal vesicles are the main source of PGE2 in the semen [24,25]. A species-specific difference in mPGES-1 expression was also observed in the seminal vesicles. In the male monkey reproductive tract, mPGES-1 expression was the strongest in the epithelial cells of the seminal vesicles; whereas mPGES-1 was only weakly expressed in seminal vesicles of various other species, i.e., mouse, rat, rabbit, and pig, as compared with its level in other organs of the reproductive tract (Fig. 4; [13]). The high expression of mPGES-1 in monkey seminal vesicles may explain why the 19-hydroxy derivative of PGE2 (19-OH PGE2) is remarkably abundant in the semen of primates [26–28] and acts as a potent immunosuppressor in the female tract following insemination [29–31]. Other reports also address the ability of both PGE2 and 19OH PGE2 supplied by the seminal plasma to facilitate sperm transport by regulating uterine contractility in the female reproductive tract [32] or to contribute to the acrosome reaction of the sperm [33]. In 1934, von Euler discovered a new group of hormones in the human seminal fluid and named these compounds PGs based on the assumption that they were produced in the prostate gland [1]. Later studies have indicated that the seminal vesicles are the preferential site of production [24,25] rather than the prostate gland. Presently, we showed by Western blotting and immunohistochemistry the absence of mPGES-1 in the
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monkey prostate, and thus, this finding supports the idea that the prostate of primates is not capable of producing PGE2. On the other hand, mPGES-1 was expressed in the epithelium of the rat prostate (Fig. 4), which is in good agreement with studies that have previously shown that PGE2 can be synthesized in the rat prostate gland [18]. These results suggest that the physiological function of the prostate is different between rodents and primates. Our Western blotting and immunohistochemical results revealed that mPGES-1 was expressed coordinately with COX-1 in the epithelial cells of the monkey caput and cauda epididymis and vas deferens (Fig. 3), but with COX-2 in epithelial cells of the monkey seminal vesicles (Fig. 4). Previously, the expression of COX-2 was detected immunohistochemically in epithelial cells of the human ejaculatory duct and seminal vesicles, by the use of monoclonal COX-2 antibody [34]; although, in monkeys, this antibody cross-reacted with both COX1 and COX-2 (Fig. 1B). COX-2 has long been recognized as the inducible COX isozyme [35]. However, COX-2 is constitutively expressed in the monkey seminal vesicles (Figs. 1 and 4), similarly as in the vas deferens of rodents [13,36] and the leptomeningeal cells in the rat brain [37], suggesting that COX-2 may contribute to the production of PGs in these tissues and cells. Recently, PGH2 19-hydroxylase (CYP4F8), catalyzing the conversion of PGH2 to 19-OH PGH2, was identified in human seminal vesicles, and COX-2, CYP4F8, and mPGES-1 were proposed to be closely linked for the production of 19-OH PGE2 [28]. Therefore, constitutive COX-2 may also be functionally coupled to other downstream enzymes, such as CYP4F8, rather than to mPGES-1 in the arachidonic acid cascade. In conclusion, the results of our immunohistochemical study on mPGES-1 and COXs in male monkey reproductive organs suggest that mPGES-1 is coordinately expressed with COX-1 and COX-2 to produce PGE2 and 19-OH PGE2, respectively, in the male reproductive system of primates. We also found highly species-specific expression patterns of mPGES-1 and COXs among monkeys, rodents (mice and rats), rabbits, and pigs, suggesting the need for critical consideration of animal models to study the physiological functions of COXs and mPGES-1.
Acknowledgements We thank Dr. Osamu Hayaishi for his encouragement of and during this study. We also acknowledge Mr. T. Deguchi, Mr. T. Hirado, and Mr. H. Osama for technical assistance; and Ms. S. Sakae, Ms. M. Yamaguchi, and Ms. T. Nishimoto for their secretarial assistance.
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