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Regular Article |
a Department of Physiology, Chinese University of Hong Kong, Shatin, N.T., Hong Kong
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, cyclooxygenase, epididymis, prostaglandins
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, PGD2, prostacyclin, and thromoboxane A from arachidonate. Sixty-one percent of the amino acids in the 2 isoforms of COX are identical. COX-1 is constitutively expressed by most tissues and generally is considered a "housekeeping" enzyme associated with unstimulated prostanoid formation [1, 2]. Conversely, COX-2 is an inducible enzyme and produces prostanoids in response to various inflammatory stimuli or growth factors [3–6]. However, constitutive expression of both COX-1 and COX-2 has recently been reported in certain tissues, including kidney, lung, brain, vas deferens, and epididymis [3, 7–11]. Novel nonsteroidal anti-inflammatory drugs (NSAIDs) that act specifically on either isozyme have been developed. The well-known gastric and renal side effect of nonselective COX inhibitors appears to be attributable to their inhibition of COX-1 [12], whereas the anti-inflammatory effects are due to selective inhibition of COX-2. In addition to its inflammatory response, COX-2 plays a role in several physiological events, such as female reproductive processes [13]. The constitutive expression of COX-1 and COX-2 in rat epididymis indicates that these isoforms are of functional importance in the epididymis. Recently, a number of vasoactive peptides, e.g., angiotensin, endothelin, and bradykinin, have been found to stimulate anion secretion in the epididymis via COX-1. According to the proposed model, agonists via COX-1 stimulate basal cells to release PGE2, which diffuses out of the cells and acts on the EP2/4 receptor to increase intracellular cAMP. The latter activates an apical anion channel (CFTR), resulting in secretion of anions and water in the epididymis. Fluid secretion by the epithelial cells provides an optimal microenvironment for maturation and storage of spermatozoa [11]. However, the physiological role of COX-2 in rat epididymis remains obscure.
Cumulative evidence suggests that selective inhibitors of COX-2 induce apoptosis in a variety of cancer cells, including those of colon [14], stomach [15], and prostate [16], and in normal cells including renal medullary interstitial cells [8] and intestinal epithelial cells [17]. In the present study, we attempted to clarify the role of COX-1 and COX-2 in epididymis by comparing the apoptotic effects of various selective COX-1 and COX-2 inhibitors and the potential antiapoptotic effects of supplementation with various PGs in cultured rat epididymal cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All experiments were carried out according to guidelines from the Laboratory Animal Services Centre of the Chinese University of Hong Kong. The procedures of tissue culture have been described previously [18, 19]. Immature male Sprague-Dawley rats weighing 150 g were used as a source of cauda epididymides. The rats were killed by CO2 inhalation, the lower abdomen was opened, and the caudal part of the epididymis was separated from the rest. The tissue was then finely chopped with scissors and placed in sterile Hanks balanced salt solution (HBSS) containing 0.25% (w/v) trypsin. Tissue was incubated for 30 min at 32°C with vigorous shaking (150 strokes/min). The tissue was separated by low-speed centrifugation (800 x g for 5 min), the supernatant was discarded, and the pellet was resuspended in HBSS containing 0.1% (w/v) collagenase and incubated for 60 min at 32°C with vigorous shaking. Cells were separated by centrifugation at 800 x g for 5 min, and the pellet was resuspended in Eagle minimum essential medium containing nonessential amino acids (0.1 mM), sodium pyruvate (1 mM), glutamine (4 mM), 5-dihydrotestosterone (1 nM), 10% fetal bovine serum, penicillin (100 IU/ml), and streptomycin (100 µg/ml). The cell suspension was incubated for 4 h at 32°C in 5% CO2. During this period, fibroblasts and smooth muscle cells attached to the bottom of the culture flask and the epididymal epithelial cells remained suspended. The cell suspension was decanted and seeded into 6-well trays at a cell density of 20 000 cells/well. Cultures were incubated for 3 days at 32°C in 5% CO2. Monolayers that reached confluency were ready for the drug treatment. After incubation with various agents for the indicated period, epithelial cells were washed with HBSS and ready for DNA extraction and in situ apoptosis detection. Control epididymal epithelial cells received dimethyl sulfoxide (DMSO) as vehicle for the COX inhibitors and were incubated for the same amount of time as the COX inhibitor-treated cells.
Detection of DNA Fragmentation Using Capillary Electrophoresis with Laser-Induced Fluorescence Detector
Capillary electrophoresis applied to the analysis of apoptosis is emerging as a novel method with advantages of fast analysis time, automation, reproducible analysis, and high resolving power for separation of double-stranded (ds) DNA fragments [20–22]. Fragmented DNA, a hallmark of apoptosis, was detected and analyzed by capillary electrophoresis with laser-induced fluorescence detector (CE-LIF; Bio-Rad Laboratories, Mississauga, ON, Canada) using the dsDNA1000 kit (Bio-Rad) following a procedure previously described [21]. Epithelial cells in culture were harvested by trypsinization, and floating cells were recovered by centrifugation at 800 x g for 3 min. Both adherent and floating cells were combined for the assessment of cell apoptosis. DNA was extracted by the following procedure. Epithelial cells were incubated with lysis buffer (5 mM Tris-HCl, 0.5% Triton X-100, and 20 mM EDTA, pH 8.0). The homogenates were centrifuged at 14 000 rpm (10 min, 4°C), and supernatants were collected. After addition of lysis buffer, phenol:chloroform:isoamyl alcohol (25:24:1) (Sigma, St. Louis, MO) and chloroform:isoamyl alcohol (24:1) extractions were performed, and sodium acetate (3 M, pH 5.2) was added (1:10) to the aqueous phase. Nucleic acids were precipitated by adding isopropanol (1:1, -20°C overnight). The DNA pellet was washed in ethanol (70%), air dried, dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8), and treated with 10 mg/ml RNase (Boehringer Mannheim, Indianapolis, IN) for 1 h 37°C. DNA samples were then stored frozen at -20°C until assayed. After spectrophotometric quantification of DNA concentration, the amount of DNA fragmentation in the DNA samples were analyzed by CE-LIF. The procedure used a coated capillary (24 cm x 75 µm), electrophoretic injection at 10 kV for 2 sec, electrophoresis at 2.5 V for 30 min at 40°C, and an argon laser at 488 nm. The kits included SYBR Green I (Bio-Rad) as the dsDNA-specific fluorescent label. Standards of known amounts of DNA fragments of 100–1000 base pairs were run in the CE-LIF using identical conditions as samples. The resulting standard curve of peak area versus DNA concentration was a straight line. This curve was used to calculate the percentage of fragmented DNA in total DNA tested in CE-LIF. Because DNA fragmentation is regarded as a hallmark of apoptosis, the percentage of apoptotic DNA fragments in the total DNA extracted from tested epithelial cells was used as an index of apoptosis.
TUNEL Method
In situ detection of apoptotic cells was performed by direct immunoperoxidase detection of digoxigenin-labeled genomic DNA using the ApopTag peroxidase kit (S7100 kit; Oncor, Gaithersburg, MD) as previously described [23]. The cultured epididymal cells were spun in a cytocentrifuge (Cytospin; Shandon Scientific, Cheshire, Wales, U.K.) and then fixed with 4% paraformaldehyde for 15 min. For antigen retrieval, cells were heated by microwave treatment in 0.01 M citrate buffer for 2 min, and endogenous peroxidase was quenched with 3% hydrogen peroxide in methanol for 15 min. The presence of apoptosis was detected by the TUNEL technique [24]. Epididymal cells were stained using reagents and instructions provided with the kit. Cells were incubated with terminal deoxynucleotidyl transferase (TdT) enzyme (1 h at 37°C) and anti-digoxigenin peroxidase (30 min at room temperature), and 3,3'-diaminobenzidine substrate solution (Vector Laboratories, Burlingame, CA) was used to stain the cells. Lillie-Mayer hematoxylin (Merck, Darmstadt, Germany) was used as counterstain. For negative control, TdT enzyme was replaced by an equal volume of water.
Cells were regarded as TUNEL positive if their nuclei were stained dark brown and displayed typical apoptotic morphology, such as apoptotic bodies with fragmented nuclei. Between 50 and 100 cells were individually observed for each treatment. The average percentage of apoptotic cells labeled by the TUNEL method was determined as the number of stained cells versus the total number of cells. Typically, 5–8 randomly selected nonoverlapping fields were examined on each slide.
Solutions and Drugs
Most of the solutions and chemicals used in culture were obtained from the usual commercial sources. PGE2, PGF2, and PGD2 were purchased from Sigma, COX-1 inhibitor SC-560 and COX-2 inhibitor NS-398 were from Calbiochem (San Diego, CA), PGI2 was from Cayman (Ann Abor, MI), COX-2 inhibitor DFU was a gift from Merck Frosst Canada (PQ, Canada), and the PGDP-receptor agonist BWA868C was a gift from Professor R. Jones (Department of Pharmacology, Chinese University of Hong Kong, Hong Kong).
DFU, SC-560, and NS-398 were dissolved in DMSO, and PGE2, PGF2, PGD2, PGI2, and BWA868C was dissolved in ethanol. In the in vivo experiments, NS-398 was dissolved in 0.9% NaCl instead of DMSO for oral administration.
Statistical Analysis
All experiments were performed at least 4 times, with 2 or 3 replicates per treatment in each experiment. Results are expressed as mean ± SEM. Comparisons between groups were made by the unpaired Student t-test; differences at P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The time courses of the effects of SC-560 and NS-398 on cell apoptosis were first examined by performing CE-LIF. Figure 1A and 1B show the representative CE-LIF electropherograms of the DNA fragments (measured in relative fluorescence units) isolated from cultured rat epididymal epithelial cells exposed to 50 µM COX-1 inhibitor SC-560 or 30 µM COX-2 inhibitor NS-398 for 0, 6, 12, 24, 36, or 48 h.
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Figure 2 shows the time-dependent effects of SC-560 and NS-398 on the percentage of DNA fragmentation. No significant apoptosis, in terms of percentage of DNA fragmentation, was seen in epididymal epithelia treated with the SC-560 at 48 h incubation. In contrast, 40% DNA fragmentation was seen when epididymal epithelia were incubated with 30 µM NS-398 for 24 h.
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Figure 3 shows the dose-dependent effects of SC-560, NS-398, and COX-2 inhibitor DFU [25] on percentage of fragmented DNA as determined by CE-LIF (Fig. 3A) and percentage of apoptotic cells visualized by the TUNEL method (Fig. 3B). Regardless of the method used, no significant apoptosis was observed in SC560-treated epididymal epithelia when compared with the normal epithelia. In contrast, there was a 5-fold increase in the percentage of DNA fragmentation and a similar increase in the percentage of apoptotic cells after incubation with 30 µM NS-398. This characteristic feature of apoptosis (DNA fragmentation by CE-LIF and TUNEL-stained cells) was also noted in epididymal epithelial cells treated with DFU. Figure 4 shows the in situ detection of apoptotic cells in cultured rat epididymal epithelia by the TUNEL method. Brownish apoptotic bodies were seen in cells treated with NS-398 for 24 h, and the number of apoptic cells increased with increasing drug concentration.
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Effect of COX Products on Fragmented DNA Induced by COX-2 Inhibitor NS-398
To determine the effect of various COX products on NS-398-induced endonucleosomal DNA fragmentation, we incubated epididymal epithelial cells with 30 µM NS-398 in the absence or presence of various PGs, including PGD2, PGE2, PGF2, and PGI2, for 24 h and then analyzed the extracted DNA by CE-LIF. The percentage of inhibition of apoptosis induced by exogenous COX products was calculated from the difference in percentage of DNA fragmentation between NS-398-treated and NS-398 plus PG-treated epithelia (Fig. 5). No significant inhibition of apoptosis was seen in NS-398-treated epithelia supplemented with PGF2 and PGI2. In contrast, the apoptotic effects of NS-398 could be reversed by 80% with 5 µM PGD2 and by 40% with 10 µM PGE2. The reversal of the apopotic effect of NS-398 by PGD2 and PGE2 was also seen using the TUNEL method (Fig. 4).
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Reversal of the Protective Effect by Specific PGD2 Receptor Antagonist
Table 1 shows the protective effect of PGD2 against apoptosis induced by 30 µM NS-398. The specific PGDP-receptor antagonist BWA868C [26] was used to confirm the involvement of PGD2 receptor. The results showed that BWA868C (1 µM) significantly attenuated the protective effect of 5 µM PGD2 against COX-2 inhibition.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To investigate the role of COX isoforms in the regulation of apoptosis, we used NSAIDs that are specific for COX-1 and COX-2. We found that COX-1 and COX-2 inhibitors have differential effects on apoptotic activity of the epididymal cells.
Because the limited amount of DNA in cultured epididymal epithelial cells precluded the use of traditional agarose gel electrophoresis, we used a recently developed ultrasensitive technique that measures the breakdown or fragmentation of chromatin DNA in apoptotic cells by capillary gel electrophoresis coupled with laser-induced fluorescence detection [21, 28, 29]. This CE-LIF technique provides a quantitative measurement of DNA fragmentation, allowing statistical analysis of the data.
COX-1 inhibitors did not cause a significant increase of DNA fragmentation in epididymal epithelial cells when compared with control cells. In contrast, exposure to the COX-2 inhibitor NS-398 for 24 h caused time- and dose-dependent increases of apoptotic DNA fragmentation. Similar results were also observed for tissues exposed to DFU, a specific COX-2 inhibitor with apparently higher IC50 value than NS-398 [30]. The COX-2 inhibitor-induced DNA fragmentation was associated with morphological changes characteristic of apoptosis, as revealed by the TUNEL technique, including detachment of cells from the plates, nuclear condensation, and appearance of apoptotic bodies. These changes follow a typical pattern of response associated with the effect of COX-2 inhibitors on cell growth in a number of epithelial cells. [8, 15, 17, 31]. The TUNEL technique is a very sensitive method for preferentially labeling apoptotic cells over necrotic cells, thereby discriminating apoptosis from necrosis. Thus, the DNA fragmentation induced by COX-2 inhibition was most likely caused by apoptosis and not necrosis. Regardless of which method was used, there was a very low level of apoptosis in the control (untreated) cauda epididymal epithelia. The apoptotic cells present in control tissue presumably resulted from the normal processes of cell death. In the present study, the COX-2 inhibitor-induced DNA fragmentation measured by CE-LIF was consistent with the results obtained by the TUNEL method.
Although PGs have been well studied with respect to mechanisms of production and functions in a wide variety of tissues, little is known about their roles in the epididymis. PGD synthase (PGDS) and PGD2 are heavily concentrated in the epididymis [32, 33]. However, the physiological significance of PGD2 in the epididymis remains obscure. In the present study, we found that PGD2 and to lesser extent PGE2, but not PGF2 or PGI2, reversed the apoptotic effects of NS-398. Similar protective effects of PGD2 and PGE2 have also been reported in other cells types [10, 14, 34, 35]. Using BWA868C (a specific PGD2 receptor antagonist), we clearly demonstrated that the effect of PGD2 on COX-2 inhibitor-induced apoptosis is mediated by a specific PGD2 receptor. Previous studies in our laboratory have indicated that the principal cells of the rat epididymis express COX-2 [11]. The difference in efficacy between PGD2 and PGE2 for protecting epididymal cells from COX-2 inhibitor-induced apoptosis may be attributed to distinct physiological roles of the 2 prostanoids. COX-1 does not control apoptosis but rather regulates anion secretion via PGE2 as a mediator. In contrast, COX-2 does not regulate anion secretion [11] but rather controls apoptosis. The biochemical mechanism by which COX-2 inhibition alters apoptotic activity in epididymal epithelial cells remains elusive. However, the mechanism probably involves inhibition of PGD2 formation, because exogenous PGD2 could markedly reverse the apoptotic response. Because apoptosis in the rat epididymis has been shown to be induced by androgen withdrawal [27], an involvement of PGD2-regulated androgen receptor expression in the epididymal cells as an explanation of the observed effects of COX-2 inhibitors cannot be excluded.
Prolonged NSAID use has been associated with necrosis of the renal papillae [36, 37] and apoptosis of renal medullary interstitial cells [8]. By blocking PGD2 formation, NSAIDs can directly induce apoptosis in epididymal epithelial cells. Given these findings, prolonged use of NSAIDs to combat cancer and inflammation may have undesirable effects on the male reproductive tissue, especially the epididymis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by an RGC Earmarked grant (CUHK4293/99M) to P.Y.D.W. 
2 Correspondence. FAX: 852 2603 5022; patrickwong{at}cuhk.edu.hk
Accepted: September 17, 2001.
Received: July 12, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and 30 µM NS-398 (
) on the percentage of DNA fragmentation obtained from CE-LIF. The percentage of fragmented DNA isolated from control cultured epithelial cells is also shown (
). Each point represents mean ± SEM of 6 experiments
), and SC-560 (
