Glucocorticoid hormone can suppress apoptosis of rat testicular germ cells induced by testicular ischemia

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1 FERTILITY AND STERILITY VOL. 75, NO. 5, MAY 2001 Copyright 2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Glucocorticoid hormone can suppress apoptosis of rat testicular germ cells induced by testicular ischemia Hiroshi Yazawa, M.D., Isoji Sasagawa, M.D., Yasuhiro Suzuki, M.D., and Teruhiro Nakada, M.D. Yamagata University School of Medicine, Yamagata, Japan Objective: To evaluate the effect of dexamethasone, a potent synthetic glucocorticoid hormone, on apoptosis of testicular germ cells and vascular neutrophil adhesion after repair of testicular torsion in rats. Design: Controlled laboratory study. Setting: Department of Urology, Yamagata University School of Medicine, Yamagata, Japan. Animal(s): Fifty-one male Sprague-Dawley rats. Intervention(s): Dexamethasone, 10 mg/kg of body weight. Main Outcome Measure(s): Testicular germ-cell apoptosis (percentages of apoptotic tubules and apoptotic cells) and vascular neutrophil adhesion were assessed by using DNA nick-end labeling and the endothelialneutrophil adhesion score, respectively. Result(s): Intravenous administration of dexamethasone at repair of 90-minute testicular torsion significantly inhibited testicular germ-cell apoptosis and vascular neutrophil adhesion. This inhibition was suppressed by intravenous injection of mifepristone, a glucocorticoid-receptor antagonist. Conclusion(s): Glucocorticoids can be administered for torsion in addition to conventional torsion repair. (Fertil Steril 2001;75: by American Society for Reproductive Medicine.) Key Words: Apoptosis, testis, glucocorticoid, torsion Received August 16, 2000; revised and accepted November 17, Reprint requests: Isoji Sasagawa, M.D., Department of Urology, Yamagata University School of Medicine, Iidanishi, Yamagata-shi, Yamagata , Japan (FAX: ; E- mail: isasaga@med.id. yamagata-u.ac.jp) /01/$20.00 PII S (01) Testicular torsion is a medical emergency that occurs in 1 of 4000 men younger than 25 years of age (1). If testicular torsion is not treated within 4 to 6 hours, germ-cell loss will occur (2). However, even in men who have undergone surgical detorsion within this time period, the ipsilateral testis often becomes permanently dysfunctional (3). Permanent loss of spermatogenesis has been induced in rats by a period of ischemia-inducing torsion during which infarction does not occur and after which mean organ blood flow returns to pretorsion values (4). Thus, permanent aspermatogenesis is not necessarily from permanent loss of blood flow. Turner et al. (5) reported that neutrophils play an important role in the modulation of inflammatory immune reactions in testes after torsion and that apoptosis of testicular germ cells is induced by reactive oxygen species arising from neutrophils. Neutrophils themselves are known to be regulated by many substances. Adherence of neutrophils to endothelial cells is inhibited by glucocorticoid hormones (6). We investigated the effect of dexamethasone, a potent synthetic glucocorticoid hormone, on apoptosis of testicular germ cells induced by testicular torsion in rats. These findings permit insights into new approaches to treatment of testicular torsion in humans. MATERIALS AND METHODS Animals This work was conducted in accordance with the principles and procedures of the National Institutes for Health Guide for the Care and Use of Laboratory Animals. In addition, all regulations of experimental animal studies recommended by Japanese law were followed. The study was approved by the institutional review board. 980

2 Young adult male Sprague-Dawley rats ( g) were purchased from Clea Japan, Inc. (Tokyo, Japan). The animals were housed in hanging wire mesh cages, two or three per cage, under controlled lighting conditions (14 hours of light beginning at 6:00 a.m. and 10 hours of darkness) at a temperature of 20 C 24 C. They were handled daily for 1 week before the experiment. Experimental Testicular Torsion Animals were intraperitoneally anesthetized with injection of sodium pentobarbital (50 mg/kg of body weight) for surgery and the duration of ischemia. Testicular torsion was induced as reported elsewhere (5, 7). The testes were retracted through a low midline laparotomy and freed from the epididymes. Testes were turned 720 degrees along the longitudinal axis for 1.5 hours. They were then repaired by counter-rotation and replaced into the scrotum while blood flow return was observed. Sham-operated animals underwent the same operation, but testicular torsion was relieved immediately. Effect of Dexamethasone on Testicular Germ- Cell Apoptosis and Vascular Neutrophil Adhesion After Torsion Repair The animals were divided into five groups. In group 1, 11 rats were given 10 mg/kg of dexamethasone (Wako Junyakukogyo, Osaka, Japan) by intravenous injection into the tail vein at repair of testicular torsion. In group 2, 10 rats were administered 10 mg/kg of mifepristone (Excelgyn, Paris, France) by intravenous injection at torsion repair. In group 3, 10 rats were given both 10 mg/kg of dexamethasone and 10 mg/kg of mifepristone (a glucocorticoid-receptor antagonist) by intravenous injection at repair of torsion. In group 4, saline was administered intravenously to 10 rats at the time of torsion repair. Group 5 consisted of 10 shamoperated rats (controls). Evaluation of In Situ Germ-Cell Apoptosis (terminal deoxynucleotidyl transferase mediated dideoxyuridine triphosphate nick-end labeling method) At 12 hours after repair of 1.5 hours of torsion, the testes were removed. Tissue sections were excised from the testes, embedded in Tissue-tek (Miles Inc., Elkhart, IN), and stored at 70 C. Frozen 5- m thick sections were mounted on silan-coated glass slides (Dako Japan, Tokyo, Japan) and fixed for minutes at room temperature in freshly prepared 4% paraformaldehyde buffered with 0.1 M sodium phosphate (ph, 7.4). Endogenous peroxidase was inactivated by 0.3% H 2 O 2 for minutes at room temperature. To make the sections permeable, they were incubated with a permeabilization buffer for 5 minutes at 4 C (8). In situ end-labeling was performed by using an in situ apoptosis detection kit (Takara Biomedicals, Tokyo, Japan) composed of nonradioactive fluorescein-dideoxyuridine triphosphate. The sections were incubated with terminal deoxynucleotidyl transferase and fluorescein-dideoxyuridine triphosphate at 37 C for minutes in a humidified chamber, and 3 -OH ends of the DNA fragments were tailed with fluorescein. The sections were then washed three times in phosphate-buffered saline. After being incubated with anti-fluorescein isothiocyanate horseradish peroxidase conjugate for 30 minutes at 37 C, the slides were washed three times in phosphate-buffered saline, developed with 0.05% diaminobenzidine, and stained for minutes at room temperature. The specimens were then washed three times in distilled water, counterstained with Mayer hematoxylin solution for 5 10 minutes, dehydrated, and mounted. For evaluation of apoptosis, microscopic fields were selected at random. The percentage of apoptotic germ cells was determined by counting 1,000 germ cells from apoptotic tubule cross sections of each specimen. The percentage of apoptotic tubules was determined by counting 100 tubule cross sections from each specimen (9). Evaluation of Vascular Neutrophil Adhesion Removed testes were fixed in 4% paraformaldehyde overnight. They were alcohol-dehydrated, paraffin-embedded, sectioned, and stained with hematoxylin and eosin so that the morphology of the adhering neutrophils could be observed. Quantification of neutrophil adhesion was not practical because of the differing size and orientation of sectioned vessels and the different amount of surface area exposed by each vessel cross-section. Therefore, we qualitatively evaluated neutrophil adhesion. Parenchymal and capsular vessels were independently scored in a blinded manner for endothelial-neutrophil adhesion as follows: 0, no neutrophil adhesion in any vessels; 1, occasional adhesion in some vessels; 2, adhesion commonly seen, with multiple neutrophils per vessel cross-section; 3, many neutrophils adhering in many vessels; 4, intense neutrophil staining of the complete endothelial circumference in multiple vessels (5). Data Analysis Results were analyzed for statistical significance (P.05) by using the unpaired Student t-test. RESULTS The control testes showed a normal architecture of seminiferous epithelia with slight signs of apoptosis (Fig. 1A). Testes that underwent torsion but no treatment frequently stained positive for apoptotic DNA (Figs. 1B and 1C), whereas tested in rats treated with dexamethasone showed a reduction in in situ end-labeled germ cells in seminiferous epithelia (Fig. 1D). As shown in Table 1, the percentages of apoptotic tubules and apoptotic germ cells were significantly higher in nontreated rats, rats treated with dexamethasone, those treated with mifepristone, and those treated with dexamethasone FERTILITY & STERILITY 981

3 FIGURE 1 In situ end-labeling of DNA fragmentation of testis section. A, Testis from control rat. In situ end-labeled germ cells were not frequently observed in the seminiferous epithelium (arrows). B, Testis after repair of 90-minute torsion. Labeled cells were frequently found. C, Positivity was mainly observed in spermatogonia (arrowheads) of testis after repair of torsion. D Testis treated with dexamethasone after repair of torsion. The proportion of in situ end-labeled germ cells is smaller than that in a nontreated testis. E, Testis treated with glucocorticoid receptor antagonist after repair of torsion. Germ cells are seriously affected. F, Testis treated with dexamethasone and glucocorticoid-receptor antagonist after repair of torsion. Apoptotic germ cells were frequently found. Magnification 150 in panels A, B, D, E, and F and 600 in panel C. Yazawa. Testicular germ cell apoptosis and ischemia. Fertil Steril plus mifepristone than in controls (P.001). However, dexamethasone-treated rats showed a significant decrease in the percentage of apoptotic tubules and apoptotic germ cells compared with nontreated rats, those treated with mifepristone, and those treated with dexamethasone plus mifepristone (P.05 and P.001, respectively) (Fig. 1E,F). 982 Yazawa et al. Testicular germ cell apoptosis and ischemia Vol. 75, No. 5, May 2001

4 TABLE 1 Effect of dexamethasone on percentages of apoptotic tubules and apoptotic cells after repair of testicular torsion. Group No. of animals Apoptotic tubules (%) Apoptotic germ cells (%) Control a a Torsion No treatment b c Dexamethasone Mifepristone b c Dexamethasone plus mifepristone b c Note: Data are means SD. a P.001 compared with all torsion groups. b P.05 compared with dexamethasone group. c P.001 compared with dexamethasone group. FIGURE 2 Neutrophil adhesion to testicular subtunical veins. A, The testis from a control rat had a few neutrophils (arrowheads) adhering to the vein. B, After repair of 90-minute torsion, increasing numbers of neutrophils adhered to the vein (arrowheads). C, Testis treated with dexamethasone after repair of torsion. Neutrophil adhesion in the subtunical vein decreased (arrowheads). All staining was done with hematoxylin and eosin. Magnification 100 in panel A and 1,200 in panels B and C. Yazawa. Testicular germ cell apoptosis and ischemia. Fertil Steril. Testicular vasculature in controls contained few neutrophils adhering to the vascular margins (Fig. 2A, Table 2). Some venules had multiple leukocytes adhering to the vascular endothelium. Compared with controls, the percentage of neutrophils adhering to the subtunical venous endothelium was significantly increased in nontreated rats, those treated with dexamethasone, those treated with mifepristone, and those treated with dexamethasone plus mifepristone (Table 2, Fig. 2B). However, the percentage of neutrophils adhering to the subtunical venous endothelium was significantly decreased in rats treated with dexamethasone compared with nontreated rats, those treated with mifepristone, and those treated with dexamethasone plus mifepristone treated rats (Table 2, Fig. 2C). DISCUSSION Experimental testicular torsion induces testicular ischemia during torsion (4, 7). Repair of torsion allows reperfusion of the tissue, but germ-cell loss occurs in the torqued testis after acute torsion. Loss of germ cells after reperfusion is from germ-cell specific apoptosis (5). According to Weins and Lucchesi (10), reperfusion injury is induced by reactive oxygen species, which arise either from activation of the xanthine oxidase system in parenchymal cells or from leukocytes that first adhere to the reperfusing venule wall before undergoing diapedesis into the tissue itself. Recently, reactive oxygen species have been thought to be downstream mediators of p53-dependent apoptosis (11). We found that dexamethasone, a synthetic glucocorticoid, was significantly reduced in testicular germ-cell apoptosis and neutrophil adhesion in testicular subtunical veins when it was administered 12 hours after initiation of reperfusion. This reduction was significantly lessened by administration of the glucocorticoid-receptor antagonist mifepristone. Yazawa. Testicular germ cell apoptosis and ischemia. Fertil Steril FERTILITY & STERILITY 983

5 TABLE 2 Effect of dexamethasone on neutrophil adhesion score in the intertubular and subtunical vein after repair of testicular torsion. Group No. of animals Intertubular Subtunical Control a Torsion No treatment b Dexamethasone Mifepristone Dexamethasone plus mifepristone Note: Data means SD. a P.001 compared with all torsion groups. b P.05 compared with all other torsion groups. Yazawa. Testicular germ cell apoptosis and ischemia. Fertil Steril. According to Turner et al. (5), repair of torsion causes a large increase in neutrophils adhering to the subtunical venous endothelium. However, some neutrophils adhere to the intratubular veins. Our study produced similar results. It remains unclear why neutrophil adhesion is unremarkable in these vessels. The subtunical veins are larger than the intratubular veins, and this difference in size may influence neutrophil adhesion. Mifepristone alone has no effect on spontaneous and tumor necrosis factor induced apoptosis of human neutrophils in vitro (12, 13). Nor does treatment with mifepristone alone influence testicular germ-cell apoptosis (14). We therefore observed no effects of mifepristone alone on testicular germ-cell apoptosis. Wheller and Perretti (15) showed that dexamethasone reduces intercellular adhesion molecule-1 expression in endothelial cell lines and inhibits transendothelial passage of neutrophils. In addition, neutrophil transmigration is selectively inhibited by dexamethasone in the postcapillary venule of the hamster (16). Thus, dexamethasone inhibits the production of reactive oxygen species and neutrophil adhesion after torsion repair and subsequently suppresses testicular germ-cell apoptosis. In rats, exogenous glucocorticoid induces testicular germcell apoptosis, and a potent glucocorticoid receptor antagonist completely suppresses glucocorticoid-induced apoptosis of testicular germ cells (17). Glucocorticoid hormone binds to the glucocorticoid receptor in the cytoplasm of target cells, and causes formation of a complex between the ligand and the receptor regulates gene expression of the glucocorticoid response element, a specific DNA sequence in the chromosome (12). Therefore, the regulation of transcription of genes mediated by the glucocorticoid receptor may enhance apoptosis of testicular germ cells. In acutely ischemic testes, however, germ-cell apoptosis is mainly because of production of reactive oxygen species by adhering neutrophils after torsion repair. Dexamethasone inhibits neutrophil diapedesis in the postcapillary venule of the hamster (16). Dexamethasone appears to inhibit production of reactive oxygen species and subsequently reduce germ-cell apoptosis. We did not study germ-cell apoptosis at different doses of dexamethasone or administration of dexamethasone at different times before and after torsion repair. Because these factors may influence germ-cell apoptosis, further studies should be done to clarify the mechanism of dexamethasone on germ-cell apoptosis after torsion repair. Despite improvement in techniques for immediate testicular salvage, long-term morbidity following unilateral testicular torsion remains high. Other than aggressive surgical repair, no other significant advances in the treatment of testicular torsion have emerged. Our results show that dexamethasone can suppress testicular germ-cell apoptosis after torsion repair. Administration of glucocorticoids may thus be a new treatment for torsion in addition to conventional torsion repair. However, glucocorticoid itself induces germ-cell apoptosis (14). Further studies are required to determine the adequate dose of glucocorticoid for treatment of testicular torsion. Acknowledgement: The authors thank Dr. Regine Sitruk-Ware, Laboratoires Exelgyn, Paris, France, for providing mifepristone. References 1. Barada JH, Weingarten JL, Cromie WJ. Testicular salvage and agerelated delay in the presentation of testicular torsion. J Urol 1989;142: Anderson JB, Williamson RCN. The fate of the human testes following unilateral torsion of the spermatic cord. Br J Urol 1986;58: Williamson RCN. The continuing conundrum of testicular torsion. Br J Surg 1985;72: Turner TT, Brown KJ. Spermatic cord torsion. Loss of spermatogenesis despite return of blood flow. Biol Reprod 1993;49: Turner TT, Tung KSK, Tomomasa H, Wilson LW. Acute testicular ischemia results in germ cell-specific apoptosis in the rat. Biol Reprod 1997;57: Bochsler PN, Slauson DO, Neilsen NR. Modulation of an adhesionrelated surface antigen on equine neutrophils by bacterial lipopolysaccharide and antiinflammatory drugs. J Leukocyte Biol 1990;48: Turner TT. Acute experimental testicular torsion. No effect on the contralateral testis. J Androl 1985;6: Technical Insert. Takara Biomedicals. In situ Apoptosis Detection Kit. Otsu, Japan: Takara Biochemicals Corporation, Yin Y, DeWolf WC, Morgentaler A. Experimental cryptorchidism induces testicular germ cell apoptosis by p53-dependent and -independent pathways in mice. Biol Reprod 1998;58: Weins SW, Lucchesi BR. Free radicals and ischemic tissue injury. Trends Physiol Sci 1990;11: Johnson TM, Yu ZX, Ferrans VJ, Lowenstein RA, Finkel T. Reactive oxygen species are downstream mediators of p53-dependent apoptosis. Proc Natl Acad Sci U S A 1996;93: Kato T, Takeda Y, Nakada T, Sendo F. Inhibition by dexamethasone of human neutrophil apoptosis in vitro. Nat Immun 1995;14: Yazawa H, Kato T, Nakada T, Sendo F. Glucocorticoid hormone suppression of human neutrophil-mediated tumor cell cytostasis. Int J Cancer 1999;81: Yazawa H, Sasagawa I, Nakada T. Apoptosis of testicular germ cells 984 Yazawa et al. 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6 induced by exogenous glucocorticoid in rats. Hum Reprod 2000;15: Wheller SK, Perretti M. Dexamethasone inhibits cytokine-induced intercellular adhesion molecule-1 up-regulation on endothelial cell lines. Eur J Pharmacol 1997;331: Mancuso F, Flower RJ, Perretti M. Leukocyte transmigration, but not rolling or adhesion, is selectively inhibited by dexamethasone in the hamster post-capillary venule. J Immunol 1995;155: Yazawa H, Sasagawa I, Nakada T. Effect of immobilization stress on testicular germ cell apoptosis in rats. Hum Reprod 1999;14: FERTILITY & STERILITY 985