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Effect of dexamethasone on germ cell apoptosis in the contralateral testis after testicular ischemia−reperfusion injury in the rat
Effect of dexamethasone on germ cell apoptosis in the contralateral testis after testicular ischemiaⴚreperfusion injury in the rat Jorge G. Mogilner, M.D.,a Yigal Elenberg, M.D.,a Michael Lurie, M.D.,b Eitan Shiloni, M.D.,c Arnold G. Coran, M.D.,d and Igor Sukhotnik, M.D.a a
Departments of Pediatric Surgery and b Pathology, Bruce Rappaport Faculty of Medicine, Technion, Bnai Zion Medical Center, Haifa, Israel; c Department of Surgery, Bruce Rappaport Faculty of Medicine, Technion, Carmel Medical Center, Haifa, Israel; and d Section of Pediatric Surgery, C. S. Mott Children’s Hospital and University of Michigan Medical School, Ann Arbor, Michigan
Objective: To evaluate the effect of dexamethasone on spermatogenesis and germ cell apoptosis in the ipsilateral and contralateral testis after testicular ischemia–reperfusion (IR) in rats. Design: Laboratory study. Setting: Medical research laboratory in a university setting. Animal(s): Forty adult Sprague-Dawley rats weighing 250 –280 g. Intervention(s): Testicular IR, treatment with dexamethasone (10 mg per kilogram of body weight). Main Outcome Measure(s): Testicular germ cell apoptosis was assessed by deoxyuridine nick-end labeling immunohistochemical assay. Result(s): Testicular ischemia in rats led to histological damage in the ipsilateral testis. In the contralateral testis, minimal damage was observed. Germ cell apoptosis in both the ischemic and the contralateral testis increased significantly after IR. Treatment with dexamethasone did not change germ cell apoptosis in ischemic testis but decreased germ cell apoptosis in the contralateral testis. Conclusion(s): Glucocorticoids may be beneficial for spermatogenesis after testicular IR by decreasing germ cell apoptosis in the contralateral testis. (Fertil Steril威 2006;85(Suppl 1):1111–7. ©2006 by American Society for Reproductive Medicine.) Key Words: Testis, ischemia, germ cells, apoptosis, dexamethasone
Despite improvement in early diagnosis and changes in clinical management (i.e., earlier surgical intervention), infertility remains one of the main sequelae of testicular torsion (1). The degree of fertility loss in an individual with testicular torsion depends on the extent of the ischemia and the subsequent damage to the contralateral testis (2). The mechanisms of testicular injury in the ischemic testis include nonspecific damage induced by ischemia per se and damage caused by testicular reperfusion. Restoration of blood flow after a period of testicular ischemia is necessary to maintain cell function and viability; however, the reintroduction of oxygen can initiate a cascade of events that exacerbates testicular tissue injury in ischemic testis via the formation of reactive oxygen species (3). The mechanism of injury to the contralateral testis after testicular ischemia– reperfusion (IR) remains unclear. The following mechanisms have been proposed to explain the contralateral testicular damage: autoimmunization against the spermatogonia, decrease in testicular blood flow caused by a reflex sympathetic response, free oxygen radical formation after detorsion, and overproduction of nitric oxide (2, 4, 5). Received April 19, 2005; revised and accepted October 6, 2005. Reprint requests: Jorge G. Mogilner, M.D., Department of Pediatric Surgery B, Bnai Zion Medical Center, 47 Golomb St., P.O.B. 4940, Haifa, 31048, Israel (FAX: ⫹972-4-8359620; E-mail: [email protected]).
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Necrosis has been assumed to be synonymous with germ cell death after an ischemic insult. However, more recent reports have noted that apoptosis is a significant, and perhaps the principal, contributor to cell death after IR. Apoptosis of germ cells plays an important role in the normal testicular physiology. Germ cell apoptosis is a common event during embryonic development of the human gonads (6). Apoptosis control is critical for normal spermatogenesis in the adult testes (7). Recent evidence suggests that accelerated apoptosis of the primary spermatocytes might partially account for the mechanism of germ cell loss in aging men (8). Besides its role in normal testicular physiology, apoptosis has been identified as important in the development of a variety of testicular disorders, including undescended testes (9), varicocele (10), and torsion of the testes (11). Although the effect of testicular IR on germ cell apoptosis in the ischemic testis has been reported by several investigators (11, 12), an understanding of the mechanisms that regulate this pathway in the contralateral testis is lacking. In a recent experiment, we examined the time-dependent effect of testicular ischemia (1, 2, 3, and 24 hours) on germ cell apoptosis in the contralateral testis in a rat. Our results have shown that germ cells in the contralateral testis undergo apoptosis 24 hours after testicular IR and that the extent of
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apoptosis increases with the duration of the ischemia. Both the number of apoptotic cells and the number of tubules containing apoptotic cells first increased after 1 hour of IR, achieved statistical significance after 2 and 3 hours of ischemia, and reached a peak after 24 hours of ischemia (13). We concluded that increased germ cell apoptosis in the contralateral testis, combined with ischemia-induced cell death in the ischemic testis, may lead to significant decrease in the germ cell mass and may cause loss of fertility. In this context, identification of those agents or drugs that can decrease germ cell apoptosis may offer novel, scientifically relevant, and practical approaches to alleviating testicular IR injury. Glucocorticoids are the major steroid hormones secreted by the adrenal gland. They play an important role in many physiologic and biologic processes. The main mechanisms of action exerted by glucocorticoids include profound antiinflammatory and immunosuppressive effects, direct inhibition of different types of cell proliferation, and apoptosis. Dexamethasone, a synthetic glucocorticoid, has anti-inflammatory and immunomodulatory properties when used as a therapeutic agent at higher doses. In a recent study, Yazawa and coworkers (14) have shown that intravenous administration of dexamethasone at a dose of 10 mg/kg after 90 minutes of testicular torsion significantly inhibited testicular germ cell apoptosis and vascular neutrophil adhesion in ischemic testes. The purpose of the present study was to evaluate the effect of dexamethasone on germ cell apoptosis and spermatogenesis in both the ischemic and the contralateral testis, 96 hours after testicular IR injury in rats. MATERIALS AND METHODS Animals Rappaport Faculty of Medicine (Technion, Haifa, Israel) Institutional Animal Care and Use Committee and Review Board approved the animal facilities and protocols. Briefly, male rats weighing 240 –260g were kept in individual stainless steel cages under standardized conditions (constant temperature and humidity, 12-hour light-dark cycle) for 3–5 days. Experimental Design Forty rats were divided randomly into four experimental groups of 10 rats each: group A, sham-operated animals (sham); group B, sham-operated rats who were treated with dexamethasone SC at a dose of 10 mg/kg, once daily, at 24 hours, 48 hours, and 72 hours after operation (sham-dex); group C, rats who underwent 90 minutes of unilateral testicular IR (IR); and group D, rats who underwent 90 minutes of unilateral testicular IR and were treated with dexamethasone given SC at a dose of 10 mg/kg, once daily, at 24 hours, 48 hours, and 72 hours after operation (IR-dex). 1112
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Surgical Procedure After an overnight fast, the animals were anesthetized with an intraperitoneal injection of pentobarbital (45 mg/kg). The abdomen was opened through a midline incision, and the left testis was retracted outside the incision. In groups A and B, the testis was replaced immediately into the scrotum, and the incision of the abdominal wall was covered with a warm moist dressing for 90 minutes to prevent hypothermia. In groups C and D, the vascular bundle of the extracted testis was occluded by an atraumatic microvascular clamp and was returned into the abdominal cavity to avoid hypothermia. After 90 minutes of testicular ischemia, the clamp was removed and the ischemic testis was replaced into the scrotum. Before closure of the abdomen, the rats were resuscitated with a 3-mL intraperitoneal injection of warm 0.9% saline. For all operations, the abdominal cavity was closed in two layers with a running suture of Dexon S Polyglycolic Acid 3– 0 (TYCO Healthcare, Mansfield, MA). Rats were allowed free access to water and food. Ninety-six hours later, the rats were anesthetized with intraperitoneal pentobarbital (75 mg/kg) and were killed by open pneumothorax. Both testes were removed, washed with cold saline, and dried. Tissue samples from both testes were fixed in 10% formalin and embedded in paraffin. Deparaffinized, 5-m-sized sections were stained with hematoxylin and eosin. Histological alterations were studied by using a graded eye piece at ⫻10 magnification. Histologic Examination of Spermatogenesis Johnsen’s criteria and the number of germinal cell layers were used to categorize the spermatogenesis (15). Johnsen’s method applies a score of 1 to 10 for each tubule crosssection, according to the following criteria: 10, complete spermatogenesis and perfect tubules; 9, many spermatozoa present and disorganized spermatogenesis; 8, only a few spermatozoa present; 7, no spermatozoa but many spermatids present; 6, only a few spermatids present; 5, no spermatozoa or spermatids but many spermatocytes present; 4, only a few spermatocytes present; 3, only spermatogonia present; 2, no germ cells but only Sertoli cells present; and 1, no germ cells and no Sertoli cells present. The number of germinal epithelial layers was counted in the 10 seminiferous tubules as described elsewhere (16), and the mean number was calculated. Measurement of Apoptosis The deoxyuridine nick-end labeling (TUNEL) assay for apoptotic cell detection was performed with the I.S. Cell Death Detection kit (Boehringer Mannheim GmbH, Mannheim, Germany). Briefly, serials (5-m-thick paraffin-embedded sections) were deparaffinized, rehydrated in graded alcohol, and microwave pretreated in 10 mM citrate buffer (pH 6.0) for 10 minutes. After washing in phosphate-buffered saline (PBS), the specimens were incubated in buffer containing a nucleotide mixture with fluorescein-labeled deoxy-UTP and
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TdT at 37°C for 1 hour. After washing, the slides were incubated with blocking solution (3% H2O2 in methanol) for 10 minutes and were stained with anti-fluorescein antibody, Fab fragment from sheep, conjugated with horseradish peroxidase (converter-POD) at 37°C for 30 minutes. Aminoethyl carbazole substrate (Zymed Laboratories) was applied for color development. For each group, the number of stained cells was counted in 10 tubules in the areas without necrosis. For each group, the number of stained germ cells was counted. The apoptotic index AI-1 was defined as the number of apoptotic TUNEL-positive cells per 100 tubules, and the index AI-2, as the number of tubules containing apoptotic cells per 100 tubules. Pathologists blinded to the source of testicular tissue performed all measurements. Statistical Analysis The data are expressed as the mean ⫾ SEM. Statistical analysis was performed using the nonparametric KruskalWallis analysis of variance test, with P⬍.05 considered statistically significant. RESULTS Testicular Parameters of Spermatogenesis Table 1 compares the histologic changes in the ischemic testes and in the contralateral testes among the four experimental groups. As expected, the sham-operated animals (group A) demonstrated a normal architecture of the seminiferous tubules and interstitium in both testes and had intact germinal epithelium with an average thickness of about four to five cell layers. Treatment with dexamethasone (group B) did not significantly change the histologic manifestations of spermatogenesis compared with sham animals (group A). Ninety minutes of testicular ischemia led to severe damage in the ischemic testis. Impaired spermatogenesis in IR rats (group C) was manifested by a threefold decrease in germ cell layer count (P⬍.05) and a threefold decrease in the
mean testicular score (Johnsen criteria; P⬍.05) compared with sham animals (group A). Treatment with dexamethasone did not significantly change testicular parameters of spermatogenesis. IR-dex rats (group D) demonstrated a mean score and germ cell layer count similar to that of IR rats (group C) and demonstrated lower values with compared with sham (group A) and sham-dex (group B) rats. Evaluation of spermatogenesis in the contralateral testis 96 hours after IR did not show marked histologic changes. The IR (group C) rats showed a slight decrease in the number of germ cell layers compared with sham animals (group A); however, this trend was not statistically significant. Administration of dexamethasone (groups B and D) did not affect testicular parameters of spermatogenesis compared with sham (group A) and IR rats (group C). Assessment of Germ Cell Apoptosis Apoptosis was characterized by a TUNEL technique that specifically detects apoptotic germ cells in the testis. As shown in Figures 1 and 2, dexamethasone in sham rats (group B) did not significantly change germ cell apoptosis in both testes compared with the case of sham animals (group A). Figure 1 demonstrates changes in germ cell apoptosis in the ischemic testis after testicular IR and administration of dexamethasone. As expected, testicular IR (group C) resulted in a significant increase in germ cell apoptosis in the ischemic testis, expressed as the number of apoptotic cells per 100 tubules (33.4 ⫾ 8.5 vs. 1.1 ⫾ 0.7 TUNEL-positive cells per 100 tubules; P⬍.05) and the number of positive tubules (11.3 ⫾ 4.8 vs. 0.5 ⫾ 0.3 TUNEL-positive tubules per 100 tubules; P⬍.05) compared with sham animals (group A). Treatment with dexamethasone (group D) did not change significantly germ cell apoptosis in the ischemic testis compared with the case of IR-untreated animals (group C). The majority of the tubules were severely damaged in
TABLE 1 Effect of dexamethasone on spermatogenesis in ipsilateral and contralateral tests after testicular IR in rat. Ischemic testis
Contralateral testis
Experimental groups
Johnsen’s criteria
Number of layers
Johnsen’s criteria
Number of layers
Sham Sham-dex IR IR-dex
7.6 ⫾ 0.9 8.7 ⫾ 0.2 2.8 ⫾ 1a,b 3.3 ⫾ 0.8a,b
3.7 ⫾ 4.3 4.3 ⫾ 0.3 1.3 ⫾ 0.6a,b 1.5 ⫾ 0.5a,b
9.1 ⫾ 0.1 9.3 ⫾ 0.3 9.2 ⫾ 0.3 9.2 ⫾ 0.3
4.5 ⫾ 0.1 4.8 ⫾ 0.2 4.2 ⫾ 0.2 4.4 ⫾ 0.2
Note: Values are mean ⫾ SEM. a P⬍.05 vs. sham rats. b P⬍.05 vs. sham-dex rats. Mogilner. Dexamethasone and germ cell apoptosis. Fertil Steril 2006.
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FIGURE 1 The effect of testicular ischemia-reperfusion (IR) and administration of dexamethasone on germ cell apoptosis (apoptotic cells per 100 tubules and positive tubules per 100 tubules) in the ischemic testis. Values are mean ⫾ SEM. Bars indicate experimental groups: , sham; , sham-dex; , IR; , IR-dex. *P⬍.05 vs. sham-operated animals.
Mogilner. Dexamethasone and germ cell apoptosis. Fertil Steril 2006.
IR-dex rats and displayed a number of apoptotic cells that was significantly greater than that in sham (group A) and sham-dex (group B) animals.
physiological mechanisms. The anti-inflammatory efficacy of corticoids is explained by their inhibition of the synthesis of numerous cytokines, enzymes and mediators of inflammation. Glucocorticoid hormones are also regulators of cell growth and differentiation. In particular, glucocorticosteroids have been shown to be potent antiproliferative agents in many organ and cell types (17) and also are known to induce gastrointestinal cell arrest and programmed death of several tumor cell lines (18). The understanding of testicular physiology and pathology requires knowledge of the regulation of cell proliferation and programmed cell death. Extensive studies in various experimental models have established that apoptosis control in the testis is crucial for the evolution of normal spermatogenesis (6 – 8). The dynamic process of germ cell turnover is regulated by several hormones: these include T, LH, and FSH. Recent evidence suggests that exogenously administered corticosteroids induce apoptosis in rat Leydig cells in the interstitium (19). Several investigators consider this pathway to be the main mechanism responsible for the decrease in serum T levels that is seen in animals that have been administered glucocorticoids (20). In a recent experiment, Yazawa and coworkers (21) have shown that dexamethasone induces significant testicular germ cell apoptosis in rats. Little is known about the effect of glucocorticoids on germ cell proliferation and apoptosis after testicular IR. Extensive studies in various experimental models have established a beneficial effect of glucocorticoids in IR events
Figures 2 and 3 summarize the data on germ cell apoptosis in the contralateral testis. Consistent with the result of our first experiment (13), testicular ischemia (group C) resulted in a significant increase in germ cell apoptosis in the contralateral testis. The IR rats demonstrated a significant increase in the number of apoptotic cells per 100 tubules (6.6 ⫾ 1.3 vs. 2.2 ⫾ 1.1 TUNEL-positive cells per 100 tubules; P⬍.05) and in the number of positive tubules per 100 tubules (4.7 ⫾ 1.0 vs. 1.3 ⫾ 0.7 TUNEL-positive tubules per 100 tubules; P⬍.05) compared with sham animals. Treatment with dexamethasone (group D) resulted in a significant decrease (vs. IR rats, group C) in the number of positive tubules per 100 tubules (2.0 ⫾ 0.7 vs. 4.7 ⫾ 1.0 TUNELpositive tubules per 100 tubules; P⬍.05) and a trend toward a decrease in the number of apoptotic cells per 100 tubules (3.8 ⫾ 1.9 vs. 6.6 ⫾ 1.3 TUNEL-positive cells per 100 tubules; P⫽.14); however, this trend did not achieve statistical significance. DISCUSSION Glucocorticosteroids are 12-carbon molecules derived from cholesterol and are produced by the adrenals in response to adrenocorticotropin hormone, which in turn is secreted by the anterior pituitary in response to corticotropin-releasing factor. Glucocorticoids have far-ranging effects on almost every tissue in the body and play an important role in the regulation of a large number of immune and metabolic 1114
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Dexamethasone and germ cell apoptosis
FIGURE 2 The effect of testicular ischemia-reperfusion (IR) and administration of dexamethasone on germ cell apoptosis (apoptotic cells per 100 tubules and positive tubules per 100 tubules) in the contralateral testis. Values are mean ⫾ SEM. Bars indicate experimental groups: , sham; , sham-dex; , IR; , IR-dex. *P⬍.05 vs. sham-operated animals, † IR-dex vs. IR rats.
Mogilner. Dexamethasone and germ cell apoptosis. Fertil Steril 2006.
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FIGURE 3 Immunohistochemistry for apoptosis in the contralateral testis in the four experimental groups. Single apoptotic cells (arrows) are present in sections from sham-treated (A) and sham-dex–treated rats (B). Labeled cells frequently are found in IR (C) rats. The number of apoptotic cells decreases after administration of dexamethasone (D).
Mogilner. Dexamethasone and germ cell apoptosis. Fertil Steril 2006.
in many organs and tissues. Dexamethasone pretreatment significantly reduces the occurrence of reperfusion-induced arrhythmias and improves cardiac recovery after myocardial IR (22). Dexamethasone decreases lung and systemic effects of intestinal IR in a rat (23). Dexamethasone combined with mannitol has beneficial effects in preventing brain IR injury in a rat (24). Methylprednisolone has a beneficial effect against IR injury in cremaster muscle flaps in rats (25). The main mechanisms of the positive effects of glucocorticoids remain unclear. It is widely accepted that glucocorticoid hormones decrease reperfusion injury by suppressing the production of oxygen-derived free radicals from polymorphonuclear neutrophils (14); by reducing the tissue levels of several proinflammatory mediators (26); by attenuation of neutrophil infiltration and intercelFertility and Sterility姞
lular adhesion molecule-1 (27); and by reducing the synthesis of eicosanoids, edema formation, and leukocyte infiltration (24). Few experimental studies have examined the effect of glucocorticoid hormones in testicular IR. In a recent experiment, Yazawa and coworkers (14) examined the effect of dexamethasone on germ cell apoptosis in the ischemic testis after testicular IR. Those investigators showed that intravenous administration of dexamethasone inhibits vascular neutrophil adhesion and decreases testicular germ-cell apoptosis in the ischemic testis. The current study was an extension of our previous work in which the effect of testicular IR on germ cell apoptosis in the contralateral testis was examined 24 hours after an IR 1115
event (13). The present study was conducted to evaluate whether germ cell apoptosis in the contralateral testis remains increased 96 hours after IR and whether administration of dexamethasone could affect germ cell apoptosis in both the ischemic and contralateral testis. Our data demonstrate that testicular ischemia induces degeneration of germ cells and impairs spermatogenesis. The average number of germinal epithelial cell layers and Johnsen’s criteria decreased significantly in the ischemic testis compared with the case of sham-operated animals. Administration of dexamethasone did not significantly affect spermatogenesis in control rats. In addition, treatment with dexamethasone did not prevent damage caused by testicular IR in the ischemic testis and did not improve the recovery of testicular tissue from the ischemic injury. Groups C (IR rats) and D (IR-dex rats) had similar histologic appearances. The number of germinal epithelial cell layers and Johnsen’s criteria were not statistically significantly different. Our data suggest that testicular IR with or without the administration of dexamethasone does not produce contralateral testicular histologic damage. All experimental groups showed normal histologic architecture of the seminiferous tubules in the contralateral testis. There was a tendency toward a decrease in the number of germ cell layers in the contralateral testis after IR compared with in sham animals; this change was not statistically significant; however, a trend was observed and may be clinically important; therefore, it was included in the Discussion section. These data fit with reports of other investigators who also have noted no contralateral damage after ipsilateral experimental torsion (3, 11). The finding of normal seminiferous tubule architecture with decreased number of germ cell layers may suggest that spermatogenesis is affected without direct damaging effect of IR. The measurement of apoptosis demonstrated an increase in germ cell programmed death after testicular IR. The present data are consistent with the results of other investigators who demonstrated that loss of germ cells after IR is from germ cell–specific apoptosis (3, 11, 14). Stimulation of germ cell apoptosis in the ischemic testis mainly is attributed to increased leukocyte adhesions (14) and production of reactive oxygen species (3, 11). We demonstrated in this experiment that germ cell apoptosis in the contralateral testis was increased after IR. We previously have reported increases in germ cell apoptosis in the contralateral testis 24 hours after IR (13). These data indicate that 96 hours after IR, germ cell apoptosis in the contralateral testis remains significantly increased. In addition to ischemia-induced cell death in the ischemic testis, increased germ cell apoptosis in the contralateral testis may lead to significant decrease in the germ cell mass and may cause loss of fertility. Despite the increased loss of germ cells, the majority of seminiferous tubules maintain normal architecture. It may be reasonable to presume that histolog1116
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ical manifestation of impaired spermatogenesis will appear after a long period of enhanced programmed cell death. Our experiment demonstrates that treatment with dexamethasone did not significantly change germ cell apoptosis in the ischemic testis. These results conflict with the report of Yazawa and colleagues (14), who emphasize evidence of decreased germ cell apoptosis in the ischemic testis after dexamethasone administration. However, the measurement of programmed germ cell death in the contralateral testis in our study demonstrates beneficial effects of dexamethasone on spermatogenesis through decreases in cell apoptosis. This is evident from the decrease in the number of apoptotic cells compared with the case of animals not treated with IR. Further experiments are needed to clarify the mechanisms of the effect of dexamethasone on germ cell apoptosis in the contralateral testis after IR as well as to determine whether a longer time interval of treatment causes more significant changes in the cell apoptosis and seminiferous tubule architecture. In conclusion, the present data are consistent with the interpretation that testicular ischemia results in fertility disturbances, including spermatogenesis arrest and increase in germ cell death. The use of glucocorticoids decreases germ cell apoptosis in the contralateral testis. Extrapolation of these data to the human situation is not appropriate; however, this information does provide a stimulus for true clinical investigations. REFERENCES 1. Bartsch G, Frank S, Marberger H, Mikuz G. Testicular torsion: late results with special regard to fertility and endocrine function. J Urol 1980;124:375– 8. 2. Harrison RG, Lewis-Jones DI, Moreno de Marval MJ, Connolly RC. Mechanism of damage to the contralateral testis in rats with an ischemic testis. Lancet 1981;2:723–5. 3. Lysiak JJ, Nguyen QA, Turner TT. Peptide and nonpeptide reactive oxygen scavengers provide partial rescue of the testis after torsion. J Androl 2002;23:400 –9. 4. Sarica K, Kupeli B, Budak M, Kosar A, Kavukcu M, Durak I, et al. Influence of experimental spermatic cord torsion on the contralateral testis in rats. Evaluation of tissue free oxygen radical scavenger enzyme levels. Urol Int 1997;58:208 –12. 5. Shiraishi K, Naito K, Yoshida K. Nitric oxide promotes germ cell necrosis in the delayed phase after experimental testicular torsion of rat. Biol Reprod 2001;65:514 –21. 6. Modi DN, Sane S, Bhartiya D. Accelerated germ cell apoptosis in sex chromosome aneuploid fetal human gonads. Mol Hum Reprod 2003; 9:219 –25. 7. Bartke A. Apoptosis of male germ cells, a generalized or cell typespecific phenomenon. Endocrinology 1995;136:3– 4. 8. Kimura M, Itoh N, Takagi S, Sasao T, Takahashi A, Masumori N, et al. Balance of apoptosis and proliferation of germ cells related to spermatogenesis in aged men. J Androl 2003;24:185–91. 9. Tomomasa H, Adachi Y, Oshio S, Umeda T, Irie H, Ishikawa H. Germ cell apoptosis in undescended testis: the origin of its impaired spermatogenesis in the TS inbred rat. Urology 2002;168:343–7. 10. Barqawi A, Caruso A, Meacham RB. Experimental varicocele induces testicular germ cell apoptosis in the rat. J Urol 2004;171:501–3. 11. Turner TT, Tung KS, Tomomasa H, Wilson LW. Acute testicular ischemia results in germ cell-specific apoptosis in the rat. Biol Reprod 1997;57:1267–74. 12. Oh SJ, Kwak C, Baek M, Kim CS, Kim KS, Choi H. Histologic and
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molecular changes in the ipsilateral and contralateral epididymides of the rat in response to unilateral testicular torsion followed by detorsion. Fertil Steril 2004;81(Suppl 1):882–7. Sukhotnik I, Miselevich I, Lurie M, Nativ O, Coran AG, Mogilner JG. The time relationship between ipsilateral testicular ischemiareperfusion and germ cell apoptosis in the contralateral testis in rat. Pediatr Surg Int 2005;21:512– 6. Yazawa H, Sasagawa I, Suzuki Y, Nakada T. Glucocorticoid hormone can suppress apoptosis of rat testicular germ cells induced by testicular ischemia. Fertil Steril 2002;75:980 –5. Johnsen SG. Testicular biopsy score count—a method for registration of spermatogenesis in human testes: normal values and results in 335 hypogonadal males. Hormones 1970;1:2–25. Miller DC, Peron SE, Keck RW, Kropp KA. Effects of hypothermia on testicular ischemia. J Urol 1990;143:1046 – 8. Hochberg Z. Mechanisms of steroid impairment of growth. Horm Res 2002;58(Suppl 1):33– 8. Strauss G, Osen W, Debatin KM. Induction of apoptosis and modulation of activation and effector function in T cells by immunosuppressive drugs. Clin Exp Immunol 2002;128:255– 66. Gao HB, Tong MN, Hu YQ, Guo QS, Ge R, Hardy MP. Gluccorticoid induces apoptosis in rat Leydig cells. Endocrinology 2002; 143:130 – 8. Orr TE, Mann DR. Role of glucocorticoid in the stress-induced suppression of testicular steroidogenesis in adult male rats. Horm Behav 1992;26:350 – 63.
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21. Yazawa H, Sasagawa I, Nakada T. Apoptosis of testicular germ cells induced by exogenous glucocorticoid in rats. Hum Reprod 2000;15: 1917–20. 22. Varga E, Nagy N, Lazar J, Czifra G, Bak I, Biro T, et al. Inhibition of ischemia/reperfusion-induced damage by dexamethasone in isolated working rat hearts: the role of cytochrome c release. Life Sci 2004;75: 2411–23. 23. Cavriani G, Oliveira-Filho RM, Trezena AG, da Silva ZL, Domingos HV, de Arruda MJ, et al. Lung microvascular permeability and neutrophil recruitment are differently regulated by nitric oxide in a rat model of intestinal ischemia-reperfusion. Eur J Pharmacol 2004;494: 241–9. 24. Wang Z, Xu X, Wu X. Therapeutic effect of dexamethasone and mannitol on global brain ischemia-reperfusion injury in rats. Beijing Da Xue Xue Bao 2003;18:303– 6. 25. Askar I, Bozkurt M. Protective effects of immunosuppressants and steroids against ischemia-reperfusion injury in cremaster muscle flap at microcirculatory level. Microsurgery 2002;22:361– 6. 26. Willemart G, Knight KR, Ayad M, Wagh M, Morrison WA. The beneficial antiinflammatory effect of dexamethasone administration prior to reperfusion on the viability of cold-stored skin flaps. Int J Tissue React 1999;21:71– 8. 27. Wheller SK, Perretti M. Dexamethasone inhibits cytokine-induced intracellular adhesions molecule-1 up-regulation on endothelial cell lines. Eur J Pharmacol 1997;331:65–71.
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Departments of Pediatric Surgery and b Pathology, Bruce Rappaport Faculty of Medicine, Technion, Bnai Zion Medical Center, Haifa, Israel; c Department of Surgery, Bruce Rappaport Faculty of Medicine, Technion, Carmel Medical Center, Haifa, Israel; and d Section of Pediatric Surgery, C. S. Mott Children’s Hospital and University of Michigan Medical School, Ann Arbor, Michigan
Objective: To evaluate the effect of dexamethasone on spermatogenesis and germ cell apoptosis in the ipsilateral and contralateral testis after testicular ischemia–reperfusion (IR) in rats. Design: Laboratory study. Setting: Medical research laboratory in a university setting. Animal(s): Forty adult Sprague-Dawley rats weighing 250 –280 g. Intervention(s): Testicular IR, treatment with dexamethasone (10 mg per kilogram of body weight). Main Outcome Measure(s): Testicular germ cell apoptosis was assessed by deoxyuridine nick-end labeling immunohistochemical assay. Result(s): Testicular ischemia in rats led to histological damage in the ipsilateral testis. In the contralateral testis, minimal damage was observed. Germ cell apoptosis in both the ischemic and the contralateral testis increased significantly after IR. Treatment with dexamethasone did not change germ cell apoptosis in ischemic testis but decreased germ cell apoptosis in the contralateral testis. Conclusion(s): Glucocorticoids may be beneficial for spermatogenesis after testicular IR by decreasing germ cell apoptosis in the contralateral testis. (Fertil Steril威 2006;85(Suppl 1):1111–7. ©2006 by American Society for Reproductive Medicine.) Key Words: Testis, ischemia, germ cells, apoptosis, dexamethasone
Despite improvement in early diagnosis and changes in clinical management (i.e., earlier surgical intervention), infertility remains one of the main sequelae of testicular torsion (1). The degree of fertility loss in an individual with testicular torsion depends on the extent of the ischemia and the subsequent damage to the contralateral testis (2). The mechanisms of testicular injury in the ischemic testis include nonspecific damage induced by ischemia per se and damage caused by testicular reperfusion. Restoration of blood flow after a period of testicular ischemia is necessary to maintain cell function and viability; however, the reintroduction of oxygen can initiate a cascade of events that exacerbates testicular tissue injury in ischemic testis via the formation of reactive oxygen species (3). The mechanism of injury to the contralateral testis after testicular ischemia– reperfusion (IR) remains unclear. The following mechanisms have been proposed to explain the contralateral testicular damage: autoimmunization against the spermatogonia, decrease in testicular blood flow caused by a reflex sympathetic response, free oxygen radical formation after detorsion, and overproduction of nitric oxide (2, 4, 5). Received April 19, 2005; revised and accepted October 6, 2005. Reprint requests: Jorge G. Mogilner, M.D., Department of Pediatric Surgery B, Bnai Zion Medical Center, 47 Golomb St., P.O.B. 4940, Haifa, 31048, Israel (FAX: ⫹972-4-8359620; E-mail: [email protected]).
0015-0282/06/$32.00 doi:10.1016/j.fertnstert.2005.10.021
Necrosis has been assumed to be synonymous with germ cell death after an ischemic insult. However, more recent reports have noted that apoptosis is a significant, and perhaps the principal, contributor to cell death after IR. Apoptosis of germ cells plays an important role in the normal testicular physiology. Germ cell apoptosis is a common event during embryonic development of the human gonads (6). Apoptosis control is critical for normal spermatogenesis in the adult testes (7). Recent evidence suggests that accelerated apoptosis of the primary spermatocytes might partially account for the mechanism of germ cell loss in aging men (8). Besides its role in normal testicular physiology, apoptosis has been identified as important in the development of a variety of testicular disorders, including undescended testes (9), varicocele (10), and torsion of the testes (11). Although the effect of testicular IR on germ cell apoptosis in the ischemic testis has been reported by several investigators (11, 12), an understanding of the mechanisms that regulate this pathway in the contralateral testis is lacking. In a recent experiment, we examined the time-dependent effect of testicular ischemia (1, 2, 3, and 24 hours) on germ cell apoptosis in the contralateral testis in a rat. Our results have shown that germ cells in the contralateral testis undergo apoptosis 24 hours after testicular IR and that the extent of
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apoptosis increases with the duration of the ischemia. Both the number of apoptotic cells and the number of tubules containing apoptotic cells first increased after 1 hour of IR, achieved statistical significance after 2 and 3 hours of ischemia, and reached a peak after 24 hours of ischemia (13). We concluded that increased germ cell apoptosis in the contralateral testis, combined with ischemia-induced cell death in the ischemic testis, may lead to significant decrease in the germ cell mass and may cause loss of fertility. In this context, identification of those agents or drugs that can decrease germ cell apoptosis may offer novel, scientifically relevant, and practical approaches to alleviating testicular IR injury. Glucocorticoids are the major steroid hormones secreted by the adrenal gland. They play an important role in many physiologic and biologic processes. The main mechanisms of action exerted by glucocorticoids include profound antiinflammatory and immunosuppressive effects, direct inhibition of different types of cell proliferation, and apoptosis. Dexamethasone, a synthetic glucocorticoid, has anti-inflammatory and immunomodulatory properties when used as a therapeutic agent at higher doses. In a recent study, Yazawa and coworkers (14) have shown that intravenous administration of dexamethasone at a dose of 10 mg/kg after 90 minutes of testicular torsion significantly inhibited testicular germ cell apoptosis and vascular neutrophil adhesion in ischemic testes. The purpose of the present study was to evaluate the effect of dexamethasone on germ cell apoptosis and spermatogenesis in both the ischemic and the contralateral testis, 96 hours after testicular IR injury in rats. MATERIALS AND METHODS Animals Rappaport Faculty of Medicine (Technion, Haifa, Israel) Institutional Animal Care and Use Committee and Review Board approved the animal facilities and protocols. Briefly, male rats weighing 240 –260g were kept in individual stainless steel cages under standardized conditions (constant temperature and humidity, 12-hour light-dark cycle) for 3–5 days. Experimental Design Forty rats were divided randomly into four experimental groups of 10 rats each: group A, sham-operated animals (sham); group B, sham-operated rats who were treated with dexamethasone SC at a dose of 10 mg/kg, once daily, at 24 hours, 48 hours, and 72 hours after operation (sham-dex); group C, rats who underwent 90 minutes of unilateral testicular IR (IR); and group D, rats who underwent 90 minutes of unilateral testicular IR and were treated with dexamethasone given SC at a dose of 10 mg/kg, once daily, at 24 hours, 48 hours, and 72 hours after operation (IR-dex). 1112
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Surgical Procedure After an overnight fast, the animals were anesthetized with an intraperitoneal injection of pentobarbital (45 mg/kg). The abdomen was opened through a midline incision, and the left testis was retracted outside the incision. In groups A and B, the testis was replaced immediately into the scrotum, and the incision of the abdominal wall was covered with a warm moist dressing for 90 minutes to prevent hypothermia. In groups C and D, the vascular bundle of the extracted testis was occluded by an atraumatic microvascular clamp and was returned into the abdominal cavity to avoid hypothermia. After 90 minutes of testicular ischemia, the clamp was removed and the ischemic testis was replaced into the scrotum. Before closure of the abdomen, the rats were resuscitated with a 3-mL intraperitoneal injection of warm 0.9% saline. For all operations, the abdominal cavity was closed in two layers with a running suture of Dexon S Polyglycolic Acid 3– 0 (TYCO Healthcare, Mansfield, MA). Rats were allowed free access to water and food. Ninety-six hours later, the rats were anesthetized with intraperitoneal pentobarbital (75 mg/kg) and were killed by open pneumothorax. Both testes were removed, washed with cold saline, and dried. Tissue samples from both testes were fixed in 10% formalin and embedded in paraffin. Deparaffinized, 5-m-sized sections were stained with hematoxylin and eosin. Histological alterations were studied by using a graded eye piece at ⫻10 magnification. Histologic Examination of Spermatogenesis Johnsen’s criteria and the number of germinal cell layers were used to categorize the spermatogenesis (15). Johnsen’s method applies a score of 1 to 10 for each tubule crosssection, according to the following criteria: 10, complete spermatogenesis and perfect tubules; 9, many spermatozoa present and disorganized spermatogenesis; 8, only a few spermatozoa present; 7, no spermatozoa but many spermatids present; 6, only a few spermatids present; 5, no spermatozoa or spermatids but many spermatocytes present; 4, only a few spermatocytes present; 3, only spermatogonia present; 2, no germ cells but only Sertoli cells present; and 1, no germ cells and no Sertoli cells present. The number of germinal epithelial layers was counted in the 10 seminiferous tubules as described elsewhere (16), and the mean number was calculated. Measurement of Apoptosis The deoxyuridine nick-end labeling (TUNEL) assay for apoptotic cell detection was performed with the I.S. Cell Death Detection kit (Boehringer Mannheim GmbH, Mannheim, Germany). Briefly, serials (5-m-thick paraffin-embedded sections) were deparaffinized, rehydrated in graded alcohol, and microwave pretreated in 10 mM citrate buffer (pH 6.0) for 10 minutes. After washing in phosphate-buffered saline (PBS), the specimens were incubated in buffer containing a nucleotide mixture with fluorescein-labeled deoxy-UTP and
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TdT at 37°C for 1 hour. After washing, the slides were incubated with blocking solution (3% H2O2 in methanol) for 10 minutes and were stained with anti-fluorescein antibody, Fab fragment from sheep, conjugated with horseradish peroxidase (converter-POD) at 37°C for 30 minutes. Aminoethyl carbazole substrate (Zymed Laboratories) was applied for color development. For each group, the number of stained cells was counted in 10 tubules in the areas without necrosis. For each group, the number of stained germ cells was counted. The apoptotic index AI-1 was defined as the number of apoptotic TUNEL-positive cells per 100 tubules, and the index AI-2, as the number of tubules containing apoptotic cells per 100 tubules. Pathologists blinded to the source of testicular tissue performed all measurements. Statistical Analysis The data are expressed as the mean ⫾ SEM. Statistical analysis was performed using the nonparametric KruskalWallis analysis of variance test, with P⬍.05 considered statistically significant. RESULTS Testicular Parameters of Spermatogenesis Table 1 compares the histologic changes in the ischemic testes and in the contralateral testes among the four experimental groups. As expected, the sham-operated animals (group A) demonstrated a normal architecture of the seminiferous tubules and interstitium in both testes and had intact germinal epithelium with an average thickness of about four to five cell layers. Treatment with dexamethasone (group B) did not significantly change the histologic manifestations of spermatogenesis compared with sham animals (group A). Ninety minutes of testicular ischemia led to severe damage in the ischemic testis. Impaired spermatogenesis in IR rats (group C) was manifested by a threefold decrease in germ cell layer count (P⬍.05) and a threefold decrease in the
mean testicular score (Johnsen criteria; P⬍.05) compared with sham animals (group A). Treatment with dexamethasone did not significantly change testicular parameters of spermatogenesis. IR-dex rats (group D) demonstrated a mean score and germ cell layer count similar to that of IR rats (group C) and demonstrated lower values with compared with sham (group A) and sham-dex (group B) rats. Evaluation of spermatogenesis in the contralateral testis 96 hours after IR did not show marked histologic changes. The IR (group C) rats showed a slight decrease in the number of germ cell layers compared with sham animals (group A); however, this trend was not statistically significant. Administration of dexamethasone (groups B and D) did not affect testicular parameters of spermatogenesis compared with sham (group A) and IR rats (group C). Assessment of Germ Cell Apoptosis Apoptosis was characterized by a TUNEL technique that specifically detects apoptotic germ cells in the testis. As shown in Figures 1 and 2, dexamethasone in sham rats (group B) did not significantly change germ cell apoptosis in both testes compared with the case of sham animals (group A). Figure 1 demonstrates changes in germ cell apoptosis in the ischemic testis after testicular IR and administration of dexamethasone. As expected, testicular IR (group C) resulted in a significant increase in germ cell apoptosis in the ischemic testis, expressed as the number of apoptotic cells per 100 tubules (33.4 ⫾ 8.5 vs. 1.1 ⫾ 0.7 TUNEL-positive cells per 100 tubules; P⬍.05) and the number of positive tubules (11.3 ⫾ 4.8 vs. 0.5 ⫾ 0.3 TUNEL-positive tubules per 100 tubules; P⬍.05) compared with sham animals (group A). Treatment with dexamethasone (group D) did not change significantly germ cell apoptosis in the ischemic testis compared with the case of IR-untreated animals (group C). The majority of the tubules were severely damaged in
TABLE 1 Effect of dexamethasone on spermatogenesis in ipsilateral and contralateral tests after testicular IR in rat. Ischemic testis
Contralateral testis
Experimental groups
Johnsen’s criteria
Number of layers
Johnsen’s criteria
Number of layers
Sham Sham-dex IR IR-dex
7.6 ⫾ 0.9 8.7 ⫾ 0.2 2.8 ⫾ 1a,b 3.3 ⫾ 0.8a,b
3.7 ⫾ 4.3 4.3 ⫾ 0.3 1.3 ⫾ 0.6a,b 1.5 ⫾ 0.5a,b
9.1 ⫾ 0.1 9.3 ⫾ 0.3 9.2 ⫾ 0.3 9.2 ⫾ 0.3
4.5 ⫾ 0.1 4.8 ⫾ 0.2 4.2 ⫾ 0.2 4.4 ⫾ 0.2
Note: Values are mean ⫾ SEM. a P⬍.05 vs. sham rats. b P⬍.05 vs. sham-dex rats. Mogilner. Dexamethasone and germ cell apoptosis. Fertil Steril 2006.
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FIGURE 1 The effect of testicular ischemia-reperfusion (IR) and administration of dexamethasone on germ cell apoptosis (apoptotic cells per 100 tubules and positive tubules per 100 tubules) in the ischemic testis. Values are mean ⫾ SEM. Bars indicate experimental groups: , sham; , sham-dex; , IR; , IR-dex. *P⬍.05 vs. sham-operated animals.
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IR-dex rats and displayed a number of apoptotic cells that was significantly greater than that in sham (group A) and sham-dex (group B) animals.
physiological mechanisms. The anti-inflammatory efficacy of corticoids is explained by their inhibition of the synthesis of numerous cytokines, enzymes and mediators of inflammation. Glucocorticoid hormones are also regulators of cell growth and differentiation. In particular, glucocorticosteroids have been shown to be potent antiproliferative agents in many organ and cell types (17) and also are known to induce gastrointestinal cell arrest and programmed death of several tumor cell lines (18). The understanding of testicular physiology and pathology requires knowledge of the regulation of cell proliferation and programmed cell death. Extensive studies in various experimental models have established that apoptosis control in the testis is crucial for the evolution of normal spermatogenesis (6 – 8). The dynamic process of germ cell turnover is regulated by several hormones: these include T, LH, and FSH. Recent evidence suggests that exogenously administered corticosteroids induce apoptosis in rat Leydig cells in the interstitium (19). Several investigators consider this pathway to be the main mechanism responsible for the decrease in serum T levels that is seen in animals that have been administered glucocorticoids (20). In a recent experiment, Yazawa and coworkers (21) have shown that dexamethasone induces significant testicular germ cell apoptosis in rats. Little is known about the effect of glucocorticoids on germ cell proliferation and apoptosis after testicular IR. Extensive studies in various experimental models have established a beneficial effect of glucocorticoids in IR events
Figures 2 and 3 summarize the data on germ cell apoptosis in the contralateral testis. Consistent with the result of our first experiment (13), testicular ischemia (group C) resulted in a significant increase in germ cell apoptosis in the contralateral testis. The IR rats demonstrated a significant increase in the number of apoptotic cells per 100 tubules (6.6 ⫾ 1.3 vs. 2.2 ⫾ 1.1 TUNEL-positive cells per 100 tubules; P⬍.05) and in the number of positive tubules per 100 tubules (4.7 ⫾ 1.0 vs. 1.3 ⫾ 0.7 TUNEL-positive tubules per 100 tubules; P⬍.05) compared with sham animals. Treatment with dexamethasone (group D) resulted in a significant decrease (vs. IR rats, group C) in the number of positive tubules per 100 tubules (2.0 ⫾ 0.7 vs. 4.7 ⫾ 1.0 TUNELpositive tubules per 100 tubules; P⬍.05) and a trend toward a decrease in the number of apoptotic cells per 100 tubules (3.8 ⫾ 1.9 vs. 6.6 ⫾ 1.3 TUNEL-positive cells per 100 tubules; P⫽.14); however, this trend did not achieve statistical significance. DISCUSSION Glucocorticosteroids are 12-carbon molecules derived from cholesterol and are produced by the adrenals in response to adrenocorticotropin hormone, which in turn is secreted by the anterior pituitary in response to corticotropin-releasing factor. Glucocorticoids have far-ranging effects on almost every tissue in the body and play an important role in the regulation of a large number of immune and metabolic 1114
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Dexamethasone and germ cell apoptosis
FIGURE 2 The effect of testicular ischemia-reperfusion (IR) and administration of dexamethasone on germ cell apoptosis (apoptotic cells per 100 tubules and positive tubules per 100 tubules) in the contralateral testis. Values are mean ⫾ SEM. Bars indicate experimental groups: , sham; , sham-dex; , IR; , IR-dex. *P⬍.05 vs. sham-operated animals, † IR-dex vs. IR rats.
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FIGURE 3 Immunohistochemistry for apoptosis in the contralateral testis in the four experimental groups. Single apoptotic cells (arrows) are present in sections from sham-treated (A) and sham-dex–treated rats (B). Labeled cells frequently are found in IR (C) rats. The number of apoptotic cells decreases after administration of dexamethasone (D).
Mogilner. Dexamethasone and germ cell apoptosis. Fertil Steril 2006.
in many organs and tissues. Dexamethasone pretreatment significantly reduces the occurrence of reperfusion-induced arrhythmias and improves cardiac recovery after myocardial IR (22). Dexamethasone decreases lung and systemic effects of intestinal IR in a rat (23). Dexamethasone combined with mannitol has beneficial effects in preventing brain IR injury in a rat (24). Methylprednisolone has a beneficial effect against IR injury in cremaster muscle flaps in rats (25). The main mechanisms of the positive effects of glucocorticoids remain unclear. It is widely accepted that glucocorticoid hormones decrease reperfusion injury by suppressing the production of oxygen-derived free radicals from polymorphonuclear neutrophils (14); by reducing the tissue levels of several proinflammatory mediators (26); by attenuation of neutrophil infiltration and intercelFertility and Sterility姞
lular adhesion molecule-1 (27); and by reducing the synthesis of eicosanoids, edema formation, and leukocyte infiltration (24). Few experimental studies have examined the effect of glucocorticoid hormones in testicular IR. In a recent experiment, Yazawa and coworkers (14) examined the effect of dexamethasone on germ cell apoptosis in the ischemic testis after testicular IR. Those investigators showed that intravenous administration of dexamethasone inhibits vascular neutrophil adhesion and decreases testicular germ-cell apoptosis in the ischemic testis. The current study was an extension of our previous work in which the effect of testicular IR on germ cell apoptosis in the contralateral testis was examined 24 hours after an IR 1115
event (13). The present study was conducted to evaluate whether germ cell apoptosis in the contralateral testis remains increased 96 hours after IR and whether administration of dexamethasone could affect germ cell apoptosis in both the ischemic and contralateral testis. Our data demonstrate that testicular ischemia induces degeneration of germ cells and impairs spermatogenesis. The average number of germinal epithelial cell layers and Johnsen’s criteria decreased significantly in the ischemic testis compared with the case of sham-operated animals. Administration of dexamethasone did not significantly affect spermatogenesis in control rats. In addition, treatment with dexamethasone did not prevent damage caused by testicular IR in the ischemic testis and did not improve the recovery of testicular tissue from the ischemic injury. Groups C (IR rats) and D (IR-dex rats) had similar histologic appearances. The number of germinal epithelial cell layers and Johnsen’s criteria were not statistically significantly different. Our data suggest that testicular IR with or without the administration of dexamethasone does not produce contralateral testicular histologic damage. All experimental groups showed normal histologic architecture of the seminiferous tubules in the contralateral testis. There was a tendency toward a decrease in the number of germ cell layers in the contralateral testis after IR compared with in sham animals; this change was not statistically significant; however, a trend was observed and may be clinically important; therefore, it was included in the Discussion section. These data fit with reports of other investigators who also have noted no contralateral damage after ipsilateral experimental torsion (3, 11). The finding of normal seminiferous tubule architecture with decreased number of germ cell layers may suggest that spermatogenesis is affected without direct damaging effect of IR. The measurement of apoptosis demonstrated an increase in germ cell programmed death after testicular IR. The present data are consistent with the results of other investigators who demonstrated that loss of germ cells after IR is from germ cell–specific apoptosis (3, 11, 14). Stimulation of germ cell apoptosis in the ischemic testis mainly is attributed to increased leukocyte adhesions (14) and production of reactive oxygen species (3, 11). We demonstrated in this experiment that germ cell apoptosis in the contralateral testis was increased after IR. We previously have reported increases in germ cell apoptosis in the contralateral testis 24 hours after IR (13). These data indicate that 96 hours after IR, germ cell apoptosis in the contralateral testis remains significantly increased. In addition to ischemia-induced cell death in the ischemic testis, increased germ cell apoptosis in the contralateral testis may lead to significant decrease in the germ cell mass and may cause loss of fertility. Despite the increased loss of germ cells, the majority of seminiferous tubules maintain normal architecture. It may be reasonable to presume that histolog1116
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ical manifestation of impaired spermatogenesis will appear after a long period of enhanced programmed cell death. Our experiment demonstrates that treatment with dexamethasone did not significantly change germ cell apoptosis in the ischemic testis. These results conflict with the report of Yazawa and colleagues (14), who emphasize evidence of decreased germ cell apoptosis in the ischemic testis after dexamethasone administration. However, the measurement of programmed germ cell death in the contralateral testis in our study demonstrates beneficial effects of dexamethasone on spermatogenesis through decreases in cell apoptosis. This is evident from the decrease in the number of apoptotic cells compared with the case of animals not treated with IR. Further experiments are needed to clarify the mechanisms of the effect of dexamethasone on germ cell apoptosis in the contralateral testis after IR as well as to determine whether a longer time interval of treatment causes more significant changes in the cell apoptosis and seminiferous tubule architecture. In conclusion, the present data are consistent with the interpretation that testicular ischemia results in fertility disturbances, including spermatogenesis arrest and increase in germ cell death. The use of glucocorticoids decreases germ cell apoptosis in the contralateral testis. Extrapolation of these data to the human situation is not appropriate; however, this information does provide a stimulus for true clinical investigations. REFERENCES 1. Bartsch G, Frank S, Marberger H, Mikuz G. Testicular torsion: late results with special regard to fertility and endocrine function. J Urol 1980;124:375– 8. 2. Harrison RG, Lewis-Jones DI, Moreno de Marval MJ, Connolly RC. Mechanism of damage to the contralateral testis in rats with an ischemic testis. Lancet 1981;2:723–5. 3. Lysiak JJ, Nguyen QA, Turner TT. Peptide and nonpeptide reactive oxygen scavengers provide partial rescue of the testis after torsion. J Androl 2002;23:400 –9. 4. Sarica K, Kupeli B, Budak M, Kosar A, Kavukcu M, Durak I, et al. Influence of experimental spermatic cord torsion on the contralateral testis in rats. Evaluation of tissue free oxygen radical scavenger enzyme levels. Urol Int 1997;58:208 –12. 5. Shiraishi K, Naito K, Yoshida K. Nitric oxide promotes germ cell necrosis in the delayed phase after experimental testicular torsion of rat. Biol Reprod 2001;65:514 –21. 6. Modi DN, Sane S, Bhartiya D. Accelerated germ cell apoptosis in sex chromosome aneuploid fetal human gonads. Mol Hum Reprod 2003; 9:219 –25. 7. Bartke A. Apoptosis of male germ cells, a generalized or cell typespecific phenomenon. Endocrinology 1995;136:3– 4. 8. Kimura M, Itoh N, Takagi S, Sasao T, Takahashi A, Masumori N, et al. Balance of apoptosis and proliferation of germ cells related to spermatogenesis in aged men. J Androl 2003;24:185–91. 9. Tomomasa H, Adachi Y, Oshio S, Umeda T, Irie H, Ishikawa H. Germ cell apoptosis in undescended testis: the origin of its impaired spermatogenesis in the TS inbred rat. Urology 2002;168:343–7. 10. Barqawi A, Caruso A, Meacham RB. Experimental varicocele induces testicular germ cell apoptosis in the rat. J Urol 2004;171:501–3. 11. Turner TT, Tung KS, Tomomasa H, Wilson LW. Acute testicular ischemia results in germ cell-specific apoptosis in the rat. Biol Reprod 1997;57:1267–74. 12. Oh SJ, Kwak C, Baek M, Kim CS, Kim KS, Choi H. Histologic and
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21. Yazawa H, Sasagawa I, Nakada T. Apoptosis of testicular germ cells induced by exogenous glucocorticoid in rats. Hum Reprod 2000;15: 1917–20. 22. Varga E, Nagy N, Lazar J, Czifra G, Bak I, Biro T, et al. Inhibition of ischemia/reperfusion-induced damage by dexamethasone in isolated working rat hearts: the role of cytochrome c release. Life Sci 2004;75: 2411–23. 23. Cavriani G, Oliveira-Filho RM, Trezena AG, da Silva ZL, Domingos HV, de Arruda MJ, et al. Lung microvascular permeability and neutrophil recruitment are differently regulated by nitric oxide in a rat model of intestinal ischemia-reperfusion. Eur J Pharmacol 2004;494: 241–9. 24. Wang Z, Xu X, Wu X. Therapeutic effect of dexamethasone and mannitol on global brain ischemia-reperfusion injury in rats. Beijing Da Xue Xue Bao 2003;18:303– 6. 25. Askar I, Bozkurt M. Protective effects of immunosuppressants and steroids against ischemia-reperfusion injury in cremaster muscle flap at microcirculatory level. Microsurgery 2002;22:361– 6. 26. Willemart G, Knight KR, Ayad M, Wagh M, Morrison WA. The beneficial antiinflammatory effect of dexamethasone administration prior to reperfusion on the viability of cold-stored skin flaps. Int J Tissue React 1999;21:71– 8. 27. Wheller SK, Perretti M. Dexamethasone inhibits cytokine-induced intracellular adhesions molecule-1 up-regulation on endothelial cell lines. Eur J Pharmacol 1997;331:65–71.
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