ABSTRACT
The objective of this study was to examine the relationship between time of reperfusion and bax/bcl-2-dependent germ cell apoptosis after testicular ischemia-reperfusion injury in the rat. In ischemic testis, bax/bcl-2 ratio did not change significantly, and the elevation of germ cell apoptosis was not marked; in the contralateral testis, germ cell apoptosis increased after 6 hours of reperfusion, achieved statistical significance after 24 hours, and decreased after 72 hours of reperfusion and was initiated by decreased bcl-2 messenger RNA levels and elevated bax/bcl-2 ratio within the first 6 hours of reperfusion.
Subject(s)
Apoptosis/physiology , Germ Cells/physiology , Ischemia/pathology , Proto-Oncogene Proteins c-bcl-2/physiology , Reperfusion , bcl-2-Associated X Protein/physiology , Animals , Apoptosis/genetics , Disease Models, Animal , Germ Cells/metabolism , Germ Cells/pathology , Ischemia/etiology , Ischemia/genetics , Ischemia/metabolism , Male , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Rats , Rats, Sprague-Dawley , Reperfusion/rehabilitation , Signal Transduction/genetics , Signal Transduction/physiology , Spermatic Cord Torsion/complications , Spermatic Cord Torsion/genetics , Spermatic Cord Torsion/metabolism , Spermatic Cord Torsion/pathology , Testis/blood supply , Testis/metabolism , Testis/pathology , Time Factors , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
OBJECTIVE: To examine the relationship between the time of reperfusion and neutrophil recruitment, E-selectin expression, and germ cell apoptosis in the ischemic and contralateral testis after testicular ischemia-reperfusion (IR) injury in a rat. DESIGN: Laboratory study. SETTING: Research laboratory in a faculty of medicine at Technion-institute of technology in Israel. ANIMAL(S): Sixty adult Sprague-Dawley rats weighing 250-280 g. INTERVENTION(S): Testicular IR. MAIN OUTCOME MEASURE(S): Testicular germ cell apoptosis was assessed by terminal deoxynucleotide transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end labeling immunohistochemical assay, using an in situ cell death detection kit. The recruitment of polymorphonuclear (PMN) cells was calculated per 100 venules. Expression of E-selectin was determined by using immunohistochemical analysis. RESULT(S): E-selectin expression and polymorphonuclear-cell recruitment in the contralateral testis increased significantly after 1 hour of reperfusion and then remained unchanged during the first 24-48 hours, followed by a gradual decrease. Germ cell apoptosis in the contralateral testis increased after 6 hours of reperfusion, achieved statistical significance after 24 hours, and decreased after 72 hours of reperfusion. CONCLUSION(S): Germ cell apoptosis in the contralateral testis increases most significantly within the first 24-48 hours, followed by a gradual decrease, after IR injury. E-selectin expression and neutrophil recruitment increases within the first 6 hours and apparently may initiate the increase in germ cell apoptosis.
Subject(s)
Apoptosis , E-Selectin/genetics , Germ Cells/physiology , Neutrophil Infiltration , Reperfusion Injury/physiopathology , Testicular Diseases/physiopathology , Animals , Disease Models, Animal , E-Selectin/metabolism , Gene Expression , Humans , Male , Random Allocation , Rats , Rats, Sprague-Dawley , Reperfusion , Reperfusion Injury/immunology , Reperfusion Injury/surgery , Testicular Diseases/immunology , Testicular Diseases/surgery , Testis/blood supply , Testis/immunology , Testis/metabolism , Testis/surgeryABSTRACT
Recent evidence suggests that apoptosis is involved in germ cell loss following testicular ischemia-reperfusion (IR) injury. Allopurinol (Allo) is as a free radical scavenger which prevents tissue damage caused by reperfusion and oxygenation after ischemia; however, its effect on apoptosis in this type of injury has not been studied. To examine the effect of allopurinol on germ cell apoptosis following testicular IR in a rat. Forty rats were divided randomly into 4 experimental groups of 10 rats each: group A (Sham)-Sham operated animals; group B (Sham-Allo)-Sham operated rats treated with allopurinol given PO (by gavage) at a dose of 200 mg/kg, once daily, immediately before and 24 h following operation; group C (IR)-rats underwent 90 min of unilateral testicular ischemia and 48 h of reperfusion; group D (IR-Allo)-rats underwent IR and were treated with allopurinol similar to group B. The ipsilateral and contralateral testes were harvested 48 h following operation. Johnsen's criteria and the number of germinal cell layers were used to categorize spermatogenesis. TUNEL assay was used to determine germ cell apoptosis. Statistical analysis was performed using one-way ANOVA test, with P < 0.05 considered statistically significant. Testicular ischemia in rats led to histological damage in the ipsilateral testis. In the contralateral testis minimal damage was observed. Treatment with allopurinol increased significantly Johnsen's score in both the ischemic (7.3 +/- 0.5 vs 5.6 +/- 0.5, P < 0.05) and contralateral (8.9 +/- 0.1 vs 8.3 +/- 0.2, P < 0.05) testis, compared to IR-animals. Germ cell apoptosis in both the ischemic and the contralateral testis increased significantly after IR. Treatment with allopurinol resulted in a significant decrease in germ cell apoptosis in the ipsilateral testis, expressed as the number of positive tubules per 100 tubules (AI-1, (apoptotic index) threefold decrease, P < 0.005) and the number of apoptotic cells per 100 tubules (AI-2, fivefold decrease, P < 0.005) as well as a significant decrease in germ cell apoptosis in the contralateral testis (AI-1, 3.5-fold decrease, P < 0.05, AI-2- sixfold decrease, P < 0.005) compared to IR animals. In a rat model of testicular IR, treatment with allopurinol decreases germ cell apoptosis in both ischemic and contralateral testes and improves spermatogenesis.
Subject(s)
Allopurinol/therapeutic use , Apoptosis/drug effects , Free Radical Scavengers/therapeutic use , Reperfusion Injury/drug therapy , Spermatozoa/pathology , Testis/blood supply , Animals , Disease Models, Animal , In Situ Nick-End Labeling , Male , Rats , Rats, Sprague-Dawley , Reperfusion Injury/pathology , Spermatogenesis/drug effects , Spermatozoa/drug effects , Treatment OutcomeABSTRACT
Recent evidence suggests that neutrophil recruitment may initiate germ cell apoptosis in the ischemic testis. The purpose of the present study was to examine the relationship between germ cell apoptosis and neutrophil recruitment in the contralateral testis following testicular ischemia-reperfusion (IR) injury in a rat. Adult male Sprague-Dawley rats were divided randomly into two experimental groups: Group A: Sham operated animals; Group B: IR rats underwent 90 min of unilateral testicular ischemia following by 96 h of reperfusion. The rats were sacrificed and testes were harvested. Johnsen's criteria and the number of germinal cell layers were measured to categorize the spermatogenesis. TUNEL assay was used to determine germ cell apoptosis in both the ischemic and contralateral testis. The recruitment of neutrophils was calculated per 100 venules. Expression of E-selectin was determined using immunohistochemical analysis. Statistical analysis was performed using Student's t test, with P less than 0.05 considered statistically significant. Germ cell apoptosis in both the ischemic and the contralateral testis increased significantly after IR. E-selectin expression was significantly greater in ischemic testis from IR rats compared to sham animals. The small increase in E-selectin expression and the concomitant increase in neutrophil recruitment in the contralateral testis of the IR rats (vs. sham animals) were not statistically significant. In conclusion, testicular ischemia causes an increase in germ cell apoptosis in the contralateral testis. Mechanisms other than neutrophil recruitment apparently initiate this process.
