Search

Purpose: The primary toxic effects of methotrexate (MTX) are myelosuppression and/or intestinal mucositis. The objective of the present study is to investigate the effect of MTX on germ cell apoptosis and spermatogenesis in a rat., Methods: Male Sprague-Dawley rats were divided into three experimental groups: control rats treated with vehicle; MTX-2 rats treated with one dose (20 μg/kg) of MTX given IP and killed on the second day; and MTX rats treated with IP MTX (20 μg/kg) and killed on day 4. Johnsen's criteria and the number of germinal cell layers in the testes were used to categorize the spermatogenesis. TUNEL assay was used to determine germ cell apoptosis. Western blotting was used to determine Bax and Bcl-2 protein levels. Statistical analysis was performed using the non-parametric Kruskal-Wallis ANOVA test, with p less than 0.05 considered statistically significant., Results: On day 2, MTX-treated animals demonstrated minimal changes in the histological parameters of spermatogenesis, but germ cell apoptosis increased significantly (threefold increase, p = 0.002) compared to control rats. On day 4, MTX-treated rats demonstrated a trend toward a decrease in germ cell apoptosis, compared to day 2, and showed histological signs of impaired spermatogenesis (decreased number of germ cell layers and Johnsen's criteria). A significant increase in cell apoptosis in MTX-treated rats was correlated with higher Bax/Bcl-2 protein levels., Conclusions: MTX induced germ cell apoptosis and impaired spermatogenesis in rat testes.

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.

Recent evidence suggests that enhanced cell apoptosis is responsible for germ cell loss following testicular ischemia-reperfusion (IR) injury. A nonsteroidal anti-inflammatory drug diclofenac sodium (Voltaren) is a prostaglandin-synthesis inhibitor, which is widely used in many testicular disorders. The purpose of the present study was to examine the effect of diclofenac (DIC) on germ cell apoptosis in the ischemic and contralateral testes following testicular IR in a rat. Forty rats were divided randomly into four experimental groups of ten rats each: group A (Sham)-Sham operated animals; group B (Sham-DIC)-Sham operated rats that were treated with DIC given subcutaneously at a dose of 10 mg/kg, once daily, 24, 48 and 72 h following operation; group C (IR) underwent 90 min of unilateral testicular IR; group D (IR-DIC)-rats underwent 90 min of unilateral testicular IR and were treated with DIC similarly to group B. Ninety-six hours following operation, the rats were sacrificed and testes were harvested. Johnsen's criteria and the number of germinal cell layers were used to categorize the spermatogenesis. TUNEL assay was used to determine germ cell apoptosis in both the ischemic and contralateral testes. Statistical analysis was performed using the non-parametric Kruskal-Wallis ANOVA test, with P less than 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. Germ cell apoptosis in both the ischemic and the contralateral testes increased significantly after IR. Treatment with DIC did not change histologic parameters of spermatogenesis in both the ischemic and contralateral testes, but decreased germ cell apoptosis in both testes following testicular IR. We conclude that testicular ischemia causes an increase in germ cell apoptosis in the contralateral testis. Diclofenac may be beneficial for spermatogenesis following testicular IR by decreasing germ cell apoptosis.

Unilateral testicular ischemia-reperfusion (IR) in the rat is followed by histologic damage in the contralateral testis, which has been previously explained on immunologic grounds. There is evidence to suggest that apoptosis in the contralateral testis is involved in germ cell loss following IR injury to the testis. We examined the time-dependent effect of testicular ischemia on germ cell apoptosis in the contralateral testis in a rat. Adult Sprague-Dawley rats weighing 250-280 g, were subjected to testicular ischemia for 1, 2, 3 or 24 h. Twenty-four hours following onset of the ischemic insult, testes were harvested for immunohistochemical studies. Apoptosis was detected using TUNEL immunohistochemical assay. Testicular ischemia in rats led to histological damage, which was related to the duration of the ischemia. In the contralateral testis, the minimal damage included a decrease in number of germ cell layers, mild disorganization, and single cell apoptosis. Apoptosis in the contralateral testes increased significantly after 2, 3, and 24 h of ischemia and showed direct, time-related correlation with the duration of ischemia. We conclude that testicular ischemia causes an increase in germ cell apoptosis in the contralateral testis. The extent of apoptosis increases with the duration of the ischemia.