Six-week old male Sprague-Dawley rats weighing between 180 and 200 g were used in the experiments. All animals were housed in our special Trace Metal Animal Facilities at a temperature range of 24±1℃, relative humidity of 45±5% and a 12 hr light-cycle. Investigations were conducted in accordance with the Guide for Care and Use of Laboratory Animals (1996, National Academy of Science).

These male rats were randomly assigned to receive cadmium at doses of 0 (control), 1, 2, 4 or 8 mg/kg either alone (Group A) or after α-tocopherol treatment for 5 weeks (Group B). The numbers of rats included in Group A for final analysis were 10, 11, 8, 14 and 17 at 0, 1, 2, 4 and 8 mg/kg, respectively; those included in Group B were 8, 8, 8, 8 and 7. The corresponding cadmium dose was administered as a 0.5mL solution of cadmium chloride (Sigma C-3141, St. Louis, MO, U.S.A.) in 0.9 % normal saline by the intraperitoneal route using a 23-gauge syringe. In both groups, control rats (i.e., cadmium dose of 0 mg/kg) received normal saline only. In Group B, rats received 0.1 mL α-tocopherol (Sigma T-3251) daily for 5 days by the intraperitoneal route before administering normal saline alone or the corresponding cadmium dose one hour after the last dose of α-Tocopherol.

One week after normal saline (control) or cadmium injections, each rat was sacrificed by cervical dislocation, and the abdomen was incised to remove both testes. Each testis was then washed with normal saline to separate the surrounding fat and connective tissues. After drying the surface with filter paper, the dimensions and weight of the testis were recorded.

The left testicle of each rat was serially sectioned and fixed in Bouin solution for 48 hr. The specimen was then dehydrated through a graded series of alcohol and cleared in three changes of xylene before embedded in paraffin. Serial sections, each of 4 µm thickness, were made and stained with hematoxylin and eosin according to standard method. Histological assessment was performed under light microscopy in terms of seminiferous tubular diameter (STD: in every H&E section a minimum of 25 circular tubule were measured in two axes drawn perpendicular to each other) using an image analyzer (Image Proplus Version 3.0, Media Cybernetics, Silver Spring, MD, U.S.A.).

For flow cytometric analysis, testicular samples were prepared and stained with minor modification according to the method described by Zante et al. (18). The right testis of each rat was collected in Dulbecco's phosphate-buffered saline (PBS), minced and filtered through a 30 µm nylon filter to form a cellular suspension. After centrifugation at 500 g for 5 min, samples were fixed with 70% ethyl alcohol and kept at -20℃. Prior to flow cytometric analysis, the fixed cells were centrifuged at 500 g for 5 min. The cells were then resuspended (1×10⁶ cells/mL) in PBS containing propidium iodide (Sigma) and 100 µL of RNase (Sigma) and kept in the dark until DNA analysis. DNA analysis was performed on the FACScalibur (Becton Dickinson, San Jose, CA, U.S.A.) equipped with a 488 nm argon laser. During analysis, the flow rate was controlled at 500 cells/sec approximately, and for each sample, at least 10,000 events were recorded. The data were processed on a computer equipped with CELLQuest software (Version 3.1; Becton Dickinson). A typical DNA histogram of human adult testicular tissue is characterized by 4 peaks; 1) Peak I represents haploid mature spermatids with highly condensed chromatin that do not stain proportionally to their DNA content; 2) Peak II, represents haploid round spermatids with a 1c DNA content; 3) Peak III represents non-germ cells (Sertoli cells, Leydig cells and macrophages) and germ cells with a 2c DNA content (including G₁-spermatogonia, primary spermatocytes at preleptotene and secondary spermatocytes); and 4) Peak IV represents mainly primary spermatocytes with a small percentage of (G₂+M)-spermatogonia. It is between Peak III and IV that cells synthesizing DNA are registered. M1, M2, M3, M4, M5 and M6 cell development correspond to Peak I, Peak II, Peak III, between Peak III and Peak IV, Peak IV, and debris, respectively.

Within-group differences in testicular length, width and weight and the distribution of cells at the different stages (M1 to M6) on flow cytometry were compared by one-way analysis of variance (ANOVA). This was followed by post hoc pairwise comparison using the Bonferroni t-procedure if the overall results were significant. Statistical analysis was performed using the SPSS for Windows, Version 9.0 (Chicago, IL, U.S.A.) with statistical significance defined at a level of p<0.05.

Following treatment with cadmium alone, there was a progressive decrease in testicular length, width and weight with increasing cadmium dose beyond 1 mg/kg (p<0.01;Table 1). A similar dose-response relationship was noted in rats pretreated with α-Tocopherol. However, relative to control rats, a significant decrease in testicular weight was observed only at the higher cadmium doses of 4 and 8 mg/kg (Table 1).

At the cadmium dose of 1 mg/kg, there were no marked changes in testicular histology relative to controls irrespective of α-tocopherol treatment. Thus, normal spermatogen esis, well-preserved Sertoli cells and well-delineated tubular basement membrane were observed in both Group A and Group B. The interstitium between tubules and Leydig cells were also intact without any inflammatory cell infiltrates (Fig. 1A, B). However, at the cadmium dose of 2 mg/kg, differences in testicular histology were noted between the two groups. In rats treated with cadmium alone (Group A), there were heavy infiltrates of acute and chronic inflammatory cells in the relatively well-preserved interstitium between seminiferous tubules. Although the tubular basement membranes of seminiferous tubules were identified, tubules could exhibit focal or diffuse extensive necrosis (Fig. 1C). A few viable cells were identified in some tubules mostly composed of immature forms of germ cells and Sertoli cells. In contrast, prior α-tocopherol treatment (Group B) reduced the degree and number of necrotic seminiferous tubules (Fig. 1D). However, most of germ cells were degenerated, especially the ones involving highly differentiated germ cells (Fig. 1D inset). At the cadmium doses of 4 mg/kg, there was diffuse and marked tubular necrosis in which α-tocopherol provided no protective effects. In both groups, all the spermatogenic cells were dead. Almost all the ground substance within the interstitium disappeared and replaced by fibroblasts and inflammatory cells (Fig. 2). These features were identical in rats receiving the cadmium dose of 8 mg/kg irrespective of α-tocopherol treatment.

In flow cytometric analysis, controls (i.e., no cadmium) showed the typical 4-peak DNA dispersion observed in the normal adult testis (Fig. 3A). The cells were distributed as follows: 88.8% M1+M2 (1c: haploid spermatid), 6.1% M3 (2c: contains diploid germ cells), 0.6% M4, 3.6% M5 (including tetaploid germ cells) and 0.9% M6 (Table 2). At a cadmium dose of 1 mg/kg (Fig. 1B), the distribution was relatively unchanged (e.g., 86.1% M1+M2). However, at a cadmium dose of 2 mg/kg, M1+M2 cells decreased to 62.9% while M3 and M4 increased to 30.2% and 2.2% respectively (Fig. 3Cand Table 2). These were significantly different from control values (p<0.01).

As for Group A, the distribution in controls (i.e., no cadmium) pretreated with α-tocopherol for 5 days showed the typical 4-peak DNA dispersion observed in the normal adult testis (Fig. 3D) with 81.8% M1+M2; 10.4% M3; 1.3% M4; and 4.6% M5. Similarly, the cell distribution at a cadmium dose of 1 mg/kg (Fig. 3E) was not significantly different from that of controls. In contrast to Group A, the distribution of M1+M2, M3 and M5 at a cadmium dose of 2 mg/kg remained unchanged with prior α-tocopherol treatment (Fig. 3F and Table 2).