Amifostine reduces the seminiferous epithelium damage in doxorubicin-treated prepubertal rats without improving the fertility status

Abstract

Background: Amifostine is an efficient cytoprotector against toxicity caused by some chemotherapeutic drugs. Doxorubicin, a potent anticancer anthracycline, is known to produce spermatogenic damage even in low doses. Although some studies have suggested that amifostine does not confer protection to doxorubicin-induced testicular damage, schedules and age of treatment have different approach depending on the protocol. Thus, we proposed to investigate the potential cytoprotective action of amifostine against the damage provoked by doxorubicin to prepubertal rat testes (30-day-old) by assessing some macro and microscopic morphometric parameters 15, 30 and 60 days after the treatment; for fertility evaluation, quantitative analyses of sperm parameters and reproductive competence in the adult phase were also carried out.

Methods: Thirty-day-old male rats were distributed into four groups: Doxorubicin (5 mg/kg), Amifostine (400 mg/kg), Amifostine/Doxorubicin (amifostine 15 minutes before doxorubicin) and Sham Control (0.9% saline solution). "Standard One Way Anova" parametric and "Anova on Ranks" non-parametric tests were applied according to the behavior of the obtained data; significant differences were considered when p < 0.05.

Results: The rats killed 30 and 60 days after doxorubicin treatment showed diminution of seminiferous epithelium height and reduction on the frequency of tubular sections containing at least one type of differentiated spermatogonia; reduction of sperm concentration and motility and an increase of sperm anomalous forms where observed in doxorubicin-treated animals. All these parameters were improved in the Amifostine/Doxorubicin group only when compared to Doxorubicin group. Such reduction, however, still remained below the values obtained from the Sham Control group. Nevertheless, the reproductive competence of doxorubicin-treated rats was not improved by amifostine pre-administration.

Conclusions: These results suggest that amifostine promotes a significant reduction of the doxorubicin long-term side effects on the seminiferous epithelium of prepubertal rats, which is reflected in the epidydimal fluid parameters in the adult phase. However, fertility status results suggest that such protection may not be effective against sperm DNA content damage. Further investigation of sperm DNA integrity must be carried out using amifostine and doxorubicin-treated experimental models.

Figure 1

Photomicrographs of testicular sections of Sham Control, Amifostine, Doxorubicin and Amifostine/Doxorubicin treated rats at different ages. PAS+H method. Portions of tubular sections of 45-day-old rats. A and B: Sham Control (A) and Amifostine-treated (B) groups showing organized seminiferous epithelium containing various cell types until round spermatids. Lymphatic space (LS); primary spermatocytes (arrowheads); round spermatids (long arrows). C and D: Doxorubicin-treated group. Note in C the cellular debris and the germinal lineage cells detached from the epithelium into the tubular lumen (short arrow); see also the discontinuous seminiferous epithelium (asterisks). In D, observe the vacuole formation (v), the primary spermatocytes (arrowheads) and the round spermatids (long arrows). E: Amifostine/Doxorubicin-treated group showing the organized seminiferous epithelium with normal morphology. Spermatogonia (arrows); primary spermatocytes (PS); round spermatids (arrowheads).

Figure 2

Photomicrographs of testicular sections of Sham Control, Amifostine, Doxorubicin and Amifostine/Doxorubicin treated rats at different ages. PAS+H method. Portions of sections of seminiferous tubules of 60-day-old rats. A and B: Sham Control (A) and Amifostine (B) groups. Elongated spermatids (arrows); interstitial tissue (IT); primary spermatocytes (arrowheads). C and D: Doxorubicin-treated group displaying damaged seminiferous epithelium. In C, an accentuated epithelial depletion and various elongated spermatids, sometimes abnormally located (arrowheads) can be observed; vacuole (asterisk); multinucleated formation in degeneration (thick arrow). In D, some spermatids showing outlined condensed and ring-shaped marginal chromatin are observed; this event suggests apoptosis occurrence (thin arrows). Inset: detail of round spermatids with ring-shaped marginal chromatin. E and F: Amifostine/Doxorubicin-treated group. Note the presence of morphologically normal round (thick arrows) and elongated (long arrows) spermatids, vacuole (asterisk) and Sertoli cell nucleus dislocated from the tubular periphery and with abnormal chromatin condensation (circle); primary spermatocytes (arrowheads); tubular lumen (TL).

Figure 3

Photomicrographs of testicular sections of Sham Control, Amifostine, Doxorubicin and Amifostine/Doxorubicin treated rats at different ages. PAS+H method. Portions of seminiferous tubule sections of 90-day-old rats. A and B: Sham Control (A) and Amifostine (B) groups showing normal organization of the seminiferous epithelium. C and D: Doxorubicin-treated group; in C, tubular sections with partial depletion of the seminiferous epithelium are noted; some areas of discontinuity in the germ cell layers can be observed (arrows); in D, a tubular section with accentuated cellular depletion and showing Sertolization is shown. E and F: Amifostine/Doxorubicin-treated group; in both figures, note the concentric and normally organized germ cell layers of the seminiferous epithelium; pachytene spermatocytes (thick arrows); Sertoli cell nucleus (S); round spermatids (arrowheads); elongated spermatids (long arrows); tubular lumen (TL).

Figure 4

Seminiferous tubule diameter morphometry in rats of the SC (Sham Control), A (Amifostine), D (Doxorubicin) and AD (Amifostine/Doxorubicin) groups at the 3 ages studied. Seminiferous tubule diameter (4A) and the growth rates (4B) observed in the different groups. 4C: Box plots illustrating the median of the seminiferous epithelium height. The median for each dataset is indicated by the line in the boxes which in themselves contain 50% of the data. The upper and lower hinges of the boxes indicate the 25-75 percentiles. Values within 1.5 times the interquartil range are indicated by the lines. Mean values +/- standard deviation; P < 0.05 (a, b, c, d, e, f). Labels - 4A: a = A and AD < SC and D; b = D < SC, A and AD; c = A and AD > D; d = A > AD; e = D > AD;f = AD < SC, A and D. 4C: a = A and AD > D; b = D < SC, A and AD; c = A > D and AD; d = D < SC, A and AD; e = AD < SC and A.

Figure 5

Graphics illustrating the sperm parameters and the fertility index of 90-day-old rats, obtained from the SC (Sham Control), A (Amifostine), D (Doxorubicin) and AD (Amifostine/Doxorubicin) groups. 5A: Box plots showing the median for sperm concentration in each group indicated by the line in the boxes which in themselves contain 50% of the data. The upper and lower hinges of the boxes indicate the 25-75 percentiles. Values within 1.5 times the interquartil range are indicated by the lines. 5B: Percentage of sperm that displayed any type of movement and normal morphology; 5C: Fertility index. Mean values +/- standard deviation; P < 0.05 (a and b). Labels - 5A: a = D < SC, A and AD; b = AD < SC and A. 5B: a = D < SC, A and AD; b = AD < SC and A. 5C: a = D < SC and A; b = AD < SC, A and D.