Late morfofunctional alterations of the Sertoli cell caused by doxorubicin administered to prepubertal rats

Abstract

Background: Doxorubicin is a potent chemotherapeutic drug used against a variety of cancers. It acts through interaction with polymerases and topoisomerase II and free radical production. Doxorubicin activity is not specific to cancer cells and can also damage healthy cells, especially those undergoing rapid proliferation, such as spermatogonia. In previous studies our group showed that etoposide, another topoisomarese II poison, causes irreversible damage to Sertoli cells. Thus, the aim of this study was to address the effects of doxorubicin on Sertoli cell morphology and function and on the seminiferous epithelium cycle when administered to prepubertal rats.

Methods: Prepubertal rats received the dose of 5 mg/Kg of doxorubicin, which was fractioned in two doses: 3 mg/Kg at 15dpp and 2 mg/Kg at 22 dpp. The testes were collected at 40, 64 and 127 dpp, fixed in Bouin's liquid and submitted to transferrin immunolabeling for Sertoli cell function analysis. Sertoli cell morphology and the frequency of the stages of the seminiferous epithelium cycle were analyzed in PAS + H-stained sections.

Results: The rats treated with doxorubicin showed reduction of transferrin labeling in the seminiferous epithelium at 40 and 64 dpp, suggesting that Sertoli cell function is altered in these rats. All doxorubicin-treated rats showed sloughing and morphological alterations of Sertoli cells. The frequency of the stages of the seminiferous epithelium cycle was also affected in all doxorubicin-treated rats.

Conclusions and discussion: These data show that doxorubicin administration during prepuberty causes functional and morphological late damage to Sertoli cells; such damage is secondary to the germ cell primary injury and contributed to enhance the spermatogenic harm caused by this drug. However, additional studies are required to clarify if there is also a direct effect of doxorubicin on Sertoli cells producing a primary damage on these cells.

Figure 1

Testicular cross-sections of 40 day-old control (1A) and doxorubicin-treated (1B-1D) rats submitted to PAS + H histochemical method. The Figure  1A shows seminiferous epithelium presenting normal morphology. The nuclei of Sertoli cells show evident nucleoli and are located close to the basal membrane of the seminiferous epithelium (inset; arrowheads). In the Figure  1B, a tubular section showing germ cell depletion is observed. Figure  1C depicts a detached portion of seminiferous epithelium in which Sertoli cell nuclei are observed (arrows). The Figure  1D shows Sertoli cell nuclei into the tubular lumen (arrows).

Figure 2

Testicular cross-sections of 64 day-old control (Figure2A) and doxorubicin-treated (Figures2B-D) rats submitted to the PAS + H method. The Figure  2A depicts part of a tubular section containing Sertoli cell nuclei with normal morphology. These nuclei are located close to the basal membrane (arrows) of the seminiferous epithelium and show evident nucleolus (inset; arrowhead). In the Figure  2B, a Sertoli cell only tubular section is observed. In this tubular section, one of the Sertoli cell nuclei is far from the basal membrane (arrowhead) and another is sloughed into the tubular lumen (arrow). The Figure  2C depicts a sloughed portion of seminiferous epithelium (arrowheads) into the tubular lumen in which a Sertoli cell nucleus can be seen (arrow). In the inset, this Sertoli cell nucleus with irregular profile shows abnormal clear areas (arrow). Figure  2D shows a tubular section in stage IX of the seminiferous epithelium cycle with retention of step 19 spermatids (arrow). Note the presence of intraepithelial spaces and free primary spermatocytes associated.

Figure 3

Testicular cross-sections of 127 day-old control (Figure3A) and doxorubicin-treated (Figures3B-3D) rats submitted to the PAS + H method. The Figure  3A depicts seminiferous tubule sections showing normal morphology. The Sertoli cells show normal nuclei (inset; arrowheads). In the Figure  3B, tubular sections with severe germ cell depletion and intraepithelial vacuoles (thin arrows) are seen. A displaced Sertoli cell nucleus from the basal membrane is also observed (thick arrow). Some Sertoli cell nuclei show abnormal profile (arrowheads). The Figure  3C shows Sertoli cell nuclei with abnormal morphology, showing round shape (thick arrows); in one of them the nucleolus is not evident. A binucleated formation of round spermatids can also be noted into the lumen (thin arrow). The Figure  3D depicts a portion of a seminiferous tubule showing a clear area without germ cells (arrows) and many primary spermatocytes in the tubular lumen (asterisk).

Figure 4

Testicular cross-sections of 40 day-old control (Figure4A) and doxorubicin-treated (Figure4B-C) rats submitted to transferrin labeling. The Figure  4A shows weak transferrin labeling in the Sertoli cell cytoplasm (arrows) of the control rat and strongly transferrin-positive germ cells, probably spermatogonia, which are located in the basis of the seminiferous epithelium (arrowheads). In the Figure  4B (doxorubicin-treated rat), although Sertoli cell cytoplasm is positive (arrows), no labeling is observed in the spermatogonia (arrowheads). The Figure  4C depicts a seminiferous tubule cross-section showing germ cell depletion and no transferrin labeling. Sertoli cell nucleus (arrow). Note the intense labeling in the interstitial tissue (Figures  4A-C; asterisks).

Figure 5

Testicular cross-sections of 64 day-old control (Figure5A) and doxorubicin-treated (Figure5B-C) rats submitted to transferrin labeling. In the Figure  5A (control rat), intense transferrin labeling is observed in the Sertoli cell cytoplasm (thin arrows) in both seminiferous tubule sections (stages II-III and XIV). Sertoli cell nuclei (thick arrows) and elongated spermatids (arrowhead) are not labeled. The Figure  5B (64 day-old doxorubicin-treated rat) shows very weak transferrin labeling in the Sertoli cell cytoplasm of tubules at stages II-III and VII (thick arrow); however, no labeling is observed in the Sertoli cell nucleus (thin arrow). The Figure  5C depicts a seminiferous tubule cross-section showing intense germ cell depletion in which no transferrin labeling is observed. Interstitial tissue immunolabeling is also noted (Figures  5A; asterisks).

Figure 6

Testicular cross-sections of 127 day-old control (Figure6A) and doxorubicin-treated (Figure6B-D) rats submitted to transferrin labeling. The Figures 6A-B show intense transferrin labeling in the seminiferous epithelium (stage VII). However, in the Figure  6A (control group), the labeling is more abundant than in the Figure  6B (doxorubicin-treated group). In the control group (Figure  6A), transferrin labeling is observed in the Sertoli cell cytoplasm (thick arrow) and nucleus (inset, thin arrow) as well as in the elongated spermatids (arrowhead). In the doxorubicin-treated group (Figure  6B) transferrin labeling is observed in the Sertoli cell cytoplasm (thick arrow) and in the elongated spermatids (arrowhead), but not in the Sertoli cell nucleus (inset, thin arrow). The Figure  6C shows two seminiferous tubule cross-sections containing large portions of sloughed seminiferous epithelium (stars). In one of them, transferrin positive Sertoli cell cytoplasm is observed (arrow). In the Figure  6D, a tubular section with intense germ cell depletion shows weak Sertoli cell cytoplasm immunolabeling (thin arrows) and no labeling in the Sertoli cell nucleus (arrowheads). Intraepithelial vacuole (thick arrow). Note the intense labeling in the interstitial tissue (Figure  6A-D; asterisks).

Figure 7

Volume density of transferrin-positive total testicular tissue (VvT) in the control and doxorubicin-treated rats. The VvT showed significant reduction in the 40dpp doxorubicin-treated rats (*) when compared with the corresponding control subgroup. No alteration of this parameter was observed in the 64 and 127dpp doxorubicin-treated rats.

Figure 8

Volume density of transferrin-positive seminiferous epithelium (VvES) in the control and doxorubicin-treated rats. A significant reduction of this parameter was observed in the doxorubicin-treated rats at 40 and 64dpp (*) when compared with the corresponding control subgroups. The 127dpp doxorubicin-treated rats showed an important recovery of the VvES.

Figure 9

Volume density of transferrin-positive interstitial tissue (VvTi) in the control and doxorubicin-treated rats. Only the 64dpp doxorubicin-treated rats showed an increase of the VvTi (*) when compared with the control subgroup. Although the 40dpp doxorubicin-treated rats have shown a smaller mean value of this parameter and the 127dpp rats have shown a higher mean value of VvTi, these data were not significant.

Figure 10

Frequency of the stages of the seminiferous epithelium cycle in the 40-day-old rats. An increase in the frequency of stages I, XI and XIV and a decrease of the frequency of stages II to VI were observed at this age. Statistically significant alterations are indicated by the asterisk (*).

Figure 11

Frequency of the stages of the seminiferous epithelium cycle in the 64-day-old rats. At this age, the frequency of the stages I and VIII decreased whereas stages II-III, VII and IX showed an increase of their frequencies. Statistically significant alterations are indicated by the asterisk (*).

Figure 12

Frequency of the stages of the seminiferous epithelium cycle in the 127-day-old rats. At this age, the stage VII showed an increase of its frequency and the stages XII-XII showed a decrease of its frequency. Statistically significant alterations are indicated by the asterisk (*).