Sir,

5-Fluorouracil (5-FU) is extensively used to treat tumours of head and neck, breast and gastrointestinal system. Despite its clinical efficacy it is cytotoxic and induces the cell death (1), and generates DNA strand breaks in mammalian cells (2). It was previously reported that 5-FU affects the cytoarchitecture of spermatids (3). Recently, we have reported that 5-FU induces the formation of abnormal germ cells, epithelial sloughing (4), decrease in testis weight and tubular atrophy (5). However, the mechanism of formation, morphology and fate of abnormal germ cells have not been previously described in 5-FU treated rats.

In the present study, 5 male Wistar rats (11 weeks old; 130-150 g body weight) were housed in plastic cages with paddy husk bedding. They were maintained under controlled temperature and humidity. Rats were treated with the single dose of 100 mg/kg (i.p.; Fluracil, Biochem, Mumbai), as this dose level is known to induce the abnormal cells after 72 h post exposure (4). At 72 h, the testes and epididymes were dissected and prepared for light microscopical studies according to the procedure explained in our previous reports (4, 5). Paraffin sections were stained with H & E and periodic acid-Schiffs’s reaction–haematoxylin (PAS-H) (6). These sections were screened to evaluate the percentage incidence of tubules with abnormal cells and number of nuclei/multinucleated cell (MNC). Diameters of 10 large and 10 small sized MNCs were measured by an ocular micrometer calibrated with the stage micrometer. The processes of MNC formation in the testis and its degeneration in the testis as well as in the epididymis were studied.

Symplasts were found in the seminiferous epithelium, lumina of tubules and in the epididymis. Epithelial sloughing showed a positive correlation with the number of MNCs. Some MNCs showed 2 or 3 nuclei, representing the ongoing process of cell union in the epithelium. The MNCs were found in 39% of tubules. In 3 testis examined from 5 animals, the MNCs were observed. Incidence of MNCs ranged from 1-14/tubule and the mean nuclei/MNCs was a 7.0 ± 2.83 (SD) with a range of 3-11. Mean diameters of small a large sized MNCs were 53.2 ± 9.84 (SD) (range 13.3-66.5) and 85.12 ± 5.25 (SD) (range 79.8-156.0) m respectively. Nuclei of these MNCs resembled the spermatid nuclei, but with the chromatin margination. Between the nuclei no membrane residues were observed, however a reticulum of cytoplasm mass was present. Some cells showed empty spaces. In the testis, the MNCs showed degeneration (Fig. 1) which continued even in the epididymis. In the epididymis the symplasts and other sloughed cells showed altered morphology. All the nuclei of MNCs joined together to form a single large basophilic mass (Fig. 2) and in other exfoliated germ cells nuclei showed the degeneration.

Fig.1 click for full view

Fig.2 click for full view

5-FU imparts its cytotoxicity by incorporating into RNA or DNA and inhibiting the activity of thymidylate synthetase (7). It arrests the cell cycle, induces the abnormal germ cell development and malorientation of spermatids in the rat testis (3, 4) and epithelial sloughing (8). Epithelial sloughing is mainly due to the Sertoli cell damage and interruption of intercellular bridges. Formation of MNCs in 5-FU treated rats therefore indicates its damaging effect on the Sertoli cells and intercellular bridges. Cells with 3-11 nuclei also indicates that sloughed germ cells especially the spermatids join together to form the MNCs. Chromatin margination in these MNCs either in the testis or epididymis is an indicator of cell degeneration. The MNCs, consequently reached the epididymis and the nuclei of these cells gradually disappeared due to degeneration. These cells could further migrate into the rest of the reproductive tract, or in due course could be phagocytosed either in the epididymis or elsewhere. These degenerating cells were PAS positive bodies with well defined outline. We conclude that 5-FU affects the Sertoli cells and intercellular bridges in the rat testis. This study also concludes, that the MNCs were formed due to the cell union, and they degenerate in the testis and epididmis.

REFERENCS

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