Effect of nitrofurazone on the reproductive organs in  adult  male mice

Shio Kumar Singh, Sumana Chakravarty

Department of Zoology, Banaras Hindu University, Varanasi, India

Asian J Androl  2001 Mar; 3:  39-44

Keywords: nitrofurazone; testis; epididymis; seminal vesicles; sperm; fructose; sialic acids

Abstract

Aim: To study the effect of 5-nitro-2-furaldehyde semicarbazone (nitrofurazone), a derivative of nitrofuran, on male reproductive organs of Parkes (P) strain mice. Methods: Mice were given nitrofurazone orally at a dose of 64 mg/kg body weight per day, for 10 and 20 days, and were killed 24 h and/or 56 days after the last treatment. Histological appearance of testis, motility and number of spermatozoa in cauda epididymidis, and biochemical indices in epididymis and seminal vesicle were evaluated. Results: Histologically, testis showed marked regressive changes in the seminiferous tubules in mice treated with nitrofurazone. Ten days after treatment, there was much depletion of germ cells in the seminiferous tubules, and the germinal epithelium was lined mainly with Sertoli cells, spermatogonia, spermatocytes, and a few round spermatids; intraepithelial vacuoles and multinucleated giant cells were also observed in tubules. By 20 days, regressive changes in the seminiferous tubules were further pronounced, and pachytene spermatocytes were the most advanced germ cells noticed in the tubules. In severe cases, the tubules were lined with a thin layer of Sertoli cells and spermatogonia. The treatment also caused marked reductions in the motility and number of spermatozoa in the cauda epididymidis, in weight and the level of fructose in the seminal vesicle, and in sialic acid level in the epididymis. Fifty six days after drug withdrawal, the alterations induced in the reproductive organs returned to control levels. Conclusion: Our results suggest that nitrofurazone treatment in P mice induces marked alterations in the male reproductive organs, and that the alterations are reversible following cessation of treatment.

1 Introduction

2 Materials and methods

Twenty-five adult (age: 12-14 weeks) male mice belonging to the Parkes (P) strain, weighing 30-38 g, were used in the experiments. The animals were housed under standard laboratory conditions and maintained on pelleted food (Lipton India Ltd) and water ad libitum. They were divided into five groups, each comprising 5 individuals. Each group was housed in separate polypropylene cages (450270150 mm) and treated as follows: 
Group I: Untreated controls;
Group II: Distilled water-treated controls;
Group III: Administration of nitrofurazone, 64 mg/kg body weight/day, for 10 days; animals were sacrificed 24 h after the last treatment;
Group IV: Administration of nitrofurazone, 64 mg/kg body weight/day, for 20 days; animals were sacrificed 24 h after the last treatment;
Group V: Administration of nitrofurazone, 64 mg/kg body weight/day, for 20 days; animals were sacrificed 56 days after the last treatment.

Nitrofurazone (Sigma Chemical Company, USA) was suspended in sterile distilled water, and the suspension was given orally by means of a stomach tube. The control Group II received an equivalent volume of distilled water (1.0 mL/100 g body weight) in a similar manner, and both the controls (Groups I and II) were sacrificed with animals in Group IV. Animals were sacrificed by dislocation of the cervical vertebrae, and the testes, epididymides, and seminal vesicles were dissected out and weighed. The testes were then fixed for histological studies in freshly prepared Bouin's fluid, dehydrated in a graded ethanol series, cleared in benzene, and embedded in paraffin wax. Tissues were sectioned at 6 m, and the sections were stained with periodic acid-Schiff (PAS) and counterstained with Harris haematoxylin.

At autopsy, spermatozoa were obtained from cauda epididymidis of each mouse in physiological saline maintained at 37, and their motility and number were assessed according to the WHO laboratory manual^[9].

The concentration of sialic acid in the epididymis was determined by Aminoff's^[10] thiobarbituric acid method, while that of fructose in the seminal vesicle was estimated according to the method of Lindner and Mann^[11].

3 Results

3.1 Body weight

No significant differences were found between the initial and final body weights of the nitrofurazone-treated animals and controls (Table 1).

Table 1. Effect of nitrofurazone administration (64 mg/kg body weight) on body.

¹Values are meanSD (n=5) and refer to the weight of the single organ.
²Treatment was discontinued after 20 days, and animals were sacrificed 56 days after drug withdrawal.
^bSignificantly different from controls (P<0.05); analyzed by ANOVA followed by Newman-Keul's multiple range test.

3.2 Organ weights

3.3 Testicular histology

The testes of untreated controls (Figure 1) and distilled water-treated controls exhibited normal histological features; the seminiferous tubules showed spermatogenic activity with successive stages of transformation of spermatogonia into spermatozoa. By contrast, marked regressive changes were observed in the seminiferous tubules in testes of nitrofurazone-treated mice (Figures 2 and 3). In testes of mice treated with nitrofurazone for 10 days (Group III), there was marked depletion of germ cells in the seminiferous tubules, and the germinal epithelium was lined mainly with Sertoli cells, spermatogonia, spermatocytes, and a few round spermatids (Figure 2). The tubules showed intraepithelial vacuoles and the occurrence of multinucleated giant cells; such giant cells contained 3-9 nuclei of germ cells arranged either at the periphery or scattered in the cytoplasm of the cell body (Figure 2). Giant cells formed with nuclei of early spermatids were more common than those with nuclei of spermatocytes. In testes of mice treated with nitrofurazone for 20 days (Group IV), regressive changes in the tubules were further pronounced (Figure 3). In many tubules, there was severe depletion of germ cells, and the epithelium consisted of a thin layer of Sertoli cells and spermatogonia. The most advanced germ cells noticed in the tubules were pachytene spermatocytes and these cells presented a necrotic appearance. However, by 56 days after drug withdrawal, testes showed histological features similar to those seen in the controls. 

Figure 1.  Testis of a control mouse (Group I). Note the normal appearance of the seminiferous tubules. (175)
Figure 2.  Testis of a mouse treated with nitrofurazone, 64 mg/kg body weight per day, for 10 days and sacrificed 24 h after the last treatment (Group III). Note marked depletion of germ cells in the seminiferous tubules; the germinal epithelium is lined mainly with Sertoli cells, spermatogonia, spermatocytes, and a few round spermatids. Intraepithelial vacuoles and multinucleated giant cells (arrows) can also be seen in the tubules. (175)
Figure 3.  Testis of a mouse treated with nitrofurazone, 64 mg/kg body weight per day, for 20 days and sacrificed 24 h after the last treatment (Group IV). Note further regressive changes in the seminiferous tubules. The most advanced germ cells in the tubules are pachytene spermatocytes, and these cells show a necrotic appearance. (175)

3.4 Sperm parameters

Nitrofurazone treatment caused significant reductions in both motility and number of spermatozoa in cauda epididymidis of the treated mice (Groups III and IV) as compared with controls; by 56 days after drug withdrawal, however, these parameters in treated mice returned to control levels (Table 2). 

Table 2.   Effect of nitrofurazone administration (64 mg/kg body weight) on motility and number of spermatozoa in the cauda epididymidis.

¹Values are meanSD (n=5).
²Treatment was discontinued after 20 days, and animals were sacrificed 56 days after drug withdrawal.
^bSignificantly different from controls (P<0.05); analyzed by ANOVA followed by Newman-Keul's multiple range test.

3.5 Chemical analyses

Table 3.    Effect of nitrofurazone administration (64 mg/kg body weight) on concentrations of sialic acid in the caput and cauda epididymides, and fructose in the seminal vesicle.

¹Values are meanSD (n=5).
²Treatment  was discontinued after 20 days, and animals were sacrificed 56 days after drug withdrawal.
^bSignificantly different from controls (P<0.05); analyzed by ANOVA followed by Newman-Keul's multiple range test.

4 Discussion

As in the rat^[2-6], nitrofurazone treatment in P mice caused regressive histological changes in the seminiferous tubules resulting in the suppression of spermatogenesis. Furthermore, the antispermatogenic effects induced by nitrofurazone in testes of P mice were reversible; this is consistent with the findings of Nelson and Steinberger^[12] and Paul et al^[13] in the rat after treatment with nitrofurazone. The present results showed that the most advanced germ cells noticed in the regressed seminiferous tubules were pachytene spermatocytes, though a few round spermatids were sometimes observed in the tubules in testes of mice treated with the drug for 10 days. Nelson and Steinberger^[14] and Paul et al^[13] have also reported the inhibition of spermatogenesis at the pachytene spermatocyte stage in rat testes following nitrofurazone treatment. Multinucleated giant cells as observed in the seminiferous tubules in testes of drug-treated mice in the present study are also reported in rat testis after nitrofurazone treatment^[3-5]. It is pertinent to note that such giant cells have also been reported in mouse  testes after efferent duct ligation^[15], vasectomy^[16], and treatment with several antispermatogenic agents^[17]; in these experimental conditions, the testis exhibited regressive changes in the seminiferous tubules. The occurrence of giant cells in the testis is considered to be an expression of germ cell degeneration. In P mice, nitrofurazone treatment for 20 days caused a marked reduction in the  level of sialic acid in the epididymis, while treatment for 10 days had no such effect. Since sialic acid is known to be a true secretory product of the epididymis^[18], the results suggest that treatment with nitrofurazone for 20 days has adverse effects on the secretory functions of the organ. The treatment also caused marked reductions in motility and number of spermatozoa in the cauda epididymidis. Albert et al^[19,20]  have also shown in in vitro studies in humans that nitrofurazone causes impairment of sperm motility. Furthermore, Nissim^[7] reported the enlargement of seminal vesicles in mice after treatment with nitrofurazone. In the present study, however, enlargement of seminal vesicles was not noticed in nitrofurazone-treated mice, and the treatment, on the other hand, caused marked reductions in the weight and the level of fructose in the gland. It is relevant to mention here that Nissim^[7] fed nitrofurazone at a concentration of 0.15%-0.3% in diet for 2-5 weeks, and each mouse received 5 g of diet daily. If one extrapolates the dose of nitrofurazone used by Nissim^[7] it turns out to be 290-580 mg/kg body weight/day. However, in the present study, nitrofurazone was given at a dosage of 64 mg/kg body weight/day, for 10 or 20 days. Thus, it is probable that the differences in the dose-regimen employed in the two studies may have some bearing on differences in observations of seminal vesicle as reported by Nissim^[7] and those in the present study.

The mechanism by which nitrofurazone causes its antispermatogenic effect is not properly understood. Studies of Uematsu^[4] suggest that nitrofurazone treatment causes suppression of spermatogenesis by acting directly on the seminiferous epithelium, and that this action is not mediated via the hypophysis. Further, Hagenas et al^[5] have shown that nitrofurazone acts directly on germ cells and thereby causes arrest of spermatogenesis. In the present study, intraepithelial vacuoles were noticed in the seminiferous tubules in testes of nitrofurazone-treated mice. It is pertinent to mention here that Hoffer^[21] has also reported occurrence of intraepithelial vacuoles in affected seminiferous tubules in rat testes after gossypol treatment, and that these vacuoles occurred primarily in the Sertoli cells. Such vacuoles are also reported to occur in the Sertoli cells after several kinds of testicular injuries, and they have, however, often been interpreted as a nonspecific reaction of these cells^[22]. Thus, it is difficult to say in P mice as to how nitrofurazone treatment induces antispermatogenic effect in the testes, though the possibility of a direct action of the drug on the germ cells can not be ruled out.

Acknowledgements

References

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Correspondence to: Dr. Shio Kumar Singh, Department of Zoology, Banaras Hindu University, Varanasi 221 005, India.
 Fax: +91-542-368 174
Received 2000-10-13     Accepted 2001-02-14