Effect of tamoxifen on spermatogenesis and tubular morphology in rats

Keywords: tamoxifen; spermatozoa; seminiferous tubule; atrophy

Abstract

Aim: To observe the effect of tamoxifen citrate on spermatogenesis and tubular morphology in rats. Methods: The effect of tamoxifen citrate i.g. at doses of 400 and 800 mgkg^-1day^-1 in 0.1 mL olive oil for 30 days on seminiferous tubular morphology, seminiferous epithelial diameter (STD), epithelial height (SEH), epididymal sperm count and percent abnormal sperm were evaluated at day 1, 12 and 36 after treatment. Controls were given the vehicle. Results: The higher dose resulted in tubular atrophy on day 31. The STD, SEH and sperm count were decreased and the abnormal spermatozoa increased in a dose-dependent manner with the maximal effect on day 36. Conclusion: Tamoxifen citrate induces tubular shrinkage and atrophy and sperm abnormality at a dose-dependent manner.

1 Introduction

Tamoxifen citrate, an antiestrogen, has been used as a single prophylactic agent of choice in the treatment of hormone responsive malignancy of the mammary gland [1]. It interferes with the negative feedback of hypotha-lamo-pituitary-gonadal axis and increases the local testosterone production. The drug is also used in oligo-astheno-teratozoospermia patients to improve the semen quality and fertility [2]. Despite of its androgen boosting efficacy in the testis, it would cause disruption of seminiferous epithelium, induction of multinucleated cells and increase in the number of spermatozoa with abnormal morphology [3-5]. Tamoxifen was known to yield 4-hydroxytamoxifen during its metabolism, which is proved to be toxic to vital cells. Pagano et al [6] proved the reproductive and cytogenetic toxicity of tamoxifen in sea urchin embryos leading to early embryonic mortality and developmental defects as a result of oxidative stress. Further, it was also known to induce apoptotic and necrotic cell death in a concentration-dependent manner as evidenced by in vitro experiments [1]. Besides, recent reports on the necessity of estrogen and its alpha-receptor for fertility in male reproduction [7] question the prescription of triphenylethylene derivatives to induce spermatogenesis in oligozoospermic males. Although there are reports on the cytotoxic, genotoxic, carcinogenic and testotoxic effects of tamoxifen, in vivo studies highlighting the effect on seminiferous tubular morphology and spermatogenesis are scanty. Hence the aim of the present study was to examine the effect of tamoxifen citrate on spermatogenesis and seminiferous tubular morphology in rats.

2 Materials and methods

Male Wistar rats, 140-160 g body weight (11-12 weeks old), were maintained in polyprophylene cages with laboratory chew and water ad libitum. The animals were randomly divided into 2 experimental and 1 control groups of 15 rats each. The 2 experimental groups were gavaged tamoxifen citrate (Lyka Laboratoies, Mumbai, India) at doses of 400 or 800 mgkg^-1day^-1 in 0.1 mL olive oil and the controls with the vehicle for 30 days.

Five animals of each group were sampled on day 1, 12 or 36 after the last dosing. Rats were sacrificed by an overdose of pentobarbital sodium (45 mg/kg body weight ip, Sigma, USA) on the stipulated day of experiment, laparatomy was performed and the testis and epididymides were carefully removed and separated. Tunica vaginalis was carefully removed and the testes were fixed in fresh alcoholic Bouin's fluid for 8 hours, embedded in paraffin and 5 µm sections were cut and stained with haematoxylin and eosin. The testis histopathology was performed according to Sobarzo and Bustos-Obregon [8, 9] and Russell et al [10]. The tissue sections were observed under a light microscope for qualitative and quantitative changes in the seminiferous tubules. The seminiferous tubular diameter (STD) and seminiferous epithelial height (SEH) of 50 cross sections per animal were measured, including the peritubular cells and basement membrane, by ocular micrometer calibrated with stage micrometer (Erma opiticals, Japan) [11].

The epididymis was minced in 1 mL phosphate buffered saline (PBS, pH 7.2) and filtered through double layer of muslin cloth. The filtrate was mixed with 1 % aqueous eosin Y (10:1) and kept for 30 min for staining. An aliquot of the epididymal sperm suspension was used for spermatozoa count using Neubauer hemocytometer [12]. Smears were prepared on glass slides and observed under a microscope (1 000 for the head and tail abnormalities (1 000 spermatozoa per animal) [13].

Data were expressed in meanSEM. Statistical analysis was done by One-way ANOVA, post hoc Bonferroni test and P<0.05 was considered significant.

3 Results

The general conditions of the animals in all the groups before and after treatment were good. The higher dose resulted in the atrophy of seminiferous tubules on day 31 (Figure 1). The STD and SEH were decreased significantly (P<0.05) in a dose-dependent manner (Table 1). Spermatozoa count exhibited a dose-dependent decline (P<0.01) with a maximum effect on day 1 and day 12 samples (Table 2). The decline in sperm count was sustained for longer duration of time, showing a significant fall evident at day 36 sample. A dose-dependent increase in the percentage of abnormal spermatozoa was observed with amorphous, folded, hookless, banana and double headed/tailed types (Table 3). The frequency of abnormal spermatozoa was significant (P<0.01) at both tamoxifen levels with a higher incidence on day 1. The day 36 sample exhibited a significant decrease in percentage abnormality compared to the day 1 and day 12 sample.

Table 1. Effect of tamoxifen on seminiferous tubules. ^cP<0.01, compared with controls.

Table 2. Effect of tamoxifen on epididymal sperm count (10⁶). ^cP<0.01, compared with controls.

Table 3. Effect of tamoxifen on sperm morphology. ^cP<0.01, compared with controls.

Figure 1. Testicular section showing atrophy of tubules (800 mgkg^-1day^-1) on day 1. Arrow indicates atrophied tubule. Tubules show extensive shrinkage and sloughing of germinal cells. Other tubules also show process of degeneration with cellular disharmony. Scale bar = 10 µm, H&E stain.

Previously it has been shown that tamoxifen induced the formation of multinucleated abnormal cells with exfoliation of germ cells in a dose-dependent manner [4]. Decrease in the STD and SEH observed in the present study were due to cell-loss as a result of sloughing and resultant tubular atrophy. Since tamoxifen was known to cause apoptosis and necrosis [6], the decrease in tubular diameter and epithelial height in the present study may be due to cell loss from the germinal epithelium. These morphometric changes as well as the decline in sperm count are also dose-dependent.

A dose-dependent decrease in sperm count and increase in abnormal sperm are in consistent with the earlier report with mice [5]. The changes in the sperm density number and abnomal sperm is an evidence of interference of tamoxifen or its metabolites with the genetically controlled differentiation of spermatozoa indicating the genotoxicity of this drug in vivo at higher dose levels [5]. The fact that the adverse effect of tamoxifen on the seminiferous epithelium was sustained and recovery was not observed even 36 days post exposure, would probably suggest an effect targeting the hypothalamus or pituitary gland resulting in a decrease in LH. Decrease in sperm count 31 days post treatment may be due to an effect on efferent ductules and fluid reabsorption, causing the sperm to be diluted in the cauda epididymis. In conclusion, this study confirms that tamoxifen citrate induces the shrinkage and atrophy of seminiferous tubules with a decrease in sperm density and an increase in abnormal sperm in a dose-dependent manner in rats.

References

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Correspondence to: Dr. Urban J. A. D'Souza, Department of Physiology, School of Medical Sciences, PPSP Universiti Sains Malaysia, 16150, Kubang Kerian, Kelantan, Malaysia.
Tel: +60-9-766 4836, Fax: +60-9-765 3370
Email: dsouza@kb.usm.my
Received 2003-08-28 Accepted 2004-05-17