Tamoxifen induced multinucleated cells (symplasts) and distortion of seminiferous tubules in rat testis

Asian J Androl 2003 Sep; 5: 217-220     

Keywords: tamoxifen; smultinucleated giant cells; gonadotoxicity; Seminiferous tubules

Abstract

Aim: To evaluate the effect of tamoxifen citrate on male reproductive system of rat. Methods: Groups of male rats were gavaged with tamoxifen at doses of 200 mgkg^-1d^-1, 400 mgkg^-1d^-1 or 800 mgkg^-1d^-1 in 0.1 mL olive oil for 10 consecutive days. Controls were treated with 0.1 mL olive oil. Rats were anesthetized and killed on d 3, d 15 or d 35 after the last dose. Testes were collected, processed for paraffin embedding, sectioned at 5 mm thickness, stained with H&E and analyzed microscopically. Results: There was a dose-dependent increase in the occurrence of seminiferous tubular distortion with germinal cell sloughing. The highest dose increased the number of multinucleated giant cells on d 3 and d 15. Conclusion: Tamoxifen citrate induces multinucleated giant cells and germinal epithelial sloughing in a dose-dependent manner and these changes are detrimental to male fertility.

1 Introduction

Tamoxifen citrate is a chemotherapeutic antineoplastic agent used for the prophylactic treatment of malignancies. It is a nonsteroidal triphenylethylene derivative used to stimulate ovulation in infertile subjects[1] and is also prescribed in the treatment of oligozoospermia to improve the impaired spermatogenesis [2, 3]. Gopalkrishnan et al. indicated that tamoxifen decreased the serum testosterone level and disrupted the testicular seminiferous epithelium [4]. Further, Rai and Vijayalaxmi [5] reported that tamoxifen dose-dependently increased the number of abnormal sperm in mice. The genotoxic effects of this drug were known to induce DNA adducts, indicating a mutagenic potential in various tissues [2, 3]. There are only few literature reports depicting the early histopathological effect of tamoxifen on the seminiferous epithelial harmonization. Hence the aim of the present study was to examine the effects of tamoxifen citrate on the seminiferous epithelium.

2 Materials and methods

Male Wistar rats, 140 g~160 g body weight, were kept in polyprophylene cages under standard laboratory conditions with free access to laboratory chew and water. The rats were randomly divided into 3 experimental and 1 control groups of 15 animals each. The 3 experimental groups were gavaged tamoxifen citrate (Lyka Labora-tories, Bombay, India) at doses of 200 mgkg^-1d^-1, 400 mgkg^-1d^-1 or 800 mgkg^-1d^-1 in 0.1 mL olive oil for 10 days. The controls received 0.1 mL olive oil.

Five rats of each group were sampled on d 3, d 15 or d 35 after the last dosing. They were anesthetized with pentobarbital sodium, 40 mg/kg (Sigma, USA) ip on the stipulated day and the mediastinum was opened. Perfusion fixation was performed as described previously [6, 7]. Briefly, heparinised normal saline (1 mL/L) was injected through the left ventricle followed by injection of Bouins fluid until the testes attained a yellowish coloration. Laparatomy was performed and the testis along with the epididymis were removed and immersed in Bouins fluid for 10 h. The testis were cut into 3 mm~4 mm thick blocks and placed in 70 % alcohol for 5 days with daily change of fresh alcohol. Then the testis was processed for paraffin embedding, 5 µm sectioning and haematoxylin-eosin staining.

2.3 Microscopic observation

Slides were observed under a light microscope for qualitative changes in the seminiferous tubules. Preliminary observation revealed sloughing of germinal epithelial cells into the lumina and the presence of abnormal multinucleated giant cells in the epithelium. The incidence of sloughing was quantified as partial or extensive. Partially sloughed tubules were those in which less than 50 % of the surface cells were separated and extensively sloughed tubules, more than 50 % to complete exfoliation. From each rat, 5 sections cut at 10 micron apart were screened according to the method of Allard et al. [8]. One hundred seminiferous tubules were counted and sloughed tubules were classified.

2.4 Data processing

Data were expressed in mean SD. Statistical analysis was done by Mann Whitney U test (two tailed) [7, 9] and P<0.05 was considered as significant.

The highest dose (800 mg/kg) induced an increase in the multinucleated giant cells in the seminiferous tubules on d 3 and d 15 (Figure 1). All the 3 doses of tamoxifen induced disintegration of seminiferous epithelium with a significant (P<0.01) increase in the number of partial and extensive sloughing tubules. The extent of partial sloughing was the greatest on d 3 with a dose-dependent increase (Figure 2). The lowest dose, 200 mg/kg, produced the maximum effect on d 15 (Figure 3). In 400 mg/kg dose, the partial sloughing was the maximum on d 3. The number of tubules exhibiting partial sloughing was significantly less (P<0.01) on d 35 compared to the d 3 sample. The response of extensive sloughing in 400 mg/kg dose followed a similar pattern with that of 200 mg/kg dose, showing the maximum response on d 15. The highest dose 800 mg/kg showed greater responses compared to other doses. The effect was the maximum on d 3 exhibiting a highest number of partial and extensive sloughing. There was a dose-dependent increase in the number of sloughed tubules with a maximum effect on d 3. The extent of sloughing was less on d 35 compared to the day 3 sample, signifying an early dose-dependent toxic effect of tamoxifen on testicular epithelial harmony. In the control animals only (2 0.5) tubules were partially sloughed without any extensive sloughing.

Figure 1. Increase in multinucleated giant cells in seminiferous tubules in 800 mg/kg group (d 3). Arrows indicate multinucleated giant cells. Tubules show extensive sloughing of germ cells. H & E staining, Scale bar = 14 µm.

Figure 2. Number of partially sloughed seminiferous tubules in control and tamoxifen treated rats. 1: Control; 2: 200mg/kg; 3: 400mg/kg; 4: 800mg/kg. ^cP<0.01, compared with controls.

Figure 3. Number of extensively sloughed seminiferous tubules in control and tamoxifen treated rats. 1: Control; 2: 200 mg/kg; 3: 400 mg/kg; 4: 800 mg/kg. ^cP<0.01, compared with controls.

4 Discussion

Tamoxifen citrate was well documented for its positive effects on the improvement of spermatogenesis in patients with oligozoospermia [10]. Antiestrogenic drugs generally act via an enhancement of gonadotropin release from the hypophysis. The increased secretion of gonadotropin interferes in the normal negative feedback of sex steroids at hypothalamic and pituitary levels. As a result, FSH and LH stimulate the Leydig cells in the testis and this has been claimed to increase the local testosterone production, thereby boosting spermatogenesis with a possible improvement in fertility [11]. However, tamoxifen reduced the spermatid and sperm populations at doses of 40 µgkg^-1d^-1 and 200 µgkg^-1d^-1 [4].

Appearance of symplasts or multinucleated cells was known to be associated with the final common pathway of germinal cell degeneration in animals treated with different chemicals [12]. The mechanism of formation of giant cells was uncertain but cell fusion of damaged spermatids was speculated to be the likely reason [13]. It was also hypothesized that these multinucleated cells were formed due to karyokinesis, which was not followed by cytokinesis [12]. The absence of mitotic figures and the presence of membranous residues with residual plasma membrane in these multinucleated cells provided a support to the hypothesize that these giant cells were formed as a result of cell fusion [14]. In the present study only 800 mg/kg dose of tamoxifen induced multinucleated cells in the seminiferous tubules on day 3 and 15 and disappearing by day 35. Further sloughing of germinal cells showed a positive correlation with the presence of giant cells. The toxicity of tamoxifen was reported to occur via induction of DNA adducts [15] and oxidative stress and lipid peroxidation [16], resulting in chemical teratogenesis and carcinogenesis. Epithelial sloughing was usually due to the damage of Sertoli cells and interruption of intercellular bridges. Tamoxifen at higher dose levels might generate metabolites toxic to the Sertoli cells and intercellular bridges, thereby facilitating the appearance of multinucleated cells. The partial and extensive sloughing of seminiferous tubules was further evidence of Sertoli cell toxicity. This study reveals that tamoxifen and its metabolites exert the maximum effect immediately after exposure. The exact mechanism underlying the cytotoxicity is not known. Gopalakrishnn et al. [4] observed a similar effect where 200 µg of tamoxifen citrate treatment resulted in disorganization of tubular elements with increased intercellular spaces. Tamoxifen has both estrogenic and antiestrogenic effects in rats and mice. The biological effects are mainly mediated through the binding to the estrogenic receptors inducing alteration in DNA transcription. The endogenous substances formed may affect either Sertoli cells or intercellular bridges between germ cells and may result in the destruction of cell association. Nakai and Hess [17] reported that chemicals, which affect the microtubules in Sertoli cells, induced epithelial sloughing. Further, the destruction of vimentin filaments, which are required as an anchoring device for the germ cells, also results in exfoliation of the germinal epithelium [18]. Hence, epithelial sloughing induced by tamoxifen indicates that this drug affects the microtubules and the anchoring device in seminiferous epithelial cells. We conclude that tamoxifen citrate is gonadotoxic in male rats with germinal cell sloughing and induction of multinucleated giant cells.

References

[1] Messins IE, Nillius SJ. Comparison between tamoxifen and clomiphene for induction of ovulation. Acta Obstet Gynecol Scand 1982; 61: 377-9.
[2] Krause W, Holland-Moritz H, Schramm P. Treatment of idiopathic oligozoospermia with tamoxifen - a randomized controlled study. Int J Androl 1992; 15: 14-8.
[3] Damber JE, Abramsson L, Duchek M. Tamoxifen treatment of idiopathic oligozoospermia: effect on hCG-induced testicular steroidogenesis and semen variables. Scand J Urol Nephrol 1989; 23: 241-6.
[4] Gopalkrishnan K, Gill-Sharma MK, Balasinor N, Padwal V, D'Souza S, Parte P, et al. Tamoxifen-induced light and electron microscopic changes in rat testicular morphology and serum hormonal profile of reproductive hormones. Contraception 1998; 57: 261-9.
[5] Padmalatha Rai S, Vijayalaxmi KK. Tamoxifen citrate induced sperm shape abnormalities in the mouse. Mutat Res 2001; 492: 1-6.
[6] Hess RA, Moore BJ. Histological methods for evaluation of the testis. In: Chapin B, Heindel J, editors. Methods in toxicology. London: Academic press; 1993. p52-85.
[7] Narayana K, D'Souza UJ, Sanyal AK, Rao KP. 5-Fluorouracil (5-FU) induces the formation of giant cells and sloughing of seminiferous epithelium in rat testis. Indian J Physiol Pharmacol 2000; 44: 317-22.
[8] Allard EK, Johnson KJ, Boekeleide K. Colchicine disrupts cytoskeleton of rat testis seminiferous epithelium in a stage-dependent manner. Biol Reprod 1993; 48: 143-53.
[9] Gupta BN. Statistics - Theory & Practice. Agra: Sahitya Bhavan Publisher; 1978. p1126-40.
[10] Krause W, Hubner HM, Wichmann U. Treatment of oligozoospermia by tamoxifen: no evidence for direct testicular action. Andrologia 1985; 17: 285-90.
[11] Vandekerckhove P, Lilford R, Vail A, Hughes E. Clomiphene or tamoxifen for idiopathic oligo/asthenzoospermia. Cochrane Database Syst Rev 2000; (2): CD000151.
[12] Reddy KJ, Svoboda DJ. Alterations in rat testes due to an antispermatogenic agent. Light and electron microscopic study. Arch Pathol 1967; 84: 376-92.
[13] Jones TM, Anderson W, Fang VS, Landau RL, Rosenfield RL. Experimental cryptorchidism in adult male rats: histological and hormonal sequelae. Anat Rec 1977; 189: 1-27.
[14] Chapin RE, Morgan KT, Bus JS. The morphogenesis of testicular degeneration induced in rats by orally administered 2, 5-hexanedione. Exp Mol Pathol 1983; 38: 149-69.
[15] Hard GC, Iatropoulos MJ, Jordan K, Radi L, Kaltenberg OP, Imondi AR, et al. Major difference in the hepatocarcinogenecity and DNA adduct forming ability between toreminfene and tamoxifen in female Crl: CD(BR) rats. Cancer Res 1993; 53: 4534-41.
[16] Pagano G, de Biase A, Deeva IB, Degan P, Doronin YK, Iaccarino M, et al. The role of oxidative stress in developmental and reproductive toxicity of tamoxifen. Life Sci 2001; 68:1735-49.
[17] Nakai M, Hess RA. Morphological changes in the rat Sertoli cell induced by the microtubule poison carbendazim. Tissue Cell 1994; 26: 917-27.
[18] Amlani S, Vogl AW. Changes in the distribution of microtubules and intermediate filaments in mammalian Sertoli cells during spermatogenesis. Anat Rec 1988; 220: 143-60.

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Correspondence to: Dr. Urban J.A. D'Souza, Department of Physiology, School of Medical Sciences, PPSP University Science Malaysia, 16150, Kubang Kerian, Kelantan, Malaysia.
Tel: +60-9-766 4836 Fax: +60-9-765 3370
E-mail: dsouza@kb.usm.my
Received 2003-01-28 Accepted 2003-06-09