Methamphetamine induces abnormal sperm morphology, low sperm concentration and apoptosis in the testis of male rats

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1 ORIGINAL ARTICLE Methamphetamine induces abnormal sperm morphology, low sperm concentration and apoptosis in the testis of male rats S. Nudmamud-Thanoi & S. Thanoi Department of Anatomy, Faculty of Medical Science, Naresuan University, Phitsanulok, Thailand Keywords Apoptosis methamphetamine seminiferous tubule sperm concentration sperm morphology Correspondence Samur Thanoi, Department of Anatomy, Faculty of Medical Science, Naresuan University, Phitsanulok 65, Thailand. Tel.: ; Fax: ; Accepted: January 27, 1 doi: /j x Summary Methamphetamine has been reported to be an important drug in the field of reproductive toxicology. The aim of this study was to investigate the effects of methamphetamine administrations on sperm morphology, sperm concentration and apoptotic activity inside seminiferous tubule in male rats. Rats were administered a dose of 8 mg kg )1, intraperitoneally (IP), for acute group and a dose of 4 mg kg )1, IP, once daily for 14 days for sub-acute group. Percentage of normal sperm morphology was decreased in acute group when compared with control. Total numbers of sperm count were significantly decreased in acute and sub-acute groups. Apoptotic activities were most abundant in the seminiferous tubules of acute treated animals with a highly significant increase in the number of apoptotic cells per tubule. Those effects of methamphetamine seem to be dose-dependent. The results suggest that methamphetamine not only works as drug of abuse in central nervous system, but also in gametogenesis of males. Introduction Methamphetamine, a central nervous system (CNS) stimulant, is an addictive drug that is often abused. There are areas of interest on the effects of methamphetamine in the changes in reproductive organs and reproductive toxicology. Methamphetamine has been reported to induce apoptosis in seminiferous tubules in male mice testis. Apoptotic cells were detected in the seminiferous tubules of male mice 24 h after a single treatment with 5, 1 and 15 mg kg )1 methamphetamine (Yamamoto et al., 2). It has also been reported that a high-dose administration of methamphetamine can decrease sperm motility and induce fluctuation of plasma testosterone concentration (Yamamoto et al., 1999). Moreover, it has been reported that gonadal steroid hormones play an important role in modulating methamphetamine neurotoxicity (Dluzen et al., 2). Therefore, the aim of this study was to investigate the effects of acute and sub-acute methamphetamine administrations on sperm morphology, sperm concentration and apoptosis inside seminiferous tubule in male rats. Materials and methods Animal treatments Male Sprague Dawley rats (25 28 g) were obtained from the National Animal Center, Mahidol University, Thailand. The animals were housed 2 3 per cage and maintained at 24 ± 1 C under a 12 h light/dark cycle with free access to water and food. All animal procedures were carried out in compliance with Mahidol University Code of Practice and the National Institutes of Health (USA) Guidelines for treatment of laboratory animals. The protocol for this study was approved by the Animal Research Committee of Naresuan University, Thailand. Rats were divided into three groups as acute, sub-acute and control groups with six animals each. To examine an acute effect of methamphetamine, animals were 278 ª 11 Blackwell Verlag GmbH Æ Andrologia 43,

2 S. Nudmamud-Thanoi and S. Thanoi Methamphetamine induces abnormal sperm administered a dose of 8 mg kg )1 intraperitoneally (IP). The sub-acute effect was examined by administering methamphetamine at a dose of 4 mg kg )1, IP, once daily for 14 days, while animals in control group were administered a vehicle. After treatments, animals were killed by cervical dislocation. Statistical analysis The data were expressed as mean ± SD and compared for statistical significance using analysis of variance (anova) followed by Bonferroni s post hoc test. A value of P <.5 was considered as a significant difference. Sperm collection Each testis was separated via midline incision. The epididymis was then dissected free of the testis and placed in pre-warm phosphate buffer saline (PBS). Blood contamination was removed by washing the tissue in PBS. The tissue was minced with scissors to release spermatozoa. Sperm morphology and concentration The cauda sperm suspensions were diluted 1 : 1 with 1% neutral buffered formalin in PBS. The cauda sperm suspensions were diluted in 2% aqueous solution of eosin and sperm heads were stained, and then the spermatozoa were evaluated for individual sperm morphology. Two hundred spermatozoa per animal were evaluated by bright field microscopy (4 objective). The criteria of Wyrobek & Bruce (1975) were employed for evaluation of sperm abnormalities. The concentration of spermatozoa was determined using Neubauer s counting chamber. An aliquot of sperm suspension was charged into the counting chamber and spermatozoa were counted. The sperm count was expressed in millions ml )1. Apoptotic activity study Small pieces of testes from six animals in each group were fixed in 1% formalin in PBS. Tissues were dehydrated in graded ethanol, embedded in paraffin and sectioned (4 lm thickness) on serial coronal plane. Apoptotic activities within the seminiferous tubules were studied using the terminal deoxynucleotidyl transferase-mediated dutp-biotin nick end labelling (TUNEL) assays (Dead EndÔ Colorimetric TUNEL System for quantitative study and Dead EndÔ Fluorometric TUNEL System for qualitative study; Promega, Madison, WI, USA). The protocols were followed by manufacture instructions. Two slides from each animal were used for quantitative study. In cross section, 24 seminiferous tubules from each group were counted for the numbers of apoptotic cell under light microscope (4 ). DNA fragmentation detected as DNA ladder in nucleus of spermatogenic cells was used as an evidence of apoptotic cell in seminiferous tubules. Apoptotic index, modified from the study by Fazlioglu et al. (8), was defined as an average apoptotic cell per tubule (apoptotic index = total apoptotic cells/24). Results Sperm morphology Percentage of normal sperm morphology was decreased in animals treated with methamphetamine. It was significantly lower (P <.1) in acute treated group (71.33 ± 7.86%) when compared with untreated animals (94.5 ± 2.17%) (Fig. 1). Sperm concentration Total number of cauda epididymal sperm counts were significantly decreased in acute group (16.67 ± cells ml )1 ) and sub-acute group ( ± cells ml )1 ) when compared with control group (185. ± cells ml )1 ) (Fig. 2). Apoptotic cell studies The present study showed that methamphetamine administration can induce apoptotic cell activities within seminiferous tubules. The appearances of TUNEL-positive cells in seminiferous tubules in animals treated with methamphetamine were detected in almost every stage of sperm development especially in the spermatogonia lining along the basement membrane of the tubules in animals treated with methamphetamine in both acute and Percentage of normal sperm Control Acute Sub-acute Fig. 1 Percentage of normal sperm morphology of male rats after treatment with various doses of methamphetamine compared with the control group. Values are means and SD, n = 6. Significantly different from the control group at P <.1 by ANOVA post hoc Dunnett test. ª 11 Blackwell Verlag GmbH Æ Andrologia 43,

3 Methamphetamine induces abnormal sperm S. Nudmamud-Thanoi and S. Thanoi Epididymal sperm numbers ( 1 6 cells) Control Acute * Sub-acute (a) Fig. 2 Cauda epididymal sperm number ( 1 6 cells ml )1 ) of male rats after treatment with various doses of methamphetamine compared with the control group. Values are means and SD, n = 6. Significantly different from the control group at P <.1, and *significantly different from the control group at P =.1 by ANOVA post hoc Dunnett test. (b) sub-acute groups (Fig. 3). Qualitatively, the proportions of apoptotic cells in the tubules of animals treated with methamphetamine were larger than those in untreated animals. Quantitative data of apoptotic index showed that averages of apoptotic cells per tubule were significantly higher in acute ( ± 33.29) and sub-acute (76.77 ± 7.76) groups when compared with the control (5.93 ± 3.68) (Fig. 4). In addition, Bonferroni s test demonstrated that an average apoptotic cell per tubule in acute treated animals was also significantly higher (P <.1) than sub-acute treated animals. Discussion The results from this study indicate that methamphetamine can induce abnormal sperm morphology, decrease sperm concentration and induce apoptotic cell activities in the seminiferous tubules in male rats. The effect of methamphetamine in sperm morphology and sperm concentration seems to be dose-dependent as seen in animals treated with high dose acutely. These results are consistent with the previous report that percentage of apoptotic tubules was increased in a dose-dependent manner (Yamamoto et al., 2). Decreased normal sperm morphology and low sperm concentrations after treated with methamphetamine may be involved in its effect on inducing apoptotic activity in seminiferous tubules. Our finding also indicates that methamphetamine administration induces apoptosis within the seminiferous tubules of male rats after acute and sub-acute treatments. Numbers of apoptotic cell per tubule were significantly increased in acute and sub-acute treated animals respectively. These were in agreement with the study by Yamamoto et al. (2) who reported that methamphetamine induces (c) Fig. 3 Apoptotic activity inside the testes of rats. Control group (a), sub-acute group (b) and acute group (c). TUNEL-positive staining indicative of DNA fragmentation was detected as bright-green fluorescent signals on the nuclei. The photographs were taken by laser confocal microscopy. Bar represents 5 lm. 28 ª 11 Blackwell Verlag GmbH Æ Andrologia 43,

4 S. Nudmamud-Thanoi and S. Thanoi Methamphetamine induces abnormal sperm Apoptotic cells per tubule Control Acute Sub-acute Fig. 4 Apoptotic index (number of apoptotic cells per tubule) of male rats after treatment with various doses of methamphetamine compared with the control group. Values are means and SD, n = 6. Significantly different from the control group at P <.1 by ANOVA post hoc Dunnett test. apoptosis in seminiferous tubules in male mice testis 24 h after a single treatment with 5, 1 and 15 mg kg )1 methamphetamine. The highest numbers of apoptotic cells per tubule found in acute treated animals in this study indicated that methamphetamine can induce apoptosis in a dose-dependent manner. These were also similar to the study by Yamamoto et al. (2) which indicated that the percentage of seminiferous tubules demonstrating apoptotic cells increased dose-dependently after treatment with methamphetamine. Recently, repeated methamphetamine administration caused a decrease in cell proliferation, induce apoptosis and reduce the proliferation/apoptosis ratio in male rat germ cells. Apoptotic cells were increased in both spermatogonia and primary spermatocytes after repeated administration of methamphetamine (Alavi et al., 8). These findings may reflect that the genotoxic effect of methamphetamine seems to be involved especially in spermatogenesis (Yamamoto et al., 2). In addition, it has been suggested that serum testosterone concentration seemed to be changed during the time course of methamphetamine treatment (Shin et al., 1999). It has also been suggested that amphetamine had an effect in reducing the release of testosterone from the testis (Tsai et al., 1996). Qualitatively, the results of the present study also indicated apoptotic activity in the nuclei of Leydig cells in rats treated with methamphetamine. Apoptosis in Leydig cells may influence the changes of testosterone concentration in the testis leading to apoptosis of spermatogenic cells as testosterone has been reported to suppress apoptosis in human seminiferous tubule (Erkkila et al., 1997). A decrease in testosterone concentration can induce an increase in the number of apoptotic germ cells in most stages of the cycle of the seminiferous epithelium in rats (Henriksen et al., 1995). In addition, dihydrotestosterone, an end metabolite of testosterone, can inhibit testicular apoptosis in humans (Pentikainen et al., ). However, Yamamoto et al. (2) showed that the concentration of testosterone in serum was significantly decreased only 24 h after treatment with high dose methamphetamine (15 mg kg )1 ) when compared with untreated rats and the level of testosterone was returned to normal afterwards. No significant changes were found in low dose (1 mg kg )1 ) treated animals. These results may reflect that the decrease of serum testosterone concentration induced by methamphetamine may occur in a short period once treated with high dose methamphetamine. In conclusion, methamphetamine can induce abnormal sperm morphology, low sperm concentration and apoptosis in seminiferous tubules of rats after acute and sub-acute administrations. The results suggest that methamphetamine not only works as drug of abuse in CNS, but also in gametogenesis. The changes in testosterone concentration induced by methamphetamine during that time course may play some role in abnormalities of spermatozoa that occurred during spermatogenesis, and may also play a function in triggering apoptosis in the spermatogenic cells, especially in dose-dependent manner. This study thus provides valuable information for further investigation of the mechanism of methamphetamine in reproductive toxicity during spermatogenesis. Acknowledgements This research was supported by the Thailand Research Fund (TRF) and Commission of Higher Education (CHE), and Naresuan Research Fund, Naresuan University, Thailand. References Alavi SH, Taghavi MM, Moallem SA (8) Evaluation of effects of methamphetamine repeated dosing on proliferation and apoptosis of rat germ cells. Syst Biol Reprod Med 54: Dluzen DE, Anderson LI, Pilati CF (2) Methamphetamine gonadal steroid hormonal interactions: effects upon acute toxicity and striatal dopamine concentrations. Neurotoxicol Teratol 24: Erkkila K, Henriksen K, Hirvonen V, Rannikko S, Salo J, Parvinen M, Dunkel L (1997) Testosterone regulates apoptosis in adult human seminiferous tubules in vitro. J Clin Endocrinol Metab 82: Fazlioglu A, Yilmaz I, Mete O, Kurtulus F, Parlakkilic O, Guctas O, Cek M (8) The effect of varicocele repair on experimental varicocele-induced testicular germ cell apoptosis. J Androl 29: ª 11 Blackwell Verlag GmbH Æ Andrologia 43,

5 Methamphetamine induces abnormal sperm S. Nudmamud-Thanoi and S. Thanoi Henriksen K, Hakovirta H, Parvinen M (1995) Testosterone inhibits and induces apoptosis in rat seminiferous tubules in a stage-specific manner: in situ quantification in squash preparations after administration of ethane dimethane sulfonate. Endocrinology 136: Pentikainen V, Erkkila K, Suomalainen L, Parvinen M, Dunkel L () Estradiol acts as a germ cell survival factor in the human testis in vitro. J Clin Endocrinol Metab 85: Shin JH, Mori C, Shiota K (1999) Involvement of germ cell apoptosis in the induction of testicular toxicity following hydroxy urea treatment. Toxicol Appl Pharmacol 155: Tsai SC, Chiao YC, Lu CC, Doong ML, Chen YH, Shih HC, Liaw C, Wang SW, Wang PS (1996) Inhibition by amphetamine of testosterone secretion through a mechanism involving an increase of cyclic AMP production in rat testes. Br J Pharmacol 118: Wyrobek AJ, Bruce WR (1975) Chemical induction of sperm abnormalities in mice. Proc Natl Acad Sci USA 72: Yamamoto Y, Yamamoto K, Hayase T (1999) Effect of methamphetamine on male mice fertility. J Obstet Gynaecol Res 25: Yamamoto Y, Yamamoto K, Hayase T, Abiru H, Shiota K, Mori C (2) Methamphetamine induces apoptosis in seminiferous tubules in male mice testis. Toxicol Appl Pharmacol 178: ª 11 Blackwell Verlag GmbH Æ Andrologia 43,