Alcohol 48 (2014) 781e786

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Restraint stress exacerbates alcohol-induced reproductive toxicity in male rats P. Hari Priya a, B.P. Girish a, P. Sreenivasula Reddy b, * a b

Department of Biotechnology, Sri Venkateswara University, Tirupati 517502, India Department of Zoology, Sri Venkateswara University, Tirupati 517502, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 March 2014 Received in revised form 6 July 2014 Accepted 9 July 2014

Cumulative exposure to multiple stresses may lead to aggravating the toxicity of each stress, qualitatively or quantitatively altering biological responses because of toxicological interaction. In this study, we intended to determine the possible effects of restraint stress on reproductive toxicity due to ethanol usage in male rats. Early pubertal male Wistar rats were subjected to either restraint stress (5 h/day) or alcohol intoxication (2 mg/kg body weight) or both for 60 days. Body weights of control and experimental rats were similar during the 60 days of this study. Testes were harvested, weighed, and prepared for enzyme assays, and cauda epididymides were isolated for the determination of density, motility, and viability of stored spermatozoa. Restraint stress or alcohol treatment significantly reduced testis weight and caused significant reductions in steroidogenesis and spermatogenesis. Mean density, motility, and viability of stored spermatozoa were reduced in experimental rats. Plasma testosterone concentrations in rats subjected to restraint stress or alcohol were decreased compared with those of controls, concomitant with increased concentrations of LH and FSH in experimental rats. These data suggest that sub-chronic exposure to restraint stress or alcohol contribute to reduce testicular and epididymal function in exposed rats. The study also suggests that restraint stress exacerbates alcohol-induced reproductive toxicity in rats. Ó 2014 Elsevier Inc. All rights reserved.

Keywords: Restraint stress Alcohol Testosterone 3b-HSD and 17b-HSD Sperm Rat

Introduction Environmental, lifestyle, dietary, and occupational factors have been found to influence the endocrine system, and exposure to these factors increases the risk of major health concerns such as cancers, reproductive and developmental defects, cardiovascular problems, etc (Barlow et al., 1999). These factors may impair male fertility by interfering with spermatogenesis, spermiogenesis, motility, sperm DNA, chromatin integrity, and hormonal regulation, or by reducing the fertilizing capacity of spermatozoa (Emanuele & Emanuele, 1998; Hammoud, Gibson, Peterson, Hamilton, & Carrel, 2006; Ivell, 2007; Sharpe, 2000). Stress is considered to be one of the most influential factors affecting sexuality, personality, emotional disorders, marital conflicts, occupation, and psychopathology on male infertility (Bents, 1985). Restraint has been adopted as a standard stressor that imposes both physical and psychological

Conflict of interest statement: All authors declare that they have no conflicts of interest. * Corresponding author. Tel.: þ91 9247593000; fax: þ91 877 2249611. E-mail addresses: [email protected], [email protected] (P.S. Reddy). http://dx.doi.org/10.1016/j.alcohol.2014.07.014 0741-8329/Ó 2014 Elsevier Inc. All rights reserved.

demands on the subject. The magnitude of the response depends on the intensity and duration of the stimulus, and either short- or long-term immobilization is stressful to laboratory animals (Charpenet et al., 1981; Collu, Gibb, & Ducharme, 1984; Orr & Mann, 1992). Restraint stress impedes the male reproductive capacity by triggering the hypothalamic-pituitary-adrenal axis and hampering the hypothalamic-pituitary-testicular axis (Norman & Smith, 1992; Rai, Pandey, & Srivastava, 2004; Retana-Marquez, Salazar, & Velazquez-Moctezuma, 1996). Restraint stress results in germ cell degeneration, significant decreases in testis weight, and declines in semen quality, sperm concentration, morphology, and fertility potential of males (Almeida, Petenusci, Anselmo-Franci, Rosa e Silva, & Lamano-Carvalho, 1998; Bonde et al., 1998). The objective of the present study was to investigate the potential of restraint stress to influence the toxic effects of alcohol on reproduction. Alcohol is one of the oldest and most widely used psychoactive drugs on earth. Its use pre-dates recorded history and may go back as far as the Paleolithic age, around 8000 BC (Brick, 2003). It is generally acknowledged that both genetic and environmental factors contribute to the propensity to drink alcohol (George, 1987; Oroszi & Goldman, 2004). Exposure to stress is one environmental

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factor that has long been thought to increase alcohol-drinking behavior and predispose people toward the development of alcoholism (Pohorecky, 1991; Volpicelli, 1987). One popular theory is that stress increases alcohol consumption because alcohol relieves the psychological and physical consequences of stress, such as anxiety and/or physical pain (Brady & Sonne, 1999). Alcoholism is a serious health problem and can have pathological effects on vital systems. Due to its solubility in both water and lipids, alcohol diffuses to all tissues of the body and affects most vital functions (Lieber, 1997, 2005). Studies have shown that alcohol consumption may result in increased oxidative stress with formation of lipid peroxides and free radicals (Bjørneboe & Bjørneboe, 1993; El-Sokkary, Reither, Tan, Kim, & Cabrera, 1999; Gentry-Nielsen, Top, Snitily, Casey, & Preheim, 2004; Nordmann, 1994). Ethanol-induced oxidative stress is not restricted to the liver where the ethanol is actively oxidized, but can affect various extra-hepatic tissues including testes (Nordmann, Ribière, & Rouach, 1990). Alcohol is toxic to testicular tissue and its chronic use leads to both endocrine and reproductive failure (El-Sokkary et al., 1999; Rosenblum, Gavaler, & Van Thiel, 1989). Prolonged alcohol abuse in men is also known to cause reduction in seminal volume, sperm concentration, percentage of motile spermatozoa, testosterone deficiency, and testicular atrophy, which can lead to impotence, sterility, and feminization (Bannister & Losowsky, 1987; Martini et al., 2004; Peltola, Huhtaniemi, MetsaKetala, & Ahotupa, 1996). Although a large number of studies have reported separately on the deleterious effects of restraint stress or alcohol on reproduction, there have been no reports dealing particularly with the detrimental effects of a combination of stresses on reproductive toxicity. Though stress is an outcome of alcohol exposure, the possibility of stress being a factor in modifying the effect of alcohol on the male reproductive system cannot be ruled out. This aspect gains significance in the context that in modern societies where reproductive disorders are increasing, stressed lifestyle and consumption of alcohol go hand-in-hand. Materials and methods Chemicals NADPH, NAD, dehydroepiandrosterone, and androstenedione were purchased from Sigma Chemical Company (St. Louis, Missouri, USA). Ethanol (>99% purity) and other chemicals used in the study were of analytical grade and purchased from local commercial sources. Animals Wistar strain early pubertal rats (60 days) were purchased from an authorized vendor (M/S Raghavendra Enterprises, Bengaluru, India) and used in the present study as an experimental model. Rats were housed in polypropylene cages (1800  1000  800 ) lined with sterilized paddy husk as bedding material with ad libitum access to tap water and rat food (purchased from HLL Animal Feed, Bengaluru, India) in an air-conditioned environment (25  2  C) with a 12-h light and 12-h dark cycle. The experiments were carried out in accordance with the guidelines of the Committee for the Purpose of Control and Supervision on Experiments on Animals, Government of India (CPCSEA, 2003) and approved by the Institutional Animal Ethical Committee, Sri Venkateswara University, Tirupati, India (Regd. No. IAEC/No438/01/a/CPCSEA dt. 04.03.2002; resolution No. 05/2011-12/(i)/a/CPCSEA/IAEC/SVU/ PSR-PHP/Dt. 01-09-2011).

Experimental design The rats were randomly divided into 4 groups. Rats in group I served as controls and were allowed ad libitum access to tap water and food. The animals in group II were subjected to chronic restraint stress by introducing them into plastic tubes (diameter: 6 cm; length: 45 cm) without promoting pain for 5 h a day as described earlier (Priya & Reddy, 2012). Restraint stress was applied at the same time each day (between 0800 and 1300 h). At the end of each 5-h restraint period, rats were returned to the colony room with free access to food and water in their home cages. Undesirable stress was avoided as much as possible by gentle handling and noiselessness throughout the experiment. The rats in group III were administered a daily dose of ethanol at 2 g/kg body weight via an orogastric tube for a period of 2 months. The rats in group IV were exposed to a combination of restraint stress and alcohol simultaneously. The treatment was carried out for 60 days. The rationale for choosing a 60-day experimental period was based on earlier reports covering one complete spermatogenic cycle in Wistar rats (Robb, Amann, & Killian, 1978). Rats were observed daily for clinical signs of toxicity (increased salivation, increased/decreased urination, mydriasis or miosis, convulsions or tremors, postural abnormalities, abnormal appearance of fur, red deposits around eyes, nose, and mouth, unusual respiration, vocalization, head flicking, compulsive biting, circling and walking backward, etc.) from the first day of treatment. The body weight of the animals was recorded on the day of initiation of the treatment and on the day of sacrifice. The rats were fasted overnight, weighed, and killed by using an overdose of anesthetic ether on the day following the last treatment. Testes and other reproductive organs were isolated and weighed after removing the adhering tissues. Tissue somatic index (TSI) was calculated using the following formula: TSI ¼ weight of the tissue (g)/body weight of the animal (g)  100. Testes were used for determination of daily sperm production, and also enzyme assays whereas epididymides were used for sperm analysis. Epididymal sperm analysis Epididymal sperm count and evaluation of the motility of epididymal sperm were done by the method of Belsey et al. (1980). The epididymal fluid was obtained by mincing the cauda epididymis in physiological saline (0.9% NaCl in distilled water) at 37  C. One hundred nL of diluted epididymal fluid was placed in a Neubauer hemocytometer and total, motile, and non-motile sperm were counted. The number of motile and non-motile sperm was determined microscopically within 5 min following their isolation from the cauda epididymis at 37  C. Non-motile sperm numbers were determined, followed by counting of total sperm. The ratio of live and dead spermatozoa was determined using 1% trypan blue reagent (Talbot & Chacon, 1981). Briefly, one drop of diluted epididymal sperm suspension was mixed with one drop of 1% trypan blue solution on a microscope slide and covered with a cover slip. After incubation at 37  C for 15 min, the slides were observed under a microscope. Unstained spermatozoa were taken as viable and stained spermatozoa were counted as dead. Sperm viability was expressed as percentage of unstained sperm of the total sperm counted. The hypoosmotic swelling (HOS) test was performed by combining 0.1 mL of sperm with 1.0 mL hypoosmotic solution, following the method described by Jeyendran, Van der Ven, and Zaneveld (1992). After incubation of the mixture for 30 min at 37  C, sperm were observed for coiled tails under a phase-contrast microscope (Olympus BX41; Olympus Optical Co. Ltd., Japan). The data were expressed as millions/mL for sperm count, and for other

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sperm parameters, the data were expressed as percentage of total sperm.

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and LH, respectively. The inter-assay CV for testosterone, LH, and FSH are not available because all of the samples were analyzed at the same time.

Daily sperm production Statistical analysis Daily sperm production was determined in the testis of adult rats by the method of Blazak, Treinen, and Juniewicz (1993). Briefly, the testes were decapsulated and homogenized in 50 mL of ice-cold 0.9% NaCl solution containing 0.01% Triton X100 using a glass TeflonÒ homogenizer. The homogenate was allowed to settle for 1 min and then was gently mixed and a 10-mL aliquot was collected into a glass vial and stored on ice. After thorough mixing of each sample, the number of sperm heads was counted in 4 chambers of an improved Neubauer-type hemocytometer. The number of sperm produced per gram of testicular tissue per day was calculated (Robb et al., 1978). Assay of testicular steroidogenic enzymes The testicular tissue was homogenized in ice-cold Tris-HCl buffer (pH 6.8). The microsomal fraction was separated and used as the enzyme source. The activity levels of 3b-hydroxysteroid dehydrogenase (3b-HSD) (EC 1.1.1.51) and 17b-hydroxysteroid dehydrogenase (17b-HSD) (EC 1.1.1.61) were determined by the method described by Bergmeyer (1974). The enzyme assays were made under conditions following zero-order kinetics after preliminary standardization regarding linearity with respect to time of incubation and enzyme concentration. The enzyme activities were expressed as nmol of NAD converted to NADH/mg protein/min (3bHSD) or nmol of NADPH converted to NADP/mg protein/min (17bHSD). Protein content in the enzyme source was estimated by the method of Lowry, Rosebrough, Farr, and Randall (1951) using bovine serum albumin as standard. Radioimmunoassay of serum hormone levels Blood was collected from the heart using a heparinized syringe from each rat prior to necropsy. The serum was separated by centrifugation at 2000g for 15 min after overnight storage at 4  C, and stored at 20  C until all of the samples were collected. Radioimmunoassay (RIA) for serum testosterone level determination was performed by the method described earlier (Rao, Charaborti, Kotagi, & Ravindranath, 1990) and previously validated in our laboratory (Anjum, Sainath, Suneetha, & Reddy, 2011). The sensitivity of testosterone assay was 0.002 ng and intra-assay coefficient of variation (CV) was 6.5%. Serum FSH and LH were assayed according to the method of Lin, Kawamura, Okamura, and Mori (1988). The sensitivity of the assay was 0.08 ng for FSH and 0.06 ng for LH. The intra-assay variations were 7.0% and 8.0% for FSH

The data were statistically analyzed using 1-way analysis of variance (ANOVA) followed by Dunnet’s multiple comparison test using Student Version 16.0, SPSS Inc., Chertsey, UK. The data were presented as mean  SD. Differences were considered to be significant at p < 0.05. Results Clinical signs of toxicity The rats were observed for responses with respect to overall appearance, body position, activity, coordination or gait, and behavior. No significant changes in lacrimation, urination, respiration, vocalization, postural, or gait abnormalities were observed in any of the control and treated rats. All the animals were apparently normal and no unusual behaviors (viz., head flicking, head searching, biting, licking, self-mutilation, circling, and walking backward) were observed in any of the rats. None of the animals was excluded from the research. Body and organ weights The body weights and relative reproductive organ weights in control and experimental animals are presented in Table 1. Subjecting rats to either restraint stress or alcohol was not found to alter the body weight gain, whereas the body weights of rats simultaneously exposed to both stresses were significantly reduced as compared to rats treated with alcohol alone. The relative weights of the testes and cauda epididymis and corpus epididymis decreased significantly in all the experimental rats when compared to the control rats, whereas caput epididymis, vas deferens, seminal vesicle, and prostate weights decreased only in rats exposed to restraint stress and alcohol simultaneously. The weight of the penis was comparable among all the groups. Sperm parameters Testicular daily sperm production, sperm reserve in the cauda epididymis, epididymal sperm motility, sperm viability, and also percentage of number of HOS tail-coiled sperm were significantly lower in rats exposed to either restraint stress or alcohol for 60 days as compared to control rats. Further decline in these parameters was observed in rats exposed to restraint stress and alcohol

Table 1 Effect of restraint stress (RS) and alcohol (AL) alone or in combination (RS þ AL) on body weights (g) and reproductive tissue indices (g%) in rat. Parameter

Control

Body weight Testis Cauda Corpus Caput Vas deferens Seminal vesicles Prostate Penis

344.3a 1.99a 0.17a 0.03a 1.14a 0.12a 0.49a 0.16a 0.14a

        

RS 25.32 0.06 0.01 0.001 0.09 0.01 0.03 0.02 0.01

322.5a 1.07b 0.10b 0.02b 1.08a 0.11a 0.45a 0.15a 0.14a

RS þ AL

AL         

20.25 (6.3) 0.12 (46.2) 0.009 (41.2) 0.001 (33.3) 0.09 (5.3) 0.009 (8.3) 0.03 (8.2) 0.01 (6.3) 0.01 (0)

Values are mean  SD of 8 animals. Values in parentheses are % decrease from the control. Values with same superscript do not differ significantly from each other; p < 0.05.

321a 1.09b 0.11b 0.02b 1.09a 0.11a 0.46a 0.14a 0.14a

        

20.15 (6.8) 0.08 (45.2) 0.007 (35.3) 0.001 (33.3) 0.07 (4.4) 0.008 (8.3) 0.03 (6.1) 0.01 (12.5) 0.01 (0)

302b 1.02b 0.09b 0.02b 0.95b 0.09b 0.40b 0.12b 0.13a

        

25.43 (12.3) 0.14 (48.8) 0.007 (47.1) 0.001 (33.3) 0.06 (16.7) 0.007 (25.0) 0.03 (18.4) 0.01 (25.0) 0.01 (7.1)

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Table 2 Effect of restraint stress (RS) and alcohol (AL) alone or in combination (RS þ AL) on sperm parameters in rat. Parameter

Control

Daily sperm production (millions/gram testes) Sperm count (millions/mL) Motile sperm (%) Viable sperm (%) HOS-tail coiled sperm (%)

20.06a 71.2a 62.25a 70.75a 53.00a

    

RS 1.89 6.47 2.06 5.75 2.58

RS þ AL

AL

13.06b 44.83b 40.08b 60.83a 46.60b

    

1.19 4.53 3.77 6.23 4.67

(34.9) (47.4) (35.6) (14.0) (12.1)

7.76c 38.05bc 45.2b 51.4b 42.67bc

    

0.68 3.48 3.27 4.14 3.08

(61.3) (55.3) (27.4) (27.3) (19.6)

6.41d 35.01c 36.5c 46.86b 38.43c

    

0.55 2.18 2.66 3.06 3.59

(68.0) (58.9) (41.4) (33.8) (27.5)

Values are mean  SD of 8 animals. Values in parentheses are % decrease from the control. Values with same superscript do not differ significantly from each other; p < 0.05.

simultaneously (Table 2). No morphological abnormalities were observed in head, neck, and tail of spermatozoa of control and treated rats. Steroidogenesis and reproductive hormones The activity levels of 3b-HSD and 17b-HSD in the microsomal fractions of testes of control rats were 24.56  2.5 and 9.37  0.74, respectively. The activity levels of 3b-HSD and 17b-HSD were 12.89  1.45 (47.52%) and 3.89  0.24 (58.48%) in testes of immobilized rats, 7.66  0.47 (68.81%) and 4.22  0.45 (54.96%) in testes of alcohol-treated rats and 4.93  0.34 (79.93%) and 1.71  0.15 (81.75%) in the testes of rats exposed to restraint stress and alcohol simultaneously (Fig. 1). The circulatory levels of testosterone in the restraint stress or alcohol groups were significantly lower than in the control groups, whereas the levels of FSH and LH were significantly higher in the serum of rats exposed to restraint stress or alcohol when compared to control rats (Table 3).

consequently, a reduction in testicular mass and weight occurs when seminiferous tubular cells such as Sertoli cells undergo apoptosis due to exposure to alcohol (Grattagliano et al., 1997; Weinberg & Vogl, 1988) or restraint stress (Priya & Reddy, 2012). Daily sperm production and epididymal sperm density, motility, and viability decreased significantly in rats subjected to restraint stress or alcohol. Similar decreases in the quality and quantity of sperm were observed in rats subjected to restraint stress or alcohol (Almeida et al., 1998; Bonde et al., 1998; Carlsen, Giwercman, Keiding, & Skakkebaek, 1992; Goverde et al., 1995; Muthusami & Chinnaswamy, 2005). Androgens are very essential for the production and maturation of spermatozoa that are capable of fertilization and for developing normal progeny results (Grover et al.,

Discussion The influence of restraint stress on toxicological actions of alcohol is important in understanding the potential health impact of both classes of stresses. This study evaluated the potential for interaction between restraint stress and alcohol exposure on reproduction in the male rat. As expected, both stresses were endocrine disruptors, as indicated by their ability to alter the pituitary-testicular axis. In the present study, subjecting rats to either restraint stress or alcohol did not affect body weight gains in rats compared to control rats. These data suggest that daily exposure to restraint stress for 5 h or consumption of 2 g alcohol/kg body weight did not impose undue stress on the rats, hence the absence of significant differences in weight gains between experimental and control rats. On the contrary, the body weight of rats exposed simultaneously to both stresses was significantly reduced. We observed a significant reduction in mean testis weight among experimental rats compared to control rats, an observation similar to that made earlier for adult rats exposed to restraint stress (Almeida et al., 1998; Priya & Reddy, 2012) or alcohol (Grattagliano et al., 1997; Kelce, Ganjam, & Rudeen, 1990), suggesting testicular toxicity. It is apparent from our daily sperm production and testosterone data that exposure to either restraint stress or alcohol reduced spermatogenesis and steroidogenesis. The morphology and functional integrity of the testis and accessory sex organs are dependent on availability of androgens. Decrease in the serum level of testosterone may be a reason behind the significant decline observed in the weight of testes in rats subjected to different stresses. This is not surprising because the bulk of the testis (85%) is involved in sperm production (Lipshultz & Witt, 1993);

Fig. 1. The activity levels of 3b-HSD (A) and 17b-HSD (B) in the testes of control and experimental rats. Vertical bars represent mean  SD of 8 rats. Bars that do not share the same superscript differ significantly at p < 0.05.

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Table 3 Effect of restraint stress (RS) and alcohol (AL) alone or in combination (RS þ AL) on the serum hormone levels in rats. Hormone

Control

RS

AL

RS þ AL

Testosterone (ng/mL) FSH (ng/mL) LH (ng/mL)

2.83a  0.22 6.51a  0.65 1.54a  0.08

2.01b  0.28 (29.0) 11.73b  1.04 (80.2) 4.73b  0.31 (207.1)

1.99b  0.08 (29.7) 15.41c  1.11 (136.7) 5.43bc  0.39 (252.6)

1.62c  0.07 (42.8) 17.01c  1.01 (161.3) 6.19c  0.55 (302.0)

Values are mean  SD of 8 animals. Values in parentheses are % change from the control. Values with same superscript do not differ significantly from each other; p < 0.05.

2005). Chronic ethanol consumption is known to cause a reduction in seminiferous tubular diameter and a decrease in germ cell numbers (Grattagliano et al., 1997; Weinberg & Vogl, 1988). Both in vivo and in vitro studies indicate that ethanol has direct adverse effects on Leydig cell morphology and function (Weinberg & Vogl, 1988). Ethanol-induced impairment of spermatogenesis could reflect a toxic effect of ethanol on Sertoli cell morphology and/or function (Weinberg & Vogl, 1988). The most prominent effect of restraint stress and alcohol demonstrated in our study was related to androgen levels in circulation. Both stresses were effective in reducing the testosterone levels in rats. A sufficient level of circulatory testosterone is important not only for the maintenance of the structural integrity of testes and accessory sex organs and maintenance of spermatogenesis (Mann, 1974), but also essential for expression of secondary sex characteristics (Steinberger, 1971). The data of the present study also reveal decreased activity levels of 3b-HSD and 17b-HSD in testes of rats exposed to ethanol or restraint stress. This is in accord with the studies of Srivastava, Taylor, and Mann (1993) and Maneesh, Jayalekshmi, Dutta, Chakrabarti, and Vasudevan (2005). Suppression of testicular testosterone production and release and diminished sperm production is also attributed to the increased production of b endorphins within the testes due to alcohol consumption and increased production of glucocorticoids due to immobilization, which induces Leydig cell and seminiferous cell apoptosis (Emanuele et al., 2001; Gao et al., 2002, 2003; Gianoulakis, 1990; Nanji & Hiller-Sturmhöfel, 1997; Wang & Gao, 2006; Yin, Mufson, Wang, & Shi, 1999). Several studies also reveal that free radical generation and lipid peroxidation might be an important mechanism in the toxicity of ethanol in the testes an, Gürdöl, & (Aitken, 1994; Grattagliano et al., 1997; Oner-Iyidog Oner, 2001; Peltola et al., 1996; Rosenblum et al., 1989; Sanocka, Miesel, Jedrzejczak, & Kurpisz, 1996). At present, we do not know whether alcohol-induced reduction in steroidogenesis and spermatogenesis is indirect (mediated by radical generation) or direct or both. The data also reveal elevated levels of FSH and LH in rats exposed to restraint stress or alcohol. FSH and LH act on Sertoli cells and Leydig cells and thus regulate spermatogenesis and steroidogenesis, respectively (Skinner, 1991). The decrease in serum testosterone levels could be due to diminished responsiveness of Leydig cells to LH and/or direct inhibition of testosterone synthesis in rats exposed to restraint stress or alcohol. The increase in serum FSH levels indicates an impairment of spermatogenesis in experimental rats and reflects the germ cell loss or damage to Sertoli cells, thereby affecting the feedback regulation of FSH secretion (Charpenet, Taché, Bernier, Ducharme, & Collu, 1982; Orr & Mann, 1992). The lowered levels of serum testosterone with elevated levels of FSH and LH in experimental rats also indicate an intact pituitary-testicular axis. Finally, the effects observed for several end points were more pronounced with simultaneous exposure of restraint stress and alcohol stress than with either stress alone. While serving our purpose of providing reference end points, the stresses selected in

the present study clearly do not represent all environmental stresses experienced by humans and wildlife. The nature of interactions between such stresses may be more complex. What we believe to be the case, however, is that alcohol consumption/ exposure to restraint stress does possess the ability to affect endocrine signals, thereby suppressing reproduction. Our findings also reveal the possible role of restraint stress in exacerbating the testicular and epididymal toxicity when combined with alcohol treatment. Obviously, our comments are purely speculative because of the descriptive nature of the study, which warrants further work to determine the underlying molecular bases of these results. Conclusion In conclusion, this study provides compelling evidence of decreased steroidogenesis and spermatogenesis in adult rats that were exposed to either immobilization or alcohol from early puberty. The maximum suppression in steroidogenesis and spermatogenesis was observed in double-stressed rats. The possible potentiation of the effects of alcohol by exposure to restraint stress that we report here might be relevant for human risk assessment because individuals are simultaneously exposed to several stresses. Acknowledgments We are grateful to Head, Department of Zoology, S.V. University for providing necessary laboratory facilities. We are also grateful to UGC for financial support in the form of MRP to PSR. The research was conducted in accordance with the regulations in the country and approved by the Ethical Committee of S.V. University, Tirupati. References Aitken, R. J. (1994). A free radical theory of male infertility. Reproduction, Fertility, and Development, 6, 19e23. Almeida, S. A., Petenusci, S. O., Anselmo-Franci, J. A., Rosa e Silva, A. A., & LamanoCarvalho, T. L. (1998). Decreased spermatogenic and androgenic testicular functions in adult rats submitted to immobilization-induced stress from prepuberty. Brazilian Journal of Medical and Biological Research, 31, 1443e1448. Anjum, M. R., Sainath, S. B., Suneetha, Y., & Reddy, P. S. (2011). Lead acetate induced reproductive and paternal mediated developmental toxicity in rats. Ecotoxicology and Environmental Safety, 74, 793e799. Bannister, P., & Losowsky, M. S. (1987). Ethanol and hypogonadism. Alcohol and Alcoholism, 22, 213e217. Barlow, S., Kavlock, R. J., Moore, J. A., Schantz, S. L., Sheehan, D. M., Shuey, D. L., et al. (1999). Teratology Society Public Affairs Committee position paper: developmental toxicity of endocrine disruptors to humans. Teratology, 60, 365e375. Belsey, M. A., Moghissi, K. S., Eliasson, R., Paulsen, C. A., Callegos, A. J., & Prasad, M. R. (1980). Laboratory manual for the examination of human semen and semencervical mucus interaction. Singapore: Press Concern. Bents, H. (1985). Psychology of male infertility e a literature survey. International Journal of Andrology, 8, 325e336. Bergmeyer, H. U. (1974). b-hydroxysteroid dehydrogenase. In H. U. Bergmeyer (Ed.), Methods of enzymatic analysis (pp. 447e489). New York: Academic Press. Bjørneboe, A., & Bjørneboe, G. E. (1993). Antioxidant status and alcohol-related diseases. Alcohol and Alcoholism, 28, 111e116. Blazak, W. F., Treinen, K. A., & Juniewicz, P. E. (1993). Application of testicular sperm head counts in the assessment of male reproductive toxicity. In R. E. Chapin, & J. J. Heindel (Eds.), Male reproductive toxicology (pp. 86e94). New York: Academic Press.

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