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Effect of selenium on testicular damage induced by varicocele in adult male Wistar rats
Accepted Manuscript Title: Effect of selenium on testicular damage induced by varicocele in adult male Wistar rats Authors: Leila Taghizadeh, Akram Eidi, Pejman Mortazavi, Ali Haeri Rohani PII: DOI: Reference:
S0946-672X(17)30572-2 http://dx.doi.org/doi:10.1016/j.jtemb.2017.08.003 JTEMB 25955
To appear in: Received date: Revised date: Accepted date:
8-5-2017 29-6-2017 2-8-2017
Please cite this article as: Taghizadeh Leila, Eidi Akram, Mortazavi Pejman, Rohani Ali Haeri.Effect of selenium on testicular damage induced by varicocele in adult male Wistar rats.Journal of Trace Elements in Medicine and Biology http://dx.doi.org/10.1016/j.jtemb.2017.08.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effect of selenium on testicular damage induced by varicocele in adult male Wistar rats Running title: Effect of selenium on testicular damage
Leila Taghizadeha, Akram Eidia*, Pejman Mortazavib, Ali Haeri Rohania
a
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
b
Department of Pathology, Faculty of Specialized Veterinary Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran
* Corresponding author at: Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran. Tel: +98 21 44865323. Fax: +98 21 44865939. E-mail addresses:: [email protected], [email protected] (A. Eidi).
Abstract Project: Varicocele is an abnormal tortuosity and distension of the veins of the pampiniform plexus in the spermatic cord. It is the most common surgically correctable cause of male infertility. Several studies have revealed the effects of increased oxidative stress on serum, semen, and testicular tissues in patients with varicocele or in animal models. The aim of this study was to investigate the effects of sodium selenite on testicular damage induced by experimental left varicocele in male Wistar rats.
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Procedure: In the present study, the effects of oral administration of sodium selenite (at doses of 0.05, 0.1, 0.2, and 0.4 mg/kg bw) were assessed in normal and varicocelized rats. Results: The varicocelized control rats showed decrease in sperm quality parameters, decreased activity of testes CAT, GPX and SOD, increased levels of MDA, and damage in testicular architecture. Administration of sodium selenite significantly reduced these changes to nearly normal levels, but did not change these parameters in normal rats. Histopathological studies further confirmed the protective effects of sodium selenite on varicocele-induced testicular damage in rats. Administrations of sodium selenite did not change these parameters in normal rats. Conclusions: Taken together, the results of this study suggest that sodium selenite treatment may have beneficial effect on the testes of varicocelized rats.
Keywords: Selenium; Varicocele; Testis; Rat
Introduction Varicocele is defined as engorgement and dilation of the internal spermatic vein and pampiniform plexus above the testis, which leads to a retrograde reflux [1]. It more often occurs on the left side and has been considered as a major cause of male infertility [2]. Despite many clinical studies, the pathophysiology of varicocele is still a matter of controversy. Although, there are some hypotheses, such as hyperthermia, alterations in testicular blood flow, hormonal disorders, renal and/or adrenal reflux, autoimmunity, apoptosis, and oxidative stress, none of them can elucidate the process [3,4].
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Selenium is an essential trace element, which is involved in various biological processes in almost all tissues of animals and human [5]. It plays an important role in a variety of fundamental biochemical and physiological processes, including antioxidant defense [6], thyroid metabolism [7,8], immune function [9,10], antimicrobial and antiulcer effects [11], endocrine function [12], cardiovascular diseases [13], diabetes [14], Parkinson’s disease, Alzheimer’s disease [15], muscle development and function [16] and fertility in both males and females [17, 18]. Many studies have revealed that selenium has a prominent effect on reproductive function in males [19]. It can maintain normal testicular function, testicular cell structure [20], and sperm motility and function [21]. It has been reported that selenium deficiency may cause various reproductive disorders, including degeneration of the seminiferous tubules, decreased numbers of spermatozoa within the seminiferous tubules, low spermatozoa integrity, and reduced sperm motility [22]. The present study aims to investigate the therapeutic effect of sodium selenite on testicular damages induced by varicocele in adult male Wistar rats.
Materials and methods Chemicals and reagents Sodium selenite was obtained from Sigma-Aldrich Chemical Company (St. Louis, MO). Superoxide dismutase (Cu/Zn SOD) kit was purchased from Randox (Crumlin, UK). All other reagents used in the experiments were of analytical grade.
Animals
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Male Wistar rats, initially weighing 200 to 250 g, were used in this study. The animals were housed in groups of five per cage in a room with controlled temperature (22 ± 2 °C), controlled lighting (on, 7 AM; off, 7 PM), and relative air humidity (40-60%). The animals had free access to standard laboratory chow (35% carbohydrates, 25% proteins, 7% lipids, and 3% vitamins) and tap water. The diet was purchased from Pars-Dam food service, Tehran, Iran. The investigation was carried out according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and our institutional Animal Ethics Committee.
Induction of varicocele Varicocele was induced according to standard protocols [23] as follows: Each animal was intraperitoneally anesthetized by 10% ketamine hydrochloride (Alfasan- Netherland, 60 mg/kg) and xylazine (Alfasan- Netherland, 10 mg/kg); then, an abdominal midline incision was made and the left renal vein, inferior vena cava, and left spermatic vein were identified. A 4/0 silk ligature was loosely placed at the site of the left renal vein and left spermatic vein insertion over a rigid hydrophilic guide wire of 0.64 mm diameter, which was placed on the left renal vein. The ligature was loosely tied and the guide wire was removed, which resulted in immediate dilation of the left renal and left spermatic vein. The incision was sutured with a 4/0 silk ligature. All surgical procedures in the sham-operated control group were the same as those of the varicocele control group, except for the vein ligation step.
Experimental design
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Two months after varicocele induction, the rats were randomly divided into the following 11 experimental groups of 9 animals each: Group I: Normal control rats were administered 1 ml of distilled water intragastrically. Group II: Sham-operated control rats were administered 1 ml of distilled water intragastrically. Groups III-VI: Normal experimental rats were administered sodium selenite dissolved in distilled water (intragastrically, daily) at doses of 0.05, 0.1, 0.2, and 0.4 mg/kg bw, respectively. Group VII: Varicocelized control rats were administered 1 ml of distilled water intragastrically. Groups VIII-XI: Varicocelized experimental rats were administered sodium selenite dissolved in distilled water (intragastrically, daily) at doses of 0.05, 0.1, 0.2 and 0.4, mg/kg bw, respectively. The volume of administration was 1 ml and the treatments lasted for 60 consecutive days. The dosage of sodium selenite in this study was determined based on the study by Ren et al. [24]. The animals were carefully monitored every day and weighed every week. Twenty-four hours after the last treatment, all rats were weighted and anesthetized by ether, and then blood samples were directly taken from the heart. Whole blood was centrifuged at 3000 ×g for 15 min at 4 °C and sera were separated and stored at -80 °C. The abdominal wall was incised longitudinally and the whole left sided reproductive system was immediately removed and placed in a Petri-dish containing normal saline at 37 °C. Then, under a dissecting microscope, the left testis and epididymis were rapidly dissected at the vaso-epididymal junction from one end and at the surrounding tissues from the other end to be cleared from the surrounding non-testicular and non-epididymal tissues. The left testicular tissues were divided 5
into 2 pieces, one of which was homogenized for biochemical assays and another was fixed in Bouin's solution, post-fixed in 70 % alcohol, embedded in paraffin blocks, and routinely processed for histopathological study.
Sperm characteristics Sperm motility Briefly, left epididymal sperms were collected by slicing the epididymis in 5 ml of Ham's F10 medium (Sigma-Aldrich Chemical Company, St. Louis, MO) and incubated for 5 min at 37 °C in an atmosphere of 5 % CO2 to allow sperm to leave the epididymal tubules. One drop of sperm suspension was placed on a microscope slide, and a cover slip was placed over the drop. At least 10 microscopic fields were observed under 400× magnification with a phase-contrast microscope (Olympus-Germany), and the percentage of motile sperm was recorded based on WHO (2010) recommendations [25]. The motility of epididymal sperm was microscopically evaluated within 2-4 min of their isolation from the epididymis and expressed as a percentage of motile sperm of the total sperm count.
Sperm count The epididymal sperm counts were determined by the method described in the WHO manual [25]. Briefly, a 5 μl aliquot of epididymal sperm was diluted with 95 μl of diluents (0.35 % formalin containing 5 % NaHCO3 and 0.25 % trypan blue) and approximately 10 μl of this diluted specimen was transferred to each of the counting chambers of a hemocytometer, which was allowed to stand in a humid chamber for 5 min to prevent drying. The sedimented cells were counted using a light microscope (Olympus, Germany), at 400× magnification [25]. 6
Sperm viability A 20 μl of sperm suspension was mixed with the same volume of 0.05 % eosin-Y. After 2 min of incubation at room temperature, the semen specimen was carefully mixed with the stain and a thin layer was spread on a clean slide. The slides were observed by a bright-field microscope (Olympus, Germany), at 400× magnification. Dead sperms appeared pink and live sperms were not stained. Two hundred sperms were counted in each sample and viability percentages were calculated [25].
Sperm abnormalities Briefly, a drop of sperm suspension in saline was smeared on a slide and air-dried. Slides were fixed in 95 % ethanol and stained with hematoxylin and eosin. Morphological evaluation of sperm head abnormalities was performed using a binocular microscope. Three hundred sperms per animal were examined at 1000× magnification to determine the morphological abnormalities [26]. Sperm morphology was determined as described by Kvist and Björndahl [27] with some minor changes. Spermatozoa were classified based on the criteria of Wyrobek and Bruce [28] as follows: normal head, amorphous head, pinhead, hookless, and sperm with coiled or abnormal tails.
Measurement of biochemical parameters Testicular tissue was homogenized with ice-cold NaCl solution. The homogenate was centrifuged at 1700g for 15 min at 4 °C and the supernatant was used to assess oxidative stress
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parameters, including malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT). MDA levels were determined using thiobarbituric acid method by spectrophotometrically monitoring the MDA-reactive products. The absorbance of the organic layer was determined at 532 nm. Data were expressed as nanomoles of MDA per milligram of testis protein [29]. SOD activity was measured according to the instructions of the kit. A competitive inhibition assay was performed using xanthine-xanthine oxidase-generated O2 to reduce nitroblue tetrazolium (NBT) to blue formazan. One unit of SOD activity was considered as the amount of enzyme required to reduce NBT to 50 % of maximum. The maximum absorbance was read at 550 nm and the enzyme activity was expressed as unit/mg protein [30]. GPX activity was measured by a method based on the reaction between GSH remaining after the action of GPX and 5,5′-dithiobis-2-nitrobenzoic acid to produce a complex with maximal absorbance at 412 nm. One unit of GPX activity was defined as 1 mol/l per min decrease of GSH in an enzymatic reaction by 1 mg protein/min, deducting the effect of nonenzyme-catalyzed reaction [31]. CAT activity was measured by the method described by Aebi [32]. A 0.1 ml sample of the supernatant was added to a cuvette containing 1.9 ml of 50 mM phosphate buffer (pH 7.0). The reaction was initiated by adding 1.0 ml of freshly prepared 30 mM H2O2. The rate of H2O2 decomposition was spectrophotometrically measured at 240 nm. The CAT activity was expressed as unit/mg protein.
Histopathological examination
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Testis was processed totally for preparing serial histological sections. Six serial sections were prepared. Then, 5-μm-thick histological sections were stained with hematoxylin and eosin and a minimum of 50 cross-sections were assessed for each slide. The testis pathology was scored as described by Johnsen [33], as follows: Score 10 = Complete spermatogenesis and perfect tubules; Score 9 = Many spermatozoa present, but disorganized spermatogenesis; Score 8 = Only a few spermatozoa present; Score 7 = No spermatozoa but many spermatids present; Score 6 = Only a few spermatids present; Score 5 = No spermatozoa or spermatids present but many spermatocytes present; Score 4 = Only a few spermatocytes present; Score 3 = Only spermatogonia present; Score 2 = No germ cells present; Score 1 = Neither germ cells nor sertoli cells present. The morphology of the observed lesions was classified and recorded.
Statistical analysis Statistical analyses were carried out using SPSS 10 (SPSS, Chicago, IL, USA) program for Windows. Data were expressed as mean ± SEM. Data were analyzed by one-way analysis of variance followed by Tukey’s post hoc test. The criterion for statistical significance was considered as p<0.05.
Results 9
Assessment of varicocele All the rats that underwent partial ligation of the left renal vein and included in this study, had an objective dilation of the left internal spermatic vein when they were put down. No animals in the normal groups showed dilation of the left internal spermatic vein.
Effects of sodium selenite on sperm quality in normal and varicocelized rats Our results showed that induction of varicocele significantly diminished sperm viability, motility, and count compared to the normal control rats. Also, sperm abnormalities were significantly increased in varicocele-induced rats. Administration of sodium selenite caused a significant improvement in all parameters of semen quality and minimized the effects of varicocele induction. Moreover, treatment with sodium selenite caused no significant enhancement in sperm characteristics in normal rats (Table 1).
Effects of sodium selenite on testicular oxidative markers in normal and varicocelized rats Our data indicated that induction of varicocele significantly decreased activity of testes CAT, GPX and SOD, and increased the levels of MDA compared to normal control animals. While, varicocelized rats treated with sodium selenite showed significant increase in CAT, GPX, and SOD activities and decrease in MDA levels compared to varicocelized control rats. Moreover, administration of sodium selenite caused no significant alteration in CAT, GPX, and SOD activities and MDA levels in normal rats (Figs. 1-4).
Effects of sodium selenite on histopathological evaluation in normal and varicocelized rats
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The testis tissue architecture in the normal control rats showed normal spermatogenesis with normal architecture and cells, whereas disintegration of the germinal epithelium, disorganization, and shrinkage of seminiferous tubules were observed in the varicocele-induced rats. In some tubules, there were noncohesive and less orderly germ cells, which sloughed into the seminiferous tubules’ lumen, and spermatogenesis was prevented. The mean Johnsen’s scores of all groups are shown in Table 2. The mean Johnsen’s score was significantly lower in the varicocele control group compared to the normal control groups. The administration of sodium selenite at doses of 0.1, 0.2, and 0.4 mg/kg prevented this histological alteration and significantly increased the mean Johnsen’s score in varicocele-induced rats (Fig. 5 and Table 2).
Discussion In the present study, we indicated that sodium selenite could dose-dependently reduce changes of sperm quality parameters, increased activities of CAT, GPX, and SOD, decreased MDA levels, and attenuated testicular architecture damage in varicocele-induced testicular damage rats. Based on our results, administration of sodium selenite in varicocelized rats caused histological and biochemical improvements. There were no significant alterations in normal rats treated with sodium selenite. Our results showed that treatment with sodium selenite, even at high concentration (0.4 mg/kg), caused no toxicity symptom, and there was no record of death. Symptoms of selenium toxicity include nausea, brittleness and loss; nail discoloration, hair loss; irritability; fatigue; and foul breath odor (described as “garlic breath”) [34]. Also, histopathology of kidney and liver was evaluated. Our result showed that administration of sodium selenite caused no damage in kidney and liver tissue. This suggests that sodium selenite even at the dose of 0.4 mg/kg is not toxic to 11
rats. A small deviation from normal levels of selenium can have severe consequences. It has been reported that selenium toxicity can result from acute or chronic ingestion of excess selenium. In the EU, the permitted level of selenium supplementation in animal feeds is 0.5 mg/kg, whereas 0.1-0.3 mg/kg selenium is permitted by Polish Swine feeding standards [35]. In confirmation of the selenium doses used in this study, the use of higher doses of selenium without toxic effects has been reported. It is reported that administration of selenium at a dose of 0.945 mg/kg for 60 days has hepatoprotective effects [36]. Also, Hamza and AL-Harbi suggested that administration of selenium at a dose of 1 mg/kg for 30 days could ameliorate the glutamateinduced testicular toxicity reduce the oxidative stress on testis tissues [37]. In the present study, we successfully created experimental left varicocele in rats. Varicocele more frequently occurs on the left side. Anatomically, the left testicular vein is longer than the right one and enters the left renal vein at a right angle. In addition, it is occasionally compressed between the descending aorta and the upper mesenteric artery, causing a “nutcracker” effect. The subsequent increase in renal vein pressure would be transmitted to the testicular vein, which explains the reason why left-sided varicocele is more common than right-sided varicocele [3]. Varicocele-associated infertility is related to many factors, such as histological alterations, molecular/genetic changes (e.g., microdeletion of Y chromosome) [38], glutathione-S-transferase M1 gene polymorphism, high levels of testicular apoptosis defects in fibroblast-associated (Fas)ligand activity that regulates apoptosis at the level of plasma membrane, caspase activity at the cytoplasmic level or gene (Bax/Bcl-2) expression at the nuclear level, and expression of heme oxygenase isoenzyme-1 (HO-1) on Leydig cell [39,40]. Changes in the testicular microenvironment and hemodynamics can increase the production of ROS (reactive oxygen species) and/or decrease the local antioxidant capacity, which lead to oxidative stress. It has been 12
suggested that spermatozoal dysfunction in varicocele patients can be multifactorial, and oxidative stress-induced injury may be one of the main causes [3]. According to a review by Kim and Goldstein [41] on the role of oxidative stress in varicocele, similar to other alternative pathophysiological mechanisms, varicocele not only affects seminiferous tubular function, but also Leydig cell and androgen production. In particular, the cytoplasmic droplets (excess residual cytoplasm) found within the seminiferous tubules [42] of infertile patients with varicocele [43,44] are a major source of ROS. Considering that Zini et al. [45] have also indicated that the retention of sperm cytoplasmic droplets is negatively correlated with sperm motility, it appears that the release of spermatozoa that have not undergone the normal epididymal maturation may play a role in decreased sperm function in individuals with varicocele. The potential causative role of ROS in varicocele-associated male infertility is still a matter of intensive clinical research. For instance, in infertile men with varicocele, the percentage of spermatozoa with DNA damage due to high levels of ROS, was increased compared to healthy subjects [46, 47]. On the other hand, similar levels of ROS production were also reported in fertile donors with clinical diagnosis of varicocele [48]. In the present study, it was indicated that experimental varicocele caused a significant decrease in the sperm quality compared to the normal control group, which attenuated with administration of sodium selenite. Varicocele-induced sperm profile damage was confirmed by increase in the number of dead sperm and morphologically sperm abnormalities along with decrease in sperm count and motility. Reductions in sperm motility and count, were also observed in varicocele control group compared to the normal control group, which was similar to the results of other studies [49,50]. For example, it was reported that all sperm parameters were lower in patients with varicocele [51]. Our results showed that administration of sodium selenite 13
significantly ameliorated these changes. It has been reported that appropriate doses of selenium in the diet can increase motility of bovine spermatozoa [52]. In addition, dietary selenium affects sperm production as well as semen production and quality in boars and men [53-55]. In agreement with our results, Marai et al. [56] reported that sodium selenite improved semen quality in rams through increasing semen volume per ejaculate, spermatozoa motility and concentration and through decreasing the percentage of dead spermatozoa, spermatozoa abnormalities, and acrosomal damage. It has been reported that selenium improves male reproductive performance by enhancing semen quality and also by suppressing free radicals [57]. Selenium was also found to play a major role in sperm motility and male fertility through upregulating the CatSper genes expression in sperm, which are a family of sperm cation channels that play a major role in sperm motility and male fertility [58]. In the present study, examination of the testes homogenates of varicocelized rats, showed a significant reduction in the activities of the antioxidant enzymes (SOD, CAT, and GPX) and increased MDA levels. Our findings showed that administration of sodium selenite decreased MDA levels and increased the antioxidant enzymes activities. Jana et al. [59], reported that sodium selenite supplementation significantly protected against exercise-induced testicular gametogenic and spermatogenic disorders, prevented testicular oxidative stress, and elevated antioxidant status. Grotto et al. [60] investigated the possible antigenotoxic effect of selenium in rats that were chronically exposed to low levels of methylmercury. It was found that selenium co-administration significantly reduced methylmercury-induced genotoxicity, restored GPX activity, and decreased DNA damage. It is known that GPX prevents free radical generation during oxidative stress, reduces hydrogen peroxide to water or lipid hydroperoxides to alcohols, mediates disulphide bridging, and its failure is related to infertility [61-63]. Kara et al. [64] 14
revealed that selenium could protect rat testes against cadmium-induced oxidative damage. On the contrary, in a study by Adesiyan et al. [65], it was reported that selenium reduced the toxic effects of atrazine-induced liver change, but showed no protective effects against biochemical changes in testis and epididymis, except for testicular lactate dehydrogenase. As expected, the histological damage of the testis was associated with the presence of free radical oxidative stress in the testis, as confirmed by significant decrease in antioxidant enzymes levels. Testes in varicocelized rats had histological changes, such as severe damage in the seminiferous tubules and vascular degeneration on the spermatogenic and sertoli cells cytoplasm. Some places of the germinal epithelium of the seminiferous tubules were thinner and spermatids were almost absent, and sperm count was low in the lumen. Testes of varicocelized rats treated with sodium selenite nearly restored its normal structure and the normal picture of seminiferous tubules was significantly regained. The histological study of the varicocele testis showed that all the cell types and compartments of the testis can be involved [66,67]. The effect of varicocele on the leydig cells was hyperplasia [68,69]. These histological changes are also typical of changes seen in the adolescent with varicocele [70,72]. It is known that varicocele causes impaired spermatogenesis, which may result in reduction of tubular volume. In agreement with our findings, concomitant antioxidant and reproduction stimulatory effects of selenium-based agents, have been reported in several models of toxic stress-related reproductive disorders in both males and females [61,72-78]. It has been well documented that selenium protects cells from oxidative stress [79-82] by expression of selenoprotein genes [83] and anti-inflammatory mechanisms [84, 85]. Selenium as a central component of GSH-Px and selenoproteins plays an important role in antioxidant defense systems and degrades hydroperoxides and hydrogen peroxide. Also, testis has a priority to retain and utilize selenium, and therefore decreased selenium level in testis may 15
cause spermatozoa abnormality and membrane damage [86]. Therefore, selenium is essential for normal testicular function and has a major role in male fertility, both structurally and enzymatically, so that selenium-deficient subjects have low sperm production as well as decreased plasma testosterone level [87]. The pathophysiology of varicocele is not yet clear. It has been reported that one of the hypotheses of complication events following varicocele is free radical production [3]. It is suggested that free radical increase in varicocele causes testicular tissue damage, disruption of sperm profile, and biochemical changes [41-44]. On the other hand, selenium is able to mitigate the damage caused by its antioxidant effect [57]. It is claimed that selenium significantly prevented testicular oxidative stress, and elevated antioxidant status [79-82]. Based on the results of present study, sodium selenite, possibly due to its antioxidant effect, can reduce varicoceleinduced testicular damage.
Conclusion The results of this study indicate that selenium treatment can reduce the varicocele-induced testicular damage in rats. This effect of selenium may be attributed to its antioxidant properties hence it enhances sperm profile, activities of the antioxidant enzymes (CAT, SOD, and GPX) and reduces the level of lipid peroxidation (MDA).
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26
Figure 1.
27
Figure 2.
28
Figure 3.
29
Figure 4.
30
31
Figure 5
32
Figure 5. Histopathology of testicular tissue in experimental groups. (A) testis in group I shows normal histology of seminiferous tubule with sertoli cell (black arrow), spermatogonium (white arrow), spermatocyte (black arrowhead), and spermatozoa (white arrowhead); (B) testis in group II shows normal histology of seminiferous tubule with sertoli cell (white arrow), spermatogonium (black arrow), spermatocyte (white arrowhead), and spermatozoa (black arrowhead); (C-F) testis in groups III-VI shows normal histology of seminiferous tubule with sertoli cell (black arrow), spermatogonium (white arrow), spermatocyte (black arrowhead), and spermatozoa (white arrowhead); (G) testis in group VII shows degeneration and shrinkage of seminiferous tubules with loss of spermatogenesis and only presence of spermatogonia (arrowhead); (H-K) testis in groups VIII-XI shows an improvement in spermatogenesis and seminiferous tubule organization, so that primary spermatocyte (arrow in Fig. H), secondary spermatocyte (arrowhead in Fig. H), spermatocyte (arrowhead in Fig. I), spermatid (arrow in Figs. I and J) and spermatozoa (arrow in Fig. K) are seen (H&E).
33
Table 1- Effect of sodium selenite on sperm count and quality in normal and varicocele-induced rats (n=9) Sperm count
Groups
Viability (%)
Sperm motility (%)
(× million)
Sperm abnormalities (%)
Group I
[Normal control]
73.62 ± 1.69
90.01 ± 0.89
75.60 ± 1.42
13.61 ± 2.56
Group II
[Sham-operated control]
72.14 ± 1.28
91.18 ± 0.72
76.19 ± 1.90
14.24 ± 1.90
Group III [Normal + sodium selenite (0.05 mg/kg)]
70.06 ± 1.09
88.05 ± 0.58
74.06 ± 1.00
13.85 ± 1.64
Group IV
[Normal + sodium selenite (0.1 mg/kg)]
85.62 ± 1.18
88.80 ± 0.58
77.20 ± 0.86
13.10 ± 1.92
Group V
[Normal + sodium selenite (0.2 mg/kg)]
74.50 ± 4.55
87.40 ± 0.51
77.03 ± 3.48
14.82 ± 1.58
Group VI [Normal + sodium selenite (0.4 mg/kg)]
71.65 ± 0.94
89.09 ± 0.89
76.60 ± 2.22
13.75 ± 2.06
Group VII [Varicocelized control]
35.25 ± 3.52٭٭٭
40.83 ± 2.87 ٭٭٭
54.08 ± 0.71 ٭٭٭
32.58 ± 3.91 ***
Group VIII [Varicocelic + sodium selenite (0.05 mg/kg)]
40.03 ± 1.03٭٭٭
45.03 ± 1.87 ٭٭٭
56.05 ± 1.07 ٭٭٭
24.06 ± 2.84 ***
Group IX [Varicocelic + sodium selenite (0.1 mg/kg)]
66.33 ± 0.81 +++
80.33 ± 1.87٭٭+++
76.11 ± 0.68 +++
19.81 ± 1.61 +++
Group X
63.70 ± 2.53 +++
81.20 ± 0.66 ٭٭+++
73.80 ± 0.73 ٭٭+
15.39 ± 1.78 +++
54.16 ± 2.24 ٭٭٭+
74.66 ± 1.02 ٭٭٭+++
73.49 ± 2.34 ٭٭+
16.47 ± 1.83 +++
[Varicocelic + sodium selenite (0.2 mg/kg)]
Group XI [Varicocelic + sodium selenite (0.4 mg/kg)] Values are mean ± S.E.M.
** p<0.01, *** p<0.001 significantly different from the normal control group. + p<0.05, ++ p<0.01, +++ p<0.001 significantly different from the varicocele control group.
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Table 2- Effect of sodium selenite on histopathological evaluation of testis in normal and varicocele-induced rats (n=9) Johnsen’s scores
Groups Group I
[Normal control]
10
Group II
[Sham-operated control]
10
Group III [Normal + sodium selenite (0.05 mg/kg)]
10
Group IV
[Normal + sodium selenite (0.1 mg/kg)]
10
Group V
[Normal + sodium selenite (0.2 mg/kg)]
10
Group VI [Normal + sodium selenite (0.4 mg/kg)]
10
Group VII [Varicocelized control]
2.87 ± 0.6٭٭٭
Group VIII [Varicocelic + sodium selenite (0.05 mg/kg)]
4.61 ± 0.5٭٭٭
Group IX [Varicocelic + sodium selenite (0.1 mg/kg)]
5.63 ± 0.5 ٭٭٭+++
Group X
[Varicocelic + sodium selenite (0.2 mg/kg)]
6.72 ± 0.4٭٭٭+++
Group XI [Varicocelic + sodium selenite (0.4 mg/kg)]
7.87 ± 0.6٭٭٭+++
Values are mean ± S.E.M. ** p<0.01, *** p<0.001 significantly different from the normal control group. + p<0.05, ++ p<0.01, +++ p<0.001 significantly different from the varicocele control group.
35
S0946-672X(17)30572-2 http://dx.doi.org/doi:10.1016/j.jtemb.2017.08.003 JTEMB 25955
To appear in: Received date: Revised date: Accepted date:
8-5-2017 29-6-2017 2-8-2017
Please cite this article as: Taghizadeh Leila, Eidi Akram, Mortazavi Pejman, Rohani Ali Haeri.Effect of selenium on testicular damage induced by varicocele in adult male Wistar rats.Journal of Trace Elements in Medicine and Biology http://dx.doi.org/10.1016/j.jtemb.2017.08.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effect of selenium on testicular damage induced by varicocele in adult male Wistar rats Running title: Effect of selenium on testicular damage
Leila Taghizadeha, Akram Eidia*, Pejman Mortazavib, Ali Haeri Rohania
a
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
b
Department of Pathology, Faculty of Specialized Veterinary Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran
* Corresponding author at: Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran. Tel: +98 21 44865323. Fax: +98 21 44865939. E-mail addresses:: [email protected], [email protected] (A. Eidi).
Abstract Project: Varicocele is an abnormal tortuosity and distension of the veins of the pampiniform plexus in the spermatic cord. It is the most common surgically correctable cause of male infertility. Several studies have revealed the effects of increased oxidative stress on serum, semen, and testicular tissues in patients with varicocele or in animal models. The aim of this study was to investigate the effects of sodium selenite on testicular damage induced by experimental left varicocele in male Wistar rats.
1
Procedure: In the present study, the effects of oral administration of sodium selenite (at doses of 0.05, 0.1, 0.2, and 0.4 mg/kg bw) were assessed in normal and varicocelized rats. Results: The varicocelized control rats showed decrease in sperm quality parameters, decreased activity of testes CAT, GPX and SOD, increased levels of MDA, and damage in testicular architecture. Administration of sodium selenite significantly reduced these changes to nearly normal levels, but did not change these parameters in normal rats. Histopathological studies further confirmed the protective effects of sodium selenite on varicocele-induced testicular damage in rats. Administrations of sodium selenite did not change these parameters in normal rats. Conclusions: Taken together, the results of this study suggest that sodium selenite treatment may have beneficial effect on the testes of varicocelized rats.
Keywords: Selenium; Varicocele; Testis; Rat
Introduction Varicocele is defined as engorgement and dilation of the internal spermatic vein and pampiniform plexus above the testis, which leads to a retrograde reflux [1]. It more often occurs on the left side and has been considered as a major cause of male infertility [2]. Despite many clinical studies, the pathophysiology of varicocele is still a matter of controversy. Although, there are some hypotheses, such as hyperthermia, alterations in testicular blood flow, hormonal disorders, renal and/or adrenal reflux, autoimmunity, apoptosis, and oxidative stress, none of them can elucidate the process [3,4].
2
Selenium is an essential trace element, which is involved in various biological processes in almost all tissues of animals and human [5]. It plays an important role in a variety of fundamental biochemical and physiological processes, including antioxidant defense [6], thyroid metabolism [7,8], immune function [9,10], antimicrobial and antiulcer effects [11], endocrine function [12], cardiovascular diseases [13], diabetes [14], Parkinson’s disease, Alzheimer’s disease [15], muscle development and function [16] and fertility in both males and females [17, 18]. Many studies have revealed that selenium has a prominent effect on reproductive function in males [19]. It can maintain normal testicular function, testicular cell structure [20], and sperm motility and function [21]. It has been reported that selenium deficiency may cause various reproductive disorders, including degeneration of the seminiferous tubules, decreased numbers of spermatozoa within the seminiferous tubules, low spermatozoa integrity, and reduced sperm motility [22]. The present study aims to investigate the therapeutic effect of sodium selenite on testicular damages induced by varicocele in adult male Wistar rats.
Materials and methods Chemicals and reagents Sodium selenite was obtained from Sigma-Aldrich Chemical Company (St. Louis, MO). Superoxide dismutase (Cu/Zn SOD) kit was purchased from Randox (Crumlin, UK). All other reagents used in the experiments were of analytical grade.
Animals
3
Male Wistar rats, initially weighing 200 to 250 g, were used in this study. The animals were housed in groups of five per cage in a room with controlled temperature (22 ± 2 °C), controlled lighting (on, 7 AM; off, 7 PM), and relative air humidity (40-60%). The animals had free access to standard laboratory chow (35% carbohydrates, 25% proteins, 7% lipids, and 3% vitamins) and tap water. The diet was purchased from Pars-Dam food service, Tehran, Iran. The investigation was carried out according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and our institutional Animal Ethics Committee.
Induction of varicocele Varicocele was induced according to standard protocols [23] as follows: Each animal was intraperitoneally anesthetized by 10% ketamine hydrochloride (Alfasan- Netherland, 60 mg/kg) and xylazine (Alfasan- Netherland, 10 mg/kg); then, an abdominal midline incision was made and the left renal vein, inferior vena cava, and left spermatic vein were identified. A 4/0 silk ligature was loosely placed at the site of the left renal vein and left spermatic vein insertion over a rigid hydrophilic guide wire of 0.64 mm diameter, which was placed on the left renal vein. The ligature was loosely tied and the guide wire was removed, which resulted in immediate dilation of the left renal and left spermatic vein. The incision was sutured with a 4/0 silk ligature. All surgical procedures in the sham-operated control group were the same as those of the varicocele control group, except for the vein ligation step.
Experimental design
4
Two months after varicocele induction, the rats were randomly divided into the following 11 experimental groups of 9 animals each: Group I: Normal control rats were administered 1 ml of distilled water intragastrically. Group II: Sham-operated control rats were administered 1 ml of distilled water intragastrically. Groups III-VI: Normal experimental rats were administered sodium selenite dissolved in distilled water (intragastrically, daily) at doses of 0.05, 0.1, 0.2, and 0.4 mg/kg bw, respectively. Group VII: Varicocelized control rats were administered 1 ml of distilled water intragastrically. Groups VIII-XI: Varicocelized experimental rats were administered sodium selenite dissolved in distilled water (intragastrically, daily) at doses of 0.05, 0.1, 0.2 and 0.4, mg/kg bw, respectively. The volume of administration was 1 ml and the treatments lasted for 60 consecutive days. The dosage of sodium selenite in this study was determined based on the study by Ren et al. [24]. The animals were carefully monitored every day and weighed every week. Twenty-four hours after the last treatment, all rats were weighted and anesthetized by ether, and then blood samples were directly taken from the heart. Whole blood was centrifuged at 3000 ×g for 15 min at 4 °C and sera were separated and stored at -80 °C. The abdominal wall was incised longitudinally and the whole left sided reproductive system was immediately removed and placed in a Petri-dish containing normal saline at 37 °C. Then, under a dissecting microscope, the left testis and epididymis were rapidly dissected at the vaso-epididymal junction from one end and at the surrounding tissues from the other end to be cleared from the surrounding non-testicular and non-epididymal tissues. The left testicular tissues were divided 5
into 2 pieces, one of which was homogenized for biochemical assays and another was fixed in Bouin's solution, post-fixed in 70 % alcohol, embedded in paraffin blocks, and routinely processed for histopathological study.
Sperm characteristics Sperm motility Briefly, left epididymal sperms were collected by slicing the epididymis in 5 ml of Ham's F10 medium (Sigma-Aldrich Chemical Company, St. Louis, MO) and incubated for 5 min at 37 °C in an atmosphere of 5 % CO2 to allow sperm to leave the epididymal tubules. One drop of sperm suspension was placed on a microscope slide, and a cover slip was placed over the drop. At least 10 microscopic fields were observed under 400× magnification with a phase-contrast microscope (Olympus-Germany), and the percentage of motile sperm was recorded based on WHO (2010) recommendations [25]. The motility of epididymal sperm was microscopically evaluated within 2-4 min of their isolation from the epididymis and expressed as a percentage of motile sperm of the total sperm count.
Sperm count The epididymal sperm counts were determined by the method described in the WHO manual [25]. Briefly, a 5 μl aliquot of epididymal sperm was diluted with 95 μl of diluents (0.35 % formalin containing 5 % NaHCO3 and 0.25 % trypan blue) and approximately 10 μl of this diluted specimen was transferred to each of the counting chambers of a hemocytometer, which was allowed to stand in a humid chamber for 5 min to prevent drying. The sedimented cells were counted using a light microscope (Olympus, Germany), at 400× magnification [25]. 6
Sperm viability A 20 μl of sperm suspension was mixed with the same volume of 0.05 % eosin-Y. After 2 min of incubation at room temperature, the semen specimen was carefully mixed with the stain and a thin layer was spread on a clean slide. The slides were observed by a bright-field microscope (Olympus, Germany), at 400× magnification. Dead sperms appeared pink and live sperms were not stained. Two hundred sperms were counted in each sample and viability percentages were calculated [25].
Sperm abnormalities Briefly, a drop of sperm suspension in saline was smeared on a slide and air-dried. Slides were fixed in 95 % ethanol and stained with hematoxylin and eosin. Morphological evaluation of sperm head abnormalities was performed using a binocular microscope. Three hundred sperms per animal were examined at 1000× magnification to determine the morphological abnormalities [26]. Sperm morphology was determined as described by Kvist and Björndahl [27] with some minor changes. Spermatozoa were classified based on the criteria of Wyrobek and Bruce [28] as follows: normal head, amorphous head, pinhead, hookless, and sperm with coiled or abnormal tails.
Measurement of biochemical parameters Testicular tissue was homogenized with ice-cold NaCl solution. The homogenate was centrifuged at 1700g for 15 min at 4 °C and the supernatant was used to assess oxidative stress
7
parameters, including malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT). MDA levels were determined using thiobarbituric acid method by spectrophotometrically monitoring the MDA-reactive products. The absorbance of the organic layer was determined at 532 nm. Data were expressed as nanomoles of MDA per milligram of testis protein [29]. SOD activity was measured according to the instructions of the kit. A competitive inhibition assay was performed using xanthine-xanthine oxidase-generated O2 to reduce nitroblue tetrazolium (NBT) to blue formazan. One unit of SOD activity was considered as the amount of enzyme required to reduce NBT to 50 % of maximum. The maximum absorbance was read at 550 nm and the enzyme activity was expressed as unit/mg protein [30]. GPX activity was measured by a method based on the reaction between GSH remaining after the action of GPX and 5,5′-dithiobis-2-nitrobenzoic acid to produce a complex with maximal absorbance at 412 nm. One unit of GPX activity was defined as 1 mol/l per min decrease of GSH in an enzymatic reaction by 1 mg protein/min, deducting the effect of nonenzyme-catalyzed reaction [31]. CAT activity was measured by the method described by Aebi [32]. A 0.1 ml sample of the supernatant was added to a cuvette containing 1.9 ml of 50 mM phosphate buffer (pH 7.0). The reaction was initiated by adding 1.0 ml of freshly prepared 30 mM H2O2. The rate of H2O2 decomposition was spectrophotometrically measured at 240 nm. The CAT activity was expressed as unit/mg protein.
Histopathological examination
8
Testis was processed totally for preparing serial histological sections. Six serial sections were prepared. Then, 5-μm-thick histological sections were stained with hematoxylin and eosin and a minimum of 50 cross-sections were assessed for each slide. The testis pathology was scored as described by Johnsen [33], as follows: Score 10 = Complete spermatogenesis and perfect tubules; Score 9 = Many spermatozoa present, but disorganized spermatogenesis; Score 8 = Only a few spermatozoa present; Score 7 = No spermatozoa but many spermatids present; Score 6 = Only a few spermatids present; Score 5 = No spermatozoa or spermatids present but many spermatocytes present; Score 4 = Only a few spermatocytes present; Score 3 = Only spermatogonia present; Score 2 = No germ cells present; Score 1 = Neither germ cells nor sertoli cells present. The morphology of the observed lesions was classified and recorded.
Statistical analysis Statistical analyses were carried out using SPSS 10 (SPSS, Chicago, IL, USA) program for Windows. Data were expressed as mean ± SEM. Data were analyzed by one-way analysis of variance followed by Tukey’s post hoc test. The criterion for statistical significance was considered as p<0.05.
Results 9
Assessment of varicocele All the rats that underwent partial ligation of the left renal vein and included in this study, had an objective dilation of the left internal spermatic vein when they were put down. No animals in the normal groups showed dilation of the left internal spermatic vein.
Effects of sodium selenite on sperm quality in normal and varicocelized rats Our results showed that induction of varicocele significantly diminished sperm viability, motility, and count compared to the normal control rats. Also, sperm abnormalities were significantly increased in varicocele-induced rats. Administration of sodium selenite caused a significant improvement in all parameters of semen quality and minimized the effects of varicocele induction. Moreover, treatment with sodium selenite caused no significant enhancement in sperm characteristics in normal rats (Table 1).
Effects of sodium selenite on testicular oxidative markers in normal and varicocelized rats Our data indicated that induction of varicocele significantly decreased activity of testes CAT, GPX and SOD, and increased the levels of MDA compared to normal control animals. While, varicocelized rats treated with sodium selenite showed significant increase in CAT, GPX, and SOD activities and decrease in MDA levels compared to varicocelized control rats. Moreover, administration of sodium selenite caused no significant alteration in CAT, GPX, and SOD activities and MDA levels in normal rats (Figs. 1-4).
Effects of sodium selenite on histopathological evaluation in normal and varicocelized rats
10
The testis tissue architecture in the normal control rats showed normal spermatogenesis with normal architecture and cells, whereas disintegration of the germinal epithelium, disorganization, and shrinkage of seminiferous tubules were observed in the varicocele-induced rats. In some tubules, there were noncohesive and less orderly germ cells, which sloughed into the seminiferous tubules’ lumen, and spermatogenesis was prevented. The mean Johnsen’s scores of all groups are shown in Table 2. The mean Johnsen’s score was significantly lower in the varicocele control group compared to the normal control groups. The administration of sodium selenite at doses of 0.1, 0.2, and 0.4 mg/kg prevented this histological alteration and significantly increased the mean Johnsen’s score in varicocele-induced rats (Fig. 5 and Table 2).
Discussion In the present study, we indicated that sodium selenite could dose-dependently reduce changes of sperm quality parameters, increased activities of CAT, GPX, and SOD, decreased MDA levels, and attenuated testicular architecture damage in varicocele-induced testicular damage rats. Based on our results, administration of sodium selenite in varicocelized rats caused histological and biochemical improvements. There were no significant alterations in normal rats treated with sodium selenite. Our results showed that treatment with sodium selenite, even at high concentration (0.4 mg/kg), caused no toxicity symptom, and there was no record of death. Symptoms of selenium toxicity include nausea, brittleness and loss; nail discoloration, hair loss; irritability; fatigue; and foul breath odor (described as “garlic breath”) [34]. Also, histopathology of kidney and liver was evaluated. Our result showed that administration of sodium selenite caused no damage in kidney and liver tissue. This suggests that sodium selenite even at the dose of 0.4 mg/kg is not toxic to 11
rats. A small deviation from normal levels of selenium can have severe consequences. It has been reported that selenium toxicity can result from acute or chronic ingestion of excess selenium. In the EU, the permitted level of selenium supplementation in animal feeds is 0.5 mg/kg, whereas 0.1-0.3 mg/kg selenium is permitted by Polish Swine feeding standards [35]. In confirmation of the selenium doses used in this study, the use of higher doses of selenium without toxic effects has been reported. It is reported that administration of selenium at a dose of 0.945 mg/kg for 60 days has hepatoprotective effects [36]. Also, Hamza and AL-Harbi suggested that administration of selenium at a dose of 1 mg/kg for 30 days could ameliorate the glutamateinduced testicular toxicity reduce the oxidative stress on testis tissues [37]. In the present study, we successfully created experimental left varicocele in rats. Varicocele more frequently occurs on the left side. Anatomically, the left testicular vein is longer than the right one and enters the left renal vein at a right angle. In addition, it is occasionally compressed between the descending aorta and the upper mesenteric artery, causing a “nutcracker” effect. The subsequent increase in renal vein pressure would be transmitted to the testicular vein, which explains the reason why left-sided varicocele is more common than right-sided varicocele [3]. Varicocele-associated infertility is related to many factors, such as histological alterations, molecular/genetic changes (e.g., microdeletion of Y chromosome) [38], glutathione-S-transferase M1 gene polymorphism, high levels of testicular apoptosis defects in fibroblast-associated (Fas)ligand activity that regulates apoptosis at the level of plasma membrane, caspase activity at the cytoplasmic level or gene (Bax/Bcl-2) expression at the nuclear level, and expression of heme oxygenase isoenzyme-1 (HO-1) on Leydig cell [39,40]. Changes in the testicular microenvironment and hemodynamics can increase the production of ROS (reactive oxygen species) and/or decrease the local antioxidant capacity, which lead to oxidative stress. It has been 12
suggested that spermatozoal dysfunction in varicocele patients can be multifactorial, and oxidative stress-induced injury may be one of the main causes [3]. According to a review by Kim and Goldstein [41] on the role of oxidative stress in varicocele, similar to other alternative pathophysiological mechanisms, varicocele not only affects seminiferous tubular function, but also Leydig cell and androgen production. In particular, the cytoplasmic droplets (excess residual cytoplasm) found within the seminiferous tubules [42] of infertile patients with varicocele [43,44] are a major source of ROS. Considering that Zini et al. [45] have also indicated that the retention of sperm cytoplasmic droplets is negatively correlated with sperm motility, it appears that the release of spermatozoa that have not undergone the normal epididymal maturation may play a role in decreased sperm function in individuals with varicocele. The potential causative role of ROS in varicocele-associated male infertility is still a matter of intensive clinical research. For instance, in infertile men with varicocele, the percentage of spermatozoa with DNA damage due to high levels of ROS, was increased compared to healthy subjects [46, 47]. On the other hand, similar levels of ROS production were also reported in fertile donors with clinical diagnosis of varicocele [48]. In the present study, it was indicated that experimental varicocele caused a significant decrease in the sperm quality compared to the normal control group, which attenuated with administration of sodium selenite. Varicocele-induced sperm profile damage was confirmed by increase in the number of dead sperm and morphologically sperm abnormalities along with decrease in sperm count and motility. Reductions in sperm motility and count, were also observed in varicocele control group compared to the normal control group, which was similar to the results of other studies [49,50]. For example, it was reported that all sperm parameters were lower in patients with varicocele [51]. Our results showed that administration of sodium selenite 13
significantly ameliorated these changes. It has been reported that appropriate doses of selenium in the diet can increase motility of bovine spermatozoa [52]. In addition, dietary selenium affects sperm production as well as semen production and quality in boars and men [53-55]. In agreement with our results, Marai et al. [56] reported that sodium selenite improved semen quality in rams through increasing semen volume per ejaculate, spermatozoa motility and concentration and through decreasing the percentage of dead spermatozoa, spermatozoa abnormalities, and acrosomal damage. It has been reported that selenium improves male reproductive performance by enhancing semen quality and also by suppressing free radicals [57]. Selenium was also found to play a major role in sperm motility and male fertility through upregulating the CatSper genes expression in sperm, which are a family of sperm cation channels that play a major role in sperm motility and male fertility [58]. In the present study, examination of the testes homogenates of varicocelized rats, showed a significant reduction in the activities of the antioxidant enzymes (SOD, CAT, and GPX) and increased MDA levels. Our findings showed that administration of sodium selenite decreased MDA levels and increased the antioxidant enzymes activities. Jana et al. [59], reported that sodium selenite supplementation significantly protected against exercise-induced testicular gametogenic and spermatogenic disorders, prevented testicular oxidative stress, and elevated antioxidant status. Grotto et al. [60] investigated the possible antigenotoxic effect of selenium in rats that were chronically exposed to low levels of methylmercury. It was found that selenium co-administration significantly reduced methylmercury-induced genotoxicity, restored GPX activity, and decreased DNA damage. It is known that GPX prevents free radical generation during oxidative stress, reduces hydrogen peroxide to water or lipid hydroperoxides to alcohols, mediates disulphide bridging, and its failure is related to infertility [61-63]. Kara et al. [64] 14
revealed that selenium could protect rat testes against cadmium-induced oxidative damage. On the contrary, in a study by Adesiyan et al. [65], it was reported that selenium reduced the toxic effects of atrazine-induced liver change, but showed no protective effects against biochemical changes in testis and epididymis, except for testicular lactate dehydrogenase. As expected, the histological damage of the testis was associated with the presence of free radical oxidative stress in the testis, as confirmed by significant decrease in antioxidant enzymes levels. Testes in varicocelized rats had histological changes, such as severe damage in the seminiferous tubules and vascular degeneration on the spermatogenic and sertoli cells cytoplasm. Some places of the germinal epithelium of the seminiferous tubules were thinner and spermatids were almost absent, and sperm count was low in the lumen. Testes of varicocelized rats treated with sodium selenite nearly restored its normal structure and the normal picture of seminiferous tubules was significantly regained. The histological study of the varicocele testis showed that all the cell types and compartments of the testis can be involved [66,67]. The effect of varicocele on the leydig cells was hyperplasia [68,69]. These histological changes are also typical of changes seen in the adolescent with varicocele [70,72]. It is known that varicocele causes impaired spermatogenesis, which may result in reduction of tubular volume. In agreement with our findings, concomitant antioxidant and reproduction stimulatory effects of selenium-based agents, have been reported in several models of toxic stress-related reproductive disorders in both males and females [61,72-78]. It has been well documented that selenium protects cells from oxidative stress [79-82] by expression of selenoprotein genes [83] and anti-inflammatory mechanisms [84, 85]. Selenium as a central component of GSH-Px and selenoproteins plays an important role in antioxidant defense systems and degrades hydroperoxides and hydrogen peroxide. Also, testis has a priority to retain and utilize selenium, and therefore decreased selenium level in testis may 15
cause spermatozoa abnormality and membrane damage [86]. Therefore, selenium is essential for normal testicular function and has a major role in male fertility, both structurally and enzymatically, so that selenium-deficient subjects have low sperm production as well as decreased plasma testosterone level [87]. The pathophysiology of varicocele is not yet clear. It has been reported that one of the hypotheses of complication events following varicocele is free radical production [3]. It is suggested that free radical increase in varicocele causes testicular tissue damage, disruption of sperm profile, and biochemical changes [41-44]. On the other hand, selenium is able to mitigate the damage caused by its antioxidant effect [57]. It is claimed that selenium significantly prevented testicular oxidative stress, and elevated antioxidant status [79-82]. Based on the results of present study, sodium selenite, possibly due to its antioxidant effect, can reduce varicoceleinduced testicular damage.
Conclusion The results of this study indicate that selenium treatment can reduce the varicocele-induced testicular damage in rats. This effect of selenium may be attributed to its antioxidant properties hence it enhances sperm profile, activities of the antioxidant enzymes (CAT, SOD, and GPX) and reduces the level of lipid peroxidation (MDA).
16
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Figure 1.
27
Figure 2.
28
Figure 3.
29
Figure 4.
30
31
Figure 5
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Figure 5. Histopathology of testicular tissue in experimental groups. (A) testis in group I shows normal histology of seminiferous tubule with sertoli cell (black arrow), spermatogonium (white arrow), spermatocyte (black arrowhead), and spermatozoa (white arrowhead); (B) testis in group II shows normal histology of seminiferous tubule with sertoli cell (white arrow), spermatogonium (black arrow), spermatocyte (white arrowhead), and spermatozoa (black arrowhead); (C-F) testis in groups III-VI shows normal histology of seminiferous tubule with sertoli cell (black arrow), spermatogonium (white arrow), spermatocyte (black arrowhead), and spermatozoa (white arrowhead); (G) testis in group VII shows degeneration and shrinkage of seminiferous tubules with loss of spermatogenesis and only presence of spermatogonia (arrowhead); (H-K) testis in groups VIII-XI shows an improvement in spermatogenesis and seminiferous tubule organization, so that primary spermatocyte (arrow in Fig. H), secondary spermatocyte (arrowhead in Fig. H), spermatocyte (arrowhead in Fig. I), spermatid (arrow in Figs. I and J) and spermatozoa (arrow in Fig. K) are seen (H&E).
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Table 1- Effect of sodium selenite on sperm count and quality in normal and varicocele-induced rats (n=9) Sperm count
Groups
Viability (%)
Sperm motility (%)
(× million)
Sperm abnormalities (%)
Group I
[Normal control]
73.62 ± 1.69
90.01 ± 0.89
75.60 ± 1.42
13.61 ± 2.56
Group II
[Sham-operated control]
72.14 ± 1.28
91.18 ± 0.72
76.19 ± 1.90
14.24 ± 1.90
Group III [Normal + sodium selenite (0.05 mg/kg)]
70.06 ± 1.09
88.05 ± 0.58
74.06 ± 1.00
13.85 ± 1.64
Group IV
[Normal + sodium selenite (0.1 mg/kg)]
85.62 ± 1.18
88.80 ± 0.58
77.20 ± 0.86
13.10 ± 1.92
Group V
[Normal + sodium selenite (0.2 mg/kg)]
74.50 ± 4.55
87.40 ± 0.51
77.03 ± 3.48
14.82 ± 1.58
Group VI [Normal + sodium selenite (0.4 mg/kg)]
71.65 ± 0.94
89.09 ± 0.89
76.60 ± 2.22
13.75 ± 2.06
Group VII [Varicocelized control]
35.25 ± 3.52٭٭٭
40.83 ± 2.87 ٭٭٭
54.08 ± 0.71 ٭٭٭
32.58 ± 3.91 ***
Group VIII [Varicocelic + sodium selenite (0.05 mg/kg)]
40.03 ± 1.03٭٭٭
45.03 ± 1.87 ٭٭٭
56.05 ± 1.07 ٭٭٭
24.06 ± 2.84 ***
Group IX [Varicocelic + sodium selenite (0.1 mg/kg)]
66.33 ± 0.81 +++
80.33 ± 1.87٭٭+++
76.11 ± 0.68 +++
19.81 ± 1.61 +++
Group X
63.70 ± 2.53 +++
81.20 ± 0.66 ٭٭+++
73.80 ± 0.73 ٭٭+
15.39 ± 1.78 +++
54.16 ± 2.24 ٭٭٭+
74.66 ± 1.02 ٭٭٭+++
73.49 ± 2.34 ٭٭+
16.47 ± 1.83 +++
[Varicocelic + sodium selenite (0.2 mg/kg)]
Group XI [Varicocelic + sodium selenite (0.4 mg/kg)] Values are mean ± S.E.M.
** p<0.01, *** p<0.001 significantly different from the normal control group. + p<0.05, ++ p<0.01, +++ p<0.001 significantly different from the varicocele control group.
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Table 2- Effect of sodium selenite on histopathological evaluation of testis in normal and varicocele-induced rats (n=9) Johnsen’s scores
Groups Group I
[Normal control]
10
Group II
[Sham-operated control]
10
Group III [Normal + sodium selenite (0.05 mg/kg)]
10
Group IV
[Normal + sodium selenite (0.1 mg/kg)]
10
Group V
[Normal + sodium selenite (0.2 mg/kg)]
10
Group VI [Normal + sodium selenite (0.4 mg/kg)]
10
Group VII [Varicocelized control]
2.87 ± 0.6٭٭٭
Group VIII [Varicocelic + sodium selenite (0.05 mg/kg)]
4.61 ± 0.5٭٭٭
Group IX [Varicocelic + sodium selenite (0.1 mg/kg)]
5.63 ± 0.5 ٭٭٭+++
Group X
[Varicocelic + sodium selenite (0.2 mg/kg)]
6.72 ± 0.4٭٭٭+++
Group XI [Varicocelic + sodium selenite (0.4 mg/kg)]
7.87 ± 0.6٭٭٭+++
Values are mean ± S.E.M. ** p<0.01, *** p<0.001 significantly different from the normal control group. + p<0.05, ++ p<0.01, +++ p<0.001 significantly different from the varicocele control group.
35