Influence of selenium induced oxidative stress on spermatogenesis and lactate dehydrogenase-X in mice testis
Parminder Kaur, M. P. Bansal
Department of Biophysics, Panjab University, Chandigarh-160014, India
Asian J Androl 2004 Sep; 6: 227-232
Keywords: testis; lactate dehydrogenase X; selenium; oxidative stress
Aim: To evaluate the effect of oxidative stress on the spermatogenesis and lactate dehydrogenase-X (LDH-X) activity in mouse testis. Methods: For creating different levels of oxidative stress in mice, three selenium (Se) level diets were fed in separate groups for 8 weeks. Group 1 animals were fed yeast-based Se-deficient (0.02 ppm) diet. Group 2 and Group 3 animals were fed with the same diet supplemented with 0.2 ppm and 1 ppm Se as sodium selenite, respectively. After 8 weeks, biochemical and histopathological observations of the testis were carried out. LDH-X levels in the testis were analyzed by western immunoblot and ELISA. Results: A significant decrease in testis Se level was observed in Group 1 animals, whereas it was enhanced in Group 3 as compared to Group 2. The glutathione peroxidase (GSH-Px) activity was significantly reduced in both the liver and testis in Group 1, but not in Group 2 and 3. A significant increase in the testis glutathione-S-transferase (GST) activity was observed in Group 1, whereas no significant change was seen in Groups 2 and 3. Histological analysis of testis revealed a normal structure in Group 2. A significant decrease in the germ cell population in Group 1 was observed as compared to Group 2 with the spermatids and mature sperm affected the most. Decrease in the lumen size was also observed. In the Se-excess group (Group 3), displacement of germ cell population was observed. Further, a decrease in the LDH-X level in testis was observed in Group 1. Conclusion: Excessive oxidative stress in the Se deficient group, as indicated by changes in the GSH-Px/GST activity, affects the spermatogenic process with a reduction in mature sperm and in turn the LDH-X level.
The changes produced by the reactive oxygen species (ROS), i.e., superoxide (O2.-), hydrogen peroxide (H2O2) and hydroxyl radical (OH.), are the major contributor to aging and degenerative processes, including cancer, heart disease and brain dysfunction . ROS are also involved in the peroxidative damage of human spermatozoa, which may result in male infertility . Human spermatozoa are particularly susceptible to peroxide damage because they contain an extremely high concentration of polyunsaturated fatty acids, exhibit no capacity for membrane repair and possess a significant ability to generate ROS, chiefly superoxide anion and hydrogen peroxide .
Testis germ cells as well as maturing spermatozoa are endowed with enzymatic and non-enzymatic scavenger system to prevent damage caused by lipid peroxi-dation . Antioxidant enzymes play a crucial role in protecting male germ cells from oxidative damage [5,6].
Various studies indicate that selenium (Se), a potent antioxidant, to be essential for male fertility . As a constituent of selenoproteins, Se has structural and enzymatic role, in the latter context being best known as an antioxidant. Biological function of Se in mammals is expressed through biologically active compounds including GSH-Px . The toxicity of the product of lipid peroxidation in a cell is reduced in part by GSH-Px and in part by GST . The contribution of GST in detoxifying the products of oxidative stress becomes quite significant under Se deficient condition, where the GSH-Px activity is greatly reduced.
LDH-X is one of the best-characterized germ cell specific isozyme  that plays an important role in the process of spermatogenesis and has been shown to be vital for sperm survival and motility. This enzyme is only found in germ cells and is present in cells of the mouse gametogenic line from pachytene primary spermatocytes to spermatozoa . Synthesis of LDH-X in the testis takes place during sexual maturation and the predominant lactate dehydrogenase fraction is present in the mature spermatozoa . LDH-X is the predominant isozyme in the pachytene spermatocyte and round and condensing spermatids, whereas spermatozoa contain only LDH-X. The present study was designed to observe the effect of oxidative stress created by various levels of Se feeding on the process of spermatogenesis and hence the LDH-X levels in the testis.
2 Materials and methods
Male Balb/c mice weighing 25 g were procured from the Central Animal House, Panjab University, Chandigarh. All procedures involving animals were approved by the Institutional Animal Ethical Committee.
To obtain different levels of selenium in three different groups viz. 0.02 ppm, 0.2 ppm and 1 ppm, the mice were kept on yeast based diet which contain 0.02 ppm Se and hence animals fed on this diet for 8 weeks were considered Se deficient animals (Group 1). Animals in Group 2 and Group 3 were fed Se deficient diet supplemented with sodium selenite at 0.2 ppm (adequate) and 1ppm Se level (excess level). All the animals were fed with the respective diet for 8 weeks and were allowed water ad libitum.
2.2 Selenium estimation
Selenium concentration in the testis was determined by the flourimetric method  and GSH-Px activity measured in liver and testis post mitochondrial fraction (PMF) as described . Glutathione-S-transferase activity was measured in the testis PMF using 1-chloro-2, 4-dinitrobenzene (CDNB) as the substrate .
2.3 LDH-X purification and antibody preparation
Murine LDH-X was purified by a modified procedure of Lee et al . Briefly, 20 % homogenate of frozen testis was prepared in 20 mmol/L Tris-Cl buffer(pH 7.4) and centrifuged at 27 000 g for 20 min at 4 . Supernatant was collected by passage through cheese cloth and heated at 60 for 15 min. Supernatant was centrifuged again at 27 000 g for 20 min at 4 to remove precipitated proteins. The pH of the supernatant was adjusted to 6.5 with KH2PO4. Enzyme solution was then loaded on a 8-(G-aminohexyl)-AMP-sepharose column and the enzyme was eluted biospecifically with reduced NAD+ pyruvate adduct. Polyclonal antibodies were raised in female rabbit by initially injecting (intra-dermal, multiple sites) 200 g of purified LDH-X protein emulsified in complete freunds adjuvant. After one week of initial injection, response was boosted thrice with 50 g LDH-X protein in incomplete adjuvant with one-week interval each. After completion of booster doses, blood was collected through ear vein of rabbit and serum was separated. Serum from non-immunized female rabbit served as control.
2.4 LDH-X analysis by western immunoblot and ELISA
LDH-X analysis in testis from all the three experimental groups was done using western immunoblot and ELISA. SDS-PAGE (10 % gel) of testis cytosol from all the three groups (10 g protein) was carried out and blotted on to the PVDF membrane. Immunoblot was prepared using LDH-X antibody as primary antibody and biotin labeled anti-mouse IgG as secondary antibody. Peroxidase and DAB/H2O2 detection system was used.
Enzyme linked immunosorbent assay was carried out for LDH-X levels. Microtitre plate was coated with 5 mg of protein in 100 L carbonate buffer (0.05 mol/L, pH 9.6) overnight at 4 . Wells were then blocked with 100 L of PBS containing 1 % BSA for 1 h at 37 . Wells were then washed thrice with 200 mL of PBS containing 0.05 % (v/v) tween-20. 100 L of anti LDH-X polyclonal primary antibody (diluted 1:1000 in PBS containing 0.05 % tween-20 and 1 % BSA) was added to each well and kept for 2 h at 37 . Plate was again washed and then incubated with anti-rabbit secondary antibody again for 2 h at 37 . Wells were washed further three times as described above and color was developed by addition of 2, 2'-azino-di- (3-ethyl- benzo-thiozolin sulfonic acid) reagent.
2.5 Histopathological studies
Fresh pieces of testis were taken randomly immediately after the sacrifice of the animals from all the three experimental groups, fixed in Zenker fixative for 24 h, processed with different grades of alcohol, embedded in paraffin wax, sectioned (8 m thick) and stained with haemotoxylin/eosin.
3.1 Selenium level
The testis Se level was found to be significantly low in Group 1 compared with that in Group 2 after 8 weeks of respective diet feeding (Table 1). In animals fed on 1 ppm Se diet (Group 3), testis Se level was found to be significantly high as compared to that in adequate Se diet fed group (Group 2).
Table 1. Selenium, glutathione peroxidase, glutathione-s-transferase and LDH-X levels (meanSEM) in testis/liver of mice (n=6) kept on various diets. cP<0.01, compared with Group 2.
3.2 Glutathione peroxidase activity
A significant decrease in the enzyme activity in both liver and testis of Group 1 animals as compared to Group 2 was observed. However there was no significant change in the enzyme activity in Group 3 as compared to that in Group 2 (Table 1).
3.3 Glutathione-S-transferase activity
A significant increase in the GST activity was observed in testis of Group 1 animals. In Group 3 the enzyme activity was found to be comparable to Group 2 (Table 1).
3.4 LDH-X analysis
The western immunoblot for the testis specific LDH-X level in the three different groups showed a single band at 35 kDa in all the samples on western transfer of SDS-PAGE (Figure 1, lane 1, 2, 3). Antibody did not recognize any band in the liver and kidney cytosol (lane 5, 6). Immunoblot intensity showed high in Group 2 and 3 compared to Group 1.
Figure 1. LDH-X level in cytosol of selenium deficient (Lane 1), Se adequate (Lane 2) and Se excess (Lane 3) mice. Lanes 4, 5 and 6 represent testis, kidney and liver cytosol in normal mice, respectively.
LDH-X was quantitated in testis cytosol of all the three experimental groups using ELISA. Table 1 shows the absorbance (A405) in wells on ELISA reaction which is taken as a direct assay of LDH-X concentration. No significant change was observed in the LDH-X level in the testis sample from animals exposed to high concentration of Se (Group 3). However, LDH-X decreased significantly in Se deficient group as compared to the adequate Se (Group 2).
3.5 Histopathological analysis
Results are shown in photomicrographs of testis section from all the three groups (Figure 2-7). Shrinkage of seminiferous tubules was seen in Group 1 as compared to Group 2 testis. Decrease in the germinal height and increase in the central lumen size was also observed in Group 1 compared to the adequate group. There is appreciable decrease in the spermatid and mature sperm number in Group 1 along with a reduction in the pachytene spermatocytes.
Figure 2. Testis of selenium deficient mice showing decrease in spermatids and mature sperm 312.
Figure 3. Testis of selenium deficient mice 500.
Figure 4. Testis of selenium adequate mice showing all stages of seminiferous epithelium 312.
Figure 5. Testis of selenium adequate mice 500.
Figure 6. Testis of selenium excess mice showing all stages from spermatogonia to mature spermatozoa with displacement of germ cell population 312.
Figure 7. Testis of selenium excess mice 500.
In the excess Se diet fed group (Group 3), shrinkage of tubule was quite apparent. The central lumen decreased in size and the displacement of the germ cell population was quite frequent. However no appreciable decrease was seen in the spermatid and the mature sperm as compared to the adequate group.
Recent advances in the understanding of male infertility has implicated oxidative stress to be a major causative factor [16, 17]. The high rate of mitosis and the various stages of meiosis in the seminiferous tubule expose the germinal cell chromosome to the potentially damaging influence of free radicals in the local environment, thus creating a need for an effective antioxidant system . Selenium, a potent antioxidant is essential for male reproduction . It is integral cofactor of GSH-Px, which removes hydrogen peroxide and lipid peroxide and thus protect testicular germ cells from peroxidative damage . Decrease in the GSH-Px level in Se deficient conditions will therefore expose germ cells to the oxidative stress. Studies have reported GST activity directly takes part in the elimination of products of lipid peroxidation and there is enhancement of GST activity in spermatogenic cells after H2O2 exposure . Increased GST activity observed in Group 1 suggests increased oxidative stress in the testis.
The present study indicated histopathological changes in the testis sections, both in Group 1 and Group 3, as compared to Group 2. A marked decrease in the spermatid and mature sperm was observed in the Se deficient Group 1 animals. Decrease in the pachytene spermatocytes was also observed. This decrease could be attributed to the oxidative stress since H2O2 and lipid peroxides have been shown to be toxic for spermatozoa . Marked alteration in the physicochemical state of the DNA protein complex in spermatozoa chromatin has been observed by various researchers during spontaneous oxidation .
Selenium excess group, also shows the displacement of germ cell population, which may be due to the ability of all Se compounds at the dose range of 1 mg/kg - 3 mg/kg of diet to generate oxidative stress through production of selenopersulfide (a selenide) which then reacts with GSH to generate superoxide within cell .
LDH-X comprises 90 % of the total LDH-X activity of the spermatozoa. It plays an essential role in the metabolism of spermatozoa and is involved in the processes specific for these cells that generate energy for their survival, differentiation and motility . There is a link between LDH-X and spermatogenesis since prepubertal males are lacking in this enzyme and amount of LDH-X increases with testis maturity . LDH-X is a testis specific enzyme and has been used as a chemical marker for the germ cell status in seminiferous epithelium . It has also been suggested to be of diagnostic value in case of testicular toxicity . In the present study, the decrease in the LDH-X level both with the western immunoblot and ELISA in animals fed on Se deficient diet was observed. This apparently is a reflection of a decrease in the number of mature spermatocytes which otherwise have high level of LDH-X. However, previous studies have indicated that toxic conditions affected the activity of LDH-X in the testis . Inverse relation has been found between LDH/LDH-X activities and ROS production . In conclusion, the oxidative stress generated due to the deficient Se in the diet may be responsible for affecting directly the process of spermatogenesis and in turn reflecting the reduced LDH-X level in the seminiferous tubule.
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Correspondence to: Dr.
M.P. Bansal, Department of Biophysics, Panjab University, Chandigarh-160014,