Prevention of di (2-ethylhexyl) Phthalate-induced Testicular Disturbance in Mice by Co-administration of L-carnitine

In this experimental study,male NMRI mice were maintained in 22 ± 2°C with a 12 hours light:dark cycle and with access to food and water ad libitum during the course of the study. All animal experiments were approved by the Animal Care and Use Committee of Baqiyatallah University of Medical Sciences.

The mice (mean age: 4-6 weeks) were divided into four groups with ten mice per group which were treated daily by gavage for 2 weeks. Control mice in group I were administered vehicle (100 μl corn oil). Animals in group II were treated with 2g DEHP (CAS NO: 117-81-7, Osaka, Japan)/100 μl corn oil/ kg. Group III received 1mg LC (Sigma-Aldrich Chemie, Italy) GmbH/100 μl deionized water and group IV were treated with a 1mg LC/μl deionized water combination 2g DEHP/100μl corn oil per kg body weight. Our previous study showed that corn oil is ineffective in spermatogenesis (19).

The initial body weight on the first day and final body weight on the termination day (prior to cervical dislocation) of the experiment and testis weights of both sides were recorded for each animal. Testes were trimmed of fat prior to recording their weights. After weighing, the left testis was fixed in Bouinś fluid for histopathological examination, and the right testis was frozen at -20°C, until thawed for daily sperm production analysis.

Fixed testis was embedded in paraffin, sectioned and stained with hematoxylin-eosin (H&E). The histopathlogical specimens were examined under light microscopy. Twelve cross-sections of stage VII-VIII seminiferous tubules from each animal were analyzed by Motic software for tubular diameter (from basal lamina to the other side basal lamina), epithelial height (basal lamina to neck of elongated spermatids), and luminal diameter. In addition, with a ×100 magnification light microscope, the number of sertoli cells and round spermatids were counted for each animal in the 12 cross-sections of stage VII-VIII seminiferous tubules. Data were expressed as the mean of the sertoli cells and round spermatids. These stages were chosen as representative to include morphologically identifiable stages.

The right testis was thawed at room temperature, decapsulated, and homogenized in 2 ml of 0.9 % NaCl solution for 6 minutes using a tissue homogenizer set at low speed. The homogenate was allowed to settle for 1 minute and then gently mixed. After thorough mixing of each sample, the number of homogenization-resistant spermatid heads was counted by a hemocytometer. The testicular spermatid number per gram testis (TSN) was calculated according to the respective formula. DSP was calculated by dividing the TSN by 4.84 (the duration of step 14-16 spermatids in the mouse seminiferous epithelial cycle) (20).

The right cauda epididymis of each animal was minced, suspended in 1 ml of T6 medium that contained 4 mg/ml bovine serum albumin (BSA) for 1 hour at 37°C and 5% CO2.

For the sperm count in the caudal epididymis, 5 μl of the sperm suspension was observed with an optical microscope using a haemocytometer with a cover glass of 0.1 mm thickness. The total number of sperm was counted and the mean was calculated from two counts.

For assessing the sperm motility, 5 μl of sample was placed on a clean microscope slide and covered with a coverslip. Immediately, with ×400 light microscope magnification, 100 sperms were analyzed per sample. Each spermatozoa was classified as: A .rapid linear progressive motility, B .slow or sluggish linear or non-linear motility, C .nonprogressive motility or D .immotile sperm. Percent motile sperm (A+B+C) and percent progressive sperm (A+B) were also calculated.

For studying the sperm morphology, a drop of sperm suspension was smeared onto a clean glass slide. The smear was then air dried and fixed in a mixture of equal parts ethanol and ether. The slides were then stained with Papanicolaou stain. Dried stained slides were scanned under oil immersion (100 objectives) for morphological abnormalities. A total of 100 sperms per sample were classified according to their morphology; such as normal, coiled mid piece, hair pin (a kink at the annulus, usually 180°), bent tail (a kink at the annulus, usually 90°), coiled tail, double head, amorphous head, triangular head, pin head and cytoplasmic droplet. Sperm abnormality was expressed as percent.

For sperm vitality, a drop of sample was put on a clean glass slide and mixed with one droplet of eosin B (0.5% in normal saline). A total of 100 sperm were assessed per animal. Each spermatozoa was classified as motile living sperm, immotile living sperm, or dead sperm.

For the sperm chromatin quality, a drop of sample was smeared, dried, and fixed. Then, slides were stained with 5% aniline blue. For each animal, 100 sperm were counted with 100x objective and classified as low-staining sperm, mid-staining sperm, or high-staining sperm.

Analyses were conducted with SPSS 15.0 for Windows software. Comparison between initial and final body weights was done by paired sample ttest. The viability, motility, progressiveness, and morphology of epididymal sperm and the chromatin quality in treated groups were analyzed by Kruskal- Wallis followed by Mann-Whitney U test. Body and testis weight, sperm count, TSN, DSP, seminiferous tubular and luminal diameter, seminiferous epithelium height, sertoli, and the round spermatid number between groups were compared statistically using analysis of variance (ANOVA), followed by Tukey’s post-hoc test for multiple comparison. Statistical significance was set at p<0.05.

Tubular diameter decreased in all experimental groups compared to the control group. The decrease was significant (p<0.05), except for group III. In addition, tubular diameter in group IV significantly increased compared to group II (p<0.05). The seminiferous epithelial height of groups II and IV decreased compared to the control. The decrease was significant (p<0.05) only in group II.

In group IV, epithelial height significantly increased when compared to group II. No significant change was detected in the luminal diameter among the groups (Table 2).

The number of sertoli cells and round spermatids per each seminiferous tubule significantly decreased in both groups II and IV compared to the control group (p<0.05). These parameters in group IV increased compared to group II, but only the increase in the number of round spermatids was significant (p<0.05) (Table 2).

Table 3 shows that the number of homogenization- resistant spermatids and daily sperm production per gram testis were not significantly changed in groups III and IV compared to control values. Those of group II were significantly altered by DEHP exposure (p<0.05). TSN and DSP of group IV significantly increased compared to group II (p<0.05).

Results of the sperm count, percentage of motility, and progressiveness of all groups are summarized in tables 3 and 4. Sperm number, percentage of motility and progressiveness of group III were not significantly altered, and those of group II were significantly diminished when compared to the control group. In group IV, decrease in sperm count, motility (%), and progressiveness (%) were seen in comparison with the control; these alternations were significant (p<0.05) except for the parameter of sperm count. There was a significant increase in the sperm count of group IV as compared with that in the control group (p<0.05).

Data of sperm vitality and chromatin quality analyses are shown in Table 4. Regarding the sperm vitality, the percentage of motile living sperm of groups II and IV significantly reduced and percentage of dead sperm in groups II and IV were significantly increased compared to the control group (p<0.05). No remarkable alternations were observed in the other cases.

Regarding sperm chromatin quality, low-staining sperm (%) of group II was significantly diminished, and those of group III was significantly increased compared to the control group (p<0.05).

In group IV, the percentage of low-staining sperm was increased from group I and II, but this increase was significant only in group II (p<0.05). A significant decrease in percentage of mid-staining sperm and high-staining sperm of group IV were seen when compared to group II (p<0.05). No significant changes were detected in these parameters of group IV compared to the control group (Fig 1).

The normal sperm morphology (%) of group II was significantly reduced compared with the control group (p<0.05). There were no remarkable differences among the groups in the other cases.