The Effects of Olive Leaf Extract on The Testis, Sperm Quality and Testicular Germ Cell Apoptosis in Male Rats Exposed to Busulfan

Olive leaves were collected from the olive tree farms from Kazeroon, Iran, authenticated by an expert and kept at the herbarium of pharmacognosy department, faculty of pharmacy, Kerman University of medical sciences, Kerman, Iran. The leaves were washed and dried at room temperature. Dried leaves (500 g) were milled and passed through a sieve (mesh 300). Plant extraction was performed using warm maceration with ethanol 80% for 72 hours. Obtained extract was concentrated under vacuum and finally dried in an oven at 40°C for 24 hours. The extract was stored at -20°C for subsequent experiments. The extract was dissolved in distilled water before use (11).

Total phenolic content of OLE was determined by Folin- Ciocalteu assay. Gallic acid was used for calibration. A stock solution of gallic acid (1000 ppm) was prepared; next, 0.1 ml stock solution was added to 0.4 ml sodium carbonate, 0.5 ml Folin reagent and 3 ml distilled water after 40 minutes incubation at room temperature. Absorbance was measured at 765 nm. The calibration curve was plotted for gallic acid based on 7 serial dilutions. After measurement of absorbance, calibration curve was plotted. By determination of extract absorbance as mentioned above and using the curve equation, total phenolic content was expressed as mg gallic acid equivalents per g of the extract (12). Each experiment was done in triplicate.

Total flavonoid content was measured by the aluminum chloride colorimetric assay. Rutin as the major flavonoid compound of the plant, was assessed for standardization using thin layer chromatography. First, 1 ml rutin (50 ppm) was added to 1 ml aluminum chloride 2%. After 30 minutes of incubation at room temperature, absorbance was recorded at 200-400 nm and maximum wavelength was 275 nm. Calibration curve was prepared using different dilutions of rutin. As mentioned above , total flavonoid content of the plant was expressed as mg rutin equivalents per g of the extract (12). Each experiment was done in triplicate.

Adult male Wistar rats (8-10 weeks old) were obtained from animal house of the university. Animals were kept in a temperature-controlled room (at 22°C) with 12 hours/12 hours light/dark cycles. Food and water were readily available. All chemicals were purchased from Sigma-Aldrich, unless otherwise noted.

Forty adult rats were randomly divided into 5 groups of control (n=8), BU (n=9) and BU co-administrated with three doses of OLE 250 mg/kg (n=8), 500 mg/kg (n=6) and 750 mg/kg (n=8) (BU+OLE 250, BU+OLE 500 and BU+OLE 750, respectively). In this study, BU was diluted in dimethyl sulfoxide (DMSO) and distilled water (D.W.) as solvent. The OLE was dissolved in the D.W. The animals in the control group (CTL) received a single intraperitoneal (i.p.) injection of BU solvent (i.e. DMSO+D.W.) and then D.W. was administrated orally by gavage for 5 weeks. The BU group received a single i.p. injection of BU (10 mg/kg) (13), the BU+OLE 250 group received OLE (250 mg/kg) orally for 5 weeks after receiving a single BU injection (10 mg/kg, i.p.), the BU+OLE 500 group received OLE (500 mg/kg) orally for 5 weeks after receiving a single BU injection (10 mg/kg, i.p.), the BU+OLE 750 group received OLE (750 mg/kg) (14) orally for 5 weeks after receiving a single BU injection (10 mg/kg, i.p.). Some rats died during the study, especially in the BU+OLE 750 group. At the beginning and end of the experiment, all rats were weighted.

After the end of the treatment period, the rats were deeply anesthetized by chloral hydrate (400 mg/kg) (15). After making an incision in their chests, the heart blood samples were collected from the left ventricle for biochemical and hormone analysis. The left testis and vas deferens were removed and separated from surrounding tissue. The testes’ weight and diameter were recorded and then tissues were fixed in formalin 10% (16) for histologic analysis. Left vas deferens was dissected and sperms were collected.

The blood (1.5 ml) collected from the heart was centrifuged at 3000 rpm for 30 minutes. Serum was carefully separated from plasma and immediately stored in a freezer at -20°C until analyzed. The level of serum testosterone and liver enzymes including alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in duplicate samples by an enzyme-linked immunosorbent assay (ELISA) using IBL kit (IBL company, Germany) and Biorex kit (Biorex company, UK) , respectively, according to the manufacturer’s instructions.

Under sterile conditions, the inferior part of rat abdomen was incised, the left vas deferens was removed and placed in a petri dish containing pre-warmed alpha MEM medium (2 ml), supplemented with 10% bovine serum albumin. It was dissected into several fragments and then, incubated at 37°C with CO₂ 5% in humidified air for 30 minutes to permit the migration of all spermatozoa from the reproductive duct to supplemented medium (15). Thereafter, the medium containing spermatozoa was collected and sperm quality was evaluated in terms of sperm motility, count, morphology and viability according to WHO guideline (17).

Immediately, 10 µl of sperm suspension (including the supplemented medium and spermatozoa) was placed on a slide and covered by a coverslip. Sperm motility was classified as fast progressive motility, slow progressive motility and immotile according to WHO guideline (17) and expressed as the percent of each one per total sperm number in at least 10 fields (300 sperms).

First, 10 µl of sperm suspension was added into 10 µl of fixative solution (formalin/sodium bicarbonate). Next, 10 µl of this mixture was placed on Neubauer haemocytometer and covered by a coverslip. The counting chamber was then placed on the light microscope stage (Nikon TS-100, Japan) at ×200 magnification, and sperms were counted in four large squares. The average of counted sperms was multiplied and was expressed as million/ml of suspension (18).

Sperm viability was assessed using eosin-nigrosin staining. Sperm suspension (5 µl) was added to eosin-nigrosin stain (5 µl). Smear was then prepared and at least 200 spermatozoa were randomly counted under a light microscope (at ×400 magnification). Sperm with red or pink head considered dead sperm and non-stained sperm, with white head, considered alive (17). The percentages of live spermatozoa were noted.

Body weights of rats were noted prior to start of experiment and 24 hours after the final day of treatment. The left testis was weighted by using a digital balance.

Left testis diameter, length and width were recorded by using standard digital calipers. In order to assess the alterations in spermatogonia cell (SG) population, histological evaluation of the rat testis was carried out (eight testis samples in each group). After testis fixation in formalin 10%, testis was dehydrated in increasing concentrations of ethanol (70, 90 and 100%) and embedded in paraffin. Five-micron thick sections of testis (at 50 µm interval) were prepared using microtome, mounted carefully and stained with hematoxylin and eosin (H<E). The slides were examined under a light microscope (Olympus/ BX51, Japan). In each section, 15 seminiferous tubules were randomly examined. Two perpendicular diameters of each seminiferous tubule (from the basement membrane to lumen) were calculated via calibrated linear scale of the Analysis software in the 10X eyepiece of Olympus microscope (18). The average of these diameters was reported. Also, thickness of the germinal epithelium layer was measured. The number of SG, primary spermatocyte (PS) cells and also Leydig cell were recorded from 10 microscopic fields at ×400.

Terminal deoxynucleotidyl-transferase-mediated DNA nick end-labelling (TUNEL) assay is a valuable method to detect apoptotic cells by labeling the terminal end of nucleic acids. TUNEL staining was done by an in situ cell death detection kit, POD (Roche-11684817910 version 14, Germany), according to the manufacturer’s instructions.

First, the testicular slides were deparaffinized by incubation at 60°C for 30 minutes, and then rehydrated in xylene (for 30 minutes) and increasing concentrations of ethanol 70, 90, and 100%, respectively (each one for 6 minutes). Slides were washed twice with distilled water.

Next, the slides were incubated in proteinase K (20 µg/ml in 10 mM Tris buffer) at 37°C for 30 minutes and washed three times with phosphate buffered saline (PBS). Afterwards, the slides were incubated in hydrogen peroxide solution (H₂O₂) 3% at room temperature for 10 minutes in the dark and re-washed three times with PBS. Immediately, TUNEL reaction mixture (enzyme solution 50 IU and label solution 450 IU) was prepared. The sections were incubated in a moist chamber containing a TUNEL reaction mixture at 37°C temperature for 60 minutes. After three-time washing by PBS, the slides were incubated in POD (anti-fluorescein antibody, FAB fragment from sheep, conjugated with peroxidase) intra a moist chamber for 30 minutes at 37ºC. The sections re-washed three times with PBS. 3,3'-Diaminobenzidine (DAB) substrate was added to the slides and incubated at room temperature for 10 minutes in the dark. After washing with PBS (once), the slides re-washed carefully with distilled water. Next, sections were stained with hematoxylin at room temperature for 30 seconds. After washing the slide with distilled water and dehydration by ascending degrees of ethanol (70, 90 and 100% respectively), the slides were mounted using Entellan. TUNEL-positive cells per tubule in at least 20 tubules from the testes were counted under a light microscope (Olympus/BX51, Japan) (18).

Statistical analysis was carried out by using Statistical Package for the Social Sciences software, version 21 (SPSS, Chicago, IL, USA). All data were expressed as means ± standard errors of the mean (SEM). At first, one-sample Kolmogorov-Smirnov test was used to check the normality of variables. Next, the differences in normal-distributed variables among five experimental groups were analyzed by using one-way ANOVA test followed by Tukey post hoc test. For nonparametric variables, non-parametric Kruskal- Wallis test (TUNEL SG, germinal epithelium height, alive and fast progressive sperm, and length of testis) was used. The level of significance was set at P<0.05.

The total flavonoid content of the OLE, calculated using calibration curve of rutin (R2=0.9635) was 1.43 g ruin equivalent/g plant extract. Total phenolic content of the plant, calculated from gallic acid standard curve (R2=0.9857) was 1.44 g gallic acid equivalents in 1000g OLE (Fig .1).

There was no significant difference in blood levels of testosterone among different groups. Analysis of the liver enzyme level showed that ALP level increased significantly in BU+OLE 750 compared to control (P<0.001), BU (P<0.001), BU+OLE 500 (P=0.003) and BU+OLE (P=0.030). Also, ALP level in BU+OLE 250 was significantly elevated versus the control (P=0.011). Compared to the BU group, AST and ALT enzyme levels did not vary significantly between BU+OLE 250 and BU+OLE 500 groups. Furthermore, AST level in rats treated with 750 mg/kg OLE showed a significant increase as compared to BU (P=0.005) and CTL (P=0.024, Table 1).

There were no significant differences in testis weight and changes in rat body weight in any group (P>0.05). The testis width, length and diameter in OLE-treated animals (all doses) remained unchanged and were similar to those of the control and BU-exposed animals (P>0.05) (data not shown).

The effects of BU and different doses of OLE on sperm count, motility, morphology, and viability are summarized in Table 2. Comparing sperm count among 5 groups by using one way-ANOVA test, showed that although exposure to BU could non-significantly decrease the sperm count (3.23 ± 0.62, P=0.086) as compared to that of CTL (5.75 ± 0.82), all doses of OLE caused an increase in sperm count as compared to BU group. Fast progressive motility decreased non-significantly in BU group (4.74 ± 1.33) vs. the CTL (11.67 ± 4.97) (P=0.19).

Oral administration of OLE at different doses of 250 (17.91 ± 2.85), 500 (28.75 ± 5.86) and 750 (20 ± 4.10) mg/kg could significantly improve the proportion of sperms to the fast progressive motility versus BU group (P=0.002, P=0.009, P=0.003, respectively).

The percentage of viable sperms in the BU group was significantly lower than that of the CTL (P=0.017). Compared with BU group, a significant change was observed viable sperm percentage in all OLE-treated groups (250 mg/kg, P<0.001, 500 mg/kg, P=0.003, and 750 mg/kg, P<0.001) (Table 2).

No statistically significant difference was observed in the mean diameter of seminiferous tubules among different groups (P>0.05). Comparison of the mean of germinal epithelium thickness between BU-treated testis and control, BU+OLE 250 and BU+OLE 500 testes showed no significant differences (P>0.05). However, significant decreases in thickness of the germinal epithelium of BU+OLE 750 group were observed as compared to the BU (P =0.033) and OLE 250 and 500 (P=0.019) treatment groups (Table 2).

In the present study, apoptotic germ cells were distinguished by TUNEL staining (Fig .3). The apoptotic index was calculated as follows: TUNEL positive nuclei (dark brown cells) /number of total germ cells. As summarized in Figure 4, a significant increase in the mean number of apoptotic SG (P<0.001) and PS (P=0.029) cells was observed in rats exposed to BU as compared the CTL. However, BU-mediated increase in apoptotic SG was significantly reduced by administration of OLE 250 and 500 mg/kg (P<0.001). This decline was markedly higher in the group administrated with OLE 500 mg/kg compared to the other doses of OLE (P<0.001), and nearer to that of the CTL. In contrast, the percentage of apoptotic SG was enhanced in rats which received OLE 750 mg/ kg, compared to the other groups (P<0.001). Also, the TUNEL-positive PS counts in the seminiferous tubules showed that OLE 250 mg/kg could not decrease the level of apoptotic PS cells significantly compared to BU group. Interestingly, only a small number of apoptotic cells were observed in the testicular sections of OLE 500 group, which was significantly different from that of the BU (P=0.029) and the other OLE groups (P=0.029). However, this reduction was more marked compared to the CTL. In contrast, the group which received OLE 750 mg/kg was shown to have significantly higher levels of apoptotic PS cells, in comparison to OLE 500 and CTL (P=0.029).