Cherry laurel fruit extract counters dimethoate-induced reproductive impairment and testicular apoptosis

Dimethoate (Korumagor 40 EC, 40 % emulsion) was purchased from Koruma Agriculture Co. (Kocaeli, Turkey). Other reagents and materials were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), unless otherwise specified.

Cherry laurel fruits were collected from Akçaabat in the Turkish province of Trabzon. A voucher specimen (AEF 26257) has been deposited in the Herbarium of the Ankara University Faculty of Pharmacy, Turkey. We first washed the fruits with distilled water and separated the seed and the pulp. Only pulp was used to prepare CLE by drying it at 40 °C for five days. Twenty grams of dried fruits were macerated with 200 mL of methanol (MeOH) at room temperature with constant magnetic stirring for 8 h, and filtered on Whatman No. 1 filter paper. This procedure was repeated twice with 200 mL of MeOH. The collected filtrates were dried under vacuum using a rotary evaporator at 40 °C. They were then frozen and lyophilised to obtain the extract. Before experiments, the lyophilisate was dissolved in saline (0.9 % NaCl).

For the experiment we used 60 male Wistar rats weighing approximately 200–250 g. The animals were kept in the Experimental and Clinical Research Centre of Erciyes University (Kayseri, Turkey) under standard laboratory conditions (12:12 h light and dark cycle, 22–24 °C temperature, and 55–60 % humidity), and had free access to commercial pellet diet (Optima Food Company, Bolu, Turkey) and water. The experimental procedures were approved by the Ethics Committee for Animal Research at Erciyes University and were carried out in compliance with international standards and national animal welfare legislation.

The animals were randomly divided into six groups of 10 to receive 4 mg/kg bw of CLE and/or 7 mg/kg bw of dimethoate or 100 mg/kg bw of vitamin C by oral gavage every day for eight weeks. The tested dimethoate dose was calculated from the percentage of the active ingredient in the commercial preparation, and the doses of all three substances were based on earlier studies (1, 15). All were freshly dissolved in 1 mL of saline before use in the experiments. We opted for the eight-week treatment to cover the complete spermatogenic cycle in rats, which spans between 49 and 52 days (16).

Group 1 (control) received only 1 mL of saline. Group 2 received dimethoate alone. Group 3 received CLE alone. Group 4 received CLE 30 min before dimethoate administration. Group 5 received vitamin C 30 min before dimethoate administration (positive control), and group 6 received dimethoate alone for the first four weeks, and then a combination of dimethoate and CLE for the other four weeks.

At the end of treatment, the rats were euthanised with an anaesthetic cocktail containing 8 mg/kg bw xylazine (Rompun^® 2 %, Bayer, Istanbul, Turkey) and 75 mg/kg bw ketamine (Ketasol^® 10 %, Interhas, Ankara, Turkey). Their whole body and absolute reproductive organ weights (testes, epididymides, seminal vesicle, and ventral prostate) were recorded in grams immediately and used to calculate relative organ weights (organ weight x 100 / whole body weight). One testis of each animal was fixed in Bouin’s solution (a mixture of 15 mL picric acid + 5 mL formaldehyde + 1 mL glacial acetic acid) for 16 h for histopathological analysis. The other testis was stored at -20 °C for oxidative stress analysis.

To determine sperm concentration, the right cauda epididymis was cut at the junction with the corpus epididymis, thoroughly dissected into small pieces with a scalpel and scissors into a Petri dish containing 1 mL saline, and the mixture was poured into an Eppendorf tube and left to incubate at room temperature for 4 h in order for all spermatozoa to pass into fluid. The left cauda epididymis was used to determine sperm motility and abnormality in a 5–10 μL suspension of spermatozoa (17).

Frozen testicular tissues were put into 9-fold phosphate buffered saline (PBS), dissected into small pieces and homogenised in cold saline. We measured the levels of malondialdehyde (MDA) as an indicator of LPO and the activities of superoxide dismutase (SOD), glutathione-peroxidase (GPx), and catalase (CAT) as described earlier by Eken et al. (15) using an ultraviolet-visible spectrophotometer (Shimadzu, Tokyo, Japan) at the following absorbances: 532 nm for MDA, 505 nm for SOD, 340 nm for GPx, and 240 nm for CAT. MDA levels were expressed in nmol/mg protein, and antioxidant enzyme activities in U/mg protein.

Semen analyses followed the methods described by Türk et al. (17). Sperm concentration in the right cauda epididymis was determined by a haemocytometer. Motility percentage in the left epididymal spermatozoa diluted with Tris buffer solution was visually determined at 400x magnification with the help of light microscope (Nikon Instruments Inc., Melville, NY, USA) with heated stage at 37 °C. Abnormal spermatozoa were identified under the same microscope with 400x magnification on slides containing a mixture of eosin-nigrosin stain and sperm samples. Abnormalities included head (banana-shaped, broken-, flat-, double-, and curved-headed sperm) and tail defects (curved-, broken-, short-, and double-tailed sperm). A total of 200 spermatozoa were examined per slide, and the findings expressed as the percentage of abnormal spermatozoa. Total abnormality was also calculated as head + tail.

We relied on the method described by Sarıözkan et al. (18). Briefly, diluted sperm samples were centrifuged (CN 090, Nüve, Ankara, Turkey) at 3000 x g at 4 °C for 10 min and supernatant removed. The remaining spermatozoa were washed with PBS without Ca²⁺ and Mg²⁺ to yield a concentration of 1 x 10⁵ spermatozoa per mL. For DNA damage analysis we used the alkaline comet (single-cell gel electrophoresis) assay. Each microscope slide was pre-coated with a layer of 1 % normal melting point (NMP) agarose in PBS and thoroughly dried at room temperature. Next, we mixed 100 μL of 0.7 % low melting point (LMP) agarose with 10 μL of cell suspension at 37 °C and dropped on top of the first slide layer. The slides were allowed to solidify in a moist box at 4 °C for 5 min. Cover slips were removed and the slides immersed in a freshly prepared cold lysis buffer containing 2.5 mol/L NaCl, 100 mmol/L Na₂- EDTA, 10 mmol/L Tris, 1 % Triton X-100, 10 % dimethyl sulphoxide (DMSO), and 40 mmol/L dithiothereitol (pH10) at 4 °C for 1 h. The slides were incubated in the lysis buffer with 100 μg/mL proteinase K at 37 °C overnight. They were then removed from the lysis buffer, drained, and placed in a horizontal electrophoresis unit filled with fresh alkaline electrophoresis solution containing 300 mmol/L NaOH and 1 mmol/L EDTA (pH 13) for 20 min to allow the DNA to unwind. Electrophoresis (12 V, 250 mA) at 8 °C lasted 20 min. The slides were then washed with a neutralising solution of 0.4 mol/L Tris (pH 7.5) to remove alkali and detergents, stained with 50 μL of 2 μL/mL ethidium bromide, and covered with a cover slip. All steps were performed under a dim light to prevent further DNA damage. For imaging and image analysis of 50 random nuclei per animal we used the Comet Assay Software Project for Windows (CASP-1.2.2, CaspLab, University of Wrocław, Wrocław, Poland).

Fixed testicular tissues were cut to 5 μm thick slices and stained with Mayer’s haematoxylin and eosin. Lesions like degeneration, congestion, and haemorrhage were examined semi-quantitatively in 20 seminiferous tubules, which were randomly selected in each section under a high-resolution microscope (Olympus BX51, Tokyo, Japan) at 400x magnification. Two independent histologists analysed preparations in a blind fashion and photographed them with the same microscope.

Testicular apoptosis was determined using the terminal deoxynucleotide-transferase (TdT)-mediated dUTP nick end labelling (TUNEL) assay with in situ Cell Death Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany) following the manufacturer’s recommendations (19). Fixed testicular tissues were embedded in paraffin, sliced at 4 μm thickness, deparaffinised in xylene, dehydrated with graded alcohol, washed in PBS, and treated with 0.05 % proteinase K for 5 min. Followed the treatment with 3 % hydrogen peroxide for 5 min to inhibit endogenous peroxidase. After re-washing in PBS, slices were incubated with the TUNEL reaction mixture containing TdT enzyme and digoxigenin-11-dUTP in a humidified chamber at 37 °C for 1 h, and then with a stop/wash buffer for another 30 min. For visualisation we used a diaminobenzidine substrate. Negative controls were treated with distilled water instead of TdT. For evaluation, 20 seminiferous tubules were randomly selected in each slide and TUNEL-positive apoptotic germ cells were counted on images obtained with the Olympus microscope (at 400x magnification) using the Image J program (Image J, Bethesda, MA, USA).

For statistical analysis we used the SPSS for Windows v. 22.0 (IBM Corp., Armonk, NY, USA). The inter-group comparisons were made with the non-parametric Kruskal-Wallis and non-parametric Mann-Whitney U tests. The results are expressed as means ± standard error of mean (SEM). The level of statistical significance was set at P<0.05.

CLE treatment alone significantly increased only seminal vesicle weight compared to control (P<0.001). Dimethoate, in turn, significantly decreased whole body, and organ weights (P<0.001). CLE pre- and post-treatment or vitamin C significantly countered these effects of dimethoate in the organs, but not in respect to the whole body weight (Tables 1 and 2).

MDA levels and the activities of SOD, GPx, and CAT are presented in Table 3. Dimethoate significantly increased MDA levels (P<0.001) and significantly lowered SOD (P<0.01) and GPx (P<0.05) activities in comparison to control. These effects were significantly countered by pre-and post-treatment with CLE or vitamin C (P<0.001 for MDA, P<0.01 for SOD, and P<0.05 for GPx). CAT activities did not change significantly between the groups.

CLE alone significantly increased sperm motility and concentration, while dimethoate significantly reduced sperm motility and concentration and significantly increased head, tail, and total abnormal sperm rate compared to control (P<0.001). Again, these effects were significantly countered by pre-and post-treatment with CLE or vitamin C (P<0.001) (Table 4).

Dimethoate significantly increased sperm DNA damage versus control (P<0.001). This time only CLE pre-treatment significantly countered dimethoate-induced DNA damage (P<0.001, Table 4).

Figure 1 shows the microphotography of histopathological findings in the testes of each experimental group. The control group (Figure 1A) and the CLE alone group (Figure 1C) showed normal testis histology with regular seminiferous tubular morphology and germ cells with normal polarity at various levels of differentiation. The Sertoli and Leydig cells also showed normal structure and polarity. Dimethoate (Figure 1B) deteriorated the histological architecture of the testes, which included abnormal seminiferous tubules with damaged walls, capillary congestion, and haemorrhage in the testis. Compared to the dimethoate-treated group pre- and post-treatment with CLE (Figures 1D and F, respectively) and vitamin C (Figure 2E) resulted in fewer atrophic and degenerative changes in the tubular epithelium. In these experimental groups, the epithelium of the germ cell layer was well coordinated, and the interstitial tissue contained blood vessels with normal structure.

Figure 1

Figure 2

Figure 2 and Table 4 show the presence of apoptotic cells in the testes of each group. Apoptotic germ cell count in the dimethoate-treated group was significantly higher than in all other groups (P<0.001). Pre- and post-treatment with CLE or vitamin C significantly reduced this count (P<0.001).