Ethics committee approval was obtained from Çanakkale 18 Mart University Ethics Committee of Animal Tests (council number: 2019/01 and decision number: 2019/01-13). A total of 29 healthy, adult female Wistar rats (aged 3–4 months and weighing 200–250 g) were used. Animals were kept in 12 hours of light and dark cycle, in a room at 24°C±2°C with sufficient air conditioning. Standard food and water were given ad libitum. A total of 29 rats with menstrual cycles were divided to three groups: Control group (saline group, Group 1, n = 7), cisplatin group (Group 2, n = 11), and cisplatin+melatonin group (Group 3, n = 11). While rats in Groups 2 and 3 were given single dose of 5 mg/kg cisplatin (Koçak Farma Drug Company, İstanbul, Turkey) via intra-peritoneal (IP) route, the rats in Group 3 were given 20 mg/kg melatonin (Sigma-Aldrich Chemical) IP route 30 minutes (min) before cisplatin administration and continued for 3 consecutive days. The rats in Group 1 were given only equal dose of saline IP. Melatonin dose was estimated based on the protective effect in previous studies [20]. One week after chemotherapy, each rat was given ketamin (50 mg/kg IM) and xylazine (10 mg/kg IM) anesthesia and underwent bilateral oophorectomy through a 3-cm midline incision; intra-cardiac blood samples were obtained and the rats were sacrificed with cervical dislocation thereafter.
Parenteral administration rules were followed during IP administration to avoid from the direct toxic effect of the chemotherapy agent.
Histological examination
The rat ovaries tissues were fixed for 24 h in 10% formalin solution. The tissues were dehydrated in graded series of ethanol and embedded in paraffin. After fixation in paraffin blocks, 4-µm serial sections were cut from ovary blocks with a microtome, and the sections were stained with routine hematoxylin and eosin (H&E). All the sections were analyzed and photographed using Zeiss Axio Scope. A1 light microscope (Carl Zeiss 3708, Göttingen, Germany). At least five randomly selected ovarian sections were examined for histopathologic examinations from each group.
Follicles were classified based on the Pedersen and Peters’s [21] morphological criteria, as described previously, in primordial, primary, secondary, antral, and atretic follicles. Additonally, these follicles were classified individually as histologically normal, when an intact oocyte was present and surrounded by granulosa cells that were well-organized in one or more layers and had no pyknotic nuclei. To avoid double counting of the same follicles, a follicle was counted on the first section in which the centrally located nucleus of the oocyte appeared.
Hormonal assays
E2 and AMH were quantitatively estimated in rat serum sample using enzyme immunassay technology (EIA) kits (catalog number: 33540 and 813127 respectively; Beckman Coulter Acces II device and Beckman Coulter Inc., USA).
RNA extraction and real-time PCR methodology
In the present study, Rat Ovary samples were contained in a −80oC refrigerator. RNA extraction was performed on these samples with Thermo Scientific® GeneJET RNA Purification Kit according to their manual. Extracted RNA’s were used to synthesize cDNA with Thermo Scientific® RevertAid First Strand cDNA Synthesis Kit, immediately afterward.
For this study, gene Act (Actin) was chosen as the control gene. ActB Forward Primer, ActB Reverse Primer, TP63 Prob, TP63 Forward Primer, and TP63 Reverse Primer (Sigma-Aldrich®) was used. For target gene, new primers and probes were designed accordingly. The following mix was used for real-time PCR, 1 µl of the selected probe; 1 µl of selected forward Primer; 1 µl of selected Reverse Primer; 2 µl ddH2O; and 2x LightCycler 480 Probes Master were added. 15 µl of this mix was put in LightCycler® Capillers and 5 µl of cDNA was added with a pipette. For real-time PCR, the following protocol was used, 10 min at 95oC for denaturation; 45 cycles of 10 s at 95oC; 30 s at 60oC; 1 s at 72oC for amplification; and 30 min of 40oC of cooling. The cycle was performed in LightCycler® 2.0 Instrument (Mannheim, Germany); Absolute Quantification module of Lightcycler2 Software 4.1.1.21 was used for analysis.
Statistical analysis
Data were expressed as mean±standard deviation (SD) and median (minimum-maximum) due to nonnormality. The Shapiro–Wilk test was used to assess the normality. The Levene test was used to evaluate homogeneity of variances. Analysis of variance (ANOVA) and Kruskal–Wallis H test was run to determine if there were differences between groups. Pairwise comparisons were performed using Dunn’s procedure with a Bonferroni correction for multiple comparisons. Adjusted p-values are presented. All statistical analyses were performed using SPSS 19.0. All p-values of less than 0.05 were considered to indicate statistical significance.