Chemicals
Penicillin, streptomycin, sodium bicarbonate, sodium pyruvate, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), epidermal growth factor (EGF), recombinant human follicle-stimulating hormone (hFSH), fetal bovine serum (FBS), ethylene glycol (EG), dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich Company (Steinheim, Germany). α-MEM (Minimum Essential Medium) and fetal bovine serum (FBS) were obtained from Gibco (Gibco, England). The rest of the materials and the tools are listed in the text.
Animals and study design
FemaleNational Medical Research Institute mice (age 6-8 weeks, n=63) were obtained from Pasteur institute (Amol branch, Iran) and kept in the animal house of Damghan University under standard conditions based on the National Institutes of Health Guide for 12 hours light/dark cycle at 21 ± 2℃ and 40-50% humidity with adequate access to water and food. The ethics committee of Damghan University approved All procedures based on the ethical principles of the Helsinki Declaration as revised in Tokyo 2004 with the ethic reference code: IR. BSDU.REC.1397.16. Mice were randomly distributed into five experimental groups, as follows: Control (n=15): Fresh ovarian tissue transplantation without any treatment, Sham (n=15): cryopreserved/warmed ovarian tissue transplantation, NAC (n=15): Cryopreserved/warmed ovarian tissue transplantation with NAC treatment, E2 (n=9): Cryopreserved/warmed ovarian tissue transplantation with E2 treatment and NAC+E2 (n=9): Cryopreserved/warmed ovarian tissue transplantation with NAC and E2 treatment. Some of the mice were sacrificed on the 2nd and 7th days after transplantation, and the ovaries were disparted to evaluate the molecular and biochemical parameters, and the reset candidate for vagina smears and blood sampling to assess the function of the transplanted ovary on the 28th day after transplantation as depicted in Figure 1.
Ovariectomy
The mice were anesthetized using ketamine (100 mg/kg/BW) and xylazine (10 mg/kg/BW) via intraperitoneal injection. Linear sections were made on both sides of the spine column. The ovaries were disparted and posited in 200 μl drops of α-MEM medium supplemented with 10% of the FBS, 2.2 g/l sodium bicarbonate, 100 IU/ml penicillin, 75 μg/ml streptomycin, and 25 mM HEPES, under mineral oil and located in incubator with the condition of 5% CO2, 37° C and 98% humidity for at least 30 min to adapt with the new condition.
Vitrification/warming and transplantation
The ovaries were vitrified and warmed according to Hatami et al. [17] with some modification. In brief, ovaries were transferred to equilibration solution (7.5% ethylene glycol + 7.5% dimethylsulfoxide) for 10 minutes and moved to vitrification solution (20% ethylene glycol + 20% dimethylsulfoxide + 0.5 M sucrose) for 5 minutes. Ovaries were placed into 1.5 ml cryovials and directly plunged into liquid nitrogen. In the warming process, vials containing vitrified ovaries were held for 20 seconds at room temperature and then filled with the warming solution (1.0 M sucrose in basal medium: fetal bovine serum + Dulbecco’s phosphate buffered saline) at room temperature for 30 seconds. Ovaries were moved to the 4-well dishes and washed in a stepwise manner (0.5 M, 0.25 M, and 0 M sucrose in basal medium with 3 minutes intervals). For transplantation, the mice were anesthetized by injection of ketamine intraperitonealy (100mg/kg/BW) and xylazine (10mg/kg). and then a deep incision (3-5 mm) was made on the right and left back muscle [18]. The ovaries were autografted and finally, muscle and skin were sutured.
NAC and E2 treatment
NAC (150 mg/kg) [7, 13] was injected intraperitonealy one hour before transplantation and repeated every 8 hours up to 24 hours. A single dose of E2 (Estradiol valerate; Aburaihan company, Iran) was injected intramuscularly 24 hours after transplantation [19].
Histological assessment
The ovarian tissues were fixed by 10% formalin for 48 hours and then inculcated in paraffin. Tissues were segmented in five μm thickness and stained with hematoxylin and eosin (H&E). Then stained tissue segments were evaluated by a light microscope (Olympus, BX51, Tokyo, Japan) equipped with a digital camera (Labomed, India). The number and types of follicles (primordial, primary, preantral, antral) were counted. Also, angiogenesis was evaluated by counting small, medium, and large diameter vessels.
Assessment of estrous cycles
On the 28th day after transplantation vagina smears were prepared to evaluate the function of the transplanted ovary. The estrous stages were determined by cytological evaluations of vaginal smears as said previously [20]. In brief, every morning between 8:00 and 9:00 a.m. ++++Vaginal smears were gained by a plastic pipette filled with 10 µL of phosphate buffer saline (PBS) by entering the tip into the vagina and pipetting and flushing repeatedly. Vaginal fluid was located on glass slides and was fixed and stained with methanol and 2% Methylene blue, respectively. Stages of the Estrous cycle were then determined using the cellular composition and the relative ratio between the cell types of the smear.
RNA extraction and Real-Time qPCR
Total RNA isolation was carried out using a QIAzol lysis reagent kit (Qiagen; Germany) based on the manufacturing procedure. The cDNA was produced with 500 ng total RNA using a cDNA synthesis kit (PrimeScript RT Reagent Kit, TAKARA, Japan). Primers were designed by Allele ID software (PREMIER Biosoft USA, version 7.5; Table 1) as previously described. The PCR was fulfilled in a ten μl total volume using 5μl of RealQ Plus 2x Master Mix Green (Amplicon, Denmark), 0.5 μl of each sense and antisense primer (10 pmol/reaction), and 25 ng cDNA. PCR was carried out using Rotor-gene 6000 PCR system (Corbett Life Science, Australia) as follows: initial denaturation at 95℃ for 15 minutes, then 40 cycles of denaturation at 95℃ for 15 seconds and annealing and extension at 60℃ for 45 seconds. The program was followed by a melt step at 65-94℃. The relative gene expression was evaluated through the 2-∆∆CT method. The ef-1 gene was used as the internal control.
Biochemical assay
Cellular supernatant was prepared as previously explained with some modification [21]. In brief, ovaries (n=3) were homogenized in 500 μl of lysis buffer (10mmol/L Tris HCL, 20mmol/L EDTA, and 0.25% V/V Trito, PH=8) and homogenized with sonication (50 W, 5 minutes). After that homogenized tissues were centrifuged at 12000 g for 20 minutes at 4℃. Extracted supernatants were used for evaluation of malondialdehyde (MDA) and total antioxidant capacity (TAC) levels as well as superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity as previously described [22]. Protein content was measured according to Bradford method [23].
Statistical analysis
The data were analyzed using SPSS software (version 24; Chicago, IL, USA). The Shapiro-Wilk normality test was performed and confirmed a normality distribution of the data thus, parametric tests were carried out.The statistical analysis was performed using a one-way analysis of variance (ANOVA) followed by post hoc Tukey's HSD. Statistical significance was considered at p < 0.05.The data presented as mean ± Standard Deviation.