The study was approved by the local Ethics Committee on the Use of Animals (CEUA-FMUSP 024/15). The animal group consisted of 26 adult female Wistar rats (Rattus norvegicus albinus) aged 3 months and weighing approximately 250 g each. The animals were kept under proper conditions of temperature and feeding as well as under a controlled light/dark cycle of 12/12 h. Only animals with three regular estrous cycles were included.
The animals were allocated to two study groups (n = 13 each): control and melatonin. In the control group, slow cryopreservation was performed according to the standard protocol with the M2 culture medium, dimethyl sulfoxide (DMSO) [17], and ethyl alcohol vehicle, whereas in the melatonin group, melatonin (Sigma Aldrich, Saint Louis, MO, USA) was added to the medium at a concentration of 10− 7 M [12]. Three animals from each group underwent graft analysis immediately after thawing from cryopreservation to verify the potential differences between groups induced up to the freezing-thawing process itself.
In both groups, the ovaries underwent slow cryopreservation and were kept in liquid nitrogen (N2) for 24 h. Thawing took place at room temperature (25 °C). The ovaries were implanted in the retroperitoneum of their respective donors, one on each side of the aorta, without anastomosis, and fixed by means of a simple stitch with an unabsorbable thread (nylon 4 − 0).
Estrous cycle control
At the beginning of the experiment, vaginal smears were obtained daily, always at the same time (8–10 h AM), to characterize the estrous cycle using the Shorr-Harris technique. Only animals with regular estrous cycles of 4–5 days were used; diestrus was the standard phase for the surgical procedures (oophorectomy for cryopreservation and euthanasia).
Daily (8–10 h AM) vaginal smear collection resumed from postoperative (PO) days 15 to 30; then euthanasia was performed as the animals entered the diestrus phase (PO day 30–35).
Protocol for anesthesia
After being weighed, the animals were anesthetized with xylazine (15 mg/kg) and ketamine (60 mg/kg) administered via intraperitoneal injection.
Oophorectomy protocol
After a median longitudinal opening of the abdominopelvic cavity, the ovaries were removed bilaterally and washed with saline solution (0.9% NaCl). The procedure was performed between 9 and 10 h of AM.
Cryopreservation and thawing protocol
The ovaries were placed in 1.2-mL cryotubes with 1 mL of 1.4 M DMSO as a cryoprotectant and the M2 medium with or without melatonin added to the medium, depending on the study group, and kept at room temperature for 5 min. Slow-freezing was performed using the CL-8800 temperature controller and the Cryogenesis software, and then controlling the freezer from 25–10oC at 1oC/min and to -7oC at 0.5oC/min, keeping the temperature for 5 min. Next, the temperature was lowered to -55oC at 0.5oC/min. At this point, the ovaries were transferred into liquid N2 at -196oC and kept for 24 h [17].
The cryotubes were thawed at room temperature until all the ice melted (15–20 min). The tissue was then transferred to 5 mL of TL-HEPES at room temperature for 10 min, while it was gently agitated to promote DMSO efflux. The tissue was kept in TL-HEPES at 37oC until transplantation [17].
Collection and analysis of the material
The ovarian grafts were recovered and cut in half for analysis. The rats were euthanized with a lethal dose of the previously employed anesthetics. Imaging and measurements were performed with a computer system comprised of a light microscope (Carl Zeiss) adapted to a high-resolution camera (Axio Cam MRC, Carl Zeiss) and a color video monitor. Measurements were taken with an image analysis software program (AxionVision REL 4.6, Carl Zeiss). Counting was always performed using four fields per animal.
The analyses comprised:
1) Estrous cycle
With the animal immobilized, the vaginal smear was first obtained using a swab impregnated with saline solution and then placed on a standard slide for subsequent staining using the Shorr-Harris technique [18]. The slides were subsequently analyzed under a light microscope at 10 × and 40 × magnification. The phases of the estrous cycle were determined according to the proportion of cells observed in the smears as follows: 1) proestrus: predominance of nucleated epithelial cells; 2) estrus: predominance of non-nucleated keratinized cells; 3) diestrus: equal proportion of leukocytes and nucleated keratinized epithelial cells [18].
2) Histology
To assess follicle development, the ovarian follicles were counted and then categorized into developing follicles, regardless of their stage and atretic follicles. The former were classified according to the degree of maturation as follows: immature follicles (including primordial, primary, and secondary), mature follicles (with a single voluminous antrum), and corpora lutea. For counting purposes, the ovarian follicles comprised of both viable and atretic follicles as well as both normal and degenerating corpora lutea [19].
Blood vessel count was performed in a 100-square-micrometer area in five randomly selected fields and analyzed by two independent observers.
For evaluation of fibrosis, the slides were stained with picrosirius red, and measurements were taken in eight fields per animal in the ovarian stroma, with a magnification of 400 × times, and the results were expressed as a percentage of the positive area (unit/mm2).
The evaluation of the slides was conducted at our Medical Investigation Laboratory (LIM-58). For the quantification of the parameters evaluated, the images were captured using a high-resolution camera (AxioCam-MCR, Carl Zeiss) adapted to a light microscope (Axiolab, Carl Zeiss) adjusted to the 40 × objective lens [17].
3) Immunohistochemistry
Slides with sections of the ovarian grafts were stained using immunohistochemistry to measure the von Willebrand factor (AB6994, 1:100, Abcam Inc., Cambridge, MA, USA), Ki67 (M724001-2, 1:100, Dako North America Inc., Carpinteria, CA, USA), cleaved caspase-3 (SANT-SC-1226, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling, Roche, Indianapolis, IN, USA). Hormonal receptor expression for estrogen (E1644, 1:50, Springe Bioscience Corporation, Pleasanton, CA, USA) and progesterone (AB51896, 1:30, Abcam Inc., Cambridge, MA, USA) was also studied. All preparations were performed according to the manufacturer’s instructions.
Microscopy images were obtained using a computer program (Leica DM2500), and quantifications were performed with the LeicaQWin V3 program. The red-brown coloration of the cytoplasm and nucleus of granulosa cells and antral follicles (for apoptosis and Ki-67) or stroma (for fibrosis and expression of endothelial cells) was considered as positive expression and any other color, as negative. Hormone receptors were evaluated in both the stroma and follicular cells. The analysis was performed in eight different fields per animal at 400 × magnification, and the results were expressed as a percentage of the positive area (unit/mm2). The interpretation was performed by two independent and blinded investigators.
Statistical analysis
The data from each group of animals were analyzed according to the type of variables. The ANOVA and the Tukey tests were used, and the rejection level of the null hypothesis was set at 0.05 or 5% (p < 0.05).