Animals
A total of 176 female mice 6–8 weeks-old from Institute of Cancer Research (ICR) mice (Yangzhou University Comparative Medicine Centre) were housed under standard conditions of temperature (23°C ± 2°C), relative humidity (65%±5%), and a 12-h light/dark cycle. Food and water were provided ad libitum. The experimental protocols and animal handling procedures were approved and monitored by the Institutional Animal Care and Use Committee (IACUC) of Yangzhou University (SYXK(SU)2017-0044).
Ovarian tissue vitrification and thawing
Mice (n = 6) were anesthetized by intraperitoneal (IP) injection of a solution of ketamine (0.15 mg/g of body weight) and xylazine (0.016 mg/g of body weight). Both ovaries were removed and freed of fat. Ovaries were vitrified according to the protocols described by Youmetal. [28]. Briefly, intact OT was serially exposed to equilibration solution (ES) for 10 min and vitrification solution (VS) for 5 min [ES composition: Dulbecco’s phosphate-buffered saline (DPBS) supplemented with 20% (v/v) fetal bovine serum (FBS; Sigma, USA), 7.5%(v/v) dimethylsulfoxide (DMSO; Sigma, USA) and 7.5% (v/v) ethylene glycol (EG; Sigma, USA); VS composition: D-PBS containing 20% FBS, 20% EG, 20% DMSO and 0.5 M sucrose (Sigma, USA)]. The ovaries were placed on gauze to remove the vitrification solution. Then, we placed the ovaries in a 1.5-ml cryovial (Corning, Mexico) and placed the cryovial in liquid nitrogen (LN2).
A week after vitrification, the vials were rapidly removed from LN2, incubated at room temperature for 10–20 s and warmed through stepwise dilution of sucrose at concentrations of 1.0, 0.5, 0.25, and 0 M in D-PBS medium supplemented with 20% FBS for 5 min each. In the fresh (n = 6) and vitrification-warmed control groups, the OT was immediately fixed in 4% paraformaldehyde after ovariectomy or freezing and thawing, respectively.
Experiment I: Evaluation of the best concentration of resveratrol
Autotransplantation was performed as described in Lee et al. [29]. After analgesia was provided to the mice, the dorsal fur was shaved, and the abdominal skin was sterilized with 70% (v/v) alcohol. A 1-cm incision was made on the skin, and both kidneys were externalized through the incision site. A small hole was created, vitrification-warmed ovaries from a mouse were placed inside the bilateral kidney capsules, and the incision and skin were closed and sutured. the mice were randomly divided into four groups according to the treatment: saline (n = 18); 5 mg/kg resveratrol (n = 18); 15 mg/kg resveratrol (n = 18) and 45mg/mg resveratrol (n = 18). Substances were given orally for one week. Resveratrol was insoluble in water and dissolved in 1% sodium carboxymethyl cellulose solution. Sodium carboxymethyl cellulose was diluted in saline. 1 ml of different concentration of resveratrol was given by oral gavage daily. The same volume of saline was given by oral gavage in the sham control group by using the same scheme described earlier. Ovarian grafts were retrieved 3, 7 and 21 days after transplantation to assess the morphology and graft survival. Six mice per group were used for histology and serum E2 and FSH analyses to select the most effective dose of resveratrol. Based on previous studies [30], we selected days 3, 7 and 21 for evaluation of ovarian follicle development and function. After mice were placed in airtight containers and anesthetized to death with an overdose of isoflurane.
Follicle classification and morphological analysis
OTs were fixed in 4% paraformaldehyde and embedded in paraffin. The specimens were cut into 5-µm sections for morphological analysis or immunohistochemical staining. The sections were collected and stained with hematoxylin and eosin (H&E, Weiwo, China). Ovarian follicles were classified according to Lundy et al. [31] for developmental stage. The stages were defined as follows:
Primordial follicles: a single layer of flattened granulosa;
Primary follicles: a complete single layer of granulosa cells, one or more of which is cuboidal;
Secondary follicles: two or more layers of cuboidal granulosa cells with no antrum;
Antral follicles: multiple layers of cuboidal granulosa cells with the antrum present.
Gandolfi’s [6] criteria were used for morphological integrity.
Primordial/primary follicle: G1, spherical with an even distribution of granulosa cells; G2, granulosa cells pulled away from the edge of the follicle but with spherical oocytes; G3, pyknotic nuclei, misshapen oocytes or vacuolation; Secondary/antral follicle: G1, intact spherical follicle with evenly distributed granulosa and theca cells, a small space and spherical oocytes; G2, intact theca cells, disrupted granulosa cells and spherical oocytes; G3, disruption and loss of granulosa and theca cells, pyknotic nuclei and missing oocytes.
Enzyme-linked immunosorbent assays for E2 and FSH
Whole blood was collected from OT-transplanted mice 3, 7 and 21 days after transplantation. After centrifugation (3000g for 5min), an enzyme-linked immunosorbent assay (ELISA) was performed to analyze E2 and FSH (Cusabio, China) in sera. A microplate reader capable of measuring absorbance at 450 nm was used, and the concentrations of serum E2 and FSH were calculated. The intra- and inter assay precision coefficients of variation were < 15%. The minimum detectable dose of mouse E2 is typically less than 40 pg/ml, and FSH is less than 2.5 mIU/ml. A standard curve was created by reducing the data using computer software capable of generating a four-parameter logistic curve fit. E2 and FSH levels were calculated by the professional software ‘Curve Expert 1.4’ (Curve Expert, USA).
Experiment II: Further evaluation of the effects of resveratrol and exploration of the mechanisms
From follicle histological and endocrine function, we found that 45 mg/kg resveratrol was beneficial for the grafted ovary tissue. The mice were randomly divided into two groups: saline (n = 46) and 45 mg/kg resveratrol (n = 46).
Apoptosis analysis
We observed follicle depletion in the early days of transplantation (day3 and day7). Therefore, we analyzed apoptosis on day 3 and day 7 between the two groups.
After deparaffinization and rehydration, sections were washed in PBS and treated with proteinase K (10 mg/ml, 37°C, 30 min) in 10 mM Tris-HCl buffer. Then the sections were rinsed twice with PBS, they were incubated with 50 µl of TUNEL reaction mixture for 1 h at 37°C in a humidified chamber in the dark and rinsed with PBS twice. Negative controls were prepared by treatment with 1500 U/ml DNase I (Roche Applied Science) in 50 mM Tris–HCl (pH 7.5, including 1 mg/ml bovine serum albumin) for 10 min at room temperature to induce DNA strand breaks prior to the labeling procedures. Some OT specimens were used as negative controls by substituting terminal deoxynucleotidyl transferase with distilled water in the reaction mixture following the protocol. Mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) was added (Vector Laboratories, USA), and the samples were examined under an inverted Zeiss AX10 microscope. An untreated TUNEL reaction mixture was used as a negative control, and a slide treated with 100 U/ml of DNase I was used as a positive control.
Six tissue sections from each group were obtained for analysis (n = 24). TUNEL-positive cells produced green fluorescence at excitation wavelengths. When 30% of the cells in one follicle were TUNEL positive, the follicle was regarded as apoptotic. DAPI produces fluorescence when bound to DNA.
Immunohistochemistry
Ovaries were collected from OT-transplanted mice at 3 and 7 days. To explore the molecular mechanism of resveratrol, we tested relevant markers on the grafted ovary through immunohistochemical examination to validate the selected pathways. Sections were incubated for 1 h with primary antibodies against MDA (StressMarp, SMC-515, 1:800), SOD2 (Huabio, ET1701-54, 1:50), NF-κb (Huabio, R1309-9, 1:50), IL-6 (Huabio, R1412-2, 1:50), and SIRT1 (Huabio, ER1308-11, 1:50) at RT in a humid chamber, washed, and treated with EnVision goat anti-mouse IgG (Fuzhou Maixin, KIT-5006). A peroxidase substrate kit (SGK347011, Shanghai Gene) was used as a chromogen, and hematoxylin was used as a counterstain. Finally, six tissue sections from each group were obtained for analysis. The slides were examined under an inverted Zeiss AX10 microscope (Carl Zeiss, Germany). Brown coloring of the cytoplasm/nucleus of stromal cells, granulosa cells, or oocytes was defined as positive staining (any other coloring was considered negative staining).
Gene expression analysis with real-time polymerase chain reaction
Ovaries were collected from OT-transplanted mice at 3 and 7 days, 8 mice per group were used for real-time quantitative PCR analysis (n = 32), and total RNA was isolated by using TRIzol (Invitrogen, Carlsbad, CA, USA). cDNA synthesis was performed with PrimeScriptTM RT Master Mix (Perfect Real Time). PCR analyses were performed with TB Green™ Premix Ex Taq™ II (TliRNaseH Plus) (TaKaRa, Code No. RR820A). The final PCR volume was 10 µl. The primer sequences used for real-time quantitative PCR are listed in Table2.
The cycling conditions for the PCR were as follows: 95°C for 30 s and 95°C for 5 s for 40 cycles and 60°C for 30 s for 40 cycles. Gene expression levels were evaluated using the delta-delta CT method and standardized to the GAPDH amplification levels.
Western blot analysis
Ovaries were harvested on days 3 and 7 after transplantation and rapidly frozen in LN2, and six mice per group were used for western blot analysis (n = 24). Each ovary tissue sample was homogenized in ice-cold homogenization buffer containing protease inhibitors. Tissue homogenates were centrifuged at 5000 × g for 5 min at 4°C, and the supernatants were collected. The Bradford method was used for protein quantification. Proteins solubilized in 4 × Laemmli sample buffer were subjected to SDS-PAGE and then transferred onto nitrocellulose membranes. After the membranes were blocked with T-TBS for 1 h at room temperature, they were probed with various primary antibodies overnight at 4°C [MDA (StressMarp, SMC-515, 1:1000), SOD2 (Huabio, ET1701-54, 1:1000), NF-κB (Huabio, R1309-9, 1:500), IL-6 (Huabio, R1412-2, 1:1000) and SIRT1 (Huabio, ER130811, 1:1000)]. After the membranes were washed with T-TBS, they were incubated with the corresponding secondary antibodies conjugated with horseradish peroxide for 1 h. Detection was conducted with an ECL kit (Millipore). Protein bands were scanned and analyzed with Quantity One Image Analysis Software (Bio-Rad, Hercules, CA, USA).
Oocyte retrieval, in vitro maturation, IVF and in vitro culture
Twenty-one days after OT transplantation, 12 transplanted mice (6 mice/group) were hyperstimulated via intraperitoneal injection of 10 IU of pregnant mare’s serum gonadotrophin. After 48 h, cumulus–oocyte complexes (COCs) were obtained by ovary puncture and then matured in vitro in M16 medium (Sigma, USA) at 37°C for 14 h. After in vitro maturation (IVM), nuclear maturation was examined. Extrusion of the first polar body was the maturation criterion and was scored under an inverted microscope (50× magnification). For sperm collection, mice were executed by cervical dislocation, and the abdominal skin was sterilized with 70% (v/v) alcohol. A 1.5-cm incision was made in midline abdominal wall, and the reproductive system was exposed. The head and tail of the epididymis and part of the vas deferens were cut together and then placed in culture medium in a glass dish. Fat tissue was removed under a microscope, and sperm inside the head of the epididymis and vas deferens were extruded to the cauda with ophthalmic forceps. Then, the heads of the epididymis and vas deferens were cut off. Sperm were released from the cauda of the epididymis and incubated for 1 h. The capacitation fluid containing sperm was absorbed and centrifuged at 1000 rpm for 4 min to collect sperm.
For fertilization, mature metaphase II (MII) oocytes were inseminated with sperm for 5 h, and the fertilized embryos obtained were washed with M16 medium and incubated at 37°C for 24 h. Then, the fertilized embryos were placed in M16 medium (Global Media, LifeGlobal, Belgium) for further development. Fertilization was assessed by the formation of two cells 24 h after insemination. The cleavage and blastocyst rates were calculated.
Statistical analysis
The Statistical Package for the Social Sciences version 24.0 software (SPSS, Inc., USA) and GraphPad Prism 5.0 (GraphPad Software, USA) were used for statistical analysis. Unless otherwise indicated, the results are shown as the mean ± SD. Data were analyzed by Student’s t-test (TUNEL, MII oocytes, fertilization, cleavage and blastocyst formation), one-way analysis of variance (serum FSH and E2, qRT-PCR, and western blot), and χ2 tests (the proportions of G1 follicles). P < 0.05 was considered significant.