2.1 Ethical approval
All procedures with animals were approved by the Institutional Animal Care and Use Committee of Nanchang, and were performed in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals.
2.2 The preparation of COS·Zn
COS·Zn was prepared under optimal reaction conditions, which were determined in the study of Jin Yue . COS and zinc sulfate were weighed with a mass ratio of 2:1 and respectively dissolved in two beakers with equal amounts of distilled water. Then the two solutions were mixed in equal volumes and the pH was slowly adjusted to 7 with 1% ammonium hydroxide solution. The mixture was then chelated at 40°C for about 30 minutes, and anhydrous ethanol at three times the volume of the mixture was slowly added to it. After rinsing with anhydrous ethanol, the product was dried in a 60°C oven for several hours to obtain a yellow powder, which was just COS·Zn. The zinc content of the COS·Zn prepared with this method was 10%.
2.3 Mice and treatments
Female KM mice aged 6 weeks (weighed 20-25g), purchased from the Laboratory Animal Center of Nanchang University, were allowed to adapt to the facility for at least 7 days before the experiment started. Food and water were abundant at all times. The temperature was maintained at 22±1°C. Each mouse received a 12h cycle of brightness and darkness, and mice were randomly divided into the treatment and prevention groups, with 6 mice in each.
Each group of mice, except the control group and COS·Zn+CY/BUS group, was administered CY/BUS for 21 days to build the models of premature ovarian failure (POF). The mice in the COS·Zn+CY/BUS group were prophylactically administered COS·Zn (300mg/kg.d) for 21 days. No special operations were offered to the control group.
The mass ratio of COS to Zn in the COS·Zn synthesized in our laboratory is 9:1, and all of the concentrations of COS·Zn mentioned in this article were measured by COS (Fig. 1). Based on the literature review and our actual observation results, it was determined that the daily food intake of the experimental mice was about 6g, which means that every mouse in the COS·Zn (150mg/kg.d) group requires 1mg COS·Zn per day and 2mg COS·Zn per day in the COS·Zn (300mg/kg.d) group. We took 0.04g COS·Zn powder, dissolved in 8mL normal saline, shaken and mixed, and added 2mL sodium carboxymethyl cellulose to prevent the deposition of COS·Zn powder; this was shaken and mixed again, to make a 4mg/mL COS·Zn solution. After the establishment of POF models, the mice underwent the following procedures: (1) the COS·Zn (150mg/kg.d) group was administered 0.125mg/mL COS·Zn solution per day by intragastric (IG) administration for 21 days; (2) the COS·Zn (300mg/kg.d) group was administered 0.25mg/mL COS·Zn solution per day by IG administration for 21 days; (3) the COS·Zn+CY/BUS group received CY/BUS by intraperitoneal (IP) injection; and (4) the control and CY/BUS groups received 0.25mL normal saline per day by IG administration for 21 days.
2.4 Histological analysis of ovarian tissue and ovarian follicle count
Mice were killed by cervical dislocation. The ovaries were collected, and fixed in 4% paraformaldehyde, before they were embedded in paraffin, as described previously . The paraffin-embedded ovaries were cut into serial sections and stained using hematoxylin and eosin (HE). The ovarian follicles were counted as described previously [38,39]. Immunostaining of the ovaries from mice from the treated and prevention groups was performed according to previously published procedures .
The primary antibodies used in this study were as follows: anti-MVH (1:100, ab27591), anti-OCT4 (1:100, ab18976), anti-PCNA (1:100, ab56701), anti-P53 (1:100, ab54073), anti-P16 (1:100, ab54073), anti-IL2 (1:100, ab54073), anti-IL4, anti-TNF-α, anti- NRF2, anti-Sestrin2 and anti-SOD2 (1:100, ab51134). The secondary antibodies used in this study were goat anti-mouse and goat anti-rabbit IgG conjugated with fluorescein isothiocyanate at a dilution of 1:200 (proteintech, China). All of the images were taken using a NIKON Eclipse 80i microscope.
2.5 Western blotting
Total protein was extracted from different ovarian tissues with RIPA lysis solution (Beyotime, P0013C). The proteins were subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and then transferred to PVDF membranes (Millipore Corp., Bedford, MA) before being processed according to the antibody manufacturer’s instructions. The antibodies used for western blotting were anti-MVH (1:100, ab27591), anti-OCT4 (1:100, ab18976), anti-PCNA (1:100, ab56701), anti-P53 (1:100, ab54073), anti-P16 (1:100, ab54073), anti-NRF2, anti-Sestrin2 and anti-SOD2 (1:100, ab51134). The results were analyzed by the gel imaging and analysis system and converted to semi-quantitative data by the Gelscan software. The level of β-actin was quantified at the same time as an internal quantitative control. Each experiment was repeated at least three times.
2.6 Tunnel assay
The apoptotic rates of ovarian sections were detected by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL), and the procedure was performed using an Apoptosis Detection Kit (Solarbio Biotech Co., Ltd., Beijing, China) according to the manufacturer’s instructions. After being treated with TUNEL reaction mixture for 1 h, the sections were washed twice with PBS for 10 min and counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) for 5 min. Washing was then performed three times with ddH2O. A confocal laser scanning microscope (Leica, TSP8) was used to photograph sections, and the apoptotic rate was analyzed using IPP 6.0 Software.
2.7 SOD and GSH detection
Superoxide dismutase (SOD): SOD was assessed using the WST-1 method. Blank wells, standard wells, determination wells and control wells were prepared according to the experimental requirements. Serum from mice was incubated at 37°C for 20 min. The absorbance value of each well at 450 nm was detected by a microplate reader.
Malondialdehyde (GSH): GSH was determined using the thiobarbituric acid method. According to the instructions provided by Shanghai GeneChem Biotechnology Co., Ltd. (Shanghai, China), blank wells, standard wells, determination wells and control wells were prepared. The absorbance value of each well at 450 nm was detected by a microplate reader (1 cm of optical path, set zero by distilled water).
2.8 Hormone level measurement
Blood was drawn from the orbital socket and serum was collected. The levels of E2 and FSH were measured by the biological company.
2.9 Statistical methods
All analyses were performed using GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA). The statistical comparisons between different groups were analyzed by Student’s paired t-test. The threshold of p < 0.05 was considered significant; p < 0.01 and p < 0.001 were each considered extremely significant.