Female C57BL/6 mice (body weight, 15±2 g) at 3 weeks of age were obtained from Guangdong Medical Laboratory Animal Center. All experimental animals were housed individually under a 12/12 h light/dark cycle in a specific pathogen-free animal facility at the Laboratory Animal Center of Southern Medical University (Guangdong, China), with controlled temperature (23±2°C) and relative humidity (40–60%). Food and water were available ad libitum during the experimental period. The experimental procedures and animal conditions were approved by the Animal Ethics Committee of The Third Affiliated Hospital of Guangzhou Medical University.

The mice were divided into four groups as described below (n=12 per group). Mice in the heavy metal coupling agent group (group A) were given pure water containing 0.3 mmol/l EDTA and 0.15 mmol/l sodium citrate (Sigma-Aldrich; Merck KGaA, Darmstadt Germany). Mice in the antioxidant group (group B) were given pure water supplemented with 40 mg/l lipoic acid and 100 mg/l acetyl carnitine (GNC Company, Propinsi Lampung, Indonesia). All concentrations were selected on basis of preliminary experiments (data not shown). Mice in the mixed group (group C) were given pure water supplemented with heavy metal coupling agents and antioxidants. Mice in the normal control group (group D) were given pure water. The pure water was processed using aseptic filtration. Mice were randomly selected from each group for subsequent experiments following 3, 6, 9 and 12 months of treatment.

Female mice were superovulated by intraperitoneal injection of 10 IU of pregnant mare serum gonadotropin (PMSG), followed 46–48 h later by 10 IU of human chorionic gonadotropin (HCG; both Ningbo Second Hormone Factory, Zhejiang, China). Drinking water was provided to mice from each group during the promotion of ovulation. The mice were sacrificed by cervical dislocation after 12–13 h to collect the oocytes. Briefly, the abdominal cavity was opened exposing the uterus, fallopian tubes and ovaries, followed by separation of blood vessels and adipose tissue. Then, the fallopian tubes were removed and quickly placed in human fallopian tube fluid (HTF) which was artificially prepared, and the fallopian tubes of mice in the same group were grouped together. Following rinsing with HTF three times, the magnum of the fallopian tubes was removed to release cumulus-oocyte complexes (COCs). The COCs were washed twice with HTF and incubated with hyaluronidase (1:9; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) to remove granular lymphocytes and obtain MII-stage nude oocytes. The oocytes were counted following three washes with HTF and incubated at 37°C in a humidified 5% CO2 incubator in 40 µl drops of HTF medium covered with mineral oil (Sigma-Aldrich; Merck KGaA). The mice reared for 12 months did not have a sufficient number of oocytes, thus, only the number of oocytes in this age group was counted.

A JC-1 mitochondrial membrane potential detection kit (Sigma-Aldrich; Merck KGaA) was used to evaluate the mitochondrial membrane potential. First, 1X working solution was prepared with 200X JC-1, dyeing buffer and pure water according to the manufacturer's protocol, then preheated at 37°C in a water bath. Next, 50 µl of 1X working solution was divided into several droplets, which were covered with mineral oil and preheated at 37°C. Subsequently, 15–20 MII-stage oocytes were incubated for 20–25 min with the working solution at 37°C in 5% CO2, followed by 2–3 washes. JC-1 mitochondrial fluorescence was visualized with a laser-scanning confocal microscope (Nikon-C2Si; Nikon Corp., Tokyo, Japan), and ImageJ software was used for quantitative analysis.

MII-stage oocytes were digested in 3% trypsin for 2–5 min to clear the zona pellucida and then quickly transferred to the fresh HTF droplets. Appropriately sized circles were drawn on a clean glass slide using a crayon, and each circle was filled with an appropriate amount of 1% paraformaldehyde (Sigma-Aldrich; Merck KGaA) with 5–10 oocytes. After the nuclear membrane was ruptured, the oocytes were fixed, dried and marked, and stored in a −20°C freezer. For imaging, the glass slides were removed, stained for 10 min with 10 µg/ml propidium iodide (PI) in the dark, covered with a cover glass, mounted with nail polish, and then stored at −20°C. The number of chromosomes was calculated in each oocyte.

First, oocytes were fixed. Briefly, 25 MII-stage oocytes from each group with normal morphology were fixed with 4% paraformaldehyde at room temperature for 30 min. Following permeabilization with 0.5% Triton X-100 in phosphate-buffered saline (PBS) for 20 min, the oocytes were blocked in a blocking solution (PBS containing 1% bovine serum albumin) for 1 h and then incubated with an α-tubulin antibody (Sigma-Aldrich; Merck KGaA) diluted in blocking solution for 1.5 h at room temperature or overnight at 4°C. After the oocytes were washed with a washing solution (PBS with 0.1% Tween-20 and 0.01% Triton X-100) three times (5 min/wash), the chromosomes were stained with PI (Sigma-Aldrich; Merck KGaA) for 15 min, and the oocytes were then mounted on glass slides and observed using a laser-scanning confocal microscope.

Next, the spindles were observed. In normal oocytes, the spindles exhibit a fusiform shape and have a granular structure at the two poles, with regularly arranged spindle microtubules (green) connecting the spindle poles to the chromosomes (red) that are arranged in order on the equatorial plate. However, in an abnormal state, the spindle length is contracted, the microtubules are irregular or completely absent, and the chromosomes are arranged outside of the equatorial plate, with a dispersed distribution, or only lightly stained.

Statistical analyses were performed using SPSS version 13.0 (SPSS Inc., Chicago, IL, USA). All experimental data are presented as percentages and were analyzed using the χ² test. A repeated-measures analysis of variance was also performed and post hoc multiple factor analysis of variance with Scheffé's test were applied to compare the quantitative data, with α=0.05 as a test standard. P<0.05 was considered to indicate a statistically significant difference.

To reduce individual differences, 10 mice were randomly selected from each group at each age stage, and the average number of oocytes was calculated. Analysis of variance and multiple-factor repetitive analysis revealed that the primary effect of age in each group was significantly different (P<0.05), the differences in the oocyte numbers at different ages were statistically significant (P<0.05), and different associations were observed between different groups and different age stages (P<0.05). There were significant differences in the number of oocytes between group D and the other three groups at 3 months (P<0.05). In addition, at 6, 9 and 12 months, the oocyte number was significantly different between group B and group D, and between group C and group D (both P<0.05) (Fig. 1). These results demonstrated that with prolonged treatment times, the oocyte number was better-maintained in the antioxidant group compared with that in the heavy metal coupling agent and normal control groups. Meanwhile, the oocyte number in the heavy metal coupling agent group was significantly different from that in the normal control group at 3 months, indicating that heavy metal coupling agents serve a role in maintaining oocyte numbers, but the effect was not as evident as that of antioxidants. One possible reason might be that the mice were not exposed to many heavy metals during treatment, which made the heavy metal coupling agents less useful.

In our experiment, mitochondrial activity in mouse oocytes decreased with age (Fig. 2A). As shown in Fig. 2B, in the four groups, mitochondrial activity, represented as red fluorescence, was significantly lower at 9 months compared with at 3 or 6 months. In addition, groups B and C had significantly higher mitochondrial activity levels compared with groups A and B at 9 months. Furthermore, no significant differences were observed in mitochondrial activity between the four groups at 3 and 6 months. These results suggested that antioxidants effectively alleviated the reduction in mitochondrial activity.

As shown in Fig. 3, the incidence of chromosomal abnormality increased with age and/or treatment time in each group. At 3 months, there were no significant differences in chromosome abnormalities between the four groups (P>0.05). Interestingly, groups A and D exhibited significantly increased chromosomal abnormalities compared with groups B and C at 6 and 9 months. These results indicated that antioxidants reduced the incidence of chromosomal abnormalities.

Immunofluorescence analysis was performed to detect the condition of spindles in oocytes (Fig. 4A). As shown in Fig. 4B, the incidence of spindle abnormality increased with age and/or treatment time in each group. Groups B and C exhibited no significant differences in spindle abnormality rate at any time point (P>0.05). There were statistically significant differences in spindle abnormalities between group A and group D at the same time points (P<0.05). At 6 and 9 months, the spindle abnormalities in groups B and C were remarkably lower compared with that in groups A and D. These results suggested that antioxidants reduced the incidence of spindle abnormalities.