Metformin prevents murine ovarian aging

Long-term treatment of mice with metformin is relatively safe

The aging of female ovary is accompanied by the depleted follicles numbers and diminished quality of oocytes. The female ovarian function appears to decline sharply at the age of 37 years old [18]. Previous studies have indicated that the corresponding age in mice is about 28 weeks [19]. Also, one study suggested that the health lifespan of C57/BL mice could be prolonged by feeding with 100mg/kg metformin [11]. Thus, mice of 28-week old were selected in this study, then 100mg/kg metformin was given for half a year to explore whether the intervention of recommended metformin dosage could delay the ovarian aging process and extend reproductive span.

Before we tested if metformin might affect ovarian aging, we first determined whether the long-term treatment of mice with a moderate dosage (100 mg/kg) of metformin is safe to mice or not. After this treatment for 6 months, some mice died of natural causes. We found that both of the CON and MET alive mice are quite healthy, and their body weights and organ weights showed no significant difference between the two groups (Table 1). To test the physical activities and whole body state of the mice, we conducted the average speed, average routine and latency to fall of the animals in the open-field test and in rotarod test, respectively, and found that there is no significant difference between the two groups, either. Interestingly, the MET mice tend to show a higher learning rate compared with the CON mice but with no significant difference, which indicate that MET may improve the memory of mice (Figure 1). Thus, these results indicate that the dosage of metformin used in this study is safe to the animals and thus will be used in the following experiments.

Figure 1. Behavior tests of CON and MET mice after 6-months’ feeding. (A), Rotarod Test; (B), Learning Rate of mice trained on the accelerating rotarod; (C), Open-field Test; (D), Average route of mice in the open-field. Data were represented as mean± S.E.M.

Metformin improves ovarian function

Next, we assessed the effect of metformin on mouse ovarian functions by first examining estrous cycles and hormones levels, as they can reflect the ovarian endocrine function. In this study, the 4-5 day estrous cycle was judged as a normal estrous cycle, and the estrous cycle lasting longer than 5 days or less than 4 days was considered as an unnormal estrous cycle. Only mice with normal estrous cycle for at least 4 times continuously in a month was regarded as with regular estrous cycle. Mice with the sustained estrous period or the intermittent normal estrous cycle was regarded as irregular. The criteria for the cessation of estrous cycle was the absence or little of cells in the vaginal smear for at least 20 days [20, 21].

As shown in Table 2, the estrous cycles of mice in both groups showed no significant difference after 3-month feeding, with the ratio of regular cycles of 85.7% in CON mice over 85% in MET mice. However, after feeding the animals with metformin-containing foods for 6 months, the regular cycle ratio of the CON mouse ovaries dropped to 38.5%, while that in the MET mice still maintained quite high at 78.9%. This result indicates that metformin protects the murine ovaries from aging by maintaining its ovarian regular cycle at a higher level. Consistent with this result, the level of E2 in the MET mice was significantly higher than that in the CON mice, though the levels of FSH showed no significant difference between the two groups (Figure 2). These results indicate that metformin could improve murine ovarian endocrine functions at the animals’ middle age.

Figure 2. E2 and FSH levels in both groups. Data were represented as mean± S.E.M. *Compared with the CON group, *P<0.05

Metformin increases the ovarian reserve

Then, we tested the ovarian reserve on those mice by measuring the number of healthy ovarian follicles, as the proper ovarian function depends on sufficient ovarian reserve. We found that the MET mice exhibited more primordial and primary follicles than CON mice (P<0.05, indicating much higher ovarian reserve (Figure 3). This result demonstrates that metformin can increase ovarian reserve significantly.

Figure 3. Effect of MET feeding for 6 months on follicle numbers at different stages. (A), The histological images of the ovaries in the CON and MET groups. The green and red arrows show primordial and primary follicles respectively. (B). The average number of follicles in each stage in the two groups. Data were presented as the mean± S.E.M. *indicates a significant difference from the CON group. *P<0.05, **P < 0.01.

Metformin decreases oxidative damage and P16 in ovaries

To further analyze the molecular and cellular changes in murine ovaries after treatment with metformin, we measured the levels of the proteins 8-OhdG,NTY and 4-HNE as they are widely accepted as biomarkers of oxidative DNA, protein, and lipid damage respectively [22]. We found that the levels of both 8-OHdG and 4-HNE are decreased in MET murine ovaries compared to that in CON murine ovaries, though there is no difference in the NTY expression between the two groups.

We also tested the level of senescence-associated protein P16 by immunohistochemistry. The P16 expression was significantly lower in the MET murine ovaries than that in the CON group, indicating a relatively younger state of ovary after 6-month feeding with metformin (Figure 4). These results demonstrate that metformin can reduce oxidative damage and P16 levels in murine ovaries and thus maintain the organs in a relatively young age.

Figure 4. Effects of MET on oxidative damage markers and the p16 level in both groups. (A). The representative IHC images of p16 protein in the CON and MET mouse ovaries. (B). Average IOD of the markers in the two groups. Values are expressed as mean± S.E.M. *indicates a significant difference between the two group. *P<0.05, **P < 0.01.

Metformin induces SIRT1 level, but reduces p-rpS6 level

To investigate the potential underlying mechanisms for the aforementioned metformin’s role in protecting ovarian aging, we determined the levels of ovarian aging associated proteins, such as SIRT1, p-rpS6 and Bcl2 by immunoblotting analysis, as the expression of SIRT1 is closely associated with ovarian reserve [23]. As a result, we found that the SIRT1 level in MET mice was higher than that in CON mice. The p-rps6 protein plays a vital role in the activation and exhaustion of ovarian primordial follicle pool [24]. Our results showed that the level of p-rps6 protein was significantly decreased in the MET group, compared to the CON group (Figure 5). However, there was no difference in the expression of Bcl2 between the two groups of mice. Since previous studies showed that metformin can induce the expression of SIRT1 in other tissues [25], we therefore speculated that metformin might play an anti-aging role probably by inducing the expression of SIRT1 but reducing the level of p-rps6.

Figure 5. Levels of SIRT1, Bcl2, p-rpS6 and rpS6 proteins in murine ovaries. (A) Western blotting analyses of SIRT1, Bcl2, p-rpS6 and rpS6 with antibodies as indicated. (B) Quantitative analysis of data in A. Data were presented as the mean ± S.E.M. *compared with the NC group, *P < 0.05.

Animal treatment

Female C57BL/6 mice of 27-week-old were purchased from Beijing HFK Bio-Technology Co., Ltd. (Beijing, PR China) and fed freely for one week to adapt to the environment. The vaginal smears of these mice were taken every morning for consecutive seven days. One week later, mice with regular estrous cycles and normal body weight (30.59±1.70g) were chosen and divided into two groups, the control group (CON, n=21) and the metformin-treated group (MET, n=22). Mice in the CON group were fed with food and water freely, and those in the MET group were fed with the chows supplemented with 100mg/kg metformin. Body weight was measured every week, and mice were sacrificed at the end of the experiment. Blood samples were centrifuged at 3000 rpm for 5 min, and serum was collected and stored at −80 °C until analysis. And heart, liver, spleen, kidney, uterus, ovary, and brain were separated and weighed. All procedures and protocols conducted on animals were approved by the ethics committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology in the People's Republic of China.

Open-field test

This test was conducted in a quiet environment. Mice were placed back to the operator in the center of the bottom of the wide-field reaction box, then the camera and timing were started. Five minutes later, the camera was stopped, and 75% alcohol was used to clean the box completely so as to wipe out the mice's residual urine, smell and hair. The experiment was repeated after alcohol completely volatilized. The average distance and velocity in the wide field experiment were recorded and analyzed.

Rotarod test

Mice were placed on the rotating bar and rotated in the form of uniform acceleration (10-40r.p.m., 5min). When the mice dropped or time reached 5 minutes, the experiment was stopped and time is recorded. Each mouse was tested 10 times, with 5 minutes apart, and for 5 consecutive days. In the test, mice holding the stick and turning around, or automatic jumping were determined as unqualified situations and excluded from the results. The learning rate and latency to fall of the mice were calculated. Latency to fall was calculated as the average time to fall from the rotarod during the 10 trials for each mouse. Learning rate was calculated as follows (mean latency to fall _{trials 9 and 10})-(mean latency to fall _{trials 1and 2})/9 (number of intertrial intervals).

Estrous cycle analysis

Vaginal smears of all mice were taken for consecutive seven days before the experiment began to exclude mice with irregular estrous cycles. During the experiment, vaginal smears were taken at about 9 am every day for consecutive 14 days after 3-month and 6-month feeding, respectively, to evaluate the estrous cycle condition. The vaginal secretion was taken with saline and then spread on the slides, followed by HE staining when it was dry. Then, we determined the stage of the estrous cycle by the cytology under microscope.

Follicle counts

Six mice for both groups were used for follicle counts. Left ovary of each mice was preserved in 4% paraformaldehyde, then cut into pieces of 5um and put on the glass slides. Follicles in each stage were counted by two people separately. The detailed procedures were performed as previously described [33].

Enzyme immunoassay for 17β-estradiol (E2) and progesterone (P)

Blood samples were collected when mice were sacrificed. Levels of serum E2 and P were measured by enzyme-linked immune sorbent assay (ELISA) according to the manufacturer's instructions (Cayman Chemical Company, Ann Arbor, USA).

Western blot analysis

Western blot analysis was performed as previously described [34]. GAPDH was considered to be a loading control. Immunoreactive bands were visualized using alkaline phosphatase and BCIP/NBT staining. All blots were repeated three times.

Immunohistochemical staining

Five representative sections from each of the ten selected ovaries per group were used for immunohistochemistry (IHC) to assess expression of P16, NTY, 4HNE, and 8-OdH. The ovaries were fixed in 4% paraformaldehyde for 24 h, then subsequently placed in 70% ethanol and bathed in paraffin. Ovaries were consecutively cut into 5μm, and every fifth section was transferred onto a slide. IHC analysis of ovarian tissues was performed using routine procedures as previously reported. Images were obtained using confocal microscopy (DM4000B; Leica, Germany).

Statistical analysis

SPSS 17.0 software was used to conduct the statistical analyses. Group differences in body weight, length of estrous cycles, exhausted swimming time, hormone levels, follicle number counts and protein expression were investigated using Student’ s t-tests or non-parametric tests. Differences in the expression of P16 between the experimental and control groups were examined using the non-parametric Kruskal–Wallis test Values are represented as the mean ± SEM, and P< 0.05 was considered statistically significant.