Repeated superovulation increases the risk of osteoporosis and cardiovascular diseases by accelerating ovarian aging in mice

Repeated superovulation decreased the physical strength of mice

We evaluated the general conditions and physiological function of the two groups of mice. The outcomes of the physiological parameters were analyzed and are summarized in Fig. 1. The body weight of the RS group did not increase significantly after ten cycles of RS (Fig. 1A). We used the exhausted swimming time to represent physical strength and found that the strength of the RS group mice was markedly decreased (Fig. 1B). A photo of the two groups of mice is shown in Fig. 1C.

Figure 1. The general condition of both groups of mice. (A) Body weight was not significantly different in both groups. (B) The exhausted swimming time of the RS mice was markedly decreased compared to that of the NC group. (C) Representative photos of NC and RS group mice. *significantly greater at P<0.05 compared to the NC group.

Repeated superovulation reduced the bone density of mice

Osteoporosis, which is characterized by changes in the architecture of bone, is the most common consequence of menopause. We examined the bone mass to evaluate the effect of the ovary in the repeated super-ovulated mice on the skeletal system. Six months after modeling, the architecture of bone in each group was investigated using the SCANCO Micro-CT50 (SCANCO Medical) equipped system through trabecular bone analysis (Fig. 2, Table 1). The trabecular bone volume (BV), number of trabeculae (TbN), connectivity density (ConnD), trabeculae thickness (TbTh) and structure model index (SMI) in the tibia showed no significant differences between the two groups. However, the trabeculae separation (TbSp) was outstandingly increased in the tibia of the RS group mice compared with that of the NC group. These changes agreed well with the observed reduced bone mineral density (BMD), which is directly related to the material stiffness of the bone.

Figure 2. The architecture of trabecular bone of the left tibia in the NC group and RS group. The trabeculae separation was outstandingly increased in the tibia of the RS group mice compared with that of the NC group.

Repeated superovulation decreased the heart function of mice

Gender differences exist in cardiovascular disease risk, beyond the protective effect of estrogens, mostly burdening postmenopausal females [17]. Mortality rates of cardiac vascular disease remain higher in women than in men despite the increased awareness of cardiovascular risk factors over recent decades. Estrogens have a protective role in this regard in premenopausal women. However, after the decrease in estrogen levels, women are exposed to the same, or even higher, cardiovascular risks as males [18,19]. Because of this, we examined the echocardiography of the mice, especially the ejection fraction (EF) and fractional shortening (FS), which are the two most commonly used parameters for estimating cardiac function, in the two groups using a Vevo®1100 Imaging system. The representative ultrasonic cardiogram reflected left ventricular function of the heart in both groups of mice (Fig. 3A). Our results indicated that ventricular systolic function declined in the RS group mice. We also found that EF significantly decreased in the RS group compared with the NC group (Fig. 3B). No significant difference in FS was observed between the groups of mice.

Figure 3. Ultrasound imaging planes analysis used for cardiovascular assessments of mice of the NC and RS groups. (A) The cardiovascular ultrasound imaging of the NC and RS groups. (B) Cardiac functions of the two groups were estimated by EF and FS. The EF in the RS group was decreased significantly compared with NC group.

Repeated superovulation diminished the ovarian reserve

We further tested the effect of RS on the ovarian reserve and the size of primordial follicle pool. The expression level of AMH, which was only produced by the granulosa cells of primary, preantral and small antral follicles, could represent the number of growing follicles [20]. AMH has been demonstrated to be an ideal marker of ovarian reserves [21–25]. In addition to AMH, the ovarian primordial follicle pool, which serves as a source of developing follicles, is another ideal parameter to evaluate the ovarian reserve [26,27]. Therefore, in this study, we used both the AMH expression and numbers of primordial follicles to reflect the ovarian reserve, evaluating the effect of RS on the ovarian follicular quantity.

When analyzing the glass slides that were stained with HE (Fig. 4A, B), we observed that the primordial follicle numbers in the mice treated with RS were lower than those in NC group. Compared with the NC group, the total AMH mRNA and protein expression of the ovaries in treated mice demonstrated a marked decline (Fig. 4C-E).

Figure 4. Effects of RS on the ovarian reserve of mice. (A) Photographs of the glass slides stained with HE in NC and RS mouse ovaries. Inset (upper right corner of each group) shows the number of primordial follicles (primordial follicles, red arrows). The images shown are at an original magnification of x200; insets are x400. (B) Bar graphs showed that the primordial follicle numbers of RS mice were significantly decreased compared to NC mice. (C) The mRNA expression of AMH decreased in RS mice. (D-E) The protein expression of AMH was determined using western blot analysis; bar graphs showed that the protein expression levels of AMH in RS mice were decreased compared to NC group.

Repeated superovulation weakened the ovarian endocrine and reproductive function of mice.

With the evidence that repeated ovarian stimulation reduced the ovarian reserve, we further evaluated ovarian function, including the estrous cycle length, serum estradiol and progesterone levels and reproductive capacity.

After repeated ovulation, the ovarian weight increased significantly (Fig. 5A). The estrous cycle is a direct manifestation of ovarian function. Fourteen days after the final hCG administration, all of the treated mice were restored to their independent estrous cycles. The estrous cycles of the RS mice were obviously prolonged when compared with those of the NC group (Fig. 5B).

Figure 5. The evaluation of ovarian endocrine and reproductive function. (A) The ovarian weight of the RS group mice increased significantly compared with the NC group mice. (B) The estrous cycles of the RS mice were obviously prolonged. The concentrations of estradiol (C) and progesterone (D) were determined in duplicate using an EIA kit. The mean litter size (E) and the mean number of litters (F) were analyzed during a 6-month course of breeding. The RS mice displayed lower levels of hormones and reduced litter sizes and numbers compared to the NC group. Data presented are the mean ± SEM. * P<0.05 compared to the NC.

The sex hormone levels of 17β-estradiol and proges-terone, which are secreted by granulosa and luteal cells, are usually used to evaluate ovarian endocrine function. We found that the serum levels of 17β-estradiol significantly decreased after RS with a concomitant decrease in progesterone levels (Fig. 5C, D).

As the decline in fertility and fecundity is the most obvious event of ovarian aging, we analyzed the mean litter size and mean number of litters of both groups after treatment during a 6-month course of breeding. Both the neonatal numbers per litter and the total litter numbers were remarkably decreased in the RS group compared with the NC group (Fig. 5E, F).

Repeated superovulation affected the ultrastructure of follicles in the mice ovaries

To analyze the impact of RS on organizational structure, we viewed ultrastructural changes of the ovaries using electron microscopy (Fig. 6A-F). Analysis of the two groups indicated that the mean areas of the GC and GC nuclei were not significantly different (Table 2). However, a marked difference was observed between the NC and RS groups when mitochondrial morphology was compared. In the NC group, cristae were clearly visible and appeared tubular, whereas in most cells of the RS group, the cristae morphology could not be easily identified because highly electron- dense matrix and cristae were barely visible. Mitochondria in some cells became swollen and showed vacuolization and degeneration of the cristae and matrix in the RS group. Moreover, the amounts of lipid droplets and swollen Golgi apparatuses increased significantly in the RS group.

Figure 6. Ultrastructural changes of the ovarian granulosa cells in both groups. (A-C) The normal ultrastructure in the ovaries of NC mice. Few lipids were observed in the ovaries, and most mitochondria structures were normal. (D-F) The mitochondria were swollen, exhibited decreased matrix density, and developed flocculent dense bodies in the matrix space in the RS group mice. The number of lipid droplets and swollen Golgi complexes were increased in RS group mice. (O: oocyte; N: nucleus of granulosa cells; M: mitochondrial; L: lipid; G: Golgi complex).

Repeated superovulation increased oxidative stress in the ovaries

Oxidative lipid (4-HNE), protein (NTY), and DNA (8-OHdG) damage in interstitial cells and follicles of super-ovulated mice were measured. These molecules are widely accepted as biomarkers of oxidative DNA, protein, and lipid damage, respectively, in biological systems [28]. We observed that the expression levels of 8-OHdG, 4-HNE and NTY were all increased in the ovarian interstitial cells of RS mice (Fig. 7A). The percentage of follicles with positive staining for the indicated marker in the granulosa cells or theca cells of healthy and atretic follicles was significantly increased compared with the NC group (Fig. 7B-E). Oxidative damage markers in interstitial cells also increased when compared with the NC group mice (Fig. 7F). SOD2 is one of the most important antioxidant enzymes in all tissues due to its role in defense against oxidative stress, and its expression was upregulated when oxidative stress occurred. Analysis of the expression of this enzyme in both groups showed that repeated ovulation markedly increased the ovarian expression of this enzyme (Fig. 7G).

Figure 7. Effects of repeated superovulation on oxidative damage and antioxidant enzymes in both groups. Oxidative stress related oxidative damage in interstitial cells was scored using immunostaining for oxidative damage markers, as described in the Methods section. (A) Representative images of immunostaining (dark brown) for each oxidative damage marker in interstitial cells: Oxidative DNA damage (8-OHdG), lipid peroxidation (4-HNE) and protein oxidation (NTY) in the ovaries of the NC group, RS group and negative control and positive control groups. All original magnifications are x200. (B-C) The percentages of positive stained granulosa cells in healthy or atretic follicles. (D-E) The percentages of positive theca cells in the healthy or atretic follicles. (F) The percentages of positive interstitial cells in the ovaries. (G) The protein expression of the antioxidant gene SOD2 was significantly increased in the RS group. Values are expressed as the mean ± SEM. *P<0.05 compared to the NC group.

Ethics statement

The experiments were approved by the Animal Care Committee of the Tongji Medical College at the Huazhong University of Science and Technology in China.

Repeated superovulation procedures

Forty-eight specific pathogen-free female C57BL/6J mice (6 weeks old) were obtained from Beijing HFK Bio-Technology Co., LTD. (Beijing, PR China). The mice were housed at a controlled temperature and allowed free access to rodent diet and water. Sixty mice were randomly divided into two groups: normal control (NC) group and RS group.

Mice of the RS group were injected intraperitoneally with 5 IU PMSG at 13:00 (Hangzhou Animal Medicine Factory, Hangzhou, PR China), followed in 48 h by injection of 5 IU hCG, also at 13:00 (Livzon Pharmaceutical Group Inc., Guangzhou, PR China). Twenty-four hours after hCG injection, the sequential administration was repeated ten times in 30 days [32]. The mice of the NC groups were injected intraperitoneally with normal saline at the same time.

Exhaustive swimming exercise

Ten mice in each group were then removed and subjected to exhaustive swimming exercises. Water was put in a plastic pool (93×58×58 cm) until its depth reached 40 cm, and its temperature was kept at 28±1°C. Endurance was observed by compelling the mice to swim in the plastic pool, and it was measured by the time the mice moved actively in the water until they sank to the bottom of the pool without movement for 10 seconds [44,45]. After exercise, the mice were euthanized, and the ovaries were dissected and fixed in 4% paraformaldehyde for ovarian follicle counts.

Sample collection

Mice were monitored daily during the whole treatment. Weekly, they were weighed to estimate the changes in body weight, and images were taken to observe the fur condition. Mice in both groups were euthanized by CO₂ inhalation on the morning of metestrus after one month of treatment. Ovaries and blood samples were collected from a total of twenty mice in each group, and the ovaries were dissected and weighed for real-time polymerase chain reaction, western blotting and immunohistochemistry. The left tibias of mice were isolated for bone density analysis.

Transmission electron microscopy

Immediately after dissection, six ovaries from each group were weighed and fixed by immersing them for 1 h in Karnovsky fixative (2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M Na-cacodylate buffer, pH 7.3), prepared as previously described [46]. Ovaries were cut into pieces approximately 1 mm³ using a scalpel. After dehydration in an ascending series of ethanol, the ovarian tissues were fixed in osmium tetroxide and embedded in epoxy resin. The sections were studied following Reynold lead citrate staining. Small follicles with visible oocyte nuclei were randomly selected, and images of these follicles were taken using a transmission electron microscope (CM 10 Philips, Eindhoven, The Netherlands) at 80 kV.

Ovarian follicle counts

Ten paraffin-embedded ovaries in each group were longitudinally and serially sectioned at 4 µm, and every fifth section was mounted on a glass slide, stained with hematoxylin and eosin (HE), and analyzed under a microscope by two persons who were blinded to the origin of the sections. Only follicles containing oocytes were scored by histomorphometric evaluation, as originally described [47]. The data are represented as the primordial follicle number.

Immunohistochemistry

Eight ovaries in each group were chosen for immunohistochemistry analysis, and representative sections from each ovary were selected. Immunohistochemical analysis was used to localize 8-hydroxy-2–deoxyguanosine (8-OHdG), 4-hydroxynonenal (4-HNE) and nitrotyrosine (NTY). The ovaries were first fixed in 4% paraformaldehyde for one day, and then, they were placed into 70% ethanol and were finally bathed in paraffin. The ovaries were consecutively split into 5-μm thick slices, and each fifth section was transferred onto a slide. Immunohistochemistry for the ovary was performed by routine procedures as previously reported [48]. It is worth mentioning that the primary antibodies were diluted to appropriate concentrations (8-OHdG, ab62623, 1:100 dilution, mouse monoclonal antibody, Abcam, Cambridge, MA, Britain; 4-HNE, ab46545, 1:500 dilution, rabbit polyclonal antibody, Abcam, Cambridge, MA, Britain; NTY, ab7048, 1:50 dilution, rabbit polyclonal antibody, Millipore, Temecula, CA) and were incubated at 4°C overnight. After that, the sections were washed with PBS and then incubated with secondary antibodies and streptavidin-biotin peroxidase complex (SABC) (PK4001/PK4002, Zhongshan Golden Bridge Biotechnology, Zhongshan Golden Bridge, China). Negative controls were left to incubate with PBS rather than the primary antibody, and images were obtained with confocal microscopy (DM4000B; Leica, Germany).

RNA isolation and real-time polymerase chain reaction

The ovaries were kept in storage at −80°C. The RNA was isolated from six ovaries per group to perform real-time polymerase chain reaction (PCR) analysis. Total RNA of the ovaries was extracted according to the manufacturer’s directions using TRIzol (Invitrogen, Carlsbad, CA, USA). The concentration of RNA was measured with the help of a NanoDrop spectrophotometer (λ=260/280 nm; ND 1000; NanoDrop Technologies, Wilmington, DE, USA). Real-time PCR was carried out using the AB StepOne Plus PCR machine (Applied Biosystems, Foster City, CA) as described in our previous study [48]. Relative gene amplification was measured by means of the delta-delta Ct method [49]. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh), which is the housekeeping gene, acted as the endogenous control. The primer sequences were as follows: anti-Mullerian hormone (AMH): forward, 5'-TCCTACATCTGGCTGAAGTGATATG-3, reverse, 5'-CAGGTGGAGGCTCTTGGAACT-3'; GAPDH: forward: 5'-TGTGTCCGTCGT GGATCTGA-3', reverse: 5'-TTG CTG TTG AAG TCG CAG GAG-3'.

Western Blotting

Ten ovaries were collected in each group, and the total expression levels of AMH and superoxide dismutase2 (SOD2) were analyzed. Tissue extracts (30 μg) were electrophoresed on a 10% SDS-PAGE gel and transferred to polyvinylidene fluoride membranes. The membranes were blocked using 5% nonfat milk in Tris-buffered saline (10 mM Tris, 150 mM phosphate buffered saline, pH 8.0) for 1 h at room temperature. Incubation of the membranes was then performed overnight at 4°C with appropriately diluted primary antibodies (AMH, AF1446, 1:250 dilution, goat antibody, R&D Systems, Minneapolis, USA; SOD2, ab68155, 1:1000 dilution, rabbit monoclonal antibody, Epitomics, California, USA; Actin, 1:1000 dilution, rabbit polyclonal antibody, Boster, China). The blots were subsequently incubated with secondary antibodies (1:1000 dilution). The immunoreactive bands were observed using alkaline phosphatase (ALP) and BCIP/NBT staining, and finally, every single blot was performed three more times.

Enzyme immunoassay for 17β-estradiol and progesterone

Concentrations of 17β-estradiol and progesterone in serum were determined using an enzyme linked immunosorbent assay (EIA) kit (Cayman Chemical Company, Ann Arbor, USA) according to the manufacturer's instructions. Briefly, 50 μl of the serum samples was incubated in microtiter wells that were coated with goat anti-mouse monoclonal antibodies against estradiol or progesterone. After incubation and washing, concentrations of 17β-estradiol and progesterone were determined using a spectro-photometer (Bio-Tek, Winooski, USA).

Estrous cycling and fertility status tests

The remaining ten mice in each group were used for estrous cycling and fertility status tests. Estrous cycling in individually housed mice was evaluated every morning for twenty days using vaginal cytology, and mating trials were initiated twenty days after RS treatment and lasted for six months for all of the remaining mice. Males were randomly rotated through the cages during the mating trials. Once pregnancy was detected, the animals were placed in separate cages, and after delivery, these females were subsequently placed back into the mating cage for the next mating. When mating trials were over, all mice were euthanized using CO₂ inhalation.

Ultrasound Imaging and Doppler Echocardiography

Ultrasound scanning was conducted with a Vevo®1100 Imaging system on six mice from each group after six months of mating. The mice were sedated using 1.5% isoflurane, with the body temperature maintained at 36–37°C and a heart rate of 450–550 beats per minute (bpm). The B-mode and M-mode axial resolutions were 0.05–0.1 mm, and the B-mode lateral resolution was 0.2–0.5 mm [50]. The temporal frame rate in echo-mode was set at 60 Hz. A 1.0-mm sampling gate was used to obtain inflow and outflow velocities, and the maximal sweep speed was 200 mm/sec. The spectral Doppler and M-mode images were recorded in multiple screen shots, with the Doppler and M-mode measurements obtained from individual heart beats. Ultrasound parameters acquired from spectral Doppler, 2D and M-mode imaging will be introduced in the results.

Bone density measurements

Ten mid-shaft tibias from each group were used for bone density analysis. The bone mass analysis using mouse micro-CT was carried out with a SCANCO µCT50 (SCANCO Medical). The scans were performed using instrument settings as follows: E=55 KVp, I=110 µA, increment 7.4 µm, threshold value=375.

Statistical analysis

Statistical evaluations were performed using SPSS 17.0 software. Differences in body weight, estrous cycles, exhausted swimming time, hormones, bone density, and cardiovascular ultrasound parameters were analyzed using Student’s t test. Differences in 8-OHdG, 4-HNE and NTY expression between the two groups were analyzed using the non-parametric Kruskal–Wallis test or the Mann–Whitney test as applicable. Values represent the mean ± SEM, and P<0.05 was considered significant.