Introduction

As an important anti-neoplastic therapeutic approach, chemotherapy improves the survival rates of patients with various cancers. However, it often has adverse effects, including ovarian damage in premenopausal women with cancer, resulting in ovarian endocrine dysfunction and an increased infertility rate [1]. The cryopreservation of gametes enables the preservation of fertility in female cancer survivors [2]. However, other outcomes of a loss of ovarian function after chemotherapy, including early menopause and its associated complications, have not been resolved [3]. Therefore, the development of a non-invasive medical prevention approach to protect ovarian function against chemotherapy-induced damage is needed.

The extent of ovarian damage largely depends on the chemotherapy regimen (i.e. drug family and dose) and patient’s age at treatment [4]. Among chemotherapy drug classes, alkylating agents (e. g. cyclophosphamide, Cy) cause the greatest damage to the ovaries, with dose-dependent effects [5]. Previous histological studies of human ovaries showed that the end-stage effects of chemotherapy are ovarian atrophy and depletion of the primordial follicle (PMF) stockpile [6]. The mechanisms underlying chemotherapy-induced PMF loss have not been fully elucidated, but may involve overactivation of PMFs [7,8,28]. Antibody for DDX4 (1:4000; ab270534, Abcam, Cambridge, UK) was used.

TUNEL assays

For apoptosis detection, ovarian tissues were obtained from mice (n = 5 mice per group) sacrificed at 24 h after Cy or PBS treatment and serially sectioned (5-μm sections). An In Situ Cell Death Detection Kit (Roche, Basel, Switzerland) was used for the in situ localisation of nuclei exhibiting DNA fragmentation by the TUNEL technique according to the manufacturer’s instructions, as previously described [29]. Sections were counterstained with haematoxylin. The number of apoptotic cells was determined by counting labelled cells from all follicle types in × 400 microscopic fields (four sections per ovary; five ovaries per group). Follicles presenting more than 5% labelled cells were considered unhealthy [26]. The apoptotic index was calculated as the fraction of unhealthy/total follicles in each class [26].

Histochemical and immunohistochemical analyses of ovarian tissues

For immunohistochemistry analysis, ovaries were obtained from mice (n = 5 mice per group) sacrificed at 24 h after Cy or PBS treatment. After deparaffinisation of the ovaries in xylene and rehydration by graduated ethanol washes, endogenous peroxidase activity in the sections was inactivated by 3% (vol/vol) hydrogen peroxide in PBS. After epitope retrieval (at 98 °C for 30 min in 0.01 M citrate buffer solution, pH 6.0; Dako, Glostrup, Denmark), non-specific binding was blocked with 2% bovine serum albumin for 1 h at room temperature. The sections were incubated with primary antibody overnight at 4 °C. Antibodies for Foxo3a (1:1000; 12829S, Cell Signaling Technology, Danvers, MA, USA), Ki67 (1:400; 12202S, Cell Signaling Technology), and cleaved caspase-3 (1:200; 9661S, Cell Signaling Technology) were used. After washing, slides were incubated with biotinylated anti-rabbit IgG (Dako) for 1 h. Finally, protein signals were visualised by DAB (Dako) staining. After stop** the reaction with distilled water, the slides were counterstained with haematoxylin, dehydrated, and mounted. Images were obtained using a digital camera (Olympus, Tokyo, Japan) mounted on a conventional light microscope (Olympus) at a magnification of × 40 [26].

Western blotting

Mice were sacrificed at 24 h after Cy or PBS treatment and the ovaries were removed, placed on ice, and homogenized in radioimmunoprecipitation assay lysis buffer supplemented with protease inhibitors [26]. After centrifugation (12,000×g, 4 °C for 15 min), proteins in the supernatant were measured with a BCA Protein Assay Kit (TH269580, Thermo Fisher Scientific, Waltham, MA, USA). Protein (30 μg) in loading buffer was separated by 8% sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The resolved proteins were transferred onto polyvinylidene fluoride membranes (Merck Millipore, Darmstadt, Germany), and the membranes were incubated in blocking buffer (5% non-fat milk, 0.1% Tween-20 in 20 mM TBS, pH 8.0) for 1 h at room temperature and probed with specific primary antibodies overnight at 4 °C. Primary antibodies against Akt (1:1000, 4691S), phospho-Akt (Ser473) (1:1000, 4060S), mTOR (7C10) (1:1000, 2983S), phospho-mTOR (Ser2448) (1:1000, 5536S), rpS6 (5G10) (1:1000,2217S), phospho-rpS6 (Ser235/236) (1:1000, 2211S), Foxo3a (1:1000,12829S), phospho-Foxo3a (1:1000, 9464S), and β-actin (1:1000, 4970S) were all rabbit monoclonal antibodies and purchased from Cell Signaling Technology. The blot was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10000, MR-M100; MR Biotech, Shanghai, China) for 1 h at room temperature, and an enzymatic chemiluminescence kit (Pierce, Thermo Fisher Scientific) was employed for visualisation. Signals were scanned using the Amersham600 imager system (GE, Chicago, IL, USA), and the integrated light intensity for each band was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). These values were used to compare treatment-induced changes in the concentrations of phosphoproteins as well as to calculate the ratio of phosphorylated proteins to their non-phosphorylated forms. β-Actin expression was measured to verify equal loading. Experiments were repeated three to five times and showed similar results, and three to five ovaries were pooled for each analysis, with a total of 45 animals.

Serum anti-Müllerian hormone (AMH) measurements

At 24 h after Cy or PBS treatment, the blood was collected by cardiac puncture at the time of sacrifice. This was performed using a 1 mL syringe and a 22-gauge needle. The needle was inserted 5 mm from the centre of the thorax towards the animal’s chin 5–10 mm deep, while holding the syringe 25–30 degrees away from the chest [30]. Next, 700–900 μl blood was collected, and the serum was separated by centrifugation at 1000×g for 15 min at 4 °C and stored at − 80 °C until further analysis. Serum AMH concentrations were quantified using a Mouse Mullerian-Inhibiting Factor ELISA Kit (Signalway Antibody, College Park, MD, USA) according to the manufacturer’s instruction, as described in a previous study [31] (n = 5 mice per group). The coefficient of variation (CV) for the AMH assay is as follows: intra-assay CV: ≤6.5%; inter-assay CV: ≤10.2%.

Statistical analyses

Results are expressed as the means ± standard error of the mean (SEM) and were analysed using SPSS software (version 24, SPSS Inc., Chicago, IL, USA). Differences between groups were evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s tests in all cases. Shapiro–Wilk test was used for normality before ANOVA. P < 0.05 was considered to indicate statistically significant result.

Results

Quercetin reduces cy-induced PMF loss

First, the effects of quercetin alone on different stages of follicle development were investigated. The total number of follicles after quercetin administration did not differ from that in the control group. However, as the quercetin dosage was increased, the number of PMFs increased slightly (but not significantly) and the EGF count decreased gradually, especially in the high-dose quercetin group (P < 0.05 compared with control mice, Fig. 1a). The ratio of EGF to PMF was lower in the high-dose quercetin group than in the control group (P < 0.05 compared with control mice, Fig. 1a). These results suggest that quercetin has the potential to inhibit the transition from PMFs to the next developmental stage.

Fig. 1
figure 1

Quercetin reduces Cy-induced PMF loss. a-b Effects of PBS alone, quercetin alone, Cy alone, or Cy with quercetin on different stages of follicle development (n = 5 mice/group). Data are expressed as the means ± SEM. One-way ANOVA followed by Tukey’s tests was used for comparisons (***P < 0.001 compared with the Cy group; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with the control group). c Representative images of haematoxylin and eosin staining ofthe ovarian histological sections from all experimental groups. Scale bars represent 100 μm (upper panel in each group) and 20 μm (lower panel in each group). Inset boxes denote areas shown in higher magnification to the below. Black arrows indicate the primordial follicles. d Representative images of DDX4-immunostained ovarian histological sections from all experimental groups. Inset boxes denote areas shown in higher magnification to the below. Red arrows indicate primordial follicles. Scale bars represent 500 μm

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To determine the impact of quercetin on Cy-induced follicle loss, adult female mice were treated with quercetin (20 or 40 mg/kg per mouse) daily for 14 continuous days, beginning at 1 week before Cy treatment and ending 14 days later. Histological analysis showed that Cy-treated ovaries contained fewer follicles than did control ones, presenting cortical fibrosis and altered stromal cells, while ovaries cotreated with Cy and quercetin contained a similar number of follicles to healthy control ovaries (Fig. 1c and d). Furthermore, mice treated with Cy showed dramatic reductions in the numbers of PMFs, EGFs, and total follicles compared with those in control animals (P < 0.001, Fig. 1b). Quercetin significantly attenuated the Cy-induced decrease in the number of PMFs, especially at a concentration of 40 mg/kg, which resulted in the maintenance of an ovarian reserve equivalent to that of untreated controls (616.56 ± 55.41 vs. 554.00 ± 41.60, P = 0.275). However, neither dosage of quercetin prevented the loss of EGFs caused by Cy (Fig. 1b). Accordingly, the ratio of EGFs to PMFs was low in mice after Cy and quercetin co-treatment, even lower than that in the control. Finally, the follicle sum was partially rescued in the Cy and quercetin co-treatment group, which showed higher values than did the Cy group (P < 0.001), but did not reach the level in the control group (Fig. 1b). Therefore, the quantification of follicles at different stages revealed that quercetin at both dosages prevented the loss of PMFs induced by Cy, but did not protect against Cy-induced damage to EGFs.

Quercetin prevents apoptosis in EGF and the decreased AMH induced by cy

Apoptosis is a possible mechanism underlying ovarian damage induced by chemotherapy [32,33,34,35,36]; accordingly, we explored whether the preventive effects of quercetin on Cy-induced PMF loss were mediated by the inhibition of apoptosis. However, neither cleaved caspase-3 staining nor TUNEL staining showed any evidence of apoptosis in PMFs after Cy treatment with or without quercetin. As follicle development progressed, the apoptosis of granulosa cells induced by Cy gradually increased, and the addition of quercetin may attenuate this increase slightly (Fig. 2a and b).

Fig. 2
figure 2

Quercetin prevents apoptosis in growing follicles and decreases AMH induced by Cy. a-b Representative images of mouse ovaries stained with TUNEL (a) and cleaved caspase-3 antibody (b) are shown for each treatment condition. Bars represent 50 μm. c TUNEL-based quantification of the apoptotic index per follicle class calculated as the fraction of apoptotic/total follicles for each type (n = 5 mice/group, *P < 0.05, **P < 0.01, compared with the Cy group; ## P < 0.01 compared with the control group). Primordial follicles did not present TUNEL-positive cells in any experimental group. One-way ANOVA followed by Tukey’s tests was used for comparisons. d AMH plasma concentrations were measured at 24 h after administration of PBS or Cy with or without quercetin (n = 5 mice/group). Data represent the means ± SEM. One-way ANOVA followed by Tukey’s tests was used for comparisons (***P < 0.001 compared with the Cy group)

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To further verify the anti-apoptotic effect of quercetin in growing follicles, the apoptotic index (TUNEL-positive follicles/total follicles) was quantified based on TUNEL sections (Fig. 2c). Cy treatment increased the apoptotic index of all growing follicles compared with that of control ovaries, and quercetin coadministration inhibited apoptosis in the follicles at the preantral and early antral stages, as evidenced by the significantly lower apoptotic indexes for follicles at these two stages after quercetin cotreatment than for those after Cy treatment alone (P < 0.05), similar to the value in controls (Fig. 2c). However, this protective effect of quercetin was not obvious in granulosa cells of mature antral follicles.

AMH is expressed by granulosa cells and is produced by growing follicles from the primary stage of development until selection for dominance [37]. A study of Amh−/− mice showed that AMH facilitates the maintenance of PMFs in a dormant state [38]. As an indirect indicator of the follicle reserve, AMH was measured at 24 h after Cy administration. As shown in Fig. 2d, the AMH concentration was significantly lower after Cy treatment (2.2 ng/mL) than in controls (5.4 ng/mL, P < 0.001); however, the coadministration of Cy and quercetin resulted in a similar concentration of AMH to that in the control. Quercetin alone did not alter the concentration of serum AMH.

These results indicate that Cy-induced PMF loss may not directly induce apoptosis in PMFs, but may have an indirect effect by acutely reducing growing follicles and AMH levels, resulting in increased recruitment of PMFs to the growing pool. Quercetin cotreatment may inhibit apoptosis induced by Cy in growing follicles and maintain the levels of AMH secreted by these follicles, thus maintaining the dormant state.

Quercetin inhibits cy-induced PI3K/Akt/Foxo3a signalling pathway activation

Recent studies have indicated that Cy-induced loss of the ovarian reserve is related to the acceleration of PMF activation via activation of the PI3K signalling pathway [7]. Therefore, we investigated the effects of quercetin on the PI3K signalling pathway in the ovaries after Cy treatment.

Foxo3a, a downstream effector of the PI3K signalling pathway, is highly expressed in the nuclei of oocytes of PMFs and is important for the maintenance of PMFs in the dormant state [13]. Its nuclear export results in the activation of PMFs and initiation of development [39]. In this study, Foxo3a expression was not detected by immunohistochemical staining in the PMF population after Cy administration; however, intense staining was observed in the quercetin cotreatment group, similar to that in the control group (Fig. 3c). Western blotting further showed that the ratio of phosphorylated Foxo3a to the non-phosphorylated form was 1.69-fold higher in Cy-treated ovaries than in the control group, with recovery to control levels after quercetin co-administration. However, quercetin alone did not significantly affect the expression of Foxo3a (Fig. 3a and b).

Fig. 3
figure 3

Quercetin reduces the Cy-induced phosphorylation of PI3K/Akt/Foxo3a pathway proteins in the ovary. a Western blot analyses were used to evaluate ovaries from 8-week-old mice removed at 24 h after a single dose of Cy (75 mg/kg) or an equivalent volume of PBS with or without quercetin (Que1: 20 mg/kg; Que2: 40 mg/kg). Comparisons of the concentrations of phosphorylated and total Foxo3a, Akt, mTOR, and rpS6, using β-actin as a loading control. Fold-change values for ratios for each protein are listed below each set of bands. b Ratios of phosphorylated/total protein are summarised in a bar graph. Experiments were repeated three to five times with similar results; three to five ovaries were pooled per result, with a total of 45 animals. The data are presented as the mean values ± SEM. (***P < 0.001 compared with the Cy group; # P < 0.05, ## P < 0.01, ### P < 0.001 compared with the control group). One-way ANOVA followed by Tukey’s tests was used for statistical analyses. c-d Immunohistochemical staining for Foxo3a and Ki67 demonstrates that quercetin inhibited Cy-induced primordial follicle overactivity and short-term growth. A representative image from each group is shown. c immunohistochemical staining for Foxo3a shows the localisation of Foxo3a in each group. Arrows indicate primordial follicles. d Ki67 staining of ovary tissues was used to evaluate cell proliferation. Black frames indicate primary follicles. Scale bars, 20 μm

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Moreover, following their activation, PMFs enter the stage of growth and granulosa cells begin to proliferate. Immunostaining for the proliferation marker Ki67 in control ovaries showed that granulosa cell proliferation was obvious in large growing follicles, with only occasional staining in small activated follicles. Interestingly, substantial Ki67 staining was observed in granulosa cells in transitional primordial and primary follicles from ovaries removed at 24 h after Cy administration, with fewer positive cells in the quercetin cotreatment group than in the Cy-treated group (Fig. 3d).

In addition to the suppressor Foxo3a, the key activation proteins Akt, mTOR, and rpS6 were detected. Cy-treated ovaries showed an increase in the phosphorylated forms of these proteins (1.3–1.6-fold increases compared to control group level, Fig. 3a and b). These increased levels of phosphorylation caused by Cy treatment were attenuated by cotreatment with quercetin, which also suppressed the phosphorylation of mTOR and rpS6 alone, particularly when administered at a high dosage.

These results suggest that quercetin prevents activation of the PI3K signalling pathway induced by Cy to preserve the ovarian reserve.

Discussion

The precise mechanisms of chemotherapy-induced ovarian damage remain unclear. Recently, the ‘burn-out effect’ [7,8,36] has been suggested as the possible mechanisms of chemotherapy-induced ovarian damage. Chemotherapy may disrupt the function of the PI3K/PTEN/Akt signalling pathway responsible for follicle quiescence, thereby directly inducing PMF overactivation and depletion of follicle reserve [7]. But current researches show that oocytes leave their follicular quiescence to initiate the DNA damage signalling pathway leads to oocyte apoptosis [40] and oocyte damage leading to PUMA-dependent apoptotic cell death being the predominant process involved in primordial follicle loss after treatment with Cy [41]. However, herein, apoptosis in the PMFs was not observed 24 h upon Cy treatment. In fact, oocytes are capable of efficient DNA repair in response to the inhibition of the apoptotic pathway in the ovary [42]. This contrasting result may be related to the time of the observation, as is possible that oocytes may have achieved complete DNA repair at this time point; nonetheless, this needs to be further confirmed. Furthermore, with the growth and development of follicles, granulosa cells proliferate vigorously and are, thus, more vulnerable to the damaging effects of Cy. Hence, Cy can promote massive apoptosis in growing follicles, thereby reducing the secretion of inhibitory factors and indirectly recruit PMFs to the growing pool [7].

In this study, the effects of quercetin, an inhibitor of PI3K [15], on Cy-induced follicle loss were evaluated. The administration of quercetin alone slightly and dose-dependently increased the number of PMFs and decreased the number of EGFs, particularly at a high dose. Cotreatment with quercetin and Cy rescued the number of dormant PMFs to that in control ovaries. Western blotting showed that although administration of Cy increased the phosphorylation levels of Akt, mTOR, and rpS6, which are involved in the regulation of PMF activation, quercetin decreased their levels, and the phosphorylation of Foxo3a, an indicator of PMF activation, was also decreased after quercetin administration. These results collectively indicate that quercetin may inhibit the transformation from PMFs to primary follicles induced by Cy by inhibiting the PI3K signalling pathway. However, the PI3K signalling pathway is important for various physiological processes [43]. Further studies are thus needed to determine whether quercetin negatively regulates the growth and development of normal cells via inhibition of the PI3K signalling pathway.

In addition to the direct mechanism described above, the PMF pool can be reduced indirectly via the loss of activated, growing follicles [4]. Our results revealed that chemotherapy promotes apoptosis in granulosa cells in growing follicles, with more mature follicles showing a higher rate of apoptosis. Accordingly, the suppression of follicular activation by AMH from granulosa cells of growing follicles is decreased, leading to the accelerated depletion of the PMF reserve [44]. As a result, PMF growth is initiated to replace damaged growing follicles. In this study, Foxo3a staining showed that PMFs are activated, and Ki67 staining showed that the proliferation of granulosa cells in transitional primordial and primary follicles is activated after chemotherapy. However, quercetin coadministration protected follicles at the preantral and early antral stages from apoptosis and maintained AMH levels, leading to preservation of the quiescence of the ovarian reserve. We speculate that this phenomenon is also related to the inhibitory effects of quercetin on the PI3K signalling pathway and its anti-proliferation effects that weaken the sensitivity of preantral follicles and early antral follicles to Cy-induced damage. However, quercetin had no protective effects on granulosa cells of mature antral follicles against damage induced by Cy; thus, the combination of quercetin with additional anti-apoptotic compounds should be considered in future research.

Quercetin has a variety of biological functions, such as anti-inflammatory, anti-oxidant, and anti-cancer effects [45], and a reasonable dosage is important for its biological activity [46]. Numerous in vitro studies have reported that quercetin promotes apoptosis at high dosages and shows anti-oxidant effects at low concentrations [15]. A previous study demonstrated that intraperitoneal injection of 20 mg/kg quercetin has protective effects against ovarian toxicity and POF induced by Cy, without compromising its antitumor effect [19]. However, in a xenograft tumour model, quercetin at 20 mg/kg may attenuate the therapeutic effects of chemotherapeutic drugs in ovarian cancer cells by reducing reactive oxygen species-induced damage [20]. Therefore, both 20 mg/kg and a relatively high dose of 40 mg/kg were chosen for this study. Both dosages of quercetin inhibited the activation of PMFs induced by Cy, specifically at the high dosage. Moreover, the overall follicle growth time in mice is 17 to 19 days [21] and the duration from preantral follicles to ovulatory follicles is about 12 days [22]; accordingly, the transformation from PMFs to primary follicles may occur within 7 days. Hence, quercetin can cover this process by daily administration for 1 week before Cy administration. However, because of the follicle counts observed at 7 days after Cy administration and the short biological half-life of quercetin [14], we continued quercetin administration for 7 days after Cy injection; thus, quercetin was administered for 14 continuous days, beginning at 1 week before Cy or PBS treatment and ending 14 days later, to maximise the protective effect of quercetin against Cy-induced damage to follicles.

Conclusions

In summary, this in vivo mouse study provides the first evidence that quercetin inhibits the Cy-induced overactivation of dormant PMFs via regulation of the PI3K/Akt/Foxo3a signals and maintaining AMH level through reduced apoptosis in growing follicles, thereby restoring the ovary to a state of equilibrium (Fig. 4). Because of its ability to prevent the loss of follicles induced by chemotherapy, quercetin is expected to be a non-invasive medical approach to prevent infertility as well as endocrine-related side effects in female cancer survivors. The results of our research also expand our understanding of the applications of quercetin.

Fig. 4
figure 4

Quercetin inhibits overactivation of dormant PMFs induced by Cy. In normal circumstances, the dormancy or activation of primordial follicles was regulated by PI3K signalling pathway and the inhibitor factors secreted by growing follicles, such as AMH. Cyclophosphamide disrupts the balance of the PI3K signalling pathway and decreases AMH level by inducing apoptosis in growing follicles, resulting in the overactivation of the primordial follicles and the burnout of the reserve. Coadministration of quercetin and Cy prevents the phosphorylation of PI3K/Akt/Foxo3a pathway members and maintains AMH level through reduced apoptosis in growing follicles to restore the ovary to a state of equilibrium

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