Quercetin reduces Cy - induced PMF loss
First, the effect of quercetin alone on different stage of follicle development was to investigate. The total number of follicles has no significant change after quercetin administration compared to control group. However, with the increase of quercetin dosage, the number of PMFs slightly increased, although there was no significant difference compared to control mice, and the EGF count decreased gradually, especially in the high-dose quercetin group (P < 0.05 compared to control mice, Fig. 1A). This trend is apparently in a comparison of the ratio of EGF versus PMF (P < 0.05 compared to control mice, Fig. 1A). These results suggest that quercetin may have the potential to inhibit the transition from PMF to the next developmental stage.
To study the impact of quercetin cotreatment on Cy-induced follicle loss, adult female mice were treated with quercetin (20 or 40 mg/kg per mouse) every day, beginning 1 week before Cy treatment and ending 1 week. Histological analysis showed that Cy-treated ovaries are poor in follicles in comparison to control ones, presenting cortical fibrosis and altered stromal cells, while ovaries cotreatment with Cy and Que groups are similar to healthy, control ovaries (Fig. 1C). Furthermore, mice treated with Cy showed a dramatical reduction in the number of PMFs, EGFs and total follicles compared to control animals (P < 0.001, Fig. 1B). When quercetin co-administration, the decreased number of PMFs induced by Cy was significantly recovered, especially those received quercetin at 40 mg/kg retained an ovarian reserve equivalent to untreated controls (616.56 ± 55.41 vs. 554.00 ± 41.60, P = 0.275). But unfortunately, both two dosage of quercetin cannot prevent the loss of EGFs caused by Cy (Fig. 1B). So, the ratio of EGF to PMFs was significantly decreased in mice after Cy and quercetin co-treatment, even lower than that in control. Finally, the follicle sum in Cy and quercetin co - treatment group obtained partially rescue, higher than that of Cy group (P < 0.001) but not yet returned to the level of control (Fig. 1B). Therefore, the quantification of different stage follicles revealed that quercetin in both dosages prevented the loss of PMFs induced by Cy but cannot provide protection against the damage of Cy to EGFs.
Quercetin prevents Cy - induced apoptosis in EGF and AMH decrease
Apoptosis is one of the possible mechanisms of ovarian damage induced by chemotherapy(27), so whether quercetin prevents Cy-induced PMFs loss through its anti-apoptotic effect was first to explore. However, neither cleaved caspase − 3 staining nor TUNEL staining showed any evidence of apoptosis in PMFs after Cy treatment with or without quercetin, while following by the development of follicle, the apoptosis of granulosa cells induced by Cy gradually became obvious and cotreatment with quercetin may ameliorate this phenomenon (Fig. 2A).
To further verify the anti-apoptotic of quercetin in growing follicles, apoptotic index (TUNEL - positive follicles/total follicles) were quantified based on TUNEL sections (Fig. 2B). The results show that Cy treatment increased the apoptotic index in all growing follicles compared to control ovaries and quercetin coadministration protected follicles of preantral and early antral stages from apoptosis, since the apoptotic indexes in these two stages follicles after Que cotreatment were significantly fewer and similar to controls (Fig. 2B). 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 ranging from the primary stage of development until selection for dominance(28). The study of Amh −/− mice shows that AMH facilitates maintenance of PMFs in a dormant state(29). As an indirect indicator of follicle reserve, AMH was measured 24 h after Cy administration. Figure 2C shows that AMH concentration was significantly decreased with Cy alone (from 5.4 to 2.2 ng/ml, P < 0.001), but after coadministration with quercetin, the concentration of AMH remained to normal. Quercetin alone did not alter the concentration of serum AMH.
These results suggest that Cy-induced PMFs loss may not directly induce apoptosis of PMFs, but indirectly via the acute reduction of growing follicles and AMH result in increased recruitment of PMFs into the growing pool. Quercetin cotreatment may inhibit the apoptosis induced by Cy in growing follicles and maintain the AMH level secreted by these follicles, thus keeping the dormancy of PMFs.
Quercetin inhibits Cy - induced of PI3K/ AKT /Foxo3a signaling pathway activation
In addition to AMH, the PI3K signaling pathway is also principally responsible for manipulating the dormancy and activation of mammalian PMFs(10). Recent studies indicate that the mechanism in Cy-induced loss of ovarian reserve is related with the acceleration of PMFs activation via the activation of PI3K signaling pathway(7). Therefore, whether quercetin reduces Cy - induced PMFs loss by inhibiting the activation of PI3K signaling pathway was then to investigate.
Foxo3a, a downstream effector of PI3K signaling pathway, is highly expressed in the nuclei of oocytes of PMFs and is important in maintaining PMFs in their dormant state(11). When it exports from nuclear, PMFs are activated and initial to development(30). Immunohistochemical staining in this study conducted on ovaries showed that the expression of Foxo3a was absence in the PMFs population after Cy administration, but deeply dyeing in Que cotreatment group, similar to the control group (Fig. 3B). And the result of WB shows that the ratio of phosphorylated Foxo3a to their non-phosphorylated forms elevated 1.69-fold in Cy-treated ovary compared to control, which was recovery to control level after Que co-administration. However, quercetin alone did not significantly affect the expression of Foxo3a. (Fig. 3A)
Moreover, once the PMFs are activated, they enter the stage of growth and granulosa cells begin to proliferate. Immunostaining with the proliferation marker ki67 in control ovaries showed granulosa cell proliferation was obvious in large growing follicles and only occasional staining in small activated follicles. Of particular interest, there was the substantial staining for Ki67 in granulosa cells in transitional primordial and primary follicles on ovaries removed 24 hours after Cy administration compared with a fewer positive cell in the Que cotreatment group (Fig. 3B).
Except for the suppressor protein Foxo3a, the key activation proteins Akt, mTOR, and rpS6 were also detected. Analysis of Cy treated ovaries showed an increase in the phosphorylated forms of these proteins (1.3- to 1.6-fold increase compared to that in control group, Fig. 3A). This increased phosphorylation of these three proteins caused by Cy treatment was attenuated after cotreatment with quercetin, who suppresses phosphorylation of mTOR and rpS6 alone, especially in high dosage.
These results suggested that quercetin may prevent the activation of PI3K signaling pathway induced by Cy to preserve ovarian reserve.