MitoQ Protects Ovarian Organoids against Oxidative Stress during Oogenesis and Folliculogenesis In Vitro

Ovarian organoids, based on mouse female germline stem cells (FGSCs), have great value in basic research and are a vast prospect in pre-clinical drug screening due to their properties, but the competency of these in vitro-generated oocytes was generally low, especially, in vitro maturation (IVM) rate. Recently, it has been demonstrated that the 3D microenvironment triggers mitochondrial dysfunction during follicle growth in vitro. Therefore, therapies that protect mitochondria and enhance their function in oocytes warrant investigation. Here, we reported that exposure to 100 nM MitoQ promoted follicle growth and maturation in vitro, accompanied by scavenging ROS, reduced oxidative injury, and restored mitochondrial membrane potential in oocytes. Mechanistically, using mice granulosa cells (GCs) as a cellular model, it was shown that MitoQ protects GCs against H2O2-induced apoptosis by inhibiting the oxidative stress pathway. Together, these results reveal that MitoQ reduces oxidative stress in ovarian follicles via its antioxidative action, thereby protecting oocytes and granulosa cells and providing an efficient way to improve the quality of in vitro-generated oocytes.


Introduction
Gametes are tasked with faithfully transmitting genetic and epigenetic information from one generation to the next. Oocytes, as female germ cells, provide the necessary maternally derived organelles support for the development of embryos, especially mitochondria. Generally, ovarian reserve is established early in life with a finite number of oocytes. Currently, there is no direct evidence that adult oogenesis ever occurs under physiological conditions [1]. Therefore, the regeneration of oocytes is one of the most important aims of researchers in the field of regenerative medicine. Concomitant with technological development in three-dimensional (3D) and organoid culture, the concept of oogenesis in vitro became a reality. Reconstitution ovary or ovarian organoid, derived from mouse pluripotent stem cells (PSCs) [2] and FGSCs [3], respectively, have been successfully implemented to achieve the entire process of oogenesis in vitro. However, the competency of these in vitrogenerated oocytes was markedly lower than that of in vivo-grown oocytes, and only a few of them developed to term after in vitro maturation (IVM) and in vitro fertilization (IVF) [2].
Takashima et al. [4] indicated that mouse follicles in vitro culture may be subjected to oxidative stress under conventional 3D culture conditions, which, in turn, leads to mitochondrial dysfunction in oocytes. ROS, a crucial factor in oocyte maturation, is produced in a healthy follicle under normal physiological activities [5]. While excessive ROS levels can cause mitochondrial dysfunction, thereby affecting oocyte maturation and subsequent fertilization [6]. Due to paternal mitochondria being damaged after sperm enters the oocyte, the quality of maternal mitochondria is vital for proper embryonic development [7]. Therefore, how to improve the in vitro gametogenesis environment and reduce the oxidative stress level are essential for the acquisition of oocyte competence.
MitoQ (MitoQuinone), as a derivative of ubiquinone conjugated to triphenylphosphonium, bearing a lipophilic cation, can enter and accumulate within the mitochondria as a result of the electrochemical gradient [8][9][10]. As a consequence of MitoQ's ability to maintain mitochondrial function by inhibiting intracellular and mitochondrial ROS production and normalizing mitochondrial membrane potential (∆Ψm) has been tested in clinical trials for many diseases [11][12][13]. A long-term MitoQ treatment did not affect the total level of gene expression in mammalian tissues [14], suggesting that long-term administration of MitoQ to animals is safe and that, in vivo, it accumulates within mitochondria at appropriate concentrations to exert protection. Previous studies reported that MitoQ treatment during IVM in human, mouse [15], and porcine [16] oocytes can significantly improve the maturation rates and oocyte quality, which together indicate their potential for application in the ovarian organoid culture.
In the present study, the antioxidant effect of MitoQ is investigated during oogenesis in vitro culture. The treatment performance of MitoQ to mouse ovarian organoids is demonstrated by follicles size, maturation rate, ROS production, mitochondrial content, and ∆Ψm in in vitro-generated oocytes. Meanwhile, the mechanisms of MitoQ against H 2 O 2 -induced apoptosis in GCs have been revealed. Our work provides an effective strategy for treating reproductive defects caused by 3D environmental stress.

MitoQ Enhanced the Maturation of In Vitro-Generated Oocytes
To establish FGSC lines, FGSCs were isolated and cultured as in the previous study [17]. After sorting and culturing for 48 h, numerous FGSC clusters could be seen ( Figure 1A). Next, FGSCs were aggregated with ovarian somatic cells to produce ovarian organoids and were subject to 3D culture ( Figure 1B). The experimentation timeline and the route of 3D ovarian organoid generation and culture are illustrated in Figure 1C. To explore the effect of MitoQ during oogenesis in vitro, ovarian organoids were cultured in fresh medium supplemented with different concentrations of MitoQ (50, 100, 150 nM). Ovarian organoids without the addition of MitoQ served as the control group. During the whole culture process, FGSCs differentiated and eventually formed the complete follicles after 21 days of culture ( Figure 1D). Then, the primary/secondary follicles were immediately isolated from the ovarian organoid and subjected to count the number of follicles based on follicle diameter (≥150 and <150 µm). At the MitoQ concentration of 50 nM and 150 nM, compared with the control side, the proportion of ≥150 µm follicles was not compromised (Control: 7.04% ± 4.79 vs. MitoQ 50 nM 14.54% ± 4.01, p > 0.05; vs. MitoQ 150 nM: 10.65% ± 2.12, p > 0.05, Figure 1F). Notably, when the concentration reached up to 100 nM, GCs exhibited robust growth and formed the rich external layer of oocytes. There was a significant rise in the proportion of ≥150 µm follicles by 53.54% ± 4.51 (p < 0.01, Figure 1E) in comparison with the control group.
Then, individual follicles were subjected to in vitro growth. After 11 days of culture, in vitro-generated (IVG) oocytes grew into GV oocytes among the experimental groups. When transferring them under the in vitro maturation (IVM) culture conditions, 40.75% of the GV oocytes extruded a first polar body in the control group. As the MitoQ concentration rose, the maturation rate of IVG oocytes began to rise gradually. The highest maturation rate was obtained when the concentration of MitoQ reached 100 nM, compared to the control side (MitoQ 100 nM: 56.00% ± 5.83 vs. control: 40.75% ± 5.10, p < 0.05, Figure 1F). Here, it should be remarked that, despite the maturation rate of IVG oocytes being ameliorated with MitoQ treatment, it remained significantly lower than in the in vivo-grown (in vivo) oocytes. Next, we conducted in vitro fertilization experiments to confirm the developmental competence of these mature oocytes. The fertilization rates of mature oocytes derived from organoids with MitoQ treatment did not differ significantly from those of controls (p > 0.05, Figure 1G). It is also noteworthy that the fertilization rates in the IVG group were markedly lower than those in the In vivo oocytes. Based on these results, MitoQ treatment has a positive effect on IVG oocyte growth and maturation. the developmental competence of these mature oocytes. The fertilization rates of mature oocytes derived from organoids with MitoQ treatment did not differ significantly from those of controls (p > 0.05, Figure 1G). It is also noteworthy that the fertilization rates in the IVG group were markedly lower than those in the In vivo oocytes. Based on these results, MitoQ treatment has a positive effect on IVG oocyte growth and maturation.

Reversal of ROS-Mediated Mitochondrial Defects in Oocytes by MitoQ
To further investigate the mechanism by which MitoQ improved IVG oocyte maturation, we used the probe DCFH-DA to detect ROS levels in in vivo and IVG oocytes at GV and MII stages. As shown in Figure 2A,B, at the GV stages, ROS levels were significantly higher in IVG oocytes compared to the in vivo oocytes. These differences became more critical at the MII stage as the ROS levels increased in IVG oocytes (p < 0.001, Figure 2C,D), suggesting that 3D environment exposure increases the levels of intracellular ROS in IVG oocytes. With the addition of MitoQ during the organoid culture process (IVGM), ROS production was significantly decreased in GV and MII IVGM oocytes (GV, p < 0.001, Figure 2B; MII, p < 0.001, Figure 2D).
Generally, excessive ROS generation leads to mitochondrial damage that may perturb the mitochondria distribution and ∆Ψm. Here, mitochondria distribution and ∆Ψm levels were detected between in vivo and IVG oocytes at GV and MII stages. As indicated in Figure 2E,G, mitochondria were normally distributed throughout the cytoplasm in the in vivo group, while aggregated and clustered mitochondria were observed in the IVG groups. In addition, at the GV and MII stages, the quantitative analyses of MitoTracker results showed that the fluorescence intensity was significantly decreased in IVG oocytes compared to the in vivo oocytes (GV, p < 0.001, Figure 2F; MII, p < 0.001, Figure 2H). With the inclusion of MitoQ during organoid culture, the fluorescence signals of mitochondria were significantly upregulated (GV, p < 0.01, Figure 2F; MII, p < 0.001, Figure 2H) compared with that of the IVG group. Next, we assessed changes of ∆Ψm using the JC-1 probe and measured the red/green ratio. In comparison to in vivo GV oocytes, a decrease in ∆Ψm in IVG oocytes was observed (p < 0.001, Figure 2I,J). However, after treatment of MitoQ, ∆Ψm was significantly increased in IVGM oocytes compared with the IVG oocytes (p < 0.001, Figure 2I,J). The differences in the MII stage between these groups also showed the same pattern ( Figure 2K,L). These data indicate that MitoQ can improve mitochondrial quantity in IVG oocytes.

MitoQ Promoted GCs Proliferation
The above findings indicated that MitoQ could improve follicles growth and increase follicle size during folliculogenesis in vitro, which seems to indicate that MitoQ contributes to GCs proliferation. Here, we assessed the number and morphology of follicles in ovarian organoids after 21 days of culture. The spatiotemporal profiles of DDX4 (oocytes)

MitoQ Promoted GCs Proliferation
The above findings indicated that MitoQ could improve follicles growth and increase follicle size during folliculogenesis in vitro, which seems to indicate that MitoQ contributes to GCs proliferation. Here, we assessed the number and morphology of follicles in ovarian organoids after 21 days of culture. The spatiotemporal profiles of DDX4 (oocytes) and INHIBIN-α (GCs) were examined by immunohistochemical staining ( Figure 3A). Compared with the control side, the proportion of G3 follicles was significantly higher in MitoQ-treated ovarian organoids (MitoQ: 39.89% ± 4.52 vs. control: 11.91% ±3.69, p < 0.01, Figure 3B). To characterize the follicles growth at the cellular level, we examined the proliferative rate of GCs in developing follicles based on PCNA incorporation staining. Accordingly, based on regions where PCNA staining coincided, GCs of MitoQ-treated ovarian organoids demonstrated significantly increased levels of proliferation (p < 0.001, Figure 3C,D). The results indicate that MitoQ enhances the proliferation of surrounding GCs, which, in turn, might contribute to the improvement of follicular development.

MitoQ Inhibits Mitochondria Injury in H2O2-Treated GCs
Follicular GCs are closely related to ovarian function. Here, we constructed an H2O2treated GCs oxidative stress model and used the CCK8 array to assess the effects of MitoQ

MitoQ Inhibits Mitochondria Injury in H 2 O 2 -Treated GCs
Follicular GCs are closely related to ovarian function. Here, we constructed an H 2 O 2treated GCs oxidative stress model and used the CCK8 array to assess the effects of MitoQ on GCs. The untreated control (no H 2 O 2 and no MitoQ) was set as 100%. CCK8 results indicated that various H 2 O 2 exposure (50, 100, and 200 µM) showed concentrationdependent inhibition of GCs survival. When cells were treated with 100 µM H 2 O 2 for 24 h, the cell survival rate was 59.17% ± 6.20, which was closest to the IC50 ( Figure 4A). Therefore, 100 µM was selected for subsequent experiments as the optimal concentration. Next, we investigated whether MitoQ reversed the cell viability change induced by H 2 O 2 . As shown in Figure 4B, the loss of GCs viability upon H 2 O 2 exposure was partially rescued in GCs pretreated with MitoQ (50 nM, p < 0.05). When even higher MitoQ concentrations (100 nM) were achieved, the viability defect in GCs was significantly relieved (p < 0.01) compared with the H 2 O 2 group.  Then, we further evaluate the antioxidant of MitoQ on GCs in several aspects, including ROS production, Mitochondrial distribution, and ΔѰm. Using DCFH-DA staining, we observed that the intracellular ROS production of H2O2-treated GCs was higher than those in the control group (p < 0.001, Figure.5A, B), but the effect was obviously reversed after treatment of MitoQ (p < 0.05, Figure 5A, B). To further evaluate the mitochondrial damage, we next stained GCs with MitoTracker and JC-1. As indicated in Figure 4E, the result showed a significant reduction of functional mitochondria in GCs with H2O2 treatment versus controls ( Figure 5C, D, p < 0.05), while MitoQ produced a significant mitigating effect on mitochondrial depolarization during oxidative stress (p < 0.05, Figure 5C, D). In parallel, the staining of JC-1 results showed that H2O2 treatment increased the fraction of depolarized mitochondria (p < 0.01, Figure 5E, F), which was significantly reversed by MitoQ treatment (p < 0.05, Figure 5E, F). Together, these results proved that MitoQ pretreatment effectively decreased the mitochondrial oxidative stress in GCs and contributed to the protection. Then, we further evaluate the antioxidant of MitoQ on GCs in several aspects, including ROS production, Mitochondrial distribution, and ∆Ψm. Using DCFH-DA staining, we observed that the intracellular ROS production of H 2 O 2 -treated GCs was higher than those in the control group (p < 0.001, Figure 5A,B), but the effect was obviously reversed after treatment of MitoQ (p < 0.05, Figure 5A,B). To further evaluate the mitochondrial damage, we next stained GCs with MitoTracker and JC-1. As indicated in Figure 4E, the result showed a significant reduction of functional mitochondria in GCs with H 2 O 2 treatment versus controls ( Figure 5C,D, p < 0.05), while MitoQ produced a significant mitigating effect on mitochondrial depolarization during oxidative stress (p < 0.05, Figure 5C,D). In parallel, the staining of JC-1 results showed that H 2 O 2 treatment increased the fraction of depolarized mitochondria (p < 0.01, Figure 5E,F), which was significantly reversed by MitoQ treatment (p < 0.05, Figure 5E,F). Together, these results proved that MitoQ pretreatment effectively decreased the mitochondrial oxidative stress in GCs and contributed to the protection. ol. Sci. 2023, 24, 924 8 of 17

MitoQ Suppresses the Apoptosis Caused by Oxidative Stress
It was found that the dysfunction of mitochondria often resulted in cell death, and some mitochondrial apoptosis-related genes, such as caspase family and apoptosis-related genes, were often regulated. Consistent with this, our results showed that exposure to H2O2 alone caused increased expression level of CASPASE 9 (p < 0.001, Figure 6A, B), BAX (p < 0.01, Figure 6A, C) and decreased the expression of BCL 2 (p < 0.05, Figure 6A, D) and PCNA (p < 0.01, Figure 6A, E) in GCs, in comparison to control group. Apparently, these changes were alleviated by the administration of MitoQ when compared to the H2O2-treated group. Collectively, our results revealed that MitoQ was able to greatly attenuate cell death induced by oxidative stress.

MitoQ Suppresses the Apoptosis Caused by Oxidative Stress
It was found that the dysfunction of mitochondria often resulted in cell death, and some mitochondrial apoptosis-related genes, such as caspase family and apoptosis-related genes, were often regulated. Consistent with this, our results showed that exposure to H 2 O 2 alone caused increased expression level of CASPASE 9 (p < 0.001, Figure 6A,B), BAX (p < 0.01, Figure 6A,C) and decreased the expression of BCL 2 (p < 0.05, Figure 6A,D) and PCNA (p < 0.01, Figure 6A,E) in GCs, in comparison to control group. Apparently, these changes were alleviated by the administration of MitoQ when compared to the H 2 O 2treated group. Collectively, our results revealed that MitoQ was able to greatly attenuate cell death induced by oxidative stress.

Discussion
Ovarian organoids are an emerging culture technology worth pursuing many applications. The ability of ovarian organoids to be monitored and manipulated in a controlled environment makes them an ideal model for exploring the potential mechanisms of oogenesis [18]. It also provides a tractable system for pre-clinic medicines screening [3]. At present, both PSCs and FGSC have been used to produce ovarian organoids, and these resultant oocytes have produced healthy pups [2,3]. Clearly, ovarian organoids have shown great potential in biomedical applications owing to their uniform characteristics. Nevertheless, the question remains as to the identification of the effective culture condition that permits both efficient and consistent production of competent oocytes during reconstitution culture. In this study, we investigated the role of MitoQ in the recovery of oocyte competency and identified the pathway impacting oocyte competency and GCs proliferation by molecular analyses.
Numerous biochemical processes are performed by mitochondria, including the production of ATP, the regulation of Ca 2+ levels, and the synthesis of steroid hormones [19]. The maintenance of mitochondria within the germline is a highly regulated process. At an early embryonic developmental stage in mice (E9.5 to E11.5), each PGC contains less than 1000 mitochondria [20]. Just prior to primordial follicle formation, mitochondria are transported from oocytes in the nest to one (or a small number) of oocytes in each nest. At this point, the content of mitochondria does not increase [21]. During and immediately after birth, the ovary is populated by primordial oocytes, which each contain about 5000 mitochondria [22]. Thus, to a somewhat lesser degree, the contact with the development of the follicles and the mitochondria number was interrelated. Normally, ROS are produced by oocyte metabolism under normal physiological conditions, but when ROS levels exceed physiological levels, mitochondria become unstable, resulting in mitochondrial damage. The impaired mitochondrial function causes a range of defects, including the integrity disruption of the meiotic and mitotic spindles, thereby disrupting oocyte maturation and embryo development [23][24][25].
Antioxidants are compounds that can delay or inhibit oxidation from reacting with other molecules by the transfer of electrons, thereby maintaining cellular redox homeostasis and proteostasis. In a broad sense, antioxidants can be divided into two broad categories: endogenous and exogenous [26]. The intracellular endogenous antioxidant defense system comprises enzymatic antioxidants (SODs GPx and catalase) along with some nonenzymatic antioxidants (vitamin E, glutathione, and bilirubin) [27,28]. Exogenous

Discussion
Ovarian organoids are an emerging culture technology worth pursuing many applications. The ability of ovarian organoids to be monitored and manipulated in a controlled environment makes them an ideal model for exploring the potential mechanisms of oogenesis [18]. It also provides a tractable system for pre-clinic medicines screening [3]. At present, both PSCs and FGSC have been used to produce ovarian organoids, and these resultant oocytes have produced healthy pups [2,3]. Clearly, ovarian organoids have shown great potential in biomedical applications owing to their uniform characteristics. Nevertheless, the question remains as to the identification of the effective culture condition that permits both efficient and consistent production of competent oocytes during reconstitution culture. In this study, we investigated the role of MitoQ in the recovery of oocyte competency and identified the pathway impacting oocyte competency and GCs proliferation by molecular analyses.
Numerous biochemical processes are performed by mitochondria, including the production of ATP, the regulation of Ca 2+ levels, and the synthesis of steroid hormones [19]. The maintenance of mitochondria within the germline is a highly regulated process. At an early embryonic developmental stage in mice (E9.5 to E11.5), each PGC contains less than 1000 mitochondria [20]. Just prior to primordial follicle formation, mitochondria are transported from oocytes in the nest to one (or a small number) of oocytes in each nest. At this point, the content of mitochondria does not increase [21]. During and immediately after birth, the ovary is populated by primordial oocytes, which each contain about 5000 mitochondria [22]. Thus, to a somewhat lesser degree, the contact with the development of the follicles and the mitochondria number was interrelated. Normally, ROS are produced by oocyte metabolism under normal physiological conditions, but when ROS levels exceed physiological levels, mitochondria become unstable, resulting in mitochondrial damage. The impaired mitochondrial function causes a range of defects, including the integrity disruption of the meiotic and mitotic spindles, thereby disrupting oocyte maturation and embryo development [23][24][25].
Antioxidants are compounds that can delay or inhibit oxidation from reacting with other molecules by the transfer of electrons, thereby maintaining cellular redox homeostasis and proteostasis. In a broad sense, antioxidants can be divided into two broad categories: endogenous and exogenous [26]. The intracellular endogenous antioxidant defense system comprises enzymatic antioxidants (SODs GPx and catalase) along with some nonenzymatic antioxidants (vitamin E, glutathione, and bilirubin) [27,28]. Exogenous antioxidants include carotenoids [29], flavonoids (e.g., anthocyanin) [30], and vitamins (e.g., A/C) [31][32][33]. Endogenous and exogenous antioxidants coordinately regulate the maintenance of mitochondria function and, in doing so, helps to protect cells from oxidative stress-induced damage, thus ensuring their proliferation and survival. Mechanistically, different antioxidants are differentially distributed in spaces, with the most distributed within the cytoplasm (untargeted antioxidants) and a small number localized within the mitochondria (Mt-targeted antioxidants) [28,[34][35][36]. The mechanism of action and protective effects of untargeted and Mt-targeted antioxidants has been extensively studied. Compared with untargeted cellular antioxidants, the Mt-targeted antioxidants exerts greater protection against oxidative damage in the mitochondria and eliminate ROS at the heart of the source through their ability to cross the mitochondria-phospholipid bilayer [34][35][36][37][38][39], such as MitoQ, which could be effective antioxidant therapies against the damage caused by enhanced ROS generation [12,36,40,41].
Currently, both for mice and humans, a previous study suggested that MitoQ protects oocytes against functional defects caused by ROS accumulation and aging [15]. In this study, the findings proved that MitoQ administration promoted follicle growth and oocyte maturation. Compared with in vivo oocytes, in vitro-generated oocytes exhibit higher basal ROS levels, abnormal mitochondrial distribution, less matured mitochondria, and lower ∆Ψm; however, after the treatment of MitoQ, these changes could significantly be reversed to a certain extent. Collectively, these findings revealed the antioxidant effect of MitoQ during oogenesis in vitro on the one hand and indicated that oxidative stress induced by traditional 3D culture environment may be responsible for the low competency of in vitro-generated oocytes on the other hand.
The mammalian oocyte growth and maturation in a mutually dependent relationship with adjacent somatic cells. Due to the naive mitochondrial state of immature oocytes, the growth and maturation of immature oocytes are mostly supported by the surrounding GCs [42]. Accordingly, GCs have been shown to play a central role in providing metabolic support via gap junctional communication and maintaining adequate oocyte ATP levels [43]. It has been found that GCs-enclosed oocytes matured in vitro have higher ATP contents throughout maturation than oocytes matured without their cumulus vestment [23]. In part, the observation of differing rates of follicles growth in vitro is likely to reflect oocyte quality. Differences in growth rates of immature follicles that survive during follicles in vitro culture have been reported in mice [44,45], caprine [46,47], cattle [48], and macaques [49,50]. Macaques, for instance, have only follicles that grow rapidly and are capable of forming mature oocytes and/or fertilized embryos [49]. In addition, faster-growing mice follicles also exhibited increased fertilization rates and a live birth [51]. In follicle development, the discrepancy in follicle growth may reflect the ability to synthesize and respond to autocrine/paracrine factors (AMH, GDF9, BMP15), which modulate GCs proliferation [52]. In the present experiment, our findings demonstrated that the size of the follicle with MitoQ treatment increased greatly during folliculogenesis in vitro, accompanied by a rapid proliferation of GCs, which indicated that MitoQ promotes the proliferation of GCs. Moreover, by the H 2 O 2 -oxidative injury model, we further validated that MitoQ guards GCs against the H 2 O 2 -induced apoptosis pathway (caspase 9 and Bax) via the preservation of mitochondrial function. Therefore, we have reason to believe that the beneficial effect of MitoQ on oocyte potential may be largely attributable to the GCs proliferation.
Aging is known to be associated with sub-optimal reproductive performance in females. However, the exact mechanisms by which it affects fertility are complex [53]. State-of-the-art research suggests mitochondrial dysfunction in GCs may play a key role in the age-related decline in ovarian function and reproductive capacity [54]. In studies, MitoQ, a derivative of coenzyme Q, effectively improves mitochondrial function [9] and attenuates redox-related diseases [55,56]. At present, for mice, humans [15], and porcine [16], multiple studies have shown that MitoQ promotes oocyte maturation in vitro and maintains the stability of mitochondrial function. Furthermore, a recent experimental study demonstrated that MitoQ is able to alleviate ovarian fibrosis in obese and reproductively old mice [57]. Our results revealed that MitoQ could protect GCs against oxidative stress, thereby improving in vitro-generated oocytes maturation competence. This suggests that the administration of MitoQ may be considered an intervention strategy for anti-ovarian aging therapy for humans.

Animal Breeding
The outbred ICR mouse was purchased from SPF Biotechnology (Beijing, China). Mice were bred in the mouse house of Inner Mongolia University in the standard temperature/humidity constant environment.

FGSCs Extraction and Culture
In this study, protocols for FGSCs isolation and sorting were identical to previous studies [17,58,59]. In brief, ovaries from 1-3 dpp female mice were digested to the single cell suspension using a two-step enzymatic method (0.

In Vitro Maturation and Fertilization
For in vitro maturation, cumulus oocyte complexes were maturated in vitro in a α-MEM based maturation media containing 5% FBS, 30 mg/mL sodium pyruvate, 0.1 IU/mL follicle-stimulating hormone, 4 ng/mL EGF, 1.2 IU/mL human chorionic gonadotrophin (Cat. No. 2413402X2053, ASKA, Tokyo, Japan), 4 ng/mL bFGF, 30 mg/ ml penicillin, 75 mg/mL streptomycin. After 16-18 h of maturation, MII oocytes were transferred into 90-µL human tubal fluid droplets (each containing 20-25 oocytes) covered with paraffin liquid in a 35-mm dish. Spermatozoa were collected from the cauda epididymis of adult ICR male mice and added at a final concentration of 2 × 10 6 /mL to a drop of human tubal fluid medium containing the MII oocyte. After inoculation for 6 h at 37 • C, the oocytes were transferred to KSOM medium and further cultured for 18 h.
The level of ROS, mitochondria content and ∆Ψm in GCs were detected as above. In brief, GCs were seeded on 6-well plates with a density of 8 × 10 4 /well, after treatment with H 2 O 2 or MitoQ according to the experimental design, cells were incubated with DCFH-DA (10 µM), MitoTracker (200 nM) and JC-1 (10 µM) solution for 30 min at 37 • C, respectively. After washing 3 times with PBS, 5 visual fields were randomly selected under the fluorescence microscope (Nikon, Tokyo, Japan) for observation. The fluorescence intensity of each sample was analyzed by Image J (U.S. National Institutes of Health, Bethesda, MD, USA, https://imagej.nih.gov/ij/, 1997-2018, accessed on 2 December 2020) software. ROS production and distribution of mitochondria were evaluated as the intensity of signals, respectively. ∆Ψm was evaluated as the ratio of the red to green fluorescence intensity. Three samples from each group were assayed and performed in triplicate.

Immunohistochemical Staining
Ovarian organoids were fixed in 4% paraformaldehyde, embedded in paraffin and sectioned. For immunohistochemical analyses, the sections were dewaxed and rehydrated by xylol and alcohol, respectively. The section (3-5 µm) was furtherly immersed into 10 mM sodium citrate buffer, pH 6.0 and heated for 10 min. Then, the slides were treated with 1% H 2 O 2 in PBS to inactivate endogenous peroxidase activity and incubated with blocking buffer, 10% serum in PBS, for 1 h at 37 • C. After that, the sections were incubated with the first antibody (DDX4: Cat. No. ab270534, Abcam, Cambridge, UK, 1:1000; Inhibin alpha: Cat. No. ab224798, Abcam, 1:1000, PCNA: Cat. No. 10205-2-AP, Proteintech, Wuhan, China, 1:500) overnight at 4 • C. The slides were washed three times in PBS, each time for 10 min, after which the slides were incubated with horseradish peroxidase (HRP) conjugated antirabbit IgG (Cat. No. A0279, Beyotime for 1 h at room temperature. HRP activity was detected with DAB solution (Cat. No. P0203, Beyotime. The slides were examined under a microscope and photos were taken for analysis by ImageJ. Staining was quantified by tracing the immunoreactive area (IA) and the integrated optical density (IOD). The staining intensity (SI) for each image was calculated as SI = IOD/IA. The follicles were classified according to their developmental stages based on the GCs morphology. All staining were repeated more than three times.

Granulosa Cells Culture
Primary GCs were isolated from PMSG-primed (24 h) PD23 female mice ovary according to described previously [60,61]. GCs were released from antral follicles by puncturing with a 27-gauge needle under a stereoscope. Cells were cultured at a density of 1 × 10 6 cells in DMEM/F12 medium (Cat. No. 12634010, Gibco) containing 5% FBS, 6 mg/mL Penicillinstreptomycin in 24-well culture dishes. After overnight culture, cells were washed and cultured in serum-free medium before any further treatment.

CCK8 Array
GCs with 5 × 10 3 cells per well were plated in 96-well plates. After 48 h of culture under DMEM/F12-based medium, GCs reached~70% confluency. CCK8 reagent (Cat. No. C0038, Beyotime) was added to the 96-well plate (10 µL/well) and incubated accordingly. Finally, the absorption value was measured with a microplate reader (Bio-Tek Instruments, Thermo Fisher Scientific) at 450 nm wavelength. Three samples from each group were assayed, and each reaction was performed in triplicate.

Western Blot
Proteins of GCs were obtained by RIPA lysis buffer RIPA (Cat. No. P0013B, Beyotime) supplemented with a protease inhibitor cocktail in centrifuged tubes in ice for 20 min. The concentration of the protein was measured by using the BCA protein assay (Cat. No. 23225, Thermo Fisher Scientific). 30 µg of proteins from each sample were loaded to SDS-PAGE before transferring to nitrocellulose membranes (Bio-Rad Biotechnology, Hercules, CA, USA), and probed with the indicated antibodies (

Statistical Analysis
Experimental data were expressed as the means ± SD with each experiment. Analyses were conducted with SPSS (Version 24) and consisted of independent-samples t-test when only a single variable was manipulated or one-way ANOVA with Tukey-Kramer post hoc when multiple variables were manipulated. p < 0.05 was considered as a statistical significance.

Conclusions
In summary, our results demonstrated that MitoQ supplementation during ovarian organoids culture could reverse the 3D culture environment-induced oxidative stress by directly scavenging ROS and promoting the proliferation activity of GCs. The ameliorative influence of MitoQ was reflected by the improvement of mitochondrial function, follicle size, and ultimately by enhancement of oocyte maturation. To thoroughly explore the underlying mechanisms of MitoQ protection, further studies indicated that MitoQ alleviates oxidative stress and attenuates oxidative stress-associated damage in GCs. This study is warranted to validate that MitoQ may serve as a potential therapeutic drug for anti-ovarian aging.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.