Effects of Coenzyme Q10 on Ovarian Surface Epithelium-derived Ovarian Stem Cells and Ovarian Function in 4-vinylcyclohexene Diepoxide-induced Ovarian Failure Mice.

Background: Several studies have shown that CoQ10 can rescue ovarian aging and that ovarian surface epithelium (OSE)-derived ovarian stem cells (OSCs) are useful for treating infertility with ovarian aging. However, there are few studies the effect of CoQ10 on OSCs. This study was aimed to investigate whether CoQ10 activates OSCs while recovering ovarian function using 4-vinylcyclohexene diepoxide (VCD)-induced ovarian failure mouse model. Methods: C57BL/6 female mice aged 6 weeks were randomly divided into four groups (n=10/group): (Control) saline and orally, (CoQ10) 150 mg/kg/day orally in 1 mL of saline daily for 14 days, (VCD) 160 mg/kg/day, 2.5 ml/kg ip for 5 days, (VCD+CoQ10) 5 days after VCD injection, CoQ10 (150 mg/kg/day) orally for 14 days. After nal treatment of CoQ10, follicle counts were evaluated by hematoxylin and eosin (H&E) staining, and ovarian mRNA expressions of Bmp-15, Gdf-9, and c-Kit were examined by quantitative real-time PCR. Serum FSH, AMH, and ROS levels were also measured. Oocyte-like structure count and expression of Oct-4 and MVH were evaluated from postcultured OSE for 3 weeks. In the second experiment, another 32 female mice were administered with CoQ10 in the same way as above and were superovulated by PMSG and hCG, followed by mated with males. Then, numbers of zygotes ovulated and embryo development rate were examined. Results: Postcultured OSE had signicantly increased numbers of oocyte-like structure and expression of Oct-4 and MVH in VCD+CoQ10 group compared to VCD group (p <0.05). Numbers of surviving follicles including from primordial to antral follicles, numbers of zygotes retrieved and embryo development rate to blastocyst were signicantly higher in VCD+CoQ10 group

The main cause of ovarian aging-related decline in oocyte quality has been known to be mitochondrial dysfunction of the oocytes, which leads to oxidative stress (OS) [1]). OS is caused by an excessive generation of reactive oxygen species (ROS). ROS is very unstable and highly reactive, and can easily damage to basic tissue components and various cellular events including cell death as well as major biomolecules such as proteins, lipids, and DNA [2]. Due to these characteristics, OS induces senescence of the cells and diminishing ovarian reserves, follicular atresia, and poor oocyte quality in the ovary [3]. In this respect, antioxidants have been attempted as one of the key components to prevent OS-induced ovarian aging by improving mitochondrial function [4][5][6].
Coenzyme Q10 (CoQ10) is one of the representative antioxidants [7,8]. It is synthesized by most of the normal tissues, slowly decreasing with aging process [9][10][11]. Previous literature has shown that oral intake of CoQ10 notably brings up its level in various tissues including muscle, adrenal gland, sperm and ovaries. Moreover, it is observed in the human follicular uid, suggesting its possible function of antioxidant protection in the human reproductive system [12][13][14]. Several studies have recently shown that CoQ10 can improve mitochondrial function and rescue ovarian aging by protecting ovarian reserve against oxidative damage [15][16][17].
However, there are few studies the effect of CoQ10 on OSCs. Therefore, this study was aimed to investigate whether CoQ10activates ovarian function and OSCs using 4-vinylcyclohexene diepoxide (VCD)-induced ovarian failure mouse model.

Animals
Six-week-old C57BL/6 female mice (body weight: 18.0 ± 2.0 g) were purchased from the Korea Experimental Animal Center (Daegu, Korea) and acclimated the controlled laboratory conditions for one week. The animals were appropriately housed in plastic cage at a constant temperature of 22 ± 2 °C, relative humidity of 55 ± 5%, and 12 h arti cial light/-dark cycle. All mice had free access to food pellets and water available ad libitum throughout the study period. This study is designed as a controlled experimental study using laboratory animals. All animal experiments were conducted under the guidance for the Care and Use of Laboratory Animals, approved by the Pusan National University Hospital Institutional Animal Care and Use Committee (PNUH-2020-161).

Experimental design
A total of 40 female mice rats were randomly divided into four groups (n = 10/group): group 1 (control): saline and orally; group 2 (CoQ10): 150 mg/kg/day orally in 1 mL of saline daily for 14 days; group 3 (VCD-induced model): 160 mg/kg/day,2.5 ml/kg ip for consecutive 5 days; group 4 (VCD + CoQ10): 5 days after VCD injection, CoQ10 150 mg/kg/day orally for consecutive 14 days. The control group was orally treated with normal saline for 14 days, and CoQ10 was orally administered at 150 mg/kg/day orally in 1 mL of saline daily for 14 days. VCD was purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA), dissolved in sesameoil (Sigma-Aldrich), and injected intraperitoneally with vehicle or 160 mg/kg of VCD ( nal volume of 2.5 ml/kg) daily for 5 days.

Collection of serum and both ovaries
After the nal administration of CoQ10 or saline, mice were anesthetized with the use of carbon dioxide (CO 2 ). Then 1 mL blood samples were taken from the heart. Blood samples were centrifuged at 3000 rpm/min and 4 °C for 15 min to obtain serum samples. After blood collection, both ovaries were removed and weighed. And then, the right ovary was provided for OSC culture, and the left ovary for histological examination and mRNA expression evaluation.
Scraping of ovarian surface epithelium (OSE) and in vitro culture of putative ovarian stem cells (OSCs) OSCs were retrieved from OSE. The right ovaries were gently rinsed several times in Dulbecco's phosphate-buffered saline (DPBS; Invitrogen, Carlsbad, CA, USA) containing antibiotics (penicillin 100 U/mL, streptomycin 100 mg/mL; Invitrogen) at ambient temperature and kept in serum-free plain and preincubated Dulbecco's Modi ed Eagle Medium/Ham's Nutrient Mixture F-12 (DMEM/F12; Gibco BRL, Grand Island, NY, USA) before scraping of OSE. The surface of the remaining intact ovary was gently scraped several times in the aseptic laminar ow hood with a sterile disposable surgical scalpel (Swann-Morton, She eld, United Kingdom) into plain DMEM/F12 in a60 mm dish at 37 °C on a preheated tray.
OSE was easily detached from the ovarian surface and centrifuged to retrieve a scraped suspension of cells. The suspension of scraped OSE cells were transferred to a 15 mL centrifuge tube and spun at 1,000 g for 10 minutes at room temperature. The pellet was suspended in fresh medium and cultured in DMEM/F12 supplemented with 20%fetal bovine serum (FBS; Invitrogen) and antibiotics (Invitrogen) in a 5% CO2 incubator at 37 °C for 3 weeks. The culture medium was replaced with fresh medium every 2 days. Cultures were carefully monitored dailyunder a heated stage inverted microscope (ECLIPSE2000-S, Nikon, Tokyo, Japan) equipped with a digital sight camera (Nikon, Tokyo, Japan). The postcultured OSE cells were utilized for real-time polymerase chain reaction (PCR).

Histological evaluations
The isolated ovaries were xed in 4% paraformaldehyde at 4 °C overnight, and dehydrated using ethanol series, cleared in xylene, embedded in para n, and sectioned at 5 µm and then stained with hematoxylin and eosin (H&E). After mounting, sections were analyzed histologically under a light microscope. Follicles were counted in all sections from each ovary, and results were corrected for double counting. Follicles were classi ed into primordial follicle (an oocyte surrounded by one layer of attened granulosa cells), primary follicle (an oocyte surrounded by one layer of cuboidal granulosa cells), secondary follicle (two or three layers of cuboidal granulosa cells with no antral space), and antral follicle (more than four layers of granulosa cells with one or more independent antral spaces, or with a cumulus granulosa cell layer) according to the criteria of Luo [30]. Follicles containing degenerated oocytes were deemed atresia based on the presence of apoptotic bodies in the granulosa cell layer, disorganized granulosa cells, a degenerating oocyte, or fragmentation of the oocyte nucleus.

Measurement of serum AMH, FSH, LH and ROS levels
Serum AMH and FSH were measured using ELISA kits from CUSABIO Technology LLC. Samples from each animal were determined in duplicate according to the manufacturer's guide. Serum ROS levels were measured by the OxiSelect™ In vitro ROS/RNS Assay Kit (Cell Biolabs, Inc., Sandiego, CA) following the manufacturer's instructions. Brie y, 50 µL of sample and hydrogen peroxide standard was add to wells of a 96-well plate, and then 50 µL of Catalyst was additional added to each well. The wells were mixed and incubated for 5 minutes at room temperature. Then, 100 µL of DCFH solution was added to each well. The plate reaction wells were covered to protect them from light and incubated at room temperature for 15-45 minutes. The uorescence of sample was read at 480 nm excitation/530 nm emission with a uorescence plate reader.

Statistical analysis
Statistical analysis used SPSS program (version 12.0) and all data were presented as a mean ± SD. All comparisons of numbers of follicles at each developmental stage, the number of zygotes retrieved, blastocyst formation rate, and ovarian mRNA expression level for Oct4 and MVH were analyzed by student t-test and one-way analysis of variance (ANOVA) with post hoc multiple comparisons by Bonferroni-Dunn analysis. A P-value of < .05 was considered statistically signi cant.

Results
Effects of CoQ10 on formation of oocyte-like structure and stem cell markers expression The scraped OSEs were attached to the broblast-like appearance cells by postculture processing and small oocyte-like cells resembling bubble structure began to appear in close vicinity broblasts in all groups from about 10 to 14 days after culture of scraped OSEs. After 21 days of culture, the size was also increased and oocyte-like structures with prominent nucleus (arrow head), zona pellucida-like structure (arrow) and cytoplasmic organelles were attached to the bottom of culture dish (Fig. 1A). The number of oocyte-like structures was signi cantly increased the CoQ10 group compared to the control group, but was signi cantly decreased in the VCD group. However, administration of CoQ10 to VCDtreated mice increased the number of oocyte-like structures to the level of the control group (Fig. 1B).
After 3 weeks of OSE cultures, mRNA expressions of pluripotent transcriptional marker (Oct-4) and germ cell-speci c protein (mouse vasa homolog, MVH) were also signi cantly increased in the VCD + CoQ10 group compared to the VCD group (Fig. 1C).

Effect of CoQ10 on ovarian function
To investigate whether CoQ10 improves ovarian function in VCD-induced ovarian failure mice, H&E staining was performed in ovaries. The histological characteristics of follicles at each development stage were shown in Fig. 2A. The number of surviving follicles including the primordial, primary, secondary, and antral follicles was signi cantly decreased in the VCD group compared to the control group, but it was signi cantly increased in the VCD group treated with CoQ10. On the other hand, the number of atretic follicles showed opposite results (Fig. 2B). The mean numbers of the primordial, primary, secondary and antral follicles were 48 ± 2.2, 26 ± 3.1, 19 ± 1.7 and 21 ± 1.5 in the VCD + CoQ10 group, respectively, which were signi cantly increased two times or more compared to the VCD group (21 ± 1.5, 7 ± 0.5, 19 ± 2.9, and 9 ± 2.2, respectively) except for the secondary follicle (Fig. 2C).

Effect of CoQ10 on expression of genes associated with follicular development
Bmp-15, Gdf-9, and c-Kit are representative factors that regulates the activation of primordial follicles and follicular development [31,32]. Expressions of these three genes were also signi cantly increased the CoQ10 group compared to the control group, but was signi cantly decreased in the VCD group. However, administration of CoQ10 to VCD-treated mice signi cantly increased mRNA expressions of these genes (p < 0.05) (Fig. 3).

Effect of CoQ10 on serum FSH, AMH and ROS
The mean serum FSH concentration in the control, CoQ10, VCD, and VCD + CoQ10 groups was 9.20, 9.17, 15.0, and 13.11 ng/mL, respectively (p < 0.01). It was signi cantly higher in the VCD group compared to the control and CoQ10 groups (p < 0.01), but administration of CoQ10 to VCD-treated mice signi cantly decreased serum FSH level (p < 0.05) (Fig. 4A).
The mean serum AMH concentration was 0.41, 1.26, 0.24, and 0.78 ng/mL in the control, CoQ10, VCD, and VCD + CoQ10 groups, respectively (p < 0.01). It was signi cantly lower in the VCD group compared to the control and CoQ10 groups (p < 0.05 and p < 0.01, respectively), but it was signi cantly increased in the administration of CoQ10 to VCD-treated mice (p < 0.01) (Fig. 4B). On the contrary, serum ROS level showed opposite results to serum AMH levels. It was signi cantly higher in the VCD group, but CoQ10 treatment signi cantly decreased to the level of control group (Fig. 4C).
Effect of CoQ10 on ovarian response and oocyte quality As a second experiment to investigate whether CoQ10 improves ovarian function in VCD-induced ovarian failure mice, ovarian response and oocyte quality were examined. Ovarian response was evaluated as the number of zygotes retrieved after superovulation, followed by mating, and oocyte quality was de ned as the fragmentation percentages and embryo development competency of zygotes retrieved. The number of zygotes retrieved and embryo development rate to blastocyst were signi cantly decreased in the VCD group compared to the control group. However, administration of CoQ10 to VCD-treated mice signi cantly increased the number of zygotes retrieved and embryo development rate compared to the VCD group. In contrast, the fragmentation percentages of retrieved zygotes was signi cantly increased in the VCD group, but increased again in the VCD + CoQ10 group (Table 1).

Discussions
Many studies have reported that CoQ10 may protect ovarian reserve against oxidative damage in mice [15][16][17]. Recently, some studies have attempted to prove the potential impacts of CoQ10 on ovarian function in infertile women with low prognosis undergoing assisted reproductive technology (ART) procedures [33]. To the best of our knowledge, no study has been conducted so far to examine the effect of CoQ10 on OSE-derived OSCs. The present study rstly showed that CoQ10 stimulated the differentiation of OSE-derived OSCs into cells with oocyte-like structure and ovarian expression of Oct-4 and MVH in the VCD-induced ovarian failure mice.
VCD speci cally targets primordial and primary follicles, causing depletion of these follicle populations in mice and rats by accelerating atresia [34,35]. In this respect, the VCD mouse model is an adequate ovarian failure model of perimanopause/menopause transition similar to menopause accompanying with the depletion of follicle while having intact ovaries.
Menopause is accompanied by ovarian failure with decreased ovarian function, which is characterized by lower serum levels of estradiol and elevated FSH level [36] Abnormal FSH level combined with low AMH level is strongly associated with pregnancy outcome [37]. Serum AMH level is a representative marker of ovarian aging as well as ovarian reserve in human, and it is commonly used to assess extent of ovarian follicle depletion, to diagnose premature ovarian insu ciency, and to predict age at menopause [38,39].
The present study showed extremely higher FSH levels and decreased AMH levels in the VCD group. This result means that VCD-treated mice had a typical ovarian failure. On the other hand, the present study also showed that administration of CoQ10 to the VCD-treated mice decreased serum FSH levels and increased serum AMH levels.
In addition, a decrease or failure in ovarian function also can be de ned as an ovarian function with decreased follicular development and oocyte quality, which determines embryo developmental competency. In the present study, the VCD-treated mice had a signi cant decrease in numbers of primordial follicles and antral follicles as well as the number of ovulated oocytes and embryo development rate. These results suggest that CoQ10 may restore ovarian reserve and ovarian function in the VCD-induced ovarian failure mice. These potential effects of CoQ10 have been introduced in several previous studies. Burstein et al. showed that CoQ10 treatment signi cantly improved ovarian function and mitochondrial function in aged mice of 18-weeks old, resulting in increasing the number of ovulated oocytes [40]. Ben-Meir et al. also observed that CoQ10 supplementation preserves ovarian reserve, and improves mitochondrial performance and fertility in reproductive aged mice of 9-month-old [16]. Özcanet al. introduced that CQ10 supplementation increased serum AMH level and numbers of antral follicles, and can protect the ovarian reserve against oxidative damage in cisplatin-treated rats [17].
The present study also con rmed that CoQ10 treatment stimulated the expressions of Bmp-15, Gdf-9, and c-Kit, which are representative factors that regulate the activation of primordial follicles and follicular development [31,32]. Especially, GDF-9 and BMP-15 play a critical role in early follicular development and ovarian function [41]. The KL/c-Kit system importantly regulates oogenesis and folliculogenesis [42,43].
This result suggests that KL/c-Kit plays an important role in increasing the number of actively growing follicles and/or in regulating of primordial follicle maintenance.
The possible mechanism by which CoQ10 enhances ovarian function in the present study can be explained in three aspects. The rst is the activation of OSCs derived from OSE as previously described.
As a result, ovarian reserve can be increased by CoQ10, and eventually the number of follicles from the primordial follicle to the antral follicle increases as shown in Fig. 2. The second is the stimulation of early follicular development, which can be supported by an increase in the expressions of Bmp-15, Gdf-9, and c-Kit as shown in Fig. 3). A recent study also reported that administration of CoQ10 upregulated expressions of folliclestimulating hormone receptor (FSHR) and proliferation cell nuclear antigen (PCNA), which are representative genes involved in folliculogenesis, on cyclophosphamide-induced premature ovarian failure in a mouse model [44]. Finally the third is the reduction of ROS by CoQ10 (Fig. 4D), which has been reported by many previous studies [17,40,45].
Another importance of this study is that CoQ10 can also contribute to the resolution of POI. POI is also known as premature ovarian failure and it is a clinical syndrome de ned by stop of ovarian function before the age of 40 [46]. Although the prevalence of POI is about 1%, the prevalence of POI is on the rise as the proportion of premenopausal cancer survivors with iatrogenic POI due to recent gonadotropin [47,48]. Nevertheless, POI remains an unmet need in infertility treatment. One of the characteristics of POI is the increase in generation of ROS causing ovarian tissue damage and decrease of ovarian reserve [49].
Therefore, CoQ10 administration may be able to reverse ovarian function in POI patients.

Conclusion
The present study shows that CoQ10 stimulates the differentiation of OSE-derived OSCs and reduces ROS levels, nally leading to improve follicular development, ovarian function, oocyte quality and embryo development competency in the VCD-induced ovarian failure mice. These results suggest the possibility of using CoQ10 as a new treatment strategy for ovarian aging or POI. This requires further research to determine the optimal dosage and duration for CoQ10 supplementation.

Declarations
Ethics approval and consent to participate KH participated in drafting the manuscript or revising it critically for important intellectual content, and nal approval of the version to be published.