Effects of coenzyme Q10 on ovarian surface epithelium-derived ovarian stem cells and ovarian function in a 4-vinylcyclohexene diepoxide-induced murine model of ovarian failure

Background Several studies have shown that coenzyme Q10 (CoQ10) can rescue ovarian aging and that ovarian surface epithelium (OSE)-derived ovarian stem cells (OSCs) are useful for treating infertility due to ovarian aging. However, few studies have examined the effect of CoQ10 on OSCs. This study was aimed to investigate whether CoQ10 activates OSCs and recovers ovarian function in a 4-vinylcyclohexene diepoxide (VCD)-induced mouse model of ovarian failure. Methods Forty female C57BL/6 mice aged 6 weeks were randomly divided into four groups (n = 10/group): a control group administered saline orally, a CoQ10 group administered 150 mg/kg/day of CoQ10 orally in 1 mL of saline daily for 14 days, a VCD group administered 160 mg/kg/day of VCD i.p. in 2.5 mL of saline/kg for 5 days, and a VCD + CoQ10 group administered VCD i.p. for 5 days injection and CoQ10 (150 mg/kg/day) orally for 14 days. After treatment, 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 counts and the expressions of Oct-4 and MVH were also evaluated after culturing OSE for 3 weeks. In a second experiment, 32 female mice were administered CoQ10 as described above, induced to superovulate using PMSG and hCG, and mated. Numbers of zygotes and embryo development rate were examined. Results Postcultured OSE showed significant increases in the numbers of oocyte-like structure and that the expression of Oct-4 and MVH were higher in the VCD + CoQ10 group than in the VCD group (p < 0.05). Numbers of surviving follicles from primordial to antral follicles, numbers of zygotes retrieved and embryo development rate to blastocyst were significantly greater in the VCD + CoQ10 group than in the VCD group (p < 0.01). Serum AMH level and ovarian expressions of Bmp-15, Gdf-9 and c-Kit were also significantly greater in the VCD + CoQ10 group than in the VCD group (p < 0.05). In contrast, serum ROS level was significantly lower in the VCD + CoQ10 group than in the VCD group (p < 0.05). Conclusion This study shows that CoQ10 stimulates the differentiation of OSE-derived OSCs and confirms that CoQ10 can reduce ROS levels and improve ovarian function and oocyte quality in mice with VCD-induced ovarian failure.

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 final 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 significantly 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 significantly higher in VCD+CoQ10 group compared to VCD group (p <0.01). Serum AMH level and ovarian expression of Bmp-15, Gdf-9, and c-Kit were significantly increased in VCD+CoQ10 group compared to VCD group (p <0.05). In contrast, serum ROS level was significantly decreased in VCD+CoQ10 group compared to VCD group (p <0.05). Conclusion(s): This is the first study to show that CoQ10 stimulates the differentiation of OSE-derived OSCs. Also this study confirms that CoQ10 can reduce ROS levels, leading to improve ovarian function and oocyte quality in ovarian failure mice.

205 Preliminary characterization of ovarian stem cells from bovine ovaries

Ovarian stem cells (OSCs) have been reportedly isolated from ovaries of rodents, pigs, humans, and cattle by targeting the germ cell marker protein DDX4. Although the role of OSCs in female reproduction is unknown, the ability to culture OSCs and differentiate oocytes invitro could benefit the cattle industry and the study of oogenesis. The aim of this study was to describe isolation and preliminary characterisation of putative bovine OSCs. Slaughterhouse-derived ovaries from adult cows were processed by mechanical and enzymatic dissociation into a single cell suspension followed by immunostaining. Cells were incubated in blocking solution followed by 10µgmL−1 rabbit anti-human polyclonal DDX4 antibody (#13840; Abcam) for 15min and 2µgmL−1 goat anti-rabbit IgG labelled with Alexa Fluor 647 for 15min in the dark. Next, cells were resuspended in Hanks’ balanced salt solution with 1% bovine serum albumin/25mM HEPES, filtered through a 30-µm strainer and subjected to fluorescence-activated cell sorting. Controls used to establish gates were unstained cells and cells incubated with secondary antibody only. 4’,6-Diamidino-2-phenylindole (DAPI) exclusion was used as a viability test. Putative OSCs were placed in culture in OSC medium (MEMα Glutamax containing 10% fetal bovine serum, 1mM sodium pyruvate, 1× nonessential amino acids, 103 units of leukemia inhibitory factor, 10µgmL−1 glial cell-derived neurotrophic factor, 10µgmL−1 basic fibroblast growth factor, 1µgmL−1 epidermal growth factor, 1× N2-max, penicillin/streptomycin) for expansion and characterisation by gene expression using reverse transcription-PCR and protein expression using immunolocalization and confocal microscopy. To ensure specificity against bovine DDX4, the same antibody used for cell sorting was used to label oocytes within ovarian follicles in histological sections. Two cell lines were obtained and expanded invitro. Gene expression was performed in putative OSCs at passages 1 to 3; cumulus-oocyte complexes (COCs) were used as positive controls and adult skin fibroblasts as negative controls, and ACTB was used as an endogenous control. Both putative OSC lines and COCs expressed the germ cell markers DAZL and C-KIT, and COCs also expressed BMP15. Only ACTB was detected in fibroblasts. Immunolocalization was performed in putative OSCs at passage 4, with oocytes and fibroblasts used as positive and negative controls. Additional controls were cells exposed to secondary antibody only. Both putative OSC lines and oocytes expressed DAZL and DDX4 and no marker was detected in fibroblasts. Next, OSC line #2 was transfected with a retroviral vector using the EF1α promoter for green fluorescent protein (GFP) expression. This is a critical step to ensure the success of experiments requiring cell tracking. Transfected cells were expanded and sorted to establish a pure population of GFP+ OSCs. To verify replication deficiency of the viral particles, supernatant from GFP+ OSCs was collected 1 passage after transfection and applied to GFP- OSCs. No GFP+ cells were observed after 24, 48, or 72h. These preliminary results confirm the presence of putative OSCs in the ovaries of cows of reproductive age. If these cells are capable of invitro differentiation, they could provide a powerful tool to study oogenesis and further develop assisted reproductive technologies.

In Vitro Generation of Oocytes from Ovarian Stem Cells (OSCs): In Search of Major Evidence

The existence of ovarian stem cells (OSCs) in women as well as their physiological role in post-menopausal age are disputed. However, accumulating evidence demonstrated that, besides the animal models including primarily mice, even in adult women putative OSCs obtained from ovarian cortex are capable to differentiate in vitro into oocyte-like cells (OLCs) expressing molecular markers typical of terminal stage of oogonial cell lineage. Recent studies describe that, similarly to mature oocytes, the OSC-derived OLCs also contain haploid karyotype. As proof of concept of their stem commitment, OSCs from mice differentiated to oocytes in vitro are suitable to be fertilized and implanted in sterilized animals resulting in embryo development. Despite enthusiasm for these data, which definitely require extended confirmation before considering potential application in humans for treatment of ovarian insufficiency, OSCs appear suitable for other clinical uses, restoring the endocrine derangements in premature ovarian failure or for fertility preservation in oncologic patients after anti-cancer treatments. In this context, the selection of viable oocytes generated from OSCs before chemotherapy protocols would overcome the potential adjunct oncogenic risk in women bearing hormone-dependent tumors who are repeatedly stimulated with high dose estrogens to induce oocyte maturation for their egg recruitment and cryopreservation.