Introduction
Female infertility is a significant concern today, with several factors contributing to it. As a result, an increasing number of people are using assisted reproductive technologies (ARTs), such as in vitro culture (IVC) of ovarian tissue, follicles, oocytes, and embryos. While ART has benefits for treating infertility, it can also lead to an increase in reactive oxygen species (ROS) during in vitro cultivation, resulting in oxidative stress (OS) due to the absence of an antioxidant defence system (Filatov et al., Reference Filatov, Nikishin, Khramova and Semenova2020).
One potential antioxidant that can help neutralize free radicals, recycle other antioxidants, suppress OS, and protect mitochondria from oxidative damage is Coenzyme Q10 (CoQ10). This fat-soluble compound is synthesized naturally in the human body and is one of the most consumed nutritional supplements (Arenas-Jal et al., Reference Arenas-Jal, Suñé-Negre and García-Montoya2020). Studies have confirmed that CoQ10 is beneficial for both in vivo and in vitro usage. For example, oral consumption of CoQ10 protects the ovarian follicular reserve, increases oocyte quality, and increases the number of cumulus cells (Rodríguez-Varela and Labarta, Reference Rodríguez-Varela and Labarta2021). Furthermore, CoQ10 has been shown to initiate and improve follicular development by upregulating BMP-15, GDF-9, and FSHR, while reducing the amount of ROS by activating other antioxidants (Lee et al., Reference Lee, Kang, Sohn, Kim, Yang and Han2022).
Excessive ROS can cause severe mutations in mitochondrial DNA (mtDNA), leading to mitochondrial dysfunction (Kung et al., Reference Kung, Lin, Kung and Lin2021). A sufficient mtDNA copy number is necessary for successful follicle development, and insufficient mtDNA copy numbers can result in oocytes that cannot fertilize or are aged or degenerated (Busnelli et al., Reference Busnelli, Navarra and Levi-Setti2021). It has been demonstrated that increasing mtDNA copy number in in vitro conditions can improve oocyte developmental competency (Mao et al., Reference Mao, Whitworth, Spate, Walters, Zhao and Prather2012). Additionally, there is a relationship between mtDNA copy number in granulosa cells (GCs) and oocytes, and follicle growth and maturation (Lan et al., Reference Lan, Zhang, Gong, Lu, Lin and Hu2020). Mitochondrial transcription factor A (TFAM) is the most prominent factor in regulating mtDNA transcription and protecting against OS (Mao et al., Reference Mao, Whitworth, Spate, Walters, Zhao and Prather2012). Tfam plays an essential role in regulating mtDNA copy number in ovarian follicles, which is crucial for follicular survival. A balance between mtDNA and Tfam ratio is necessary, and Tfam, with factors such as NF-E2–related factor 2 (Nrf2), Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), and Sirtuin 1 (SIRT1) genes, upregulates mitochondrial biogenesis (Popov, Reference Popov2020).
A Tfam knockdown experiment by Otten et al. showed that zebrafish embryos with knocked-down Tfam had low ATP and mtDNA content, leading to mitochondrial dysfunction and embryo abnormalities (Otten et al., Reference Otten, Kamps, Lindsey, Gerards, Pendeville-Samain, Muller, van Tienen and Smeets2020). In addition, studies have shown that upregulation of Tfam expression and subsequent elevation of mtDNA copy number can improve vitrified–thawed oocyte competency and quality (Amoushahi et al., Reference Amoushahi, Salehnia and Mowla2017, Reference Amoushahi, Salehnia and Ghorbanmehr2018; Ito et al., Reference Ito, Shirasuna, Kuwayama and Iwata2020; Moshaashaee et al., Reference Moshaashaee, Zavareh, Pourbeiranvand and Salehnia2021).
Although some studies have shown promising results regarding the effects of COQ10 on folliculogenesis during in vitro culture (Hosseinzadeh et al., Reference Hosseinzadeh, Zavareh and Lashkarboluki2015, Reference Hosseinzadeh, Zavareh and Lashkarbolouki2017; Kashka et al., Reference Kashka, Zavareh and Lashkarboluki2015, Reference Kashka, Zavareh and Lashkarbolouki2016; Heydarnejad et al., Reference Heydarnejad, Ostadhosseini, Varnosfaderani, Jafarpour, Moghimi and Nasr-Esfahani2019), there is limited knowledge about its effect on mtDNA copy number and Tfam gene expression in ovarian follicles. Therefore, the aim of this study was to investigate the effect of CoQ10 on mtDNA copy number and Tfam gene expression in mouse oocytes and GCs of preantral follicles (PFs) during in vitro culture.
Materials and methods
Chemicals
All chemicals used in this study were purchased from Sigma-Aldrich (Germany), unless otherwise specified.
Animals
Female and male National Medical Research Institute (NMRI) mice were obtained from the Pasteur Institute of Iran. They were housed and cared for in accordance with the guidelines of our university’s animal ethics committee. The animals were kept under controlled conditions (12-hour light/12-hour dark cycle, 20–25°C room temperature, and 40–50% humidity) and provided with standard water and laboratory chow. The animal research ethics committee of Damghan University approved this study (Ref. No. 34/2018).
Preantral follicle isolation
Newborn female mice (14 days old, n = 20) were sacrificed, and their ovaries were removed and placed in droplets of α-minimum essential medium (α-MEM; Gibco, UK) supplemented with 0.22g/l sodium bicarbonate, 0.0036g/l sodium pyruvate, 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 µg/ml streptomycin. Preantral follicles were mechanically isolated using an insulin syringe’s 29-gauge needle under a stereomicroscope at ×10 magnification (Talebi et al., Reference Talebi, Zavareh, Kashani, Lashgarbluki and Karimi2012). Eligible PFs were selected based on the following criteria: round-shaped follicles with diameters of 140–160 µm and containing intact oocytes with several layers of GCs. During the process, the medium was kept at 37°C.
Experimental design
The collected PFs were assigned randomly to control and CoQ10-treated groups. PFs from both groups were cultured in vitro for 12 days. The study was conducted in two parts. First, follicular development was assessed, including follicular survival, growth, antrum formation, and ovulation. Second, mtDNA copy number and Tfam gene expression in GCs and oocytes were evaluated.
In vitro culture of PFs
Isolated PFs were individually transferred using glass Pasteur pipettes into 25-µl drops of α-MEM supplemented with 1% insulin transferring selenium (ITS; Gibco, UK), 20 ng epidermal growth factor (EGF), 100 mIU recombinant human follicle-stimulating hormone (rhFSH; Cinnal-f, Iran), 5% v/v FBS, and 50 µM CoQ10 under sterile mineral oil, and then incubated under 5% CO2, 37°C, and 95% humidity conditions for 12 days. Next, 10 µl of culture medium from each drop was replaced with fresh medium every other day. Follicle diameter was measured using an inverted microscope at ×10 magnification on the initial day, second, and fourth day of culturing, as described previously (Talebi et al., Reference Talebi, Zavareh, Kashani, Lashgarbluki and Karimi2012). In brief, the diameter of the follicles was measured by calculating the average of two perpendicular diameters using an inverted microscope equipped with a pre-calibrated optical micrometer. The evaluation of GC proliferation, growth, and antrum cavity formation was observed under a stereomicroscope over the 12-day culture period. The presence of any lucent space between GCs during in vitro culture was defined as an antrum cavity. Moreover, the survival rate was determined by observing morphological changes during the culture period. On the 12th day of the culture period, ovulation was induced by adding 1.5 IU/ml of human chorionic gonadotropin (hCG; IBSA, Switzerland) to the culture medium. After 18 h, the oocytes were classified into two different types: the first type was germinal vesicle breakdown (GVBD), where the germinal vesicle was absent, and the second type was metaphase II oocytes (MII), identified by the time the first polar body was ejected.
Granulosa cell separation
For granulosa cell separation, the culture medium was supplemented with 0.02 g collagenase for 1 h. Oocytes were picked up using a mouth pipette and then transferred individually to 1.5-ml microtubes. Granulosa cells were separated from the bottom of the culture dish by adding 1 ml trypsin and centrifuging at 3000 rpm for 3 min.
Real-time polymerase chain reaction (PCR)
Real-time PCR was used to determine Tfam relative mRNA expression in both oocytes and GCs. RNA was extracted separately from oocytes and GCs using the RiboEx® protocol (PCRlab, Germany). The samples were then quantified and qualified by spectrophotometer and electrophoresis gel, respectively. cDNA was synthesized using the TaKaRa cDNA synthesis kit (TaKaRa Bio, Japan) based on the manufacturing protocol. Tfam mRNA expression was assessed using the Rotor-gene 6000 (Qiagen) and QuantiTect SYBR Green RT-PCR kit (Qiagen, Hilden, Germany). The Livak method and 2-ΔΔCT were used to analyze Tfam expression. Tfam primers were designed using AlleleID software (Premier Biosoft, USA), and elongation factor 1 (Ef1) was used as a housekeeping gene (Table 1).
Table 1. Designed primer sequences used for real-time polymerase chain reaction
Quantification of mtDNA copy number
DNA samples were extracted from oocytes and GCs following the procedure described by Ghorbanmehr et al. (Reference Ghorbanmehr, Salehnia and Amooshahi2018). Briefly, 10 µl of lysis solution containing 50 mM Tris–HCl (pH 8.5), 0.1 mM EDTA, 0.5% Tween-20, and 200 µg/ml proteinase K (Roche, Germany) were mixed with the samples and incubated at 55°C. After proteinase K deactivation at 95°C for 10 min, the mouse mitochondrial DNA sequence was obtained from NCBI (NC_005089.1) and divided into 200-bp fragments with 50-bp overlaps. Primers were designed using Primer3 plus software to avoid duplication in the nuclear genome (Table 2) and synthesized by MWG DNA sequencing service in Germany (Ebersberg, Germany). The PCR product was extracted from agarose gel and cloned into the pTZ57R/T vector (Thermo Scientific Bio). Five serial dilutions of the plasmid were prepared, and real-time PCR was performed to quantify the mtDNA copy number in oocytes and GCs. Each extracted mtDNA sample was tested in triplicate using five points on the serial standard curve without template control. The cycling programme included initial denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s.
Table 2. Mouse mitochondrial specific primer sequences
Statistical analysis
Statistical analysis was performed using the Statistical Package for the Social Sciences software (SPSS, IBM SPSS statistic 16, USA). After checking the normal distribution of data using the Shapiro test, the Mann–Whitney U-test was applied. Statistical significance was set at P < 0.05.
Results
PFs development
The growth of PFs was examined morphologically every other day during the in vitro culture period by an inverted microscope (Figure 1). The diameter of isolated PFs is summarized in Figure 2. The diameter at the initial time did not differ in both groups (P > 0.05). However, on the second and fourth day of cultivation, the follicle’s diameter showed a significant increase in the presence of CoQ10 compared with the control group (P < 0.05; Figure 2). On the second day of culture, the isolated PFs attached to the bottom of the culture dish through granulosa cell proliferation and developed a round and adhesive shape. On the fourth day, the number of GCs increased and surrounded the oocyte and formed a redundant form. From the sixth day onwards, the antrum cavity formed, and PFs with round and intact oocytes, zona pellucida, and regular perivitelline space were considered as surviving follicles. Degenerated follicles were the ones that released their oocytes before adding hCG or had a delay in granulosa cell proliferation. The survival rate of PFs in the presence of CoQ10 and the control group was 87% and 79%, respectively, which was significantly higher in CoQ10 compared with the control group (P < 0.05; see Table 3). The rate of antrum formation in the CoQ10-treated group and the control group was 71% and 58%, respectively, which was significantly higher in the treated group (P < 0.05; Table 3). Furthermore, the rate of ovulated follicles in the CoQ10-treated group (51%) was significantly higher than in the control group (40%; P < 0.05; Table 3). The maturation rate of oocytes derived from PFs is shown in Table 3. The percentage of released metaphase I (MI) and MII oocytes from PFs in the CoQ10-treated group was significantly increased compared with those in the control group (P < 0.05; Table 3).
Figure 1. Images of the in vitro cultured mouse PFs during in vitro culture; PFs on the initial day (a), fourth day (b), 12th day (c).
Figure 2. Diameter of PFs during the culture period. The stars indicate a significant difference compared with the control group (P < 0.05).
Table 3. Maturation rates of cultured preantral follicles
In all cases at least three experimental replicates were performed.
* Indicates different levels of significant difference with control groups (P < 0.05).
MI: metaphase I oocyte; MII: metaphase II oocyte.
Tfam gene expression
The relative mRNA expression of Tfam was evaluated in oocytes and GCs. The relative expression of the Tfam gene in oocytes was considerably higher in the CoQ10-treated group compared with the control group (P < 0.05; Figure 3). Also, the relative mRNA expression of the Tfam in GCs of the CoQ10-treated group was considerably higher compared with the control group (P < 0.05; Figure 3).
Figure 3. The relative mRNA expression of the Tfam gene. The stars indicate a significant difference compared with the control group (P < 0.05).
mtDNA copy number
The mtDNA copy numbers in the oocytes and GCs of both the treated and control groups are presented in Figure 4. The mtDNA copy numbers in oocytes derived from the CoQ10-treated and control groups were 506,512 and 171,887, respectively. This value was significantly higher in the CoQ10-treated group (P < 0.05; Figure 4). Additionally, the mtDNA copy number of GCs derived from the CoQ10-treated and control groups were 484,589 and 314,786, respectively. The results indicated that the mtDNA copy number of GCs was significantly higher in the CoQ10-treated group compared with the control group (P < 0.05; Figure 4).
Figure 4. The mtDNA copy number of granulosa cells and oocyte. The stars indicate a significant difference compared with the control group (P < 0.05).
Discussion
In vitro maturation is a commonly used assisted reproductive technology that faces several challenges, such as an increase in OS and ROS production during the culture period (Talebi et al., Reference Talebi, Zavareh, Kashani, Lashgarbluki and Karimi2012; Kashka et al., Reference Kashka, Zavareh and Lashkarbolouki2016). While ROS plays a crucial role in follicle development and fertilization, an excessive amount can negatively affect different signalling pathways involved in oocyte and embryo development (Lu et al., Reference Lu, Wang, Cao, Chen and Dong2018; Misrani et al., Reference Misrani, Tabassum and Yang2021). As a result, the addition of antioxidants to the culture medium is recommended. However, it is still unclear how antioxidants affect ovarian follicle formation and growth during in vitro culture. Antioxidants have been shown to be crucial for oocyte maturation and ovulation (von Mengden et al., Reference von Mengden, Klamt and Smitz2020). Therefore, this study aimed to examine the effect of CoQ10, a potent antioxidant, on Tfam gene expression and mtDNA copy number in ovarian follicles.
The study found that CoQ10 increases Tfam gene expression and mtDNA copy number in preantral follicle oocytes and GCs, thereby improving folliculogenesis during in vitro culture. Previous studies have shown that high ROS production during in vitro maturation interrupts follicle development and reduces its competency (Soto-Heras and Paramio, Reference Soto-Heras and Paramio2020). Conversely, CoQ10 can elevate follicle growth and development (Xu et al., Reference Xu, Nisenblat, Lu, Li, Qiao, Zhen and Wang2018). Lee et al. showed that CoQ10 promotes ROS reduction and oocyte quality improvement by inciting ovarian follicle stem cells (Lee et al., Reference Lee, Park, Joo, Joo, Kim, Yang, Kim and Kim2021, Reference Lee, Kang, Sohn, Kim, Yang and Han2022). The same group demonstrated that CoQ10 improves mouse embryo development in vitro by elevating bcl2 and sirt1 expression in cumulus cells, which reduces ROS (Lee et al., Reference Lee, Kang, Sohn, Kim, Yang and Han2022). Additionally, CoQ10 helps infertile individuals undergoing in vitro fertilization (IVF) by improving mitochondrial function and ovarian follicle maintenance (Özcan et al., Reference Özcan, Fıçıcıoğlu, Kizilkale, Yesiladali, Tok, Ozkan and Esrefoglu2016). A study by Ma et al. (Reference Ma, Cai, Hu, Wang, Xie, Xing, Shen, Cui, Liu and Liu2020) found that CoQ10 reduced post-meiotic aneuploidy and increased oocyte maturation rates in aged women during in vitro maturation. Yang et al. (Reference Yang, Liu, Miao, Mou, Liu, Wang, Huo and Du2021a, Reference Yang, Yu, Park, Kim, Eum, Paek, Hwang, Lyu, Kim, Lee, Yoon, Song and Lee2021b) observed that 50 µm CoQ10 suppressed OS, improved mitochondrial function, and upgraded meiotic maturation during in vitro cultivation, therefore increasing oocyte quality. These results are in line with our findings regarding the application of 50 µm CoQ10 to an in vitro culture medium.
However, Streacker and Whitaker (Reference Streacker and Whitaker2019) confirmed that 100 µm CoQ10 was deleterious during in vitro maturation, while Maside et al. (Reference Maside, Martinez, Cambra, Lucas, Martinez, Gil, Rodriguez-Martinez, Parrilla and Cuello2019) found that applying 100 µm CoQ10 negatively affected ART outcomes. In contrast, our results were compatible with studies that used 50 µm CoQ10 during in vitro culture medium. Furthermore, CoQ10 acts as an anti-apoptotic antioxidant, preventing cell death by reducing OS and balancing the amount of anti-apoptotic Bcl-2 and pro-apoptotic BAX factors (Delkhosh et al., Reference Delkhosh, Shoorei, Niazi, Delashoub, Gharamaleki, Ahani-Nahayati, Dehaghi, Raza, Taheri, Mohaqiq and Abbasgholizadeh2021).
It has been investigated that CoQ10 in oocytes and cumulus cells decreases the expression of apoptotic genes such as caspase-3 and Bax, while considerably increasing GDF9 expression (Heydarnejad et al., Reference Heydarnejad, Ostadhosseini, Varnosfaderani, Jafarpour, Moghimi and Nasr-Esfahani2019). Li et al. (Reference Li, Zhan, Hou, Chen, Hou, Xiao, Luo and Lin2019) also observed that CoQ10 activated enzymatic antioxidants such as superoxide dismutase (SOD) and glutathione (GSH), downregulated Bax and caspase-3, and increased Bcl-2 gene expression. Among the anti-apoptotic factors, nuclear respiratory factor 2 (Nrf2) is vital in maintaining redox balance and regulating antioxidants and follicle maintenance (Smolková et al., Reference Smolková, Mikó, Kovács, Leguina-Ruzzi, Sipos and Bai2020).
As a result of increased OS, Nrf2, by binding to the antioxidant response element (ARE), activates protective factors such as SOD and suppresses OS. Conversely, activated NF-κB, as a major transcription factor, controls inflammatory and apoptotic responses regulated by Nrf2 during OS situations (Khodakarami et al., Reference Khodakarami, Adibfar, Karpisheh, Abolhasani, Jalali, Mohammadi, Gholizadeh Navashenaq, Hojjat-Farsangi and Jadidi-Niaragh2022). Nrf2, via activating PGC-1α, regulates Tfam gene expression, which is essential for mtDNA transcription (Shimizu et al., Reference Shimizu, Kasai, Yamazaki, Tatara, Mimura, Engler, Tanji, Nikaido, Inoue, Suganuma, Wakabayashi and Itoh2022). Similarly, CoQ10 can suppress NF-κB and increase Nrf 2 gene expression, leading to Tfam expression regulation (Li et al., Reference Li, Zhan, Hou, Chen, Hou, Xiao, Luo and Lin2019). It is therefore reasonable to consider that CoQ10 has a beneficial effect on Tfam expression.
Based on previous studies, the Tfam level is regulated by PGC-1α and is dependent on NRF1 and NRF2, which can be stimulated by PGC-1α. It is worth mentioning that NRF2 expands the antioxidant defence system, and CoQ10, by upregulating NRF2, not only improves the antioxidant defence system but also increases Tfam expression by activating the PGC-1α gene (Li et al., Reference Li, Zhan, Hou, Chen, Hou, Xiao, Luo and Lin2019; Deng et al., Reference Deng, Lin, Fu, Xu, Luo, Jin, Liu, Sun and Su2020). Furthermore, there is a relationship between Tfam expression and mtDNA copy numbers, whereby an increase in Tfam increases the mtDNA copy number (Lan et al., Reference Lan, Zhang, Gong, Lu, Lin and Hu2020), which agrees with our results. It has been shown that good quality embryos have higher mtDNA copy numbers compared with bad quality ones (Cecchino and Garcia-Velasco, Reference Cecchino and Garcia-Velasco2019). Matured oocytes contain 150,000 copies of mtDNA, while infertile ones have fewer mtDNA copy numbers, indicating that growing follicles are in great need of mtDNA (Rahimi Darehbagh et al., Reference Rahimi Darehbagh, Khalafi, Allahveisi and Habiby2022). A lack of efficient mtDNA copy numbers may cause unpredictable ART outcomes. In this case, Yang et al. (Reference Yang, Liu, Miao, Mou, Liu, Wang, Huo and Du2021a, 2021b) found that the mtDNA and gDNA ratio was noticeable for IVF outcomes. They observed that good quality embryos had a higher rate of mtDNA/gDNA in their cumulus cells compared with those with a low mtDNA/gDNA ratio. These findings are compatible with our observation regarding mtDNA copy number and follicle development. In conclusion, supplementing CoQ10 to the PFs culture medium can improve follicle development by upregulating Tfam gene expression and increasing mtDNA copy number.
Author contributions
R.H.: Conducted experiments and collected data. S.Z.: Supervision, conceptualization, methodology, software, data curation, writing - original draft preparation. M.N.: Supervision, visualization, investigation. S.S.: Investigation and writing draft. All authors contributed to the finalization of the manuscript and approved the final draft.
Funding
This project was extracted from an MSc. thesis, and was founded and supported by Damghan University, Damghan, Iran.
Disclosure
None of the authors have any financial or other potential conflicts of interest.
