Introduction
In mammals, preantral follicles represent approximately 90% of the ovarian follicular population, but most of them (around 99.0%) became atretic during their growth and maturation (Hsueh et al., Reference Hsueh, Kawamura, Cheng and Fauser2015). Therefore, isolation and in vitro culture of these follicles open up a new possibility of maximizing the use of oocytes enclosed in preantral follicles and, consequently, have gained great scientific effect (Figueiredo et al., Reference Figueiredo, Cadenas, Lima and Santo2019). It is important to note that, in bovine species, primordial follicles are activated and begin to grow at least 100 days before ovulation (Britt, Reference Britt1991), this means that, in vitro, these follicles need to be cultured for long periods. However, one of the main obstacles during in vitro culture of preantral follicles is the excessive production of reactive oxygen species (ROS), caused by factors such as manipulation, absence of physiological protection mechanisms and increased oxygen levels in the cellular environment (Sá et al., Reference Sá, Bruno, Guerreiro, Cadenas, Alves, Cibin, Leal-Cardoso and Figueiredo2018). Such factors cause a redox imbalance, generating oxidative stress, which in turn compromises the quality of cultured follicles (Sies, Reference Sies2015; Saeed-Zidane et al., Reference Saeed-Zidane, Linden, Salilew-Wondim, Held, Neuhoff, Tholen, Hoelker, Schellander and Tesfaye2017).
During in vitro culture, follicles are exposed to supraphysiological concentrations of oxygen (up to 20%) that can trigger oxidative stress and consequently reduce the ability of follicles to develop in vitro (Sá et al., Reference Sá, Bruno, Guerreiro, Cadenas, Alves, Cibin, Leal-Cardoso and Figueiredo2018). Oxidative stress during in vitro culture can cause severe cellular damage such as lipid peroxidation, protein denaturation, alterations in nucleic acids and membrane disruption (Halliwell, Reference Halliwell2014). Kashka et al. (Reference Kashka, Zavareh and Lashkarbolouki2016) reported that lipid peroxidation of membranes, mitochondrial lesions and impaired cell integrity resulted in the low production of competent oocytes during in vitro culture. Additionally, lesions caused by ROS promote the inactivation of antioxidant enzymes, such as glutathione peroxidase 1 (GPX1), catalase (CAT), superoxide dismutase (SOD) and peroxiredoxins (PRDX) that act physiologically to balance cellular homeostasis (Sovernigo et al., Reference Sovernigo, Adona, Monzani, Guemra, Barros, Lopes and Leal2017). To minimize the damage caused by oxidative stress during in vitro culture, substances with antioxidant activity were added to the culture medium (Barberino et al., Reference Barberino, Barros, Menezes, Santos, Araújo, Queiroz, Almeida, Palheta and Matos2015).
Eugenol (4-allyl-2-methoxyphenol) contains phenolic compounds in its composition that are responsible for protecting cells and tissues against damage caused by free radicals (Nagababu et al., Reference Nagababu, Rifkind, Boindala and Nakka2010). These authors reported that, in vitro, eugenol inhibits lipid peroxidation in liver cell mitochondria, while, in vivo, eugenol reduces cell necrosis in rat liver with induced hepatotoxicity. The phenol group found in eugenol is the aromatic phenylpropanoid that is present in several groups of plants including cloves (Syzygium aromaticum, Myrtaceae) and basil (Ocimum basilicum, Lamiaceae) (Alves et al., Reference Alves, Ferreira, Santos, Silva, Rodrigues, Neto, Farah, Santos, Mendes, Sousa, Monteiro, Dos Santos, Santos, Perez, Romero, Denadai and Guzzo2017). Magalhães et al. (Reference Lopes, da Fonseca, Rocha, de Freitas, Araújo, Wong, Lima Júnior and Lea2018) showed that eugenol reduced lipopolysaccharide-induced protein oxidation in vivo, through both anti-inflammatory and anti-oxidative effects, avoiding damage to mice lung structure. Eugenol also demonstrated antioxidant activity on human cell lines cultured in vitro (Slamenová et al., Reference Slamenová, Horváthová, Wsólová, Sramková and Navarová2009). In addition, the ability of eugenol to inhibit ROS and lipid peroxidation was five times greater than that of α-tocopherol (Nagababu and Lakshmaiah, Reference Nagababu and Lakshmaiah1992; Nagababu and Lakshmaiah, Reference Nagababu and Lakshmaiah1994). Despite these studies, it is not known if eugenol influences mRNA expression of antioxidant enzymes such as GPX1, CAT, SOD and PRDX6 and if it affects bovine preantral follicles growth and viability during in vitro culture.
The objectives of this study were to evaluate the effects of different concentrations of eugenol (0.5, 5.0 and 50.0 µM) on viability, growth, antrum formation and on the levels of mRNA for SOD, CAT, PRDX6 and GPX1 of bovine secondary follicles cultured in vitro for 18 days.
Materials and methods
Chemicals
The culture medium and other chemicals used in this study were purchased from Sigma Chemical Co. (St Louis, MO, USA), unless otherwise indicated in the text.
Ovarian collection
Ovaries of multiparous cows (n = 50) were collected from a local slaughterhouse in Sobral, Ceará, Brazil. Immediately after removal, ovaries were washed in 70% ethanol for approximately 10 s, followed by two further washes in TCM-199 supplemented with penicillin (100 mg/ml), streptomycin (100 mg/ml) and HEPES. Then, the ovaries in TCM-199 at 4°C were transported within 1 h to a laboratory.
Follicle isolation and in vitro culture
In the laboratory, the ovarian cortex was fragmented (1–2 mm) with a sterile scalpel blade and the fragments were placed in HEPES-buffered TCM-199 supplemented with penicillin (100 mg/ml) and streptomycin (100 mg/ml). Secondary follicles measuring 150–250 μm in diameter were manually isolated with the aid of 26-gauge needles and using a stereomicroscope (SMZ 645 Nikon, Tokyo, Japan). After isolation, follicles containing visible oocytes surrounded by granulosa cells and intact basement membrane, and without the presence of antral cavity, were selected for culture. Then, follicles were cultured individually in 100-μl droplets of culture medium in Petri dishes (60 × 15 mm; Corning, USA). The control medium, identified as TCM-199+, consisted of TCM-199 (pH 7.2–7.4) supplemented with 3.0 mg/ml bovine serum albumin (BSA), 100 IU penicillin/streptomycin, 10 μg/ml insulin, 5.5 μg/ml transferrin and 5 ng/ml selenium (ITS), 50 μg/ml ascorbic, 100 ng/ml FSH, 2 mM glutamine and 2 mM hypoxanthine. The follicles were distributed randomly and cultured in TCM-199+ alone or supplemented with 0.5, 5.0 or 50.0 μM of eugenol. The eugenol concentrations used were based on the study of Lopes et al. (2018). In each treatment, approximately 60 follicles were cultured. Follicles culture was performed at 38.5°C with 5% CO2 in air for 18 days. Every 2 days of culture, 60 μl of medium was replaced with fresh medium. The percentage of follicles considered morphologically normal was evaluated on days 0, 6, 12 and 18 of culture. Follicles containing an opaque and/or extruded oocyte and having opaque granulosa cells were considered degenerated. For follicular diameter evaluation, two perpendicular measurements were performed in normal follicles using an inverted microscope with NIS-Elements 2.4 software (Nikon, Nikon Instruments Inc., Japan). To calculate daily growth rate, the difference in follicle diameter between days 0 and 18 was calculated and divided by 18 (days of culture).
Assessment of preantral follicles viability using fluorescence microscopy
After 18 days of culture, follicles (n = 20/treatment) were incubated in 100-μl droplets of TCM-199 supplemented with 4 mM calcein-AM and 2 mM ethidium homodimer-1 (Molecular Probes, Invitrogen, Karlsruhe, Germany) at 37°C for a period of 15 min. The follicles were then washed three times in TCM-199 and then examined under a fluorescence microscope (Nikon, Eclipse, TS 100, Japan). Oocytes and granulosa cells were considered viable if the cytoplasm was positively stained with calcein-AM (green) and non-viable if the chromatin was positively labelled with ethidium homodimer (red) (Van Den Hurk et al., Reference Van Den Hurk, Spek, Hage, Fair, Ralph and Schotanus1998).
Expression of mRNA for SOD, CAT, PRDX6 and GPX1 in cultured follicles
Normal follicles that had been cultured for 18 days in each treatment were collected and then stored at −80°C until extraction of total RNA for further analysis of the levels of mRNA for SOD, CAT, PRDX6 and GPX1. Total RNA extraction was performed using a TRIzol® purification kit (Invitrogen, São Paulo, Brazil) in accordance with the manufacturer’s instructions. Quantification of mRNA was performed using SYBR Green. PCR reactions were composed of 1 μl cDNA as a template in 7.5 μl of SYBR Green Master Mix (PE Applied Biosystems, Foster City, CA, USA), 5.5 μl of ultra-pure water, and 0.5 μM of each primer. The primers were designed to perform amplification of SOD, CAT, PRDX6, GPX1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Table 1). GAPDH was used as the reference gene. The specificity of each primer pair was confirmed using melting curve analysis of PCR products. The thermal cycling profile for the first round of PCR was initial denaturation and activation of the polymerase for 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, 30 s at 58°C, and 30 s at 72°C. The final extension was for 10 min at 72°C. All reactions were performed in a Step One Plus instrument (Applied Biosystems, Foster City, CA, USA). The ΔΔCt method was used to transform the Ct values into normalized relative expression levels (Livak and Schmittgen, Reference Livak and Schmittgen2001).
Primer pairs used for real-time PCR
Statistical analysis
Data on follicular diameters were submitted to statistical analysis using the Kolmogorov–Smirnov and Bartlett test to assess normal distribution and homoscedasticity, respectively. These data were then evaluated using ANOVA. For comparisons of follicular diameters and levels of mRNA between treatments, the Kruskal–Wallis test (GraphPad Prism software, version 5.0) was used. Percentages of follicles with different daily growth rate, follicular survival and antrum formation after in vitro culture within each treatment were compared using the chi-square test. Differences were considered significant when P < 0.05.
Results
Effects of eugenol on follicular growth, antrum formation and viability
A significant and progressive increase in follicular diameter was observed from day 0 up to day 12 of culture in all treatments (Table 2). The only exception was secondary follicles cultured in medium supplemented with 50 µM eugenol, which had significant growth between days 0 and 6, but not after 6, 12 and 18 days. When comparisons were made between treatments, no significant difference was observed. No effect of eugenol on follicular daily growth rate was observed (Table 2).
Diameters and daily growth (mean ± standard error of the mean (SEM)) of bovine secondary follicles after 0, 6, 12 and 18 days of in vitro culture in TCM-199+ alone or supplemented with 0.5, 5.0 or 50.0 μM of eugenol (EUG)
Significantly different (P < 0.05).
a,b,cLowercase letters represent statistically significant differences between days of culture (between columns).
Table 3 shows the percentage of normal follicles and antrum formation after 18 days of culture in control medium alone or supplemented with different concentrations of eugenol. The great majority of the follicles remained viable after culture in all treatments, and the percentage of normal follicles after culture in medium supplemented with 50 µM eugenol was higher than that seen in follicles cultured with 0.5 µM eugenol (P < 0.05). However, no significant differences were observed among the percentage of normal follicles after culture in control medium alone or supplemented with eugenol in the different concentrations. No differences were also observed in the percentage of antrum formation after follicle culture in the different treatments.
Percentages of morphologically normal secondary follicles and of antrum formation after 18 days of in vitro culture in TCM-199+ alone or supplemented with 0.5, 5.0 or 50.0 μM of eugenol (EUG)
Significantly different (P < 0.05).
a,bLowercase letters represent statistically significant differences between treatments (between columns).
Effects of eugenol on follicular viability
Fluorescence microscopy analysis showed that, after 18 days of culture in control medium alone or supplemented with 0.5, 5.0 and 50.0 μM eugenol, all follicles previously considered morphologically normal by light microscopy were viable, as they stained positively for calcein-AM, but not for ethidium homodimer (Fig. 1). Conversely, some stromal cells organized around the follicles cultured in all treatments were positively stained with ethidium homodimer.
Viability of bovine secondary follicles cultured for 18 days after staining with calcein-AM (green) and ethidium homodimer-1 (red). Secondary follicles were cultured in TCM-199+ alone (A, B) or supplemented with 0.5 (C, D), 5.0 (E, F) or 50.0 (G, H) μM of eugenol (EUG). Scale bars represent 100 μm.
Expression of mRNA for SOD, CAT, PRDX6 and GPX1 in cultured follicles
Figure 2 shows the levels of mRNA for SOD, CAT, PRDX6 and GPX1 in bovine secondary follicle after 18 days of culture in TCM-199+ alone or supplemented with eugenol. The presence of 5.0 and 50.0 μM eugenol in culture medium significantly increased the levels of mRNA for GPX1 in secondary follicles when compared with those cultured in control medium (P < 0.05). However, secondary follicles cultured in the presence of 0.5 μM eugenol had reduced levels of mRNA for SOD, when compared with control medium. Conversely, the presence of eugenol in culture medium did not influence the expression of mRNA for CAT and PRDX6 in cultured follicles.
Levels of mRNA (means ± standard deviation (SD)) for (A) SOD, (B) CAT, (C) GPX1 or PRDX6 (D) in secondary follicles cultured in vitro for 18 days in TCM 199+ alone or supplemented with 0.5, 5.0 or 50.0 μM of eugenol (EUG). a,b,cLowercase letters represent statistically significant differences between treatments (P < 0.05).
Discussion
This study shows that eugenol retains follicle viability and morphology, but it reduces follicle growth when used at high concentrations (50 µM). It has been reported previously that addition of 50 µM eugenol reduced the growth of human keratinocytes, but when nanoencapsulated it decreased its possible cytotoxic effect on this cell type and improved its anti-inflammatory effect (Lopes et al., 2018). These authors showed that a controlled release device for this substance in the culture medium increased the positive action of eugenol. These results are in accordance with those obtained during culture of bovine secondary follicles in medium supplemented with higher concentrations of eugenol.
In bovine ovarian follicles cultured in vitro, we showed for the first time that 0.5 and 5.0 µM eugenol influences, respectively, the expression of mRNAs for SOD and GPX1 in bovine secondary follicles cultured in vitro. Changes in the levels of antioxidant enzymes might be very important in the regulation of the response to oxidative stress (Salah et al., Reference Salah, Bouaziz, Amara, Abid-Essefi and Bacha2019). SOD catalyzes the dismutation of the extremely reactive superoxide anion to H2O2, which can further be separated into water and oxygen by CAT or GPX. Reduction in the levels of mRNA for SOD in bovine secondary follicles cultured in the presence of 0.5 µM eugenol can be associated with diminished intracellular levels of reactive superoxide anion in vitro. Salah et al. (Reference Salah, Bouaziz, Amara, Abid-Essefi and Bacha2019) reported that eugenol reduced ROS and O2 − production in intestinal cell lines (HCT116) cultured in vitro and, consequently, these cells had reduced activities of SOD and CAT. It has been reported that phenolic antioxidants including eugenol have the capacity to enhance antioxidant enzyme activities (Tiku et al., Reference Tiku, Abraham and Kale2004; Zhao et al., Reference Zhao, Xiang, Ye, Yuan and Guo2006). In vivo, eugenol also reduced peroxidation in mice liver and reduced oxidative stress (Tiku et al., Reference Tiku, Abraham and Kale2004). Reduction of lipid peroxidation in the oocyte is very important as the products of this process, such as malondialdehyde (MDA) and 4-hydroxynonenal (4HNE), have traditionally been viewed as toxic substances (Gallelli et al., Reference Gallelli, Calcagnini, Romano, Koczwara, de Ceglia, Dante, Villani, Giudetti, Cassano and Gaetani2018). Eugenol possesses antioxidant properties due to its phenolic hydroxyl group that donates electrons to quench free radicals (Fouad and Yacoubi, Reference Fouad and Yacoubi2011).
This study showed that 5 and 50 µM eugenol increased the mRNA levels of GPX1 in bovine follicles cultured in vitro. Glutathione is the main nonenzymatic cellular defence mechanism against ROS, and GPX1 catalyzes the oxidation of glutathione into hydroperoxide, playing a role in repairing damage caused by lipid peroxidation (Kurutas, Reference Kurutas2016). High expression of GPX1 in bovine granulosa cells is associated with follicle survival, as well as estradiol and progesterone secretion, as GPX1 is significantly upregulated in healthy follicles compared with small, healthy or atretic follicles (Ceko et al., Reference Ceko, Hummitzsch, Hatzirodos, Bonner, Aitken, Russell, Lane, Rodgers and Harris2015). The absence of a positive effect of eugenol on the survival of bovine secondary follicles cultured in vitro could be due to differences in the cell types cultured (isolated granulosa cells versus secondary follicles) and stage of development, as granulosa cells were isolated from antral follicles. A previous study has shown that preantral follicles cultured in the presence of sodium selenite increases GPX activities and decreases ROS levels, therefore improving the development rate of mouse follicles in vitro (Abedelahi et al., Reference Abedelahi, Salehnia, Allameh and Davoodi2010). These authors also reported that low levels of GPX activity in cultured preantral follicles and high concentrations of ROS may be the major factors responsible for follicular regression in vitro. It is important to highlight that, during in vitro culture, cells are exposed to high concentrations of oxygen (up to 20%; Sá et al., Reference Sá, Bruno, Guerreiro, Cadenas, Alves, Cibin, Leal-Cardoso and Figueiredo2018). Salavati et al. (Reference Salavati, Ghafari, Zhang and Fouladi-Nashta2012) showed that oocytes cultured in high oxygen concentrations showed increased GPX1 expression in response to high oxygen tension.
Expression of mRNA for CAT and PRDX6 was not influenced by eugenol in in vitro cultured bovine secondary follicles. CAT has an important role during follicle development as it neutralizes H2O2 and maintains the balance in the production of ROS, and influences the production of steroidal hormones (Wang et al., Reference Wang, Xu, Zhou, Wang and Zhang2017). During meiotic maturation of mouse oocytes, it has been observed that catalase protects the genome from oxidative damage (Park et al., Reference Park, You, Cho, Jeon, Lee, Cho, Kim and Oh2016). CAT acts in the oocyte nucleus to avoid chromosomal defects, and inhibition of this enzyme is associated with chromosomal misalignment and DNA damage (Park et al., Reference Park, You, Cho, Jeon, Lee, Cho, Kim and Oh2016). PRDX6 is an enzyme with antioxidant activity that is widely present in the intracellular environment to promote the protection of cells against damage caused by exposure to high levels of oxygen (Wang et al., Reference Wang, Phelan, Manevich, Feinstein and Fisher2006). Leyens et al. (Reference Leyens, Knoops and Donnay2004) reported that PRDX6 transcripts are stored in the oocyte for later use in early embryos. PRDX6 peroxide reductase activity controls the levels of hydrogen peroxides in cumulus–oocyte complexes and early embryos (Harvey et al., Reference Harvey, Kind and Thompson2002). PRDX6 can also use the glutathione accumulated both in the oocyte and cumulus cells during maturation to reduce its oxidized form during the peroxidation process (de Matos et al., Reference De Matos, Furnus and Moses1997). In addition to its peroxidase activity, PRDX6 expresses acidic calcium-independent phospholipase A2 and lysophosphatidylcholine acyl transferase activities in separate catalytic sites (Arevalo and Vázquez-Medina, Reference Arevalo and Vázquez-Medina2018). Phospholipase A2 activity in cumulus cells is linked to the production of arachidonic acid for the production of prostaglandins, involved in the oocyte maturation process (Elvin et al., Reference Elvin, Yan and Matzuk2000; Calder et al., Reference Calder, Caveney, Westhusin and Watson2001). Fernandez et al. (Reference Fernandez, Yu, Moawad and O’Flaherty2019) also reported that PRDX6 promoted the survival of human sperm in vitro through the activation of the PI3K-AKT pathway. Absence of changes in the levels of CAT and PRDX6 can be associated with maintenance of the equilibrium between antioxidants and pro-oxidants during follicular culture.
In conclusion, eugenol reduces the levels of mRNAs for SOD and increases those of GPX1 in bovine secondary follicles cultured in vitro for 18 days. Eugenol also kept follicle viability and morphology, but it reduced follicle growth when used at high concentrations (50 µM). This study showed the potential of eugenol to control oxidative stress during culture of bovine preantral follicles in vitro, as it increased the expression of mRNA for GPX1, an enzyme that catalyzes the oxidation of glutathione, and plays an important role in repairing damage caused by lipid peroxidation.
Financial support
This research was supported by grants from the National Council for Scientific and Technological Development (CNPq, Brazil) and Coordination for the Improvement of Higher Education Personnel (CAPES). J.R.V. Silva is an investigator for CNPq. E.M. Vasconcelos is the recipient of a scholarship from CAPES, Brazil.
Statement of interest
The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
Ethical standards
The authors declare that all procedures were performed according to national and institutional guides on the care and use of animals. This study was registered approved (Number: 03/2019) by the Committee of Ethics and Animal Welfare of the Federal University of Ceará.
