Introduction
Preantral follicles, i.e. primordial, primary and secondary follicles, represent approximately 90% of ovarian follicular population and are essential for the maintenance of fertility throughout the reproductive life of females (Sá et al., Reference Sá, Bruno, Guerreiro, Cadenas, Alves, Cibin, Leal-Cardoso, Gastal and Figueiredo2018). However, the vast majority of these follicles is lost due to the natural process of atresia (Figueiredo and Lima, Reference Figueiredo and Lima2017). Therefore, in vitro culture of ovarian tissue containing preantral follicles has been used in an attempt to avoid this huge follicular loss that occurs naturally in vivo. Isolation and in vitro culture of these follicles allow us to monitor follicular responses to hormones, growth factors, nutrients, vitamins, antioxidants and local factors during their development (Guerreiro et al., Reference Guerreiro, Lima, Rodrigues, Carvalho, Castro, Campello, Pessoa, Gadelha, Figueiredo, Bordignon and Rodrigues2016). However, Magalhães et al. (Reference Magalhães, Araujo, Verde, Matos, Silva, Lucci, Báo, Campello and Figueiredo2009) have shown that in vitro grown follicles have less developmental capacity than those grown in vivo. Although the birth of mice from primordial follicles grown, matured and fertilized in vitro has already been reported (O’Brien et al., Reference O’Brien, Pendola and Eppig2003), in species such as caprine (Magalhães et al., Reference Magalhães, Fernandes, Mororó, Silva, Rodrigues, Bruno, Matos, Campello and Figueiredo2011) and ovine (Arunakumari et al., Reference Arunakumari, Shanmugasundaram and Rao2010) only a few embryos have been produced in vitro. In bovine species, however, the results have been limited to the formation of early antral follicles in vitro (Mclaughlin et al., Reference McLaughlin, Bromfield, Albertini and Telfer2010). For these reasons, there is still a need to improve in vitro culture systems for preantral follicles in domestic species.
It is already know that during in vitro culture, preantral follicles are exposed to supraphysiological concentrations of oxygen (up to 20%; Sá et al., Reference Sá, Bruno, Guerreiro, Cadenas, Alves, Cibin, Leal-Cardoso, Gastal and Figueiredo2018), which can result in excessive formation of reactive oxygen species (ROS). Triggering oxidative stress can cause mitochondrial and DNA damage, peroxidation of lipid membranes, in addition to damage to cell proteins and consequent reduction in oocyte quality (Lins et al., Reference Lins, Cavalcante, Santos, Menezes, Barros, Barberino, Bezerra, Macedo and Matos2017). In addition, disturbing extracellular matrix (ECM) structure by oxidizing its compounds can have a negative effect on follicular dynamic. In fact, ECM is directly or indirectly involved in tissue remodelling, signalling, multiplication and cellular specialization during follicle development (Muncie and Weaver, Reference Muncie and Weaver2018). As a consequence of increased production of ROS, ECM homeostasis can be severely disturbed, especially by changes in the activity of matrix metalloproteinases (MMPs). These proteases are responsible for remodelling and degradation of ECM components, including collagen (Verma and Hansch, Reference Verma and Hansch2007). As a consequence, tissues and cells can be severely damaged and negatively influence follicular development (Fu et al., Reference Fu, Chen, Wang, Peng, Wang, Yuan, Gao, Jiang and Zhang2019).
Cellular defence mechanisms against oxidative stress involve enzymes such as superoxide dismutase (SOD), catalase, and peroxidases. SOD promotes a dismutation of superoxide radicals to H2O2, which is further detoxified to water and oxygen by catalase or glutathione peroxidase (GPx). Catalase is responsible for the conversion of H2O2 into water and oxygen, while GPx catalyzes the degradation of lipid peroxides, as well as H2O2 (Kala et al., Reference Kala, Shaikh and Nivsarkar2017). Peroxiredoxin 6 (Prdx6) is another enzyme with the ability to bind and reduce phospholipid hydroperoxides (Fisher et al., Reference Fisher, Vasquez-Medina, Dodia, Sorokina, Tao and Feinstein2018). In an attempt to improve in vitro culture systems, several non-enzymatic antioxidants extracted from plants have been highlighted due to their potential to combat ROS. Rutin (Lins et al., Reference Lins, Cavalcante, Santos, Menezes, Barros, Barberino, Bezerra, Macedo and Matos2017), extracts of Morus nigra (Gouveia et al., Reference Gouveia, Barberino, Menezes, Macedo, Cavalcante, Gonçalves, Almeida and Matos2019), Amburana cearensis (Gouveia et al., Reference Gouveia, Macedo, Santos, Barberino, Menezes, Müller, Almeida, Figueiredo and Matos2016) and Justicia insularis (Mbemya et al., Reference Mbemya, Cadenas, Ribeiro de Sá, Damasceno Guerreiro, Donfack, Alberto Vieira, Canafístula de Sousa, Geraldo Alves, Lobo, Santos, Lima Pinto, Loiola Pessoa, Smitz, Comizzoli, Figueiredo and Rodrigues2018) have contributed to maintaining the balance between the production of ROS and the defence systems, favouring the maintenance of follicle growth and survival in vitro. In addition, it has been shown that Aloe vera extract has more than 75 different potentially active compounds including minerals, enzymes, vitamins, polysaccharides, phenolic compounds and organic acids that have several pharmacological properties, including antioxidant activity (Sumi et al., Reference Sumi, Sikder, Rahman, Lubna, Ulla, Hossain, Jahan, Alam and Subhan2019). Aloe vera can modulate oxidative stress due to the presence of antioxidant compounds such as α-tocopherol (vitamin E), carotenoids, ascorbic acid (vitamin C), flavonoids and tannins (Cesar et al., Reference Cesar, Jozić, Begović, Vuković, Mlinarić, Lepeduš, Borović Šunjić and Žarković2018). Sumi et al. (Reference Sumi, Sikder, Rahman, Lubna, Ulla, Hossain, Jahan, Alam and Subhan2019) reported that Aloe vera increases the activity of antioxidant enzymes such as catalase, SOD and glutathione. The protective effect of Aloe vera on ECM integrity has also been reported (Saito et al., Reference Saito, Tanaka, Misawa, Yao, Nabeshima, Yamauchi, Abe, Yamamoto and Furukawa2016; Park et al., Reference Park, Son, Yoo and Lee2017). Acemannan, a beta-(1,4)-acetylated mannan, is a bioactive polysaccharide present in Aloe vera gel that promotes skin wound healing through activating the AKT/mTOR-mediated protein translation mechanism in fibroblasts, resulting in increased secretion of collagen in vivo (Xing et al., Reference Xing, Guo, Zou, Fu, Li, Zhu, Qi, Song, Dong, Li, Xiao, Yuan, Huang and Xu2015). However, it is still not known if Aloe vera influences the collagen distribution in bovine ovarian tissues cultured in vitro and has a positive effect on primordial follicle survival and development. It is hypothesized that supplementation of culture medium with Aloe vera extract improves the integrity of collagen fibres in the ECM of cultured ovarian tissue, increases the expression of antioxidant enzymes and promotes primordial follicle development and survival.
This study aims to evaluate the effect of different concentrations of Aloe vera extract (1.0, 5.0, 10.0 or 50.0%) on distribution of collagen fibres in the ECM, primordial follicles activation, development and follicular survival, as well as on expression of mRNA for antioxidant enzymes (SOD, CAT, PRDX1 and GPX6) in bovine ovarian tissue cultured in vitro for 6 days.
Materials and methods
Chemicals
Unless otherwise indicated, culture medium and other chemicals used in this study were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Plant material and extract preparation
The plant Aloe vera was cultured in a sandy clay soil type at Frecheirinha – Ce, northeast of Brazil (3°45′49.3′′S, 40°50′14.0′′W), with a semi-arid tropical climate and temperatures ranging from 24ºC to 36ºC. To obtain the crude extract of Aloe vera, leaves at an intermediate stage of maturation were collected, properly disinfected, and transported to the laboratory. Then, the leaves were washed with distilled water and their surface parts were removed to expose a colourless gel that was extracted from the parenchyma of the leaves. The gel was filtered through a sieve and stored at 4ºC in sterile 50 ml Falcon tubes (Souza et al., Reference Souza, Lima, Peixoto, Silva, Oliveira and Silva2016). Before supplementation of culture medium, the extract was filtered again through 0.45-µm membranes.
Source of ovaries
Bovine ovaries (n = 20) from cycling mixed breed cows were collected at a local slaughterhouse. Immediately post mortem, the ovaries were washed once in 70% alcohol (Dinâmica, São Paulo, Brazil), and twice in minimum essential medium (α-MEM) buffered with HEPES (MEM-HEPES) and supplemented with antibiotics (100 μg/ml penicillin and 100 μg/ml streptomycin). After washing, each pair of ovaries was individually transported to the laboratory in Falcon tubes containing α-MEM supplemented with penicillin (100 µg/ml) and streptomycin (100 µg/ml) at 4ºC, within 1 h. This study was approved by the Committee of Ethics and Animal Welfare of the Federal University of Ceará (no. 04/2019).
In vitro culture of ovarian tissue
In the laboratory, using a sterile scalpel no. 22, ovarian cortical tissue from the same ovarian pair (n = 10 pairs) was cut into 22 slices (3 mm × 3 mm × 1 mm) in dissection medium composed of α-MEM supplemented with penicillin (100 µg/ml) and streptomycin (100 µg/ml). For each animal, two cortical slices were fixed in paraformaldehyde (4%) for 24 h at 4ºC for histological analysis (uncultured control) as well as for analysis of the ECM. The remaining fragments were cultured in 24-well culture dishes for 6 days (Passos et al., Reference Passos, Costa, da Cunha, Silva, Ribeiro, de Souza, Barroso, Dau, Saraiva, Gonçalves, van den Hurk and Silva2016; Silva et al., Reference Silva, Ribeiro, Menezes, Barberino, Passos, Dau, Costa, Melo, Bezerra, Donato, Peixoto, Matos, Gonçalves, Van den Hurk and Silva2017). Culture was performed at 38.5°C in 5% CO2 in an humidified incubator. The basic culture medium consisted of α-MEM (pH 7.2–7.4) supplemented with ITS (10 μg/ml insulin, 5.5 μg/ml transferrin, and 5 ng/ml selenium), 2 mM glutamine, 2 mM hypoxanthine, antibiotics (100 IU/ml penicillin and 100 mg/ml streptomycin) and 1.25 mg/ml of bovine serum albumin (α-MEM+). The fragments were cultured in 1 ml of control medium (α-MEM+) alone or in control medium (α-MEM+) supplemented with different proportions of Aloe vera extract [1.0% (+99.0% α-MEM+), 5.0% (+95.0% α-MEM+), 10.0% (+90.0% α-MEM+) or 50.0% (+50.0% α-MEM+)]. The proportions of Aloe vera were chosen according to Souza et al. (Reference Souza, Lima, Peixoto, Silva, Oliveira and Silva2016). Every 2 days, 60% of the culture medium was replaced with fresh medium. Each treatment was repeated 10 times. After the end of the culture period, the fragments were taken for morphological analysis.
Analysis of extracellular matrix
To evaluate collagen fibres distribution in the ECM, ovarian cortical tissues were stained with picrosirius red (Abcam Kit), according to the methodology described by Rittié (Reference Rittié2017), with modifications. Briefly, 6.0-µm ovarian sections were dewaxed in xylol and incubated in Sirius Red solution (0.1%) for 1 h at room temperature. After removal of excess dye with acetic acid solution (0.5%), the sections were dehydrated and evaluated under an optical microscope (Nikon, Eclipse, TS 100, Japan) at ×400 magnification. For each treatment, the percentage of area occupied by collagen fibres in 10 different fields was measured with the aid of a DS Cooled Camera Head DS-Ri1 coupled to a microscope (Nikon, Eclipse, TS 100, Japan) and the images were analyzed using ImageJ software (v.1.51p, 2017). After staining, the collagen fibres were marked in red by picrosirius, while the follicles remained uncoloured. ImageJ software was used to quantify the percentage of collagen fibred in uncultured and cultured tissues. The staining intensity of collagen fibres was determined by measuring the average pixel intensity of the total area imaged after background subtraction.
Morphological analyses and assessment of in vitro follicular growth
Histological analysis was performed according to the methodology of Bizarro-Silva et al. (Reference Bizarro-Silva, Santos, Gerez, González, Lisboa and Seneda2018), with modifications. Fresh control (D0) and cultured ovarian fragments were fixed in paraformaldehyde (4%) for 24 h, dehydrated in increasing concentrations of ethanol, cleared with xylol and included in paraffin. Serial sections (6-µm thickness) were performed and, each fifth section was stained with haematoxylin and eosin. Only preantral follicles with oocyte nuclei in the section were analyzed to avoid double counting. Coded anonymized slides were examined under a microscope (Nikon, Tokyo, Japan) at ×100 and ×400 magnification. The developmental stages of follicles were classified as primordial (one layer of flattened or flattened and cuboidal granulosa cells around the oocyte) or growing follicles (primary: one layer of cuboidal granulosa cells, and secondary: two or more layers of cuboidal granulosa cells around the oocyte) (Figueiredo and Lima, Reference Figueiredo and Lima2017). These follicles were further classified as normal when an intact oocyte was present, surrounded by granulosa cells that were well organized into one or more layers, and had no pyknotic nucleus. Degenerated follicles were defined as those with a retracted oocyte, which had a pyknotic nucleus and/or was surrounded by disorganized granulosa cells, which are detached from the basement membrane (Telfer et al., Reference Telfer, McLaughlin, Ding and Thong2008). The percentages of healthy primordial and developing follicles were calculated before (uncultured control) and after culture in a particular medium.
Expression of mRNA for SOD, CAT, PRDX6 and GPX1 in ovarian tissue
For analysis of gene expression, ovarian tissues were stored at −80ºC immediately after the end of the culture period until the extraction of total RNA for further analysis of expression of SOD, CAT, PRDX6 and GPX1 mRNAs. Tissue cultured in control medium (α-MEM+) and those cultured in medium supplemented with 10% Aloe vera, i.e. the treatment that improved ECM tissue distribution, were selected to investigate mRNA expression. For this, ovarian tissues were macerated using scalpels no. 22 under sterile conditions and then subjected to extraction of total RNA 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. Primers were designed to perform the amplification of SOD, CAT, PRDX6, GPX1 and GAPDH (Table 1). The specificity of each primer pair was confirmed by 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.
Table 1. Primer pairs used for real-time PCR
Statistical analysis
Statistical analysis was performed using GraphPad Prism (5.0) software. Data that did not show a normal distribution, according to Shapiro–Wilk test, underwent logarithmic transformation. The percentage of normal follicles and follicular activation in each treatment were evaluated using Tukey’s test, and comparison between treatments using Fisher’s exact test. Data for collagen fibres distribution were analyzed by Kruskal–Wallis test, followed by Dunn’s comparison test. Levels of mRNA were analyzed by analysis of variance (ANOVA) and Student’s t-test. The differences were statistically significant when the P-value was < 0.05.
Results
Assessment of ovarian extracellular matrix
Ovarian tissues cultured with 10.0 or 50.0% Aloe vera had higher percentages of collagen fibres than tissues cultured in control medium, but had similar percentages of collagen fibres when compared with uncultured tissues (P < 0.05) (Figure 1A,B).
Figure 1. (A) Percentages of collagen in uncultured tissues and in tissues cultured for 6 days in control medium alone or supplemented with 1.0, 5.0, 10.0 or 50.0% Aloe vera. a–cDifferent lowercase letters indicate statistically significant differences between treatments. (B) Representative images of collagen fibres in uncultured tissues (a) and in tissues cultured for 6 days in control medium alone (b) or supplemented with 1.0, 5.0, 10.0 or 50.0% Aloe vera (c–f). The black arrow shows an unstained primordial follicle. Scale bars: 100 µm.
Effects of Aloe vera on the activation and development of primordial follicles
Ovarian tissues cultured with 10% Aloe vera showed a significant decrease in the percentage of primordial follicles and concomitant increase in developing follicles, when compared with uncultured control. Additionally, the percentages of developing follicles in tissues culture with 10% Aloe vera were higher than those seen in tissues cultured with 1.0, 5.0 or 50.0% of Aloe vera. (Figure 2A,B). The percentages of primordial follicles in ovarian tissues cultured with 1.0, 5.0 or 50.0% of Aloe vera did not differ from those observed in uncultured ovarian tissues (P < 0.05). Moreover, ovarian tissues cultured with 10% Aloe vera had similar rates of primordial/developing follicles when compared with those tissues cultured in control medium.
Figure 2. Percentages of primordial follicles (A), and developing follicles (B) in uncultured tissues and in tissues cultured for 6 days in control medium alone or supplemented with 1.0, 5.0, 10.0 or 50.0% Aloe vera. a–cDifferent lowercase letters indicate statistically significant differences between treatments.
Effects of Aloe vera on follicular morphology after in vitro culture
In total, 932 preantral follicles were analyzed. Figure 3 shows the morphology of normal and degenerate follicles in uncultured and cultured ovarian cortical tissues. After a 6-day culture period, a significant decrease in the percentage of normal follicles in ovarian tissues cultured in all treatments was observed, compared with the uncultivated control (P < 0.05). The presence of Aloe vera (all concentrations) in the culture medium maintained the percentage of morphologically normal follicles when compared with tissues cultured in the control medium. Ovarian tissues cultured with 50.0% Aloe vera showed a higher percentage of morphologically normal follicles than those grown with 10.0% Aloe vera (P < 0.05) (Figure 4).
Figure 3. Representative images of sections of bovine ovarian tissue showing morphologically normal and atretic follicles. Normal primordial follicles (A) and atretic (B); normal (C) and atretic (D) primary follicle; normal (E) and atretic (F) secondary follicle. Granulosa cells (GC); oocyte (O); oocyte nucleus (N). Scale bars: 100 µm.
Figure 4. Percentages of morphologically normal follicles in uncultured tissues and in tissues cultured for 6 days in control medium alone or supplemented with 1.0, 5.0, 10.0 or 50.0% Aloe vera. a–cDifferent lowercase letters indicate statistically significant differences between treatments.
Levels of mRNA for SOD, CAT, PRDX6 and GPX1 after in vitro culture
Ovarian cortical tissues cultured in the presence of 10% Aloe vera had significantly higher mRNA levels for PRDX6 (P < 0.05) than those tissues cultured with α-MEM+ alone (Figure 5). However, Aloe vera did not influence in the expression of mRNA for SOD, CAT and GPX1 (P > 0.05).
Figure 5. Levels of mRNA for SOD (A), CAT (B), PRDX6 (C) and GPX1 (D) in ovarian tissues cultured for 6 days in control medium alone or supplemented with 10.0% Aloe vera. a–cDifferent letters indicate significant difference between treatments (P < 0.05).
Discussion
The present study investigated for the first time the effect of Aloe vera on the maintenance of the ECM, follicular activation and levels of mRNA for SOD, CAT, PRDX6 and GPX1 in bovine ovarian cortical tissues cultured in vitro for 6 days. The presence Aloe vera extract in culture medium had no toxic effect on bovine ovarian tissues cultured in vitro. We showed that Aloe vera increased the number of collagen fibres in ECM of bovine ovarian tissue cultured in vitro, and kept ECM similar to uncultured tissues. ECM participates in the regulation of a variety of cellular functions in many tissues, inclusive the ovary. The formation, development and regression of ovarian follicles require considerable tissue remodelling, cell multiplication, and bidirectional communication among oocyte, granulosa cells, and theca cells, and the surrounding ECM (Berkholtz et al., Reference Berkholtz, Shea and Woodruff2006). Therefore, in addition to providing mechanical support to cells, ECM is very important to cell–cell and tissue-cell communication, adhesion, migration, survival and proliferation (Muncie and Weaver, Reference Muncie and Weaver2018). In addition, growth factors such as epidermal growth factor, fibroblast growth factor, transforming growth factor-β and amphiregulins can be stored in the ECM and released locally during the remodelling process through matrix cleavage (Gonella-Diaza et al., Reference Gonella-Diaza, Silveira Mesquita, Lopes, Ribeiro da Silva, Cogliati, De Francisco Strefezzi and Binelli2018). Consequently, regulation of the mechanisms that control the integrity of ECM is necessary for the growth of follicles from primordial the ovulatory stages (Franchi et al., Reference Franchi, Hernandes, Coalho Ferreira, Vieira de Lima, de Oliveira Mendes, Musa de Aquino, Scarano and César de Souza Castilho2020). Homeostasis of extracellular matrix is influenced by matrix metalloproteinases (MMPs) that are responsible for remodelling and degradation of ECM components, including collagens (Verma and Hansch, Reference Verma and Hansch2007). For these reasons, MMPs play an important role during follicular development (Kliem et al., Reference Kliem, Welter, Kraetzl, Steffl, Meyer, Schams and Berisha2007). Although MMPs are regulated at the transcriptional level, a wide variety of stresses can result in excessive degradation of ECM. Among then, oxidative stress during in vitro culture of ovarian tissues can be a critical factor in this process (Quan et al., Reference Quan, Elhousiny, Johnson and Gao2013). In fact, Ali et al. (Reference Ali, Mahmoud, Le Master, Levitan and Phillips2019) demonstrated increased expression of MMP-2 and MMP-9 in endothelial cells under oxidative stress. In our study, supplementation of culture medium with 10 or 50% Aloe vera preserved the number of collagen fibre during in vitro culture. Previous studies have already reported that Aloe vera has a protective effect on ECM. Saito et al. (Reference Saito, Tanaka, Misawa, Yao, Nabeshima, Yamauchi, Abe, Yamamoto and Furukawa2016) reported a protective effect of Aloe vera on ECM of mice skin after irradiation by X-rays. These authors showed that Aloe vera prevents the increase of MMP-2 and MMP-9. A similar effect was reported by Park et al. (Reference Park, Son, Yoo and Lee2017), i.e. Aloe vera was able to reduce the levels of mRNA and protein for MMP-9 in mouse tissues, which helped to maintain tissue structure.
In the present study, 10% Aloe vera treatment showed a similar rate of primordial follicles when compared with culture control treatment. Compared with ovarian tissue cultured with 10.0% Aloe vera, tissues cultured for 6 days in the presence of 1.0, 5.0 or 50.0% Aloe vera demonstrated a reduction in activation and follicular growth rates. These data show that 10% Aloe vera can be added to culture medium without influencing the maintenance of the primordial follicle reserve. Previous studies have shown that a 6-day culture period is sufficient to promote activation and development of primordial follicles. When fragments of ovarian cortex are cultured there is a significant shift of follicles from quiescent to the growing pool over short culture periods of 6–10 days (Telfer and Zelinski, Reference Telfer and Zelinski2013). It is known that the activation of primordial follicles is a very dynamic process and, despite enormous progress made in mapping their regulation, many molecular mechanisms are still not fully understood (Figueiredo and Lima, Reference Figueiredo and Lima2017). The reduction in follicular activation rates during culture in the presence of 1.0 or 5.0% Aloe vera can be associated with the reduction in collagen fibres described in this study. Woodruff and Shea (Reference Woodruff and Shea2007) reported that an appropriately rigid ovarian extracellular environment can be a necessary requirement for follicle survival and growth. Various growth factors, such as TGF-β superfamily members, are involved in primordial follicle activation and, considering that ECM components are capable of binding these factors (Smith et al., Reference Smith, Bryant, Coupe, Vary, Gotwals, Koteliansky and Lindner1999), reduction of ECM density can diminish the availability of these growth factors and downregulate primordial follicle activation and growth. To explain the reduction in activation in tissues cultured in 50.0% Aloe vera, Tseng et al. (Reference Tseng, Wang, Tzeng, Chen, Liao, Chiu and Hsieh2017) showed that high concentrations of bioactive components present in Aloe vera extract can block PI3K activation signalling. Additionally, Adhikari et al. (Reference Adhikari, Flohr, Gorre, Shen, Yang, Lundin, Lan, Gambello and Liu2009) reported that PI3K/Akt pathway activation in oocytes induces primordial follicle activation, while Santos et al. (Reference Santos, Santos, Menezes, Barberino, Gouveia, Cavalcante, Lins, Gonçalves, Celestino and Matos2017) showed that blocking the PI3K pathway inhibits spontaneous activation of ovine primordial follicles in vitro.
A high percentage of normal follicles (70.0–80.0%) was observed in ovarian tissues cultured either in control medium alone or supplemented with Aloe vera. In general, the presence of Aloe vera in culture medium did not influence follicle viability, based on comparisons with tissues cultured in control medium. This could be due to the presence of antioxidants in control culture medium, such as transferrin, selenium and ascorbic acid. It is also important to consider that Aloe vera has antioxidant activity. The maintenance of cell morphology of ovarian follicles in in vitro culture depends on the cellular and tissue redox balance (Sá et al., Reference Sá, Bruno, Guerreiro, Cadenas, Alves, Cibin, Leal-Cardoso, Gastal and Figueiredo2018). Elevated levels of ROS can activate apoptotic clumps that result in follicular degeneration (Agarwal et al., Reference Agarwal, Aponte-Mellado, Premkumar, Shaman and Gupta2012). Cesar et al. (Reference Cesar, Jozić, Begović, Vuković, Mlinarić, Lepeduš, Borović Šunjić and Žarković2018) reported the presence of antioxidants in Aloe vera leaves, such as ascorbic acid, carotenoids and soluble polyphenols. These authors also demonstrated that 10.0% Aloe vera increased the growth of HeLa cells in culture and reduced the cytotoxicity induced by H2O2. Aloe vera was also able to protect sperm cells against oxidative damage induced by bisphenol A (BPA) and X-rays, respectively (Bala et al., Reference Bala, Chugh, Bansal, Garg and Koul2017; Behmanesh et al., Reference Behmanesh, Najafzadehvarzi and Poormoosavi2018). In this sense, the protective effects of Aloe vera were associated with increased antioxidant defence that was enzymatic (glutathione reductase, glutathione peroxidase, catalase and SOD) and non-enzymatic (GSH) in nature. In addition, it has been shown that Aloe vera can reduce extensive damage to membrane lipids, therefore reducing apoptotic processes (Haritha et al., Reference Haritha, Ramesh and Saralakumari2014). In the present study, tissues cultured with 50% Aloe vera had higher percentages of normal follicles than those cultured with 10% Aloe vera. It is known that many locally produced growth factors regulate follicle survival. Probably, cytoprotective effects of nutrient constituents in Aloe vera such as acemannan, sugars, amino acids used in structural proteins, fats, and ions such as calcium, magnesium, potassium, and vitamins (Joseph and Raj, Reference Joseph and Raj2010) improve follicle survival when compared with tissues cultured in 10% Aloe vera, but not when compared with control medium. Sholehvar et al. (Reference Sholehvar, Mehrabani, Yaghmaei and Vahdati2016) showed that dental pulp stem cells cultured in medium supplemented with 50% Aloe vera had improved viability.
The presence of 10% Aloe vera in culture medium increased PRDX6 mRNA levels in cultured tissues. Among mammalian peroxiredoxins, PRDX6 (Prdx6, 1-cys peroxiredoxin) is capable of reducing a wide range of substrates including H2O2, peroxynitrite, alkyl peroxides, and phospholipid peroxides (Perkins et al., Reference Perkins, Poole and Karplus2014; Fisher et al., Reference Fisher, Vasquez-Medina, Dodia, Sorokina, Tao and Feinstein2018). Due to its antioxidant properties, it plays an important role in maintaining the redox balance in mammalian cells including ovarian cells. For this reason, it is an important antioxidant defence in most cells exposed to oxygen (Sharapov et al., Reference Sharapov, Novoselov and Gudkov2019). Peroxideroxin 6 is the only peroxiredoxin capable of reducing phospholipid hydroperoxides through the activity of glutathione peroxidase (GPX). Animals with knockout of the PRDX6 gene, despite the normal expression of genes encoding other antioxidant enzymes, have a high sensitivity to oxidative stress, which is accompanied by a high level of oxidative damage to organs and tissues (Sharapov et al., Reference Sharapov, Novoselov and Gudkov2019). Zha et al. (Reference Zha, Wu, Zhao, Zhou, Zhang, Li, Ma and Zhang2015) demonstrated that PRDX6 can protect ARPE-19 cells, at least partially, through the PI3K/Akt signalling pathway. In the ovary, this pathway appears to play a regulating role in the growth and differentiation of ovarian follicles (Dupont and Scaramuzzi, Reference Dupont and Scaramuzzi2016). The balance between PI3K/Akt substrates determines the acceleration, deceleration, survival and apoptosis of follicular growth (Zhou et al., Reference Zhou, Xie, Li, Liang, Qiu, Lin and Zhang2017). In our study, increased levels of PRDX6 mRNA can be associated with higher levels of collagen fibres in the ECM of cultured ovarian tissues.
In conclusion, 10.0% Aloe vera increases the number of collagen fibres in the ECM and the expression of PRDX6 mRNA in bovine ovarian tissues cultured for 6 days. This demonstrates the potential for Aloe vera to be used as a supplement of culture medium of ovarian tissue.
Financial support
This research was supported by donations from the Cearense Foundation for Support for Scientific and Technological Development (FUNCAP, Ceará, 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.
