Introduction
In recent years, much attention has focused on optimizing in vitro culture medium composition for small ovarian follicles as they can act as a source of fertilizable oocytes for further in vitro embryo production (Gupta et al., Reference Gupta, Rameshb, Nandi and Ravindra2007). Although acceptable rates of follicular viability and growth have been obtained with various media, Minimal Essential Medium alpha modified (α-MEM) has emerged as the most commonly used medium for culture of oocytes and follicular cells in caprine (Chaves et al., Reference Chaves, Duarte, Rodrigues, Celestino, Silva, Lopes, Almeida, Donato, Peixoto, Moura, Lobo, Locatelli, Mermillod, Campelo and Figueiredo2012) and ovine (Esmaielzadeh et al., Reference Esmaielzadeh, Hosseini, Hajian, Nasiri, Chamani, Afshar and Nasr-Esfahani2013; Santos et al., Reference Santos, Menezes, Barberino, Macedo, Lins, Gouveia, Barros, Santos, Gonçalves and Matos2014) species. However, for improving the developmental competence of oocytes, α-MEM needs a constant addition of supplements, such as antioxidants, hormones and growth factors (Abedelahi et al., Reference Abedelahi, Salehnia, Allameh and Davoodi2010; Andrade et al., Reference Andrade, Van Den Hurk, Lisboa, Hertel, Melo-Sterza, Moreno, Bracarense, Landim-Alvarenga, Seneda and Alfieri2012), which can make follicular cell culture more expensive. Therefore, the natural compounds presented in medicinal plants are a promising strategy for alternative sources of basic culture medium. Among the several extracts of medicinal plants, Amburana cearensis noteworthy for its numerous cytoprotective characteristics.
Amburana cearensis (Allemão) A.C. Smith (Fabaceae) is a tree commonly found in northeastern Brazil, where it is popularly known as ‘Cumaru’, ‘Amburana-de-cheiro’ or ‘Umburana’ (Albuquerque et al., Reference Albuquerque, Medeiros, Almeida, Monteiro, Neto, Melo and Santos2007; Leal et al., Reference Leal, Nobre, Cunha, Moraes, Pessoa, Oliveira, Silveira, Canuto and Viana2010), being widely used in traditional medicine for the treatment of a wide range of diseases including respiratory problems in general, influenza, cough, expectorant, thrombosis, hypertension, inflammations and healing (Cartaxo et al., Reference Cartaxo, Souza and Albuquerque2010). This plant has phenolic compounds, especially flavonoids and phenolic acids (isokaempferide, kaempferol, afromosin and quercetin) which can act as antioxidants through their ability to scavenge reactive oxygen species (ROS) (Leal et al., Reference Leal, Nobre-Júnior, Cunha, Moraes, Pessoa, Oliveira, Silveira, Canuto and Viana2005; Ruijters et al., Reference Ruijters, Weseler, Kicken, Haenen and Bast2013; Wang & Shahidi, Reference Wang and Shahidi2013). In addition, coumarin, also present in A. cearensis, reduces the lipid peroxidation activity in different types of rat cells (Neichi et al., Reference Neichi, Koshihara and Murota1983; Martín-Aragón et al., Reference Martín-Aragón, Bened and Villar1998).
It is thought that ovarian follicular atresia is initiated as a consequence of inadequate protection of cultured follicular cells from the damaging effects of ROS (Tilly & Tilly, Reference Tilly and Tilly1995). Therefore, it could be speculated that the natural compounds of A. cearensis, especially those with antioxidant properties, would protect ovarian follicles from ROS during in vitro culture. Thus, A. cearensis extract may be a useful substitute for standard cell culture medium. In this sense, there were no reports in which A. cearensis extract has been used as a culture medium for ovine ovarian follicles. Thus, this study was conducted to evaluate the effect of this extract on in vitro survival and development of sheep isolated secondary follicles. Additionally, the effectiveness of the addition of supplements to A. cearensis extract was studied.
Materials and methods
Unless otherwise mentioned, culture media, supplements and chemicals used in the present study were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Plant material and extract preparation
Fresh leaves of A. cearensis were collected in Petrolina (09°23′55″ South and 38–40°30′03″ West, Pernambuco, Brazil), from the Caatinga biome, and characterized in a semi-arid climate, with average annual temperature of 26.4ºC. A voucher specimen (5545) was deposited at the Herbário Vale do São Francisco (HVASF) of the Federal University of San Francisco Valley during the dry season, flowering and fructification stages of the plant. The leaves were dried in an oven at 40°C and pulverized and extracted at room temperature with 95% ethanol (Vetec, Duque de Caxias-RJ, Brazil) for 72 h. The extract was dried at 45°C using a rotavator and the yield was approximately 10%, obtaining the crude ethanolic extract of the leaves of A. cearensis, which was dissolved in 0.9% saline solution, corresponding to concentrations of 0.1, 0.2 or 0.4 mg/ml, which were keep at 4°C.
Source of ovarian tissue
This experiment was approved and performed under the guidelines of the Committee of Ethics and Deontology Studies and Research at the Federal University of San Francisco Valley (00508/2012).
Ovaries (n = 50) were collected at a local abattoir from 25 adult (1–3 years old) mixed-breed ovine (Ovis aries). Immediately post-mortem, pairs of ovaries were washed once in 70% alcohol and then twice in minimum essential medium buffered with HEPES (MEM-HEPES) and supplemented with antibiotics (100 μg/ml penicillin and 100 μg/ml streptomycin). Next, the ovaries were transported within 1 h to the laboratory in tubes containing MEM-HEPES with antibiotics at 4°C (Chaves et al., Reference Chaves, Martins, Saraiva, Celestino, Lopes, Correia, Lima Verde, Matos, Báo, Name, Campello, Silva and Figueiredo2008).
Isolation and selection of ovine secondary follicles
In the laboratory, the surrounding fatty tissues and ligaments were stripped from the ovaries; large antral follicles and corpora lutea were removed. Ovarian cortical slices (1–2 mm thick) were cut from the ovarian surface using a surgical blade under sterile conditions and subsequently placed in fragmentation medium consisting of MEM-HEPES with antibiotics. Ovine secondary follicles, approximately 200 μm in diameter without antral cavities, were visualized under a stereomicroscope (SMZ 645 Nikon, Tokyo, Japan; at x100 magnification) and mechanically isolated by microdissection using 26-gauge (26G) needles. These follicles were then transferred to 100 μl droplets containing basic culture medium for the quality evaluation. Only follicles that displayed the following characteristics were selected for culture: an intact basement membrane, two or more granulosa cells layers and a visible and healthy oocyte that was round and centrally located among the cells, without any dark cytoplasm. Isolated follicles were pooled and then randomly allocated to the treatment groups, with 30 follicles per group.
In vitro culture of secondary follicles
The basic control medium consisted of α-MEM (pH 7.2–7.4; Gibco, Invitrogen, Karlsruhe, Germany) supplemented with 3.0 mg/ml bovine serum albumin (BSA), 10 ng/ml insulin, 5.5 μg/ml transferrin, 5.0 ng/ml selenium, 2 mM glutamine, 2 mM hypoxanthine and 50 μg/ml ascorbic acid and then referred as α-MEM+. To verify the influence of the plant, the follicles were randomly distributed in α-MEM+ (control medium) or into medium composed of different concentrations of A. cearensis extract without supplements (Amb 0.1; 0.2 or 0.4 mg/ml) or A. cearensis extract supplemented with the same substances described above for α-MEM+ supplementation. The A. cearensis supplemented medium was named Amb 0.1+; 0.2+ or 0.4+ mg/ml (Figure 1). The follicles were individually cultured (one follicle per droplet) in 100 μl of culture medium under mineral oil in Petri dishes (60 × 15 mm; Corning, Sarstedt, Newton, NC, USA). Incubation was performed at 39°C and 5% CO2 in air for 18 days. In all the treatments 60 μl of the culture media was replaced with fresh media in each droplet at every 6 days (Magalhães et al., Reference Magalhães, Fernandes, Mororó, Silva, Rodrigues, Bruno, Matos, Campello and Figueiredo2011).
Figure 1 General experimental protocol for in vitro culture of ovine preantral follicles in α-MEM+ or different concentrations of Amburana cearensis extract in the absence (Amb 0.1; 0.2 and Amb 0.4) or presence of supplements (Amb 0.1+; Amb 0.2+ and Amb 0.4+).
Morphological evaluation of follicle development
The morphological aspects of all preantral follicles were assessed every 6 days using a pre-calibrated ocular micrometer in a stereomicroscope (SMZ 645 Nikon, Tokyo, Japan; ×100 magnification). Only those follicles showing an intact basement membrane, with bright and homogeneous granulosa cells and an absence of morphological signs of degeneration, were classified as morphologically normal follicles. Follicular atresia was recognized when a darkening of the oocytes and surrounding cumulus cells, misshapen oocytes or decreased follicle diameter was noted. The rupture of the basement membrane was also observed and characterized as oocyte extrusion. The following characteristics were analyzed in the morphologically normal follicles: (i) antral cavity formation, defined as the emergence of a visible translucent cavity within the granulosa cell layers; (ii) follicle diameter, measured from the basement membrane, which included two perpendicular measures of each follicle; and (iii) daily follicular growth rate, calculated as the diameter variation during the culture period.
Statistical analysis
Data from morphologically normal follicles, extruded follicles and antrum formation after in vitro culture were expressed as percentages and compared by the chi-squared test. Follicular diameter and daily follicular growth rates were submitted to the Shapiro–Wilk test to verify normal distribution of residues and homogeneity of variances using PROC UNIVARIATE SAS 9.0 procedure. The residues that did not show a normal distribution were logarithmically transformed [log10(X + 1)] for normal distribution adjustment. Because requirements underlying the analysis of variance were present, for follicular diameter, ANOVA was executed using PROC MIXED SAS (2003) with REPEATED statement to account for autocorrelation between sequential measurements. The model included the effects of treatment (α-MEM and A. cearensis concentrations), time of culture (0, 6, 12 or 18 days) and their interactions. Data from follicular growth were analyzed in the same way, however, using the PROC GLM procedure. Due to the presence of heterogeneity of variances for data from follicular diameter and growth rate, the Kruskal–Wallis non-parametric test was used through the PROC NPAR1WAY SAS 9.0 procedure. These results were expressed as the means ± standard error mean (SEM) and differences were considered significant when P < 0.05.
Results
Follicular survival after in vitro culture
Morphologically normal follicles showed centrally located oocytes and normal granulosa cells, which were enclosed by an intact basement membrane (Fig. 2 A). As early as day 6 of the culture, a small antral cavity (Fig. 2 B) and atretic follicles (Fig. 2 C) could be observed in all the treatments. Extruded follicles were observed only after culture in α-MEM+ (Fig. 2 D).
Figure 2 Morphologically normal secondary follicle at day 0 (A), antral (B), atretic (C), and extruded (D) follicle after 6 days of culture. GC: granulosa cell; O: oocyte. Arrow: antral cavity. Scale bar: 100 µm (× 100 magnification).
The percentage of follicular survival after 18 days of culture in the different concentrations of A. cearensis extract is shown in Fig. 3. The percentage of morphologically normal follicles decreased significantly from day 0 to day 6 only in the Amb 0.1+ mg/ml treatment. Nevertheless, the percentage of normal follicles decreased significantly from day 6 to day 12 in all the treatments, except in Amb 0.2 and Amb 0.4 mg/ml without supplements. A significant decrease in the percentage of normal follicles in these two A. cearensis concentrations was observed only after 18 days of culture.
Figure 3 Percentages of morphologically normal follicles cultured in α-MEM+ or different concentrations of Amburana cearensis extract in the absence (Amb 0.1; 0.2 and Amb 0.4) or presence of supplements (Amb 0.1+; Amb 0.2+ and Amb 0.4+). a,b,cDifferent letters denote significant differences among culture periods in the same treatment (P < 0.05). A,BDifferent letters denote significant differences among treatments in the same period (P < 0.05).
When the treatments were compared at day 18, there were more morphologically normal follicles in Amb 0.1 or Amb 0.4 mg/ml (without supplements) than in control medium (α-MEM+). In addition, there was no significant difference in the rate of follicular survival among the A. cearensis concentrations, with or without supplements (P > 0.05).
Antral cavity formation during in vitro culture
Compared with day 0, the rates of antral cavity formation increased significantly only from day 12 of culture onwards in Amb 0.2, Amb 0.4, Amb 0.2+ or Amb 0.4+ mg/ml treatments (Fig. 4). Moreover, follicles cultured in Amb 0.1 mg/ml increased significantly the percentage of antrum formation only at day 18.
Figure 4 Percentages of antral cavity formation in follicles cultured in α-MEM+ or different concentrations of Amburana cearensis extract in the absence (Amb 0.1; 0.2 and Amb 0.4) or presence of supplements (Amb 0.1+; Amb 0.2+ and Amb 0.4+). a,b,cDifferent letters denote significant differences among culture periods in the same treatment (P < 0.05). A,BDifferent letters denote significant differences among treatments in the same period (P < 0.05).
After comparing the different treatments at day 18 of culture, the rate of antrum formation was significantly higher in Amb 0.1 and Amb 0.2 mg/ml (without supplements) than in α-MEM+ (control medium) and Amb 0.1+ mg/ml, and similar to the other treatments (P > 0.05).
Follicular diameter and growth rate in vitro
All A. cearensis extract media induced a progressive and significant increase in follicular diameter throughout the culture period (Fig. 5). After 12 days, all the treatments had similar follicular diameters (P > 0.05), which was significantly higher than that found after Amb 0.4+ treatment. At the end of the culture, follicles cultured in non-supplemented 0.1, 0.2 or 0.4 mg/mL A. cearensis had larger diameters than those after Amb 0.2+ mg/ml treatment and similar to the other treatments (P > 0.05).
Figure 5 Diameter (μm) of follicles cultured for 18 days in α-MEM+ or different concentrations of Amburana cearensis extract in the absence (Amb 0.1; 0.2 and Amb 0.4) or presence of supplements (Amb 0.1+; Amb 0.2+ and Amb 0.4+). a,b,c,dDifferent letters denote significant differences among culture periods in the same treatment (P < 0.05). A,BDifferent letters denote significant differences among treatments in the same period (P < 0.05).
Follicles cultured in α-MEM+, in A. cearensis without supplements or in Amb 0.1+ (8.9 μm/day) or Amb 0.2+ (8.3 μm/day) mg/ml showed similar rates of daily growth (P > 0.05), which were significantly higher than Amb 0.4+ mg/ml (7.0 μm/day) treatment.
Discussion
To the best of our knowledge, this study constitutes the first report to demonstrate the beneficial effect of A. cearensis extract on in vitro culture of ovine isolated follicles. It is known that the levels of antioxidant defenses are lower during in vitro oocyte and embryo culture than in vivo. Therefore, the addition of an antioxidant to the medium may be important to reduce the damage caused by oxidative stress (Crocomo et al., Reference Crocomo, Marques Filho and Landim-Alvarenga2012; Rajabi-Toustani et al., Reference Rajabi-Toustani, Motamedi-Mojdehi, Mehr and Motamedi-Mojdehi2013). In the present study, after 18 days of culture, appropriate concentrations of medium composed of A. cearensis extract (0.1 or 0.4 mg/ml) showed higher percentages of morphologically normal follicles than for the control medium (α-MEM+).
A recent study performed by our group used high performance liquid chromatography (HPLC) to determine the fingerprint chromatogram of the ethanolic extract of A. cearensis (Gouveia et al., 2015). This extract was the same used in the current work and the following compounds were identified and quantified: gallic acid, protocatechuic acid, epicatechin, p-coumaric acid and kaempferol. Gallic acid (GA) and protocatechuic acid (PCA), compounds, found in large amounts in the A. cearensis extract, have antioxidant action and scavenging ROS. The GA, an endogenous plant phenol (Singh et al., Reference Singh, Rai, Upadhyay, Kumar and Singh2004), showed anti-lipid peroxidative and antioxidant effects in pancreatic tissue of Wistar rats after induced toxicity by streptozotocin (Punithavathi et al., Reference Punithavathi, Prince, Kumar and Selvakumari2011). PCA, one of the major benzoic acid derivatives from vegetables and fruits (Guan et al., Reference Guan, Ge, Liu, Ma and Cui2009), extends lifespan and increases osmotic and heat stress resistance in Caenorhabditis elegans, by elevate antioxidant enzyme activities such as catalase and superoxide dismutase (Kim et al., Reference Kim, Seo, Lee, Kim, Jeon and Cha2014).
In addition, epicatechin and kaempferol are phenols belonging to the flavonoids class. Their prominent and most useful properties are scavenge ROS and metal chelating activities (Lopez-Revuelta et al., Reference Lopez-Revuelta, Sanches-Gallego, Hernandez-Hernandez, Sanchez-Yague and Llanillo2006), being considered more efficient antioxidants than vitamin C (ascorbic acid), vitamin E (α-tocoferol) and β-carotene (Gao et al., Reference Gao, Huang and Xu2001). Recent studies have demonstrated that epicatechin protects human vascular endothelial cells against oxidative stress (Ruijters et al., Reference Ruijters, Weseler, Kicken, Haenen and Bast2013) and that kaempferol has antimicrobial and antioxidant properties (Tatsimo et al., Reference Tatsimo, Tamokou, Havyarimana, Csupor, Forgo, Hohmann, Kuiate and Tane2012).
With respect to p-coumaric acid (CA), an intermediate product of the phenylpropanoid pathway in plants, it was able to directly scavenge ROS and protect low-density lipoprotein from oxidation (Zang et al., Reference Zang, Cosma, Gardner, Shi, Castranova and Vallyathan2000). Therefore, in our study, it is possible that these natural antioxidants, especially GA and PCA, may act in isolation or in association to decrease oxidative damage during follicular cell culture in sheep, contributing to the maintenance of higher morphologically normal follicle rates.
Besides its antioxidant activity, PCA stimulated neural stem cells proliferation in vitro (Guan et al., Reference Guan, Ge, Liu, Ma and Cui2009). In the current study, it is possible that PCA may have supported follicular development in vitro without the necessity of medium supplementation, as, after 18 days, the rates of antrum formation were higher in follicles cultured in 0.1 or 0.2 mg/ml of A. cearensis extract without supplements, compared with α-MEM+. This finding is important because the ability to form an antrum may be considered as a marker of follicular functionality (Rossetto et al., Reference Rossetto, Saraiva, Santos, Silva, Faustino, Chaves, Brito, Rodrigues, Lima, Donato, Peixoto and Figueiredo2012), as the mechanisms by which small cavities of fluid develop inside the follicle to form the antral cavity are related to the secretion of osmotically active molecules into small spaces between the granulosa cells (Rodgers & Irving-Rodgers, Reference Rodgers and Irving-Rodgers2010). However, in previous reports, the rate of antrum formation was higher than that observed in our study (Saraiva et al., Reference Saraiva, Rossetto, Brito, Celestino, Silva, Faustino, Almeida, Bruno, Magalhães, Matos, Campello and Figueiredo2010; Barboni et al., Reference Barboni, Russo, Cecconi, Curini, Colosimo, Garofalo, Capacchietti, Di Giacinto and Mattioli2011). This situation could be explained because our culture media were FSH-free, which is a different formulation from the culture media used by the above-cited authors. After antrum formation, the follicle becomes dependent on follicle-simulating hormone (FSH) (Erickson & Shimasaki, Reference Erickson and Shimasaki2001) and the absence of this hormone in the basic medium may have contributed to lower rates of antrum formation in the present study. Similar results were obtained by Luz et al. (Reference Luz, Araújo, Duarte, Silva, Chaves, Brito, Serafim, Campello, Feltrin, Bertolini, Almeida, Santos and Figueiredo2013), who observed a low and constant rate of antrum formation during 18 days of culture of sheep secondary follicles in a FSH-free medium.
The presence of supplements (BSA, insulin, transferrin, selenium, glutamine, hypoxanthine and ascorbic acid) in the A. cearensis media did not improve follicular survival and antrum formation compared with the control medium (α-MEM+). Previous studies have shown that addition of supplements into the culture medium could maintain ovarian follicle viability (Silva et al., Reference Silva, van den Hurk, Costa, Andrade, Nunes, Ferreira, Lôbo and Figueiredo2004; Peng et al., Reference Peng, Yang, Wang, Tong and Guo2010), due its antioxidant capacity (selenium and transferrin) or to be effective energy substrate (glutamine and BSA) for follicular cells (Songsasen et al., Reference Songsasen, Spindler and Wildt2007; Abedelahi et al., Reference Abedelahi, Salehnia, Allameh and Davoodi2010; Rodrigues et al., Reference Rodrigues, Silva, Faustino, Bruno, Magalhães, Campello and Figueiredo2010). The concentrations of the supplements used in our study are the same previously used in the culture of isolated preantral follicles in α-MEM+ (Saraiva et al., Reference Saraiva, Rossetto, Brito, Celestino, Silva, Faustino, Almeida, Bruno, Magalhães, Matos, Campello and Figueiredo2010). Therefore, it is likely that these concentrations could be unsuitable for the A. cearensis extract medium. It is known that some antioxidant substances may act as pro-oxidants when used at high concentrations (Andrade et al., Reference Andrade, Melo-Sterza, Seneda and Alfieri2010). In fact, an excess of antioxidant compounds can increase the redox state, which may have deleterious effects on embryos (Guérin et al., Reference Guérin, El Mouatassim and Ménézo2001). In this way, some studies have shown that high concentrations of ascorbic acid, an important antioxidant added to the culture medium, can inhibit important physiological processes in the ovary, resulting in follicular degeneration (Murray et al., Reference Murray, Molinek, Baker, Kojima, Smith, Hillier and Spears2001), besides promoting oxidative damage to cellular DNA (Li & Schellhorn, Reference Li and Schellhorn2007). Thus, as the balance between antioxidants and pro-oxidants must be preserved in the cells, future studies should be done to investigate the suitable concentrations of the supplements added to the medium containing A. cearensis extract.
It is important to note that the costs for purchasing medium containing A. cearensis extract may make in vitro culture studies cheaper and more available. These costs were limited to acquisition of 95% ethanol and 0.9% saline solution for extract dilution. Adversely, MEM is an expensive commercial medium that would cost about 40 times more than plant extract. Therefore, our findings encourage future studies of follicle culture in A. cearensis because this medicinal plant is cheaper than MEM.
In conclusion, this study showed that an appropriate concentration of A. cearensis extract, without supplementation, maintains follicular survival and promotes the development of ovine isolated secondary follicles in vitro. The concentration of 0.1 mg/ml must be highlighted due to the satisfactory results in all evaluated parameters after 18 days of culture. This extract can be a cheap and efficient alternative culture medium for preantral follicle development. Therefore, a greater knowledge of A. cearensis extract's chemical composition is important because it would make this protocol efficient and reproducible as that of MEM and also allows that this protocol becomes a commercial product. In future studies, we also suggest that more techniques should be performed to permit a deeper examination of the cells, identifying even slight damage in the follicle ultrastructure caused by in vitro culture.
Acknowledgements
This work was supported by the National Council for Scientific and Technological Development (CNPq; Process 562548/2010–6). R. Sousa Barberino receives a scholarship from the CNPq/Univasf. M.H.T. Matos is supported by a grant from CNPq.
Declaration of interest
None of the authors have any conflict of interest to declare.
