Introduction
Since the 1930s, reproductive research concerning relationships between the oocyte and its environment has yet to tackle how to identify and select competent oocytes to undergo in vitro maturation (IVM), in vitro fertilization (IVF) and further development to a healthy offspring (Callensen, Reference Callensen2010; Fair, Reference Fair2010). A non-invasive and unharmed test, based on glucose-6-phosphate dehydrogenase (G6PDH) activity, was described as an indirect marker of oocyte quality in different mammalian species like porcine (Ericsson et al., Reference Ericsson, Boyce, Funahashi and Day1993), caprine (Rodríguez-Gonzáles et al., Reference Rodríguez-Gonzáles, Lópes-Bejar, Vellila, Mertens and Paramio2001), bovine (Alm et al., Reference Alm, Torner, Löhrke, Viergutz, Ghoeneim and Kanitiz2005), bubaline (Manjunatha et al., Reference Manjunatha, Gupta, Devaraj, Ravindra and Nandi2007) and canine (Rodrigues et al., Reference Rodrigues, Rodriguez, Silva, Cavalcante, Fletrin and Rodrigues2009). For a review of morphological, cellular and molecular evaluation predictors of oocyte competence see Wang & Sun (Reference Wang and Sun2007) and Ferreira et al. (Reference Ferreira, Verique, Adona, Meirelles, Ferrarini and Navarro2009).
The enzyme G6PDH is heavily synthesized in growing oocytes but its activity decreases in grown oocytes, depicting – if stained with brilliant cresyl blue stain (BCB test) – a cytoplasm with blue colouration because they do not reduce BCB to a colourless compound (Ericsson et al., Reference Ericsson, Boyce, Funahashi and Day1993). Data are, however, still scarce concerning in vitro cytoplasm maturation rate, characterized by a reorganization of cortical granules (CG) and mitochondria for instance, of immature bovine oocytes if pre-selected by BCB test (Opiela et al., Reference Opiela, Katska-Ksiazkiewicz, Lipinski, Stomski, Bzowska and Rynska2008). Thus, the aim of this study was to determine nuclear and cytoplasm in vitro maturation rates of bovine oocytes selected by BCB test and the further embryo development until the blastocyst stage.
Materials and methods
Unless otherwise indicated, all plasticware, i.e. culture dishes used in our experiments were obtained from Nunc, Roskild, Denmark, while all chemicals and medium were purchased from Sigma, St. Louis, MO, USA.
Oocyte collection
Bovine ovaries were obtained from a slaughterhouse and transported to the laboratory at 25–32°C in modified phosphate-buffered saline (PBS) solution (mPBS). In the laboratory, the ovaries were washed in fresh mPBS and cumulus–oocyte complexes (COCs) were recovered from ovarian follicles (3–8 mm in diameter) with a 21-gauge needle attached to a 10 ml disposable syringe. Oocytes with two or more compact cumulus cell layers and uniform cytoplasm were used for the experiment.
Brilliant cresyl blue (BCB) staining
After collection, COCs were washed three times in mPBS containing 0.4% BSA (A-7888), and randomly divided in two groups. The control group containing COCs was submitted directly to 90 min in mPBS without exposition to BCB stain. In the experimental group the COCs were exposed for 90 min to 26 μM of BCB diluted in mPBS at 38.5°C in humidified air atmosphere containing 5% CO2 for 90 min. Following BCB exposure, oocytes were washed three times in mPBS and classified into two groups depending on their cytoplasm colouration: BCB+ (oocytes with blue cytoplasm) and BCB− (oocytes without blue colouration).
In vitro maturation
Oocytes were matured for 24 h in TCM199 containing 0.68 mM l-glutamine and 25 mM HEPES supplemented with 0.2 mM pyruvate, 26.2 mM NaHCO3, 50 μg/ml oestradiol-17β, 0.5 μg/ml FSH (Follitropin, Vetrefarm,), 0.03 IU/ml hCG (Profasi) and 10% (v/v) heat-treated fetal calf serum (FCS; Nutricell, Nutrientes Celulares). The selected COCs were first washed three times in maturation medium and transferred into 100 μl IVM medium drops under mineral oil and matured at 38.5°C in a humidified atmosphere containing 5% CO2.
Evaluation of nuclear maturation
Oocytes containing a polar body after IVM were considered to reach metaphase II, and to have undergone nuclear maturation successfully.
Evaluation of cytoplasmic maturation
For the visualization of CGs, the method described by Yoshida et al. (Reference Yoshida, Gran and Pursel1993) was used. Briefly, after the maturation period, the oocytes were transferred to mPBS supplemented with 10% FCS, mechanically denuded from cumulus cells and the zona pellucida removed after exposure to a mPBS solution containing 0.5% pronase. Thereafter, the oocytes were washed in mPBS three times again for 5 min each time for removal of the pellucida zone remnants, and washed in blocking solution (BS; PBS supplemented with 0.1 mg/ml BSA, fraction V and 100 mM glycine). Next, oocytes were fixed in a paraformaldehyde solution (in PBS with 0.1% polyvinylalcohol (PP)) for 30 min. Then, the oocytes were permeabilized for 5 min with the BS solution containing 0.1% Triton X-100 in block and finally washed three times in BS solution. After that, oocytes were exposed during 15 min at 38.5°C (protected from light) to 1 μg/ml FTIC-conjugated Lens culinaris lectin and 10 μg/ml propidium iodide diluted in BS solution. Finally, these oocytes were washed three times in BS solution and placed between a glass slide and coverslip and evaluated under a fluorescence microscope. Oocytes with CG arranged in clusters throughout the entire cytoplasm were classified as immature and those with peripheral CG as mature. The mitochondrion distribution throughout the cytoplasm was examined after exposing denuded oocytes during 20 min to a solution containing 0.5 μM Mitotracker Red (molecular Probes) and 10 μg/ml (Hoechst 33342) and then washed three times in PP solution. Finally, they were mounted in the same way as described above for CG evaluation. Oocytes with mitochondria dispersed in the central area of the cytoplasm were classified as mature and the others containing the mitochondria localized in peripheral area were denominated immature.
In vitro fertilization (IVF) and in vitro culture (IVC)
Straws (0.5 ml) containing bovine semen from a tested bull were thawed and sperm progressive motility and vigor immediately assessed. After that, spermatozoa were selected using the swim-up procedure as described by Parrish et al. (Reference Parrish, Susko-Parrish, Winer and First1986). The IVF medium drops (100 μl) containing the COCs were inseminated with 1 × 106 spermatozoa/ml, and co-cultured during 22 h at 38.5°C in atmosphere 5% CO2 in air and saturated humidity.
After IVF, presumptive zygotes were partially stripped of cumulus cells and transferred to 100 μl drops of modified synthetic oviduct fluid (SOFaa, Holm et al., Reference Holm, Booth, Schimidt, Greve and Callensen1999) for IVC at 38.5°C in atmosphere of 5%CO2, 5% O2 and 90% N2 and saturated humidity. Cleavage rate (D2) was performed at 48 h of culture and blastocyst rates at day 7 (D7).
Experimental design
Experiment 1: Oocyte meiosis progression related to BCB test
The nuclear maturation was assessed by the polar body presence. The cytoplasmic maturation was characterized by CG peripheral localization, and by mitochondrion central distribution. The control group containing COCs was submitted directly to mPBS for 90 min without exposition to BCB staining. Nine replication trials were carried out.
Experiment 2: Competence of oocytes selected by BCB test undergo in vitro embryo development until blastocyst stage
Oocytes were first selected on the basis of BCB test, and after submitted to IVM, IVF and IVC under standard procedures. After 48 h (D2) cleavage rate was assessed, and on day 7 (D7) blastocyst rate was determined. COCs of control group were submitted into mPBS and after directly to IVM, IVF and IVC without exposition to BCB stain. Nine replication trials were carried out.
Statistical analysis
In Experiment 1, the percentages of oocytes reaching MII (metaphase II), characterized by the polar body presence, CGs and mitochondria organization were subjected to analysis of variance (ANOVA). Differences with p-values <0.05 were considered significant.
In Experiment 2, the differences in developmental competence of fertilized oocytes (cleavage and blastocyst rates) were determined using chi-squared test, with a 5% significance level.
Results
Experiment 1:
G6PDH activity in immature bovine oocytes
A total number of 1207 immature oocytes were subjected to BCB test, and 65% showed low G6PDH activity (Table 1).
Table 1 G6PDH activity in immature oocytes subjected to BCB test
Nuclear maturation after IVM
After IVM, the presence of the first polar body was higher (p < 0.05) in BCB+ (65%) than in BCB− (20%) and IVM control group oocytes (40%), (Table 2).
Table 2 Presence of first polar body inside the ZP (zona pellucida) of oocytes selected by BCB
a, b, cDifferent letters within the same column indicate differences among treatments (p < 0.05).
Cytoplasmic maturation after IVM
Following IVM, CG presenting with peripheral distribution was higher (p < 0.05) in BCB+ oocytes (72%) than in BCB− (14%), or among control (55%), (Table 3).
Table 3 Cortical granules distribution in oocytes selected by BCB
a, b, cDifferent letters within the same column indicate differences among treatments (p < 0.05).
Regarding mitochondria organization, 82% of BCB+ oocytes showed central localization, higher (p < 0.05) than the BCB− (19%), or control (64%), (Table 4).
Table 4 Distribution of mitochondria in oocytes selected by BCB
a, b, cDifferent letters within the same column indicate differences among treatments (p < 0.05).
Experiment 2:
Competence of oocytes selected by BCB test to undergo in vitro embryo development
On D2, the cleavage rate was similar (75%) among the three experimental groups (Table 5). Significantly (p < 0.05) more BCB+ pre-selected oocytes reached the stage of blastocyst (35%) than BCB− (10%) or control (28%), (Table 5).
Table 5 Embryo development rates of oocytes selected by BCB test
a, b, cDifferent letters within the same column indicate differences among treatments (p < 0.05).
Discussion
It is well known that the proportion of bovine oocytes that fails to develop to the blastocyst stage following maturation, fertilization and culture in vitro is very large. Besides culture media that may contribute to this poor development, oocyte quality is more likely to be the limiting factor. In fact, oocytes obtained from ovaries of slaughtered cows are very heterogeneous in quality as they are recovered from follicles of different growing stages or atresia (Ferreira et al., Reference Ferreira, Verique, Adona, Meirelles, Ferrarini and Navarro2009). The BCB test was described by Alm et al. (Reference Alm, Torner, Löhrke, Viergutz, Ghoeneim and Kanitiz2005) as non-invasive and an unharmful, indirect marker for bovine oocyte competence. Opiela et al. (Reference Opiela, Katska-Ksiazkiewicz, Lipinski, Stomski, Bzowska and Rynska2008) conducted one experiment dealing with the expression of apoptosis-related genes and its correlation with bovine immature oocytes competence pre-selected by the BCB test. The lack of significant differences between blastocyst rates drove them to consider the BCB test as questionable. Our findings just the contrary, the BCB oocyte selection was based on the in vitro nuclear and cytoplasm maturation. These events were characterized by mitochondria and CG migration to reach MII.
Considering the high value of using only non-invasive methods (Hillier, Reference Hillier2008) highlighted the challenge to identify one viable non-invasive test of oocyte and embryo competence. BCB test was used to pre-select developmentally competent oocytes before maturation. With a concentration of 26 μM, 65% (Table 1) of the oocytes depicted blue cytoplasm, indicating that they have finished their growth phase and could thus be used for in vitro embryo production. The same BCB concentration was used in earlier studies in prepubertal goats (Rodríguez-González et al., Reference Rodríguez-Gonzáles, Lópes-Bejar, Izquierdo and Paramio2002), heifers (Pujol et al., Reference Pujol, Lopéz-Béjar and Paramio2004); and cows (Alm et al., Reference Alm, Torner, Löhrke, Viergutz, Ghoeneim and Kanitiz2005). The percentage of BCB+ oocytes (65%) obtained in the present study, employing 26 μM BCB, seemed similar to those reported in heifer (62%) by Pujol et al. (Reference Pujol, Lopéz-Béjar and Paramio2004) and buffalo oocytes (57.2%) by Manjunatha et al. (Reference Manjunatha, Gupta, Devaraj, Ravindra and Nandi2007) and was lower than the observed value with porcine oocytes (91%, Ericsson et al., Reference Ericsson, Boyce, Funahashi and Day1993; and 81%, Roca et al., Reference Roca, Martinez, Vazquez and Lucas1998).
In the present study, the nuclear maturation rate was significantly higher in BCB+ oocytes than the BCB− oocytes, similar to previous reports in goats (Rodríguez-González et al., Reference Rodríguez-Gonzáles, Lópes-Bejar, Izquierdo and Paramio2002), heifers (Pujol et al., Reference Pujol, Lopéz-Béjar and Paramio2004); cows (Alm et al., Reference Alm, Torner, Löhrke, Viergutz, Ghoeneim and Kanitiz2005), and buffalo (Manjunatha et al., Reference Manjunatha, Gupta, Devaraj, Ravindra and Nandi2007). It has been reported that the overall nuclear maturation rate of the oocytes remains unaffected by exposure to BCB.
The proportion of oocytes reaching metaphase II after IVM in the BCB− COCs was found to be significantly lower than in the control or BCB+ COCs probably might be the result of the unfinished growth of the oocytes (Alm et al., Reference Alm, Torner, Löhrke, Viergutz, Ghoeneim and Kanitiz2005). Although the biochemical basis of BCB metabolism in COCs is not fully understood, some evidence for the ability of BCB to play a role as electron acceptor and thereby become colourless during the electron flow induced by the G6PDH-catalyzed oxidation of G6P and reduction of NADP+ has been reported (Alm et al., Reference Alm, Torner, Löhrke, Viergutz, Ghoeneim and Kanitiz2005). The G6PDH enzyme activity permits to identify oocytes not competent to undergo fertilization and embryo development.
The present bovine in vitro production experiments provide a significantly increased rate of cytoplasm maturation for the BCB+ group (72%) characterized by CG migration rate as compared with the control (55%) and the BCB− (14%) groups. The high rates of CG distributed throughout the cytoplasm in the BCB− oocytes (82%) suggested cytoplasm immaturity. These delays in organelle migration and redistribution following maturation in bovine oocytes could be due an intrinsic deficiency as well as anomaly involves different patterns of migration and dispersal of CGs, as reported by Damiani et al. (Reference Damiani, Fissore, Cibeli, Long, Balise, Robl and Duby1996) in calf oocytes and Wang et al. (Reference Wang, Abeydeera, Prather and Day1998) in pig oocytes. In the present study, only 14% (6/43), CG migration was observed in the BCB− group.
Mitochondria play a central role in ATP generation for maturation, fertilization and embryo development (Wilding et al., Reference Wilding, Dale, Marino, Di Matteo, Alviggi, Pisaturo, Lombradi and De Plácido2001). During GV, these organelles are mostly concentrated in the periphery of the oocyte, while in mature oocytes (MII) these structures migrate toward the center (Sun et al., Reference Sun, Wu, Lai, Park, Cabot, Cheong, Day, Prather and Schaten2001). A similar pattern was observed in the experimental groups in the present study. However, there was a difference in mitochondria distribution among the groups of oocytes. Only 64% of the control, 85% of the BCB+ and 19% of the BCB− oocytes that showed nuclear maturation had centrally located mitochondria, thus indicating that the other oocytes were not able to complete their mitochondria migration during the 24 h-period of in vitro maturation. We postulate, therefore, that although BCB+ oocytes had higher percentages of nuclear and cytoplasm maturation compared with BCB− oocytes and BCB is a useful test to select fully grown oocytes, that are able to mature to MII stage, most oocytes are unable to support total or normal cytoplasm maturation. Moreover, there is a delay in organelle distribution.
Regarding embryo development, differences between groups were only detected later in development because cleavage rates were the same (75%) in all treatments. Although BCB− oocytes make up their high cleavage rate during the IVF period, it is not enough to sustain embryonic development to the blastocyst stage. This was clearly reflected by lower blastocyst rates as compared with BCB+ and control oocytes. Probably, BCB− oocytes have high rates of polyspermic fertilization as reported by Rodríguez-González et al. (Reference Rodríguez-Gonzáles, Lópes-Bejar, Vellila, Mertens and Paramio2001) using the BCB test in goats and Damiani et al. (Reference Damiani, Fissore, Cibeli, Long, Balise, Robl and Duby1996) suggested that a limited Ca2+ release that could result in abnormal fertilization and decreased rates of development. Blastocyst rate on day 7 observed in BCB+ group was 35%, whereas oocytes classified as BCB− had only 10% blastocyst development. Those results are more consistent in the present study, which was attributed to the more consistent use of the BCB test to identify competent oocytes. Thus, the percentage of BCB+ oocytes that developed to blastocyst stage was significantly higher than in the control group. Conversely, blastocyst development rate was low in BCB− oocytes (10%), but higher than for development rates BCB− oocytes as reported in heifers (1.6%; Pujol et al., Reference Pujol, Lopéz-Béjar and Paramio2004), buffalo (5.2%; Manjunatha et al., Reference Manjunatha, Gupta, Devaraj, Ravindra and Nandi2007), and cows (3.9%; Alm et al., Reference Alm, Torner, Löhrke, Viergutz, Ghoeneim and Kanitiz2005).
In conclusion, this study showed that the BCB test is efficient enough to identify competent immature bovine oocytes that undergo synchronous nuclear and cytoplasm in vitro maturation thus allowing an increase in in vitro embryo development rate to blastocyst stage.
Acknowledgements
We thank ABS Pecplan for supplying the semen, Rost abattoir for supplying the bovine ovaries. The authors thank the National Council for Scientific and Technological Development (CNPq) for the provided grant and fellowships.
