Introduction
Establishment of an effective and consistent method of maturing oocytes in vitro is the foundation of most in vitro fertilization (IVF) protocols. Immature equine oocytes have been characterized morphologically to select those best suited for in vitro maturation (IVM) (Goudet et al., Reference Goudet, Bézard, Duchamp, Gerard and Palmer1997; Hinrichs & Schmidt, Reference Hinrichs and Schmidt2000). Although equine oocytes are commonly recovered from abattoir-derived ovaries and used for in vitro biotechnologies, there is little information regarding assessment of their developmental competence (Dell'Aquila et al., Reference Dell'Aquila, Fusco, Lacandra and Maritato1997).
Immature oocytes synthesize a variety of proteins, including glucose-6-phosphate dehydrogenase (G6PDH). This enzyme, a component of the pentose phosphate cycle, is active in the growing oocyte, but is relatively less active in oocytes that have completed growth (Alm et al., Reference Alm, Torner, Lohrke, Viergutz, Ghoneim and Kanitz2005). Brilliant cresyl blue (BCB) is a vital blue dye that assesses intracellular G6PDH activity. In pigs (Roca et al., Reference Roca, Martinez, Vazquez and Lucas1998), prepubertal goats (Rodriguez-Gonzalez et al., Reference Rodriguez-Gonzalez, Lopez-Bejar, Velilla and Paramio2002), cattle (Pujol et al., Reference Pujol, Lopez-Bejar and Paramio2004), buffalo (Manjunatha et al., Reference Manjunatha, Gupta, Devaraj, Ravindra and Nandi2007), dogs (Rodrigues et al., Reference Rodrigues, Rodriguez, Silva, Cavalcante, Fletrin and Rodrigues2009), the BCB test has identified oocytes that were more suitable for in vitro maturation, fertilization and embryo development. The cytoplasm of these oocytes turned blue (BCB positive/BCB+) because it did not reduce BCB to a colourless compound. In contrast, oocytes not staining blue have active G6PDH and reduce the dye to a colourless solution; it is likely that these oocytes are still growing and therefore not developmentally competent (BCB negative/BCB–) (Ericsson et al., Reference Ericsson, Boyce, Funahashi and Day1993).
The percentages of BCB-positive bovine oocytes were variable after oocyte selection solely based on morphology (Goovaerts et al., Reference Goovaerts, Leroy, Jorssen and Bols2010). A BCB dye test may facilitate identification of equine oocytes with greater potential to mature in vitro, especially those with compact cumulus cells, which interfere with morphological assessment.
The objective of the present study was to evaluate the BCB test as a criterion to assess developmental competence of equine oocytes cultured in vitro, based on intracellular activity of G6PDH. In addition, we also evaluated the effect of equine growth hormone (eGH) on IVM of equine oocytes. We tested the hypothesis that BCB dye determines the intracellular activity of G6PDH in immature equine oocytes and is predictive of developmental competence.
Materials and methods
Collection of cumulus–oocyte complexes (COCs)
Unless otherwise indicated, all chemicals used were from Sigma Chemical Co. (St. Louis, MO, USA). Fresh equine ovaries were obtained during the physiological breeding season from an abattoir near the Lethbridge Research Centre, Alberta, Canada. The ovaries were transported to the laboratory in 0.9% NaCl solution in a thermal container at 25°C. The tunica was removed and follicles <30 mm in diameter were aspirated to recover oocytes. After 20 min (to allow sedimentation to occur), the pellet containing COCs was examined under a stereomicroscope and complexes were classified based on their morphology and structural integrity as: compact (CP; having a tight, complete compact cumulus with a distinct, smooth hillock); expanded (EX; having a granular or expanded cumulus); or denuded (D; having a partial cumulus cells layer or only corona radiata present). Only oocytes with a compact cumulus and homogenous cytoplasm were used.
Selection by BCB test and IVM of COCs
After selection, COCs were exposed to 26 μM BCB in Dulbecco's phosphate-buffered saline (PBS) for 90 min at 38.5°C. Thereafter, oocytes were selected based on their cytoplasm colouration: BCB+ oocytes that had blue cytoplasmic staining, whereas BCB– oocytes had a colourless cytoplasm. Then, COCs were washed three times in modified PBS (mPBS) prior to IVM.
The basal medium was composed of TCM-199, 0.1% BSA, 100 IU/ml penicillin G, and 50 μg/ml streptomycin sulfate. The medium was filtered through a 0.22 μm pore diameter filter and allowed to equilibrate for at least 1 h under 5% CO2 in air. Equine growth hormone was provided by The Andrology Laboratory (The University of Auckland, Auckland, New Zealand) and used at a concentration of 400 ng/ml (Pereira et al., Reference Pereira, Lorenzo, Carneiro, Bilodeau-Goeseels, Kastelic, Pegoraro, Pimentel, Esteller-Vico, Illera, Silvan, Casey and Liu2006, 2011). Before incubation, COCs were randomly allocated into a four-well Petri dishes containing 500 μl of maturation media according to the treatments groups, as follows: (a) IVM group (cultured immediately after morphological selection); (b) eGH group (cultured with 400 ng/ml eGH immediately after morphological selection); (c) BCB–/IVM group (IVM medium after BCB– classification); (d) BCB+/IVM group (IVM medium after BCB+ classification; (e) BCB–/eGH (eGH medium after BCB– classification); and (f) BCB+/eGH (eGH medium after BCB+ classification).
Assessment of oocyte maturation
After 30 h of maturation at 38.5 °C in air containing 5% CO2, the surrounding cumulus cells were removed by application of 0.1% hyaluronidase solution for 3 min. Thereafter, oocytes were incubated in 10 μg/ml bis-benzamide (Hoechst 33342) for 20 min at 39 °C. To evaluate nuclear maturation, oocytes were evaluated using an epifluorescence microscope with a 365-nm exciter filter. Oocytes at the germinal vesicle (GV) stage of development were characterized by a diffuse appearance of their chromatin. Nuclear maturation was recorded as the percentage of oocytes that achieved metaphase-II stage (M-II), based on the presence of the chromosomes on the metaphase plate and the first polar body.
Experimental design and statistics
Prior to the BCB test, COCs were cultured in vitro, with or without eGH. The COCs were classified as BCB+, BCB–, or without BCB, respectively. The proportions of COCs that matured after IVM and categorized as BCB+ or BCB– were recorded. Differences in proportions among treatment groups were calculated by Fisher's Exact test analysis using JMP7 Software. The level of significance was set at P < 0.05.
Results
The percentage of mature oocytes after BCB– classification was similar in the eGH (5/11, 45.5%) group compared with the control (3/7, 42.9%). Similarly, for the BCB+ classification, the percentage of mature oocytes was not significantly different for eGH versus control (4/10, 40.0% versus 5/11, 45.5%). The proportion of M-II oocytes after eGH addition was not different between BCB– oocytes (5/11, 45.5%) and BCB+ oocytes (4/10, 40.0%). There was a higher maturation rate for equine oocytes subjected to IVM in the presence of eGH in the culture than in the control group (16/28, 57.1% versus 8/26, 30.8%, respectively; P < 0.05). Data from the maturation rates of equine oocytes and BCB exposure after IVM are shown (Table 1).
Effect of equine growth hormone (eGH; 400 ng/ml) on equine oocytes in vitro matured after 30 h of culture after selection of equine oocytes using brilliant cresyl blue stain (BCB; 26 μM)
a,bWithin a column, percentages without a common superscript differed (P < 0.05).
GV, germinal vesicle; IVM, in vitro maturation; M-II, metaphase II.
Following exposure to BCB, COCs were classified as either colourless (BCB–, Fig. 1a) or with a blue cytoplasm colouration (BCB+, Fig. 1b). In addition, equine COCs evaluated before 90 min of BCB exposure are shown (Fig. 1c). Oocytes were classified as mature based on the presence of the chromosomes on the metaphase plate and the presence of the first polar body (Fig. 1d).
(a) Equine cumulus–oocytes complexes (COCs) with a clear cytoplasm (brilliant cresyl blue (BCB) negative/BCB–). (b) Equine COCs with blue cytoplasm colouration (BCB positive/BCB+). Scale bars represent 40 μm. (c) Equine COCs evaluated prior to exposure to BCB. Scale bar represents 100 μm. (d) Equine oocytes matured with eGH: polar body (PB) and metaphase II (M-II) labelled with Hoechst 33342 stain after in vitro maturation (IVM) and BCB+ selection. Scale bar represents 20 μm.
Discussion
Based on the current study, the BCB test was not a useful criterion to assess developmental competence of equine oocytes cultured in vitro. As BCB was not consistently useful to assess developmental competence of porcine oocytes (Ishizaki et al., Reference Ishizaki, Watanabe, Bhuiyan and Fukui2009), despite the lack of a positive outcome in the present study and based on success in other species (Rodriguez-Gonzalez et al., Reference Rodriguez-Gonzalez, Lopez-Bejar, Velilla and Paramio2002; Pujol et al., Reference Pujol, Lopez-Bejar and Paramio2004), we remain steadfast in our belief that the concept still has merit. In that regard, further studies are essential to evaluate the expression of genes and G6DPH activity that might be involved on developmental competence of equine oocytes exposed to BCB.
Culture media may contribute to this poor equine oocytes development, but oocyte quality is most likely the limiting factor, due to the heterogeneous morphological characterization of various growing stages or atresia (Del Campo et al., Reference Del Campo, Donoso, Parrish and Ginther1990; Hinrichs & Schmidt, Reference Hinrichs and Schmidt2000). Alm et al. (Reference Alm, Torner, Lohrke, Viergutz, Ghoneim and Kanitz2005) described a non-invasive test using an indirect marker for bovine oocyte competence based on G6DPH activity. The expression of apoptosis-related genes and its correlation with competence of bovine immature oocytes after pre-selected evaluation using the BCB test showed the lack of significant differences between blastocyst rates (Opiela et al., Reference Opiela, Katska-Ksiazkiewicz, Lipinski, Słomski, Bzowska and Rynska2008). The need for a reliable method of predicting development competence of equine oocytes is clear.
In addition, we observed that eGH enhanced maturation in equine oocytes, consistent with previous reports in this specie (Marchal et al., Reference Marchal, Caillaud, Martoriati, Gerard, Mermillod and Goudet2003; Pereira et al., Reference Pereira, Lorenzo, Carneiro, Bilodeau-Goeseels, Kastelic, Pegoraro, Pimentel, Esteller-Vico, Illera, Silvan, Casey and Liu2006, 2011). Furthermore, in cattle, rbGH in maturation medium improved nuclear maturation of bovine oocytes and stimulated subsequent embryonic development in vitro (Izadyar et al., Reference Izadyar, Colenbrander and Bevers1996, Reference Izadyar, Hage, Colenbrander and Bevers1998). In the present study, the percentage of BCB+ oocytes that reached M-II (53.8%) seemed lower than those numbers reported with oocytes from heifers (62%) (Pujol et al., Reference Pujol, Lopez-Bejar and Paramio2004) and pigs (81%) (Roca et al., Reference Roca, Martinez, Vazquez and Lucas1998). It was noteworthy that there were no indications that BCB impaired the overall nuclear maturation rate.
Our experiment revealed that the difference between BCB– and BCB+ oocytes is often subjective and that the procedure itself requires a 90 min incubation, which can makes this technique time consuming and can affect the developmental competence of the screened oocytes. Accordingly to our IVM results, we observed an effect of eGH on equine oocyte maturation in vitro. In conclusion, despite the urgent need to assess developmental competence of equine oocytes cultured in vitro, the BCB test was not a useful criterion based on the utility of this test in other species, this test possibly could be adapted to provide predictive information.
Acknowledgements
The authors thank Paul Panich (Lethbridge Research Centre, Lethbridge) for technical assistance with recovery of COCs. This research was supported by The Del Amo Program Grant (University of California, Davis and Universidad Complutense de Madrid, Spain) and The Coordination for the Improvement of Higher Education Personnel (CAPES), Brazil. No conflicts of interest have been declared.
