Introduction
Although successful pregnancies from cryopreserved bovine oocytes have been reported (Fuku et al., Reference Fuku, Kojima, Shioya, Marcus and Downey1992), the efficiency of producing transferable blastocysts after in vitro fertilization (IVF) is still low even after application of vitrification protocols (Chian et al., Reference Chian, Kuwayama, Tan, Tan, Kato and Nagai2004). Possible reasons for high sensitivity of oocytes to cryopreservation include large cell size and low permeability of water and cryoprotective agent (Leibo, Reference Leibo1981), meiotic spindle disassembly and chromosome misalignment (Shi et al., Reference Shi, Zhu, Zhang, Wang, Tang, Hou and Tian2006), and oocyte activation prior to IVF (Larman et al., Reference Larman, Sheehan and Gardner2006). We have recently reported that vitrification of bovine oocytes induces frequent multiple aster formation and leads to impaired pronuclear migration and development before the first cleavage (Hara et al., Reference Hara, Hwang, Kagawa, Kuwayama, Hirabayashi and Hochi2012).
Glutathione (l-γ-glutamyl-l-cysteinyl-glycine; GSH), a major non-protein sulfydryl compound, plays an important role in the protection of cells against the destructive effects of reactive oxygen species and regulating syntheses of DNA and proteins (Meister, Reference Meister1983). GSH level increases during oocyte maturation in the ovary and reaches a peak at the metaphase-II stage (Perreault et al., Reference Perreault, Barbee and Slott1988). However, the GSH levels of oocytes matured under in vitro conditions are lower when compared with those of ovulated oocytes, as reported in some species (Brad et al., Reference Brad, Bormann, Swain, Durkin, Johnson, Clifford and Krisher2003; Rodríguez-González et al., Reference Rodríguez-González, López-Bejar, Mertens and Paramio2003; Kim et al., Reference Kim, Hossein, Oh, Fibrianto, Jang, Kim, Hong, Park, Kang and Lee2007; Ge et al., Reference Ge, Sui, Lan, Liu, Wang and Tan2008). GSH synthesis in oocytes during in vitro maturation (IVM) may be disturbed by a low availability of cysteine (Meister, Reference Meister1983; Furnus & de Matos, Reference Furnus and de Matos1999). Low-molecular-weight thiol compounds, such as β-mercaptoethanol (βME) and cysteamine, can promote cysteine (cystine) uptake through formation of a mixed disulfide compound (e.g. βME-cysteine; Ishii et al., Reference Ishii, Bannai and Sugita1981; Ohmori & Yamamoto, Reference Ohmori and Yamamoto1983). In addition, such thiol compounds supplemented into IVM medium can increase intracellular GSH level and the developmental potential of the oocytes in several domestic species including pig (Abeydeera et al., Reference Abeydeera, Wang, Cantley, Prather and Day1998) and cattle (de Matos et al., Reference de Matos, Furnus, Moses, Martinez and Matkovic1996).
It has been reported that GSH in bovine IVM–IVF oocytes can stimulate sperm aster formation (Sutovsky & Schatten, Reference Sutovsky and Schatten1997) and that the GSH level of porcine oocytes is adversely affected by vitrification (Somfai et al., Reference Somfai, Ozawa, Noguchi, Kaneko, Kuriani Karja, Farhudin, Dinnyés, Nagai and Kikuchi2007). Therefore, we hypothesize that the decreased level of GSH in vitrified–warmed bovine oocytes may be responsible for abnormal aster formation and poor developmental potential. In the present study, the effect of GSH content of vitrified bovine oocytes on multiple aster formation and subsequent in vitro development was investigated.
Materials and methods
In vitro maturation
Unless otherwise stated, all chemicals used in this study were purchased from Sigma-Aldrich Chemicals (St. Louis, MO, USA). Abattoir-derived bovine ovaries were transported to the laboratory in saline (maintained at 10 to 12°C) within 24 h of slaughter. The contents of 2–8 mm follicles were aspirated with an 18-G needle connected to a 10-ml syringe. Oocytes that were surrounded with at least two layers of compact cumulus cells were cultured in HEPES-buffered Tissue Culture Medium (TCM)-199 (Earle's salt; Gibco BRL, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (FBS; SAFC Biosciences, Lenexa, KS, USA), 0.2 mM sodium pyruvate, 0.02 AU/ml FSH (Kyoritsu Seiyaku Co., Tokyo, Japan), 1 μg/ml 17β-estradiol, and 50 μg/ml gentamycin sulfate for 22 h at 38.5°C under 5% CO2 in air. To increase the intracellular GSH level, 50 μM βME (Takahashi et al., Reference Takahashi, Nagai, Hamano, Kuwayama, Okamura and Okano1993) and 1 mM l-cysteine (Choe et al., Reference Choe, Shin, Kim, Sho, Kim, Choi, Han, Han, Son and Kang2010) (βME/Cys) were added to the maturation medium. After the maturation culture, cumulus cells were removed by brief vortex mixing in HEPES-buffered TCM-199 supplemented with 3 mg/ml bovine serum albumin (BSA), 0.2 mM sodium pyruvate, 1000 IU/ml hyaluronidase, and 50 μg/ml gentamycin sulfate. Oocytes with an extruded first polar body were defined as matured and were used for experiments.
Vitrification and warming
Matured oocytes were subjected to vitrification according to the method described previously by Hara et al. (Reference Hara, Hwang, Kagawa, Kuwayama, Hirabayashi and Hochi2012). Briefly, oocytes were equilibrated with 7.5% ethylene glycol (EG; Wako Pure Chemical Industries Co., Osaka, Japan) and 7.5% dimethylsulfoxide (DMSO; Wako) in HEPES-buffered TCM-199/20% FBS base medium for 3 min at room temperature, and then transferred into a vitrification solution that consisted of 15% EG, 15% DMSO and 0.5 M sucrose in the base medium for approximately 60 s at room temperature. Within this 60 s, up to eight oocytes were loaded onto the top of the polypropylene strip of a Cryotop (Kitazato BioPharma Co., Shizuoka, Japan) with a minimal amount of the vitrification solution, and then quickly immersed into liquid nitrogen (LN2).
After storage for 1 to 10 weeks in a liquid nitrogen tank, oocytes were warmed by immersing the polypropylene strip of a Cryotop into 3 ml of the base medium that contained 1 M sucrose at 38.5°C, and left for 1 min. The oocytes were transferred to the base medium at room temperature in a stepwise manner (0.5, 0.25, and 0 M sucrose for 3, 5, and 5 min, respectively). Oocytes were cultured in HEPES-buffered TCM-199 supplemented with 5% FBS, 0.2 mM sodium pyruvate and 50 μg/ml gentamycin sulfate (TCM-199/5% FBS) for 1–2 h at 38.5 °C under 5% CO2 in air before being subjected to GSH measurement or IVF.
Measurement of intracellular GSH
Intracellular GSH content was measured by a 5,5′-dithio-bis(2-nitrobenzoic acid)-GSH reductase recycling assay with a total glutathione quantification kit (Dojin Molecular Technologies Inc., Kumamoto, Japan). Oocytes were washed three times with Ca2+-/Mg2+-free phosphate-buffered saline (PBS) that contained 1 mg/ml polyvinylpyrrolidone (PVP). According to the instructions, pools of 35–40 oocytes from each treatment were transferred to 12 μl of 10 mM HCl in a 0.6-ml microfuge tube. Then the oocytes were frozen in LN2 and were thawed at room temperature. This freeze–thaw procedure was repeated twice and the oocytes were stored at –80 °C until being assayed. After the final thawing, 3 μl of 5% 5-sulfosalicylic acid was added to the samples and the tubes were centrifuged for 10 min at 8000 g. Next, 10 μl of supernatant was diluted with 40 μl purified water, and then 20 μl of the sample solution was transferred to each well of 96-well microplate preloaded with 20 μl co-enzyme working solution, 120 μl buffer solution and 20 μl enzyme working solution. After incubation for 10 min at 37.0°C, 20 μl of substrate working solution was added to the each well. The absorbance at 405 nm was determined by a microplate reader (Bio-Rad Laboratories Inc., Hercules, CA, USA) following incubation for 30 min at room temperature. The total GSH content (pmol/oocyte) was calculated by reference to a standard curve prepared with authentic GSH.
In vitro fertilization and culture
Commercially available frozen semen from a Japanese Black bull was used. After thawing in a water bath at 37°C for 30 s, the contents of a 0.5-ml straw were layered on top of a Percoll density gradient that consisted of a 2 ml layer of 45% Percoll above 2 ml of 90% Percoll in a 15-ml conical tube, and then centrifuged for 20 min at 700 g. The pellet was re-suspended in 4 ml of modified Brackett & Oliphant (mBO) medium (IVF100; Institute for Functional Peptides, Yamagata, Japan) supplemented with 5 mM theophylline, washed twice (5 min at 300 g each) and then re-suspended in the mBO medium supplemented with 5 mg/ml BSA and 10 μg/ml heparin (IVF medium) to yield a concentration of 1.5 × 107 sperm cells/ml. Ten to 12 matured oocytes in the IVF medium were co-incubated with the above sperm suspension at a final concentration of 3 × 106 sperm cells/ml for 6 h in 100-μl microdrops under mineral oil at 38.5°C under 5% CO2 in air.
Up to 30 presumptive zygotes were cultured in a 250-μl microdrop of modified synthetic oviduct fluid (mSOF; Holm et al., Reference Holm, Booth, Schmidt, Greve and Callesen1999), supplemented with 30 μl/ml essential amino acids solution (50×, Gibco-11130), 10 μl/ml non-essential amino acids solution (100×, Gibco-11140) and 5% FBS at 39.0°C under 5% CO2, 5% O2 and 90% N2 for up to 8 days. Cleavage rate was determined on day 2 (day 0 was defined as the day of IVF), and number of blastocysts were recorded on days 7 and 8.
Immunostaining of pronuclear zygotes
To assess the aster formation of pronuclear zygotes, inseminated oocytes were cultured for an additional 4 h in TCM-199/5% FBS at 38.5°C under 5% CO2 in air, and then immunostained according to the method described previously (Hara et al., Reference Hara, Abdalla, Morita, Kuwayama, Hirabayashi and Hochi2011). The zygotes were extracted for 15 min by buffer M (25% glycerol, 50 mM KCl, 0.5 mM MgCl2 0.1 mM EDTA, 1 mM EGTA and 50 mM imidazole hydrochloride, pH 6.8) that contained 5% (v/v) methanol and 1% (v/v) Triton X-100, after zonae pellucidae had been removed with 0.75% protease in M2 medium. The zygotes were then fixed with cold methanol for 10 min and permeabilized overnight in PBS that contained 0.1% (v/v) Triton X-100. Microtubules were labelled with a monoclonal antibody against α-tubulin (T5168; diluted 1:1000). The primary antibodies were detected by fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (F1010; diluted 1:200). Nuclear DNA was visualized by counterstaining with 2.5 μg/ml 4′,6-diamidino-2-phenylindole (DAPI). Preparations were mounted with coverslips in antifade agent, and digital images were collected at 2 μm distance using a confocal laser scanning microscope (FV1000-D; Olympus, Tokyo, Japan). The digital images were stacked and assessed with Image-J software (National Institutes of Health, Bethesda, ML, USA; accessed on-line). 2-PN zygotes were defined as those that fertilized normally, and the larger pronucleus was defined as male pronucleus.
Statistical analysis
Maturation rate of cumulus–oocyte complexes and morphological survival rate of vitrified oocytes were compared between non-treated and βME/Cys-treated groups by Student's t-test. Arcsin-transformed data for cleavage rate, blastocyst yield, fertilization rate, polyspermic penetration rate and aster formation rate, as well as data for GSH content, distance between male and female pronuclei and pronuclear size were compared by one-way analysis of variance (ANOVA). When the ANOVA was significant, differences among means were analyzed by Tukey's test. A value of P < 0.05 was defined as a significant difference.
Results
Effect of βME/Cys in IVM medium on intracellular GSH content
As oocyte maturation rate was assessed with presence of the first polar body, treatment of the cumulus–oocyte complexes with βME/Cys did not influence the maturation rate (62%, 634/1029 versus 66%, 658/1004 in the non-treated group, P > 0.05). The GSH content of fresh control oocytes matured in the presence of βME/Cys was significantly higher than that of non-treated oocytes (P < 0.05; Fig. 1). After vitrification and warming, all the matured oocytes (n = 318 and 326 for βME/Cys and non-treated groups, respectively) appeared morphologically normal. The GSH content of the vitrified–warmed oocytes in the βME/Cys group remained at 2.5-fold higher than that of those in the non-treated group (P < 0.05).
Figure 1 Glutathione (GSH) content of bovine oocytes treated with β-mercaptoethanol and l-cysteine (βME/Cys) during in vitro maturation. Half the total of denuded mature oocytes were subjected to vitrification before GSH measurement. Mean ± standard error of the mean (SEM). a,bDifferent letters on SEM bars denote significant difference (P < 0.05).
Effect of increased GSH content on aster formation
Incidences of normal fertilization (2-PN), assessed by DAPI staining, were comparable among all the four groups (P > 0.05; Table 1). Polyspermic penetration occurred at similar rates between non-treated and βME/Cys groups regardless of vitrification (fresh control; 17 versus 16%, vitrified; 24 versus 24%). Immunostaining for α-tubulin indicated that the percentage of 2-PN zygotes that exhibited sperm aster(s) was high as >95% in all groups (Table 1). However, ratios of zygotes exhibiting multiple asters were more than three-fold higher in the vitrified group than those in the fresh control group (P < 0.05).
Table 1 Aster formation in pronuclear-stage bovine zygotes matured in the presence of βME/Cys, vitrified-warmed, and fertilized in vitro
Percentages were expressed as mean ± standard error of the mean (SEM) of six replicates in each group.
a,bDifferent superscripts denote significant difference within a column (P < 0.05).
Pronuclear migration and development of the βME/Cys-treated 2-PN zygotes, regardless of vitrification, were comparable with those of the non-treated zygotes (Table 2), as far as zygotes with a single aster were concerned. While zygotes with multiple asters exhibited an impaired migration and development of their pronuclei, neither treatment with βME/Cys nor vitrification also did not influence the extent of these parameters.
Table 2 Migration and development of pronuclei in bovine zygotes with a single aster or multiple asters
Numbers of 2PN-zygotes analyzed correspond to those in Table 1.
a–dDifferent superscripts within a column denote significant difference (P < 0.05).
Effect of increased GSH content on embryonic development
Cleavage rates of presumptive zygotes were comparable in all four groups (P > 0.05; Table 3). On the other hand, developmental potential of vitrified oocytes into blastocysts until day 8 was not improved by increasing intracellular GSH level with βME/Cys treatment (P > 0.05) and still lower than that of fresh control oocytes (P < 0.05). Within fresh control groups, increased level of intracellular GSH did not contribute to improve the blastocyst yield (P > 0.05).
Table 3 In vitro development of bovine oocytes with an increased level of GSH after vitrification and in vitro fertilization
Percentages were expressed as mean ± standard error of the mean (SEM) of four replicates in each group.
a,bDifferent superscripts denote significant difference within a column (P < 0.05).
Discussion
The effect of addition of low-molecular-weight thiol compound during IVM (to increase ooplasmic GSH level) on the developmental potential of cryopreserved bovine oocytes has not been investigated to date; while there is only one report on this methodology, attempted without success, in pig (Gupta et al., Reference Gupta, Uhm and Lee2010). Oocyte maturation rate was similar between non-treated and βME/Cys-treated groups, but mean GSH level in βME/Cys-treated oocytes (16.2 pmol/oocyte) was significantly higher than that in non-treated oocytes (6.5 pmol/oocyte; Fig. 1). Mizushima & Fukui (Reference Mizushima and Fukui2001) reported an enhanced maturation rate of bovine oocytes in the presence of βME. The composition of IVM medium, the density of oocytes during IVM and the period of ovary storage were different from those employed in the present study. Cryotop vitrification procedure did not decrease the GSH level of post-warm oocytes in both non-treated and βME/Cys-treated groups. Somfai et al. (Reference Somfai, Ozawa, Noguchi, Kaneko, Kuriani Karja, Farhudin, Dinnyés, Nagai and Kikuchi2007) reported a significant decrease in GSH levels in porcine oocytes after a solid-surface vitrification procedure. This decrease may depend upon species difference and/or suitability of the vitrification procedure.
After vitrification and IVF, bovine oocytes with increased GSH level exhibited similar incidence of multiple aster formation compared with the oocytes without increased GSH level (Table 1). Sutovsky & Schatten (Reference Sutovsky and Schatten1997) reported that sperm aster formation in bovine IVF oocytes was disturbed when the oocytes were treated with buthionine sulfoximine, a specific inhibitor of γ-glutamyl-cysteine synthetase. Yoshida et al. (Reference Yoshida, Ishigaki, Nagai, Chikyu and Pursel1993) also reported that intracellular GSH plays an important role in male pronuclear development of porcine IVF oocytes. To make the sperm centrosome act as a microtubule-organizing centre (MTOC), reducing activity for disulfide bonds is required (Schatten, Reference Schatten1994). However, our data suggest that a GSH level of <6 μmol/oocyte is enough to support the function of the sperm centrosome as an MTOC and to form both pronuclei in our IVF system. The higher incidence of multiple aster formation observed in vitrified–warmed bovine oocytes may be triggered by change of other ooplasmic components other than the GSH. Shimizu et al. (Reference Shimizu, Nagamori, Yabuta and Nojima2009) reported that knockdown of cyclin G-associated kinase by siRNA in HeLa S3 cells caused multiple aster formation, which was due to abnormal fragmentation of the pericentriolar material.
The extent of delayed or arrested pronuclear development (Table 2) and impaired development into the blastocyst stage (Table 3) in vitrified–warmed bovine oocytes following IVF was consistent with our previous study (Hara et al., Reference Hara, Hwang, Kagawa, Kuwayama, Hirabayashi and Hochi2012). There are several reports that deal with improved yields of bovine blastocysts after treatment of oocytes with thiol compound during IVM (de Matos et al., Reference de Matos, Furnus, Moses and Baldassarre1995, Reference de Matos, Furnus, Moses, Martinez and Matkovic1996, Reference de Matos, Herrera, Cortvrindt, Smitz, Van Soom, Nogueira and Pasqualini2002; Balasubramanian & Rho, Reference Balasubramanian and Rho2007), but our study failed to improve the blastocyst yield by increasing the GSH level even in fresh control oocytes (both 41% on day 8; Table 3). In those reports (de Matos et al., Reference de Matos, Furnus, Moses and Baldassarre1995, Reference de Matos, Furnus, Moses, Martinez and Matkovic1996, Reference de Matos, Herrera, Cortvrindt, Smitz, Van Soom, Nogueira and Pasqualini2002; Balasubramanian & Rho, Reference Balasubramanian and Rho2007), the blastocyst yields of fresh oocytes without thiol treatment were all less than 20% of the cultured oocytes. It is still unclear how much the developmental loss of cryopreserved bovine oocytes is a result of the abnormal microtubule assembly. Further study is required to elucidate the mechanism responsible for multiple aster formation and poor developmental potential of vitrified–warmed bovine oocytes.
In conclusion, the vitrification procedure did not decrease the intracellular GSH level of bovine oocytes stimulated by treatment with βME/Cys. However, high content of GSH in matured oocytes did not result in suppression of the high incidence of multiple aster formation and improvement of the poor developmental potential into blastocyst stage.
Acknowledgements
This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Sciences (no. 24580407).
