Introduction
Glutamine (Gln) is the most abundant amino acid in porcine follicular fluid (Hong & Lee, Reference Hong and Lee2007) and is constantly used as an energy substrate throughout development (Swain et al., Reference Swain, Bormann, Clark, Walters, Wheeler and Krisher2002). Similarly, addition of Gln to culture media improves in vitro development of porcine embryos (Petters et al., Reference Petters, Johnson, Reed and Archibong1990; Suzuki et al., Reference Suzuki, Yoshioka, Sakatani and Takahashi2007). However, addition of Gln has a potential drawback due to its inherent instability as Gln spontaneously degrades into ammonia and 2-pyrrolidone-5-carboxylic acid (Vickery et al., Reference Vickery, Pucher and Clark1935). In the female reproductive tract, ammonia products are absorbed or neutralized by surrounding epithelial tissues, however in the case of in vitro culture they accumulate in the culture medium (Gardner & Lane, Reference Gardner and Lane1993). Accumulated ammonia is detrimental to mammalian oocytes and to embryo development (Hammon et al., Reference Hammon, Wang and Holyoak2000).
The adverse effect of Gln can be avoided by using stable dipeptide derivatives such as alanyl-glutamine (Ala-Gln) or glycyl-glutamine (Roth et al., Reference Roth, Ollenschlager, Hamilton, Simmel, Langer, Fekl and Jakesz1988). Replacement of Gln with the dipeptide derivatives reduced the rates of polyspermy and apoptosis and increased cell numbers in blastocysts and the pregnancy rates in mice (Biggers et al., Reference Biggers, McGinnis and Lawitts2004; Summers et al., Reference Summers, McGinnis, Lawitts and Biggers2005). However, information on the effects of the dipeptide derivatives on porcine oocytes and/or embryo culture is scarce. Therefore, in the present study, we compared the use of Ala-Gln and Gln during in vitro maturation (IVM) and/or in vitro culture (IVC) of porcine oocytes/embryos in order to optimize porcine in vitro production (IVP) protocols.
Materials and methods
Materials
All chemicals and media were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA) except for Ala-Gln, which was obtained from Invitrogen (GlutaMAX™-I; Carlsbad, CA, USA).
In vitro maturation of porcine oocytes
Porcine oocytes were matured in vitro following our standard protocol described elsewhere (Park et al., Reference Park, Koo, Kwon, Kang, Jang and Lee2010) but with slight modification. Briefly, cumulus–oocyte complexes (COCs) were aspirated from abattoir-derived porcine ovaries, washed three times and cultured in IVM medium supplemented with 0.5 μg/ml follicle-stimulating hormone and 0.5 μg/ml luteinizing hormone at 39°C, in 5% CO2 in air for 22 h. The cultured COCs were washed and transferred to fresh IVM medium without hormones and cultured again for another 22 h under the same conditions. The IVM medium used was Medium 199 (without l-glutamine) supplemented with 10 ng/ml epidermal growth factor, 0.57 mM cysteine and 10% (v/v) porcine follicular fluid. For the experiments, either 2 mM l-Gln or Ala-Gln was added to the IVM medium.
Assessment of nuclear maturation of oocytes
Nuclear maturation was evaluated by presence of the first polar body. After IVM culture, oocytes were transferred to 0.1% hyaluronidase in IVM medium then pipetted gently until the cumulus cells were completely removed. Denuded oocytes were stained with 5 μg/ml of Hoechst 33342 for 20 min and observed under ultraviolet light.
Parthenogenetic activation and in vitro culture
Denuded oocytes were equilibrated in 280 mM mannitol solution supplemented with 0.1 mM MgCl2, 0.1 mM CaCl2 and 0.01% PVA for 1 min, then transferred to a chamber between two electrodes 3.2 mm apart in the same mannitol solution. The oocytes were activated with a single direct current pulse of 1.5 kV/cm for 60 μs using an electrocell manipulator (BTX ECM 2001, Harvard Apparatus Inc., Holliston, MA, USA). Activated oocytes were cultured with Porcine Zygote Medium-3 (PZM-3) (Yoshioka et al., Reference Yoshioka, Suzuki, Tanaka, Anas and Iwamura2002) under mineral oil at 39°C in a mixture of 5% CO2, 5% O2 and 90% N2. Embryos were evaluated for cleavage on day 2 and for blastocyst formation and total cell numbers of blastocysts on day 7 of culture. In this study, embryos greater than 8-cell in size on day 2 were regarded as early cleavage stages. During IVC, 2 mM of l-Gln or Ala-Gln was added to PZM-3 as per the experimental design described below.
Counting blastocyst cell numbers
Blastocysts total cell numbers were counted after staining with 25 μg/ml Hoechst 33258 for 20 min. Stained blastocysts were mounted on a glass slide in a drop of glycerol, gently flattened with a cover glass and visualized under a fluorescence microscope (Nikon Corporation, Tokyo, Japan).
Statistical analysis
All experiments were replicated at least five times and analysed statistically using Prism software (GraphPad, La Jolla, CA, USA). Student's t-test was used to analyze differences in maturation and early cleavage rates after IVM. One-way analysis of variance (ANOVA) was used to analyse cleavage and blastocyst rates and total cell number of blastocyst after IVC. A P-value less than 0.05 was considered to be significant.
Experimental design
For evaluating effects of Ala-Gln on IVM, oocytes were divided into two groups and cultured with Ala-Gln or Gln, respectively. After IVM, each group was activated and then cultured with PZM-3 with Gln for cleavage timing assessment.
For evaluating effects of Ala-Gln on IVC, oocytes were divided into four groups. Group G-g was cultured with Gln during both IVM and IVC. Group G-x was cultured with Gln during IVM and Ala-Gln for IVC. Group X-g was cultured with Ala-Gln for IVM and Gln for IVC. Group X-x was cultured with Ala-Gln during both IVM and IVC.
Results and Discussion
In the present study, we found that replacement of Gln with Ala-Gln improved both IVM and IVC results with culture of porcine oocytes/embryos. Oocyte quality after IVM culture can be assessed from two points of view, nuclear maturation and cytoplasmic maturation. Nuclear maturation involves resumption and progression of meiosis I and is assessed by extrusion of the first polar body. In this study, nuclear maturation was similar between Gln- and Ala-Gln-treated groups (Table 1). Cytoplasmic maturation involves structural redistribution of cellular organelles in the oocytes and synthesis of mRNA to initiate embryo development. It is difficult to evaluate cytoplasmic maturation, however in vitro development after activation indirectly reveals the extent of cytoplasmic maturation of oocytes. In the present study, while there was no difference between the Gln- and the Ala-Gln-treated groups in the proportion of activated oocytes that cleaved at least once, the Ala-Gln group showed a significantly higher proportion of early cleavage stage embryos compared with the Gln group on day 2 after activation (Table 2; 33.0 ± 4.8% versus 16.7 ± 2.8%, respectively). It has been proven that early cleaved embryos in many mammalian species including pigs have more developmental competence (Isom et al., Reference Isom, Li, Whitworth and Prather2011). We also observed in a previous study that early cleaved porcine embryos showed higher implantation rates (Koo et al., Reference Koo, Lee, Kang, Kwon, Park, Park, Kim, Jang and Lee2010). Consistent with this finding, in the present study the blastocyst rate in the Ala-Gln-treated IVM group (X-g group in Table 3; 36.4 ± 3.6%) was significantly higher than in the Gln-treated group (G-g group in Table 3; 23.2 ± 2.7%).
Effect of glutamine or alanyl-glutamine on in vitro nuclear maturation of porcine oocytes
Effect of glutamine or alanyl-glutamine treatment during in vitro maturation on cleavage timing of porcine parthenogenetic embryos
a ,b Different superscripts within same column represent significant differences.
Effect of glutamine or alanyl-glutamine treatment during in vitro maturation and/or in vitro culture on development of porcine parthenogenetic embryos
a–cDifferent superscripts within same column represent significant differences. G-g: Gln on both IVM and IVC; G-x: Gln on IVM and Ala-Gln on IVC; X-g: Ala-Gln on IVM and Gln on IVC; X-x: Ala-Gln on both IVM and IVC.
Effects of Ala-Gln on IVC of parthenogenetic embryos were also assessed. Cleavage rates were similar among all the groups in this study (Table 3). However, blastocyst rates showed significant differences. As mentioned above, replacement of Gln with Ala-Gln during IVM led to a significantly improved blastocyst rate (between G-g and X-g groups). This significant improvement was also found between groups G-x (34.2 ± 3.8%) and X-x (51.3 ± 1.94%). In particular, the blastocyst rate in the X-x group was significantly higher compared with all the other groups.
On the other hand, total cell numbers of blastocysts were independent from IVM conditions: there were no significant differences between the G-g and X-g or between G-x and X-x groups. However, total blastocyst cell numbers were increased significantly in the G-x and X-x groups (63.5 ± 4.5 and 63.2 ± 2.7, respectively) compared with the G-g and X-g groups (41.9 ± 1.8 and 47.4 ± 1.6, respectively). This situation means that higher cell number in blastocysts is connected with IVC conditions. Previous reports have shown that embryo cell number is correlated with cell death, particularly apoptosis (Hardy, Reference Hardy1997; Yu et al., Reference Yu, Ding, Wang, Chen, Li, Zhao, Fan, Wang, Beaujean, Zhou, Jouneau and Ji2007). The concentration of ammonia gradually increased during culture of embryos in the presence of Gln (Gardner & Lane, Reference Gardner and Lane1993). Ammonia induces apoptosis of blastomeres in a dose-dependent manner (Lane & Gardner, Reference Lane and Gardner2003). Consequently, reduced cell number in G-g and X-g groups in this study seems to be affected by accumulated ammonia from degraded Gln. As high cell number in blastocysts is critical for postimplantation development (Brison & Schultz, Reference Brison and Schultz1997), use of Ala-Gln rather than Gln to increase cell numbers of blastocyst is recommended for optimization of porcine IVP systems.
In conclusion, replacement of Gln with Ala-Gln during IVM did not improve nuclear maturation but did improve developmental competence after oocyte activation. Also, use of Ala-Gln during IVC increased the total cell number of blastocysts without reducing blastocyst formation rate. We therefore recommend the use of Ala-Gln in both IVM and IVC of porcine oocytes and embryos.
Acknowledgements
This study was supported by the Korean Ministry of the Knowledge and Economy (grant #10033839, #10033805). We thank Dr Barry D. Bavister for editing the manuscript and Yoon-Sang Kwon for technical support.
