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a Centre for Early Human Development, Institute of Reproduction and Development, Monash University, Monash Medical Centre, Clayton, Victoria 3168, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A major step toward ameliorating media for the culture of bovine embryos was the discovery that the addition of Eagle's amino acids improved embryo development [1, 2, 6, 7]. Much of our understanding of the way that amino acids affect mammalian embryo development and subsequent viability has come from studies on the hamster [8–11], mouse [12–17], and rat [18]. These studies have determined that amino acids can be either stimulatory or inhibitory to embryo development in vitro and that the presence of amino acids in culture media has a significant effect on postimplantation development.
In a recent study by Partridge and Leese [19], the depletion of 19 amino acids from culture medium was measured during culture of bovine embryos. It was found that the rate of depletion of individual amino acids changed with developmental stage, suggesting that the bovine embryo changes its requirements for amino acids during development. Subsequently, Lane and Gardner [17] reported that the mouse embryo changed its requirements for amino acids as it developed from the zygote to the blastocyst. Development of the early cleavage stages was stimulated by the nonessential amino acids and glutamine but was not affected by the essential amino acids. The presence of the essential amino acids during culture of the early cleavage stages did, however, decrease subsequent blastocyst cell number and embryo viability following transfer. Furthermore, the presence of essential amino acids in the culture medium negated the beneficial effects of the nonessential group [13, 16, 17]. During development from the 8-cell stage to the blastocyst, the nonessential amino acids and glutamine stimulated blastocyst formation and hatching, while the essential amino acids increased blastocyst cell number and differentiation of cells into the inner cell mass (ICM). The study by Lane and Gardner [17] highlighted the importance of using a two-step culture system to evaluate the requirements of the developing embryo. It also demonstrated the need to assess the effect of amino acids on embryo development not only in terms of proportions reaching given stages but also on possible indicators of viability such as blastocyst cell number and the differentiation of cells into the trophectoderm (TE) and ICM.
The main objective of the present study was therefore to determine whether the bovine embryo changes its requirements for amino acids as it develops from the zygote to the blastocyst. A two-step culture system was used to determine the effect of amino acids during culture of both the early cleavage stages (zygote to Day 4 postinsemination [pi]) and the later stages of development (Day 4 to Day 7 pi). Embryo development, blastocyst cell number, and differentiation of cells into the TE and ICM were assessed. Further experiments were done to 1) determine the effect of altering the concentration of the essential amino acids and 2) assess the embryo's requirement for glutamine, and its potential role as an osmolyte, for both the early cleavage stages and later stages of development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bovine ovaries were obtained from the abattoir and transported to the laboratory in 0.9% saline. Within 5 h of collection, oocytes were aspirated from follicles with a diameter 2–5 mm, using a 19-gauge needle and hospital vacuum suction (approximately 75 mm Hg). Cumulus-oocyte complexes (COCs) were washed 3 times in Hepes-buffered TCM-199 (Sigma Chemical Company, St. Louis, MO) supplemented with 20 mM Hepes (Sigma), 5 mM NaHCO3 (Analar grade; BDH, Poole, Dorset, UK), 0.25 mM sodium pyruvate (Sigma), 60 µg/ml streptomycin (Sigma), 50 µg/ml penicillin (Sigma), and 4 mg/ml BSA (lipid-stripped, no. PSB10036IM; Life Technologies, Mulgrave, Australia); this wash is referred to below as H-199. Groups of 50 COCs with compact cumulus cells were then transferred to organ culture dishes (Falcon, Becton Dickinson, Victoria, Australia) containing 800 µl of equilibrated maturation medium and were matured for 22–24 h in 5% CO2 and air, at 39°C. Maturation medium was bicarbonate-buffered TCM-199 supplemented with 25 mM NaHCO3, 10% (v:v) fetal calf serum (Life Technologies), 0.25 mM sodium pyruvate, 60 µg/ml streptomycin, 50 µg/ml penicillin, 0.01 U/ml FSH (Sioux Biochemical, Sioux Center, IA), and 0.01 U/ml LH (Sioux Biochemical).
After maturation, COCs were washed 3 times in H-199 and transferred in groups of 10 to drops of fertilization medium in 60-mm culture dishes (non-pyrogenic; Falcon) containing 9 ml mineral oil (Sigma). Medium used for fertilization was Fert-TALP [20] containing 10 µg/ml heparin (Sigma) and 6 mg/ml BSA. A straw of frozen spermatozoa from a bull of proven fertility was thawed at 35°C for 30 sec, layered onto a Percoll (Sigma) gradient (45%/70%/90%), and centrifuged at 600 x g for 20 min. The pellet of motile sperm was then washed in 5 ml Fert-TALP and resuspended in approximately 500 µl Fert-TALP. Sperm was then added to drops of fertilization medium containing COCs to make a final volume of 50 µl, with 2 x 106/ml motile sperm per drop.
Culture Media
The base medium for embryo culture was a modified synthetic oviduct fluid (SOF [21]) containing 8 mg/ml BSA. When amino acids were added to SOF, the osmolarity of the medium was maintained at 270–280 mOsmol by adjusting the concentration of sodium chloride. All salts and glucose were of Analar grade (BDH). Sodium lactate, glutamine, sodium pyruvate, and phenol red were embryo culture tested (Sigma). Betaine and antibiotics were from Sigma. Eagle's minimum essential medium (MEM) essential amino acids without glutamine and nonessential amino acids [22] (Table 1) were supplied by ICN Biomedical (Seven Hills, Australia).
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Embryo Culture
At 18 h pi, presumptive zygotes were vortexed to remove all cumulus cells and washed 3 times in a Hepes-buffered modification of synthetic oviduct fluid (H-SOF), in which 20 mM NaHCO3 was replaced with 20 mM Hepes and 4 mg/ml BSA was added. Embryos were cultured in groups of 5 in 30-µl drops of medium, under 7 ml light mineral oil (Sigma), in a 60-mm culture dish at 39°C in an atmosphere 7% O2, 5% CO2, and 88% N2. All embryos were cultured for a period of 144 h (to Day 7 pi), with a change into fresh medium after the first 72-h culture.
Assessment of Embryo Morphology
For each experiment, embryo morphology was assessed after 72 h and 144 h of culture, using a stereomicroscope (x200). At 72-h culture, the percentage of embryos developing to at least the 8-cell stage was scored. At 144-h culture, development of embryos to the morula and blastocyst stages was scored. Development to the morula stage was determined in order for comparisons to be made with previous studies reporting the developmental competence of bovine embryos in various culture systems.
Determination of Total Cell Number and Differentiation of Cells
Embryos reaching the blastocyst stage after 144-h culture were dual stained in order to determine total cell number and the differentiation of cells into the ICM and the TE. The procedure was a modification of that used by Hardy et al. [23]. Blastocysts were treated with Pronase (0.5% [w:v]; Sigma) in protein-free H-SOF for 3–5 min, at 39°C, to remove the zona pellucida. Blastocysts were then washed in protein-free H-SOF and incubated for 10 min on ice, in 10 mM picrysulfonic acid (Sigma) in SOF with 4 mg/ml polyvinylpyrrolidone (Calbiochem Corporation, La Jolla, CA). After being washed in protein-free H-SOF, blastocysts were incubated for 60 min in H-SOF containing 0.1 mg/ml anti-dinitrophenol BSA (ICN) at 39°C. Blastocysts were again washed in protein-free H-SOF and incubated in the dark for 12–15 min in a 1/10 dilution of guinea pig complement (ICN) in H-SOF containing 20 µg/ml propidium iodide (Sigma), at 39°C. Blastocysts were then washed briefly in PBS and transferred to ethanol containing 25 µg/ml bisbenzimide (Hoechst 33258; Sigma). Blastocysts remained in bisbenzimide for at least 18 h in the dark at 4°C. Blastocysts were subsequently washed in ethanol before being mounted on a microscope slide in a small drop of glycerol. A coverslip was placed over the drop of glycerol before embryos were viewed under UV light on an inverted microscope (Leica, Sydney, Australia). Two filters were used to count ICM and TE cells. Both ICM (appeared blue) and TE (appeared orange) cells could be viewed with filter number 1 (excitation wavelength of 340–380 nm). Only TE cells could be viewed with filter number 2 (green/blue filter with an excitation wavelength of 350–460), enabling TE cells to be counted more easily.
Experiments
Experment 1a: Effect of amino acids during culture of embryos from the zygote to Day 4 pi Presumptive zygotes (18 h pi) were cultured for 72 h to Day 4 pi in one of five media: 1) SOF (control), 2) Gln (SOF with 1 mM glutamine), 3) NeGln (SOF with 1 mM glutamine and MEM nonessential amino acids), 4) EssGln (SOF with 1 mM glutamine and MEM essential amino acids), or 5) 20aa (SOF with 1 mM glutamine, MEM nonessential amino acids, and MEM essential amino acids). Embryos were then transferred in their respective treatment groups to 20aa for a further 72-h culture (to Day 7 pi).
Experiment 1b: Effect of amino acids during culture of embryos from Day 4 to Day 7 pi Because of the results of experiment 1a, presumptive zygotes (18 h pi) were cultured in NeGln for 72 h (to Day 4 pi). Embryos were then divided equally among one of five media: 1) SOF, 2) Gln, 3) NeGln, 4) EssGln, or 5) 20aa. Embryos were then cultured for a further 72 h (to Day 7 pi).
Experiment 1c: Role of nonessential and essential amino acids (in the presence and absence of glutamine) in blastocyst formation, cleavage, and cell differentiation from Day 4 to Day 7 pi. Presumptive zygotes (18 h pi) were cultured in NeGln for 72 h (to Day 4 pi). Embryos were then divided equally among one of five media: 1) Ne (SOF with MEM nonessential amino acids), 2) NeGln, 3) Ess (SOF with MEM essential amino acids), or 4) EssGln. Embryos were then cultured for a further 72 h (to Day 7 pi).
Experiment 2a: Effect of reducing the concentration of essential amino acids during culture of embryos from the zygote to Day 4 pi Presumptive zygotes (18 h pi) were randomly allocated to SOF with 1 mM glutamine and MEM nonessential amino acids, plus MEM essential amino acids at one of three concentrations (single-, half-, or quarter-strength essential amino acids). Embryos were cultured for 72 h (to Day 4 pi) and then transferred in their respective groups to 20aa for a further 72-h culture (to Day 7 pi).
Experiment 2b: Effect of reducing the concentration of essential amino acids during culture of embryos from Day 4 to Day 7 pi Presumptive zygotes (18 h pi) were cultured for 72 h to Day 4 pi in NeGln. Embryos were then divided equally among 3 groups and transferred to SOF with 1 mM glutamine and MEM nonessential amino acids, plus MEM essential amino acids at one of three concentrations (single-, half-, or quarter-strength essential amino acids) for a further 72-h culture (Day 7 pi).
Experiment 3a: Role of glutamine and betaine during culture of embryos from the zygote to Day 4 pi Presumptive zygotes (18 h pi) were randomly allocated to one of four media: 1) NeGln (control; SOF with 1 mM glutamine and MEM nonessential amino acids), 2) Ne (SOF with MEM nonessential amino acids, 3) NeBet (SOF with 1 mM betaine and MEM nonessential amino acids), or 4) Bet (SOF with 1 mM betaine). Embryos were cultured for 72 h to Day 4 pi and then transferred in their respective groups to 20aa for a further 72-h culture to Day 7 pi.
Experiment 3b: Role of glutamine and betaine during culture of embryos from Day 4 to Day 7 pi Presumptive zygotes (18 h pi) were cultured for 72 h to Day 4 pi as for experiment 1b. Embryos were then divided equally among 3 groups and transferred to one of three media: 1) 20aa (control: SOF with 1 mM glutamine, MEM nonessential amino acids, and MEM essential amino acids), 2) 19aa (SOF with MEM nonessential and MEM essential amino acids, or 3) 19aaBet (19aa with 1 mM betaine). They then underwent a further 72-h culture to Day 7 pi.
Statistical Analysis
Differences among treatment means in each experiment were determined by an ANOVA, followed by the Tukey-Kramer multiple comparison test. Bartlett's test was used to check for homogeneity of variances. When variances were found to be heterogenous, data were analyzed by the Kruskal-Wallis nonparametric test, and differences between individual treatments were determined using Dunn's multiple-comparison test. For data expressed as percentages, an ANOVA was performed after arc sine transformation of original data expressed as proportions. When the degree of association between two variables was determined, the correlation coefficient (r) was calculated. A probability of p < 0.05 was considered significant for all statistical tests.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Culture of embryos in NeGln for 72 h from the zygote significantly increased the number of embryos reaching the 8- to 16-cell stage, compared to culture in EssGln (p < 0.05) or in the absence of amino acids (SOF: p < 0.05; Table 2). Culture of embryos in EssGln neither stimulated nor inhibited embryo development with respect to culture in SOF (p > 0.05). The addition of the essential amino acids to medium containing nonessential amino acids and glutamine (20aa) did not further affect embryo development (p > 0.05). Development of embryos to the 8- to 16-cell stage in media containing different groups of amino acids was predictive of subsequent blastocyst development. Blastocyst development was significantly higher when embryos had been cultured for the first 72 h in medium containing nonessential amino acids and glutamine (NeGln and 20aa) as compared to culture in EssGln or SOF (p < 0.05). The early cleavage stage showed a requirement for the nonessential amino acids, as culture for the first 72 h in NeGln resulted in significantly more embryos reaching the blastocyst stage than culture of embryos in Gln (p < 0.05). Development of embryos to the morula and blastocyst stages, however, was equivalent after culture in Gln, NeGln, and 20aa (p > 0.05).
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Culture with amino acids during the first 72 h did not significantly affect subsequent blastocyst cell number when compared to that in the control (SOF: p > 0.05; Fig. 1a). Culture in EssGln, however, did depress subsequent blastocyst cell number and resulted in blastocysts with significantly fewer cells than after culture in Gln or NeGln (p < 0.05). The depression in total cell number after culture in EssGln was seen in both the TE and ICM and was significant in the ICM (p < 0.05; Fig. 1a). There was, however, no significant difference between any of the treatments in the proportion of cells allocated to the ICM (p > 0.05; Fig. 1b). During the first 72-h culture, the essential amino acids counteracted the stimulatory effect of glutamine on blastocyst cell number (EssGln vs. Gln: p < 0.05; Fig. 1a).
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Experiment 1b: Effect of Amino Acids During Culture of Embryos from Day 4 to Day 7 pi
From Day 4 to Day 7 pi, the bovine embryo showed a requirement for glutamine and both the nonessential and essential amino acids. While development to the morula and blastocyst stages combined was not significantly different after culture in Gln, NeGln, EssGln, and 20aa (p > 0.05), culture in 20aa resulted in the formation of significantly more blastocysts than did any other treatment (p < 0.05; Table 3). Further, culture in 20aa resulted in a significantly higher blastocyst cell number (p < 0.05; Fig. 1c). Analysis of the differentiation of cells revealed that culture in 20aa significantly increased the number of cells in both the TE and ICM (p < 0.05; Fig. 1c). Further, culture in 20aa significantly increased the proportion of total cells allocated to the ICM in comparison to that in embryos cultured in SOF, Gln, or EssGln (p < 0.05; Fig. 1d). Proportions of cells in the ICM of embryos cultured in NeGln and 20aa were not significantly different (p > 0.05).
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Experiment 1c: Role of the Nonessential and the Essential Amino Acids (in the Presence and Absence of Glutamine) in Blastocyst Formation, Cleavage, and Cell Differentiation from Day 4 to Day 7 pi
Blastocyst formation as well as development to the morula stage was significantly higher after culture with nonessential amino acids (Ne and NeGln) than after culture with essential amino acids in the absence of glutamine (Ess: p < 0.05; Table 4), but not in the presence of glutamine (EssGln: p > 0.05). There was no significant difference between the effect of the nonessential and essential amino acids on total cell number or the differentiation of cells into TE and ICM (Ne vs. Ess: p > 0.05; Fig. 1e). Further, neither the nonessential nor essential amino acids on their own affected the proportion of cells in the ICM (p > 0.05; Fig. 1f). The addition of glutamine to medium containing nonessential or essential amino acids did not significantly affect morula or blastocyst development, cell number, or cell differentiation (Ne vs. NeGln; Ess vs. EssGln: p > 0.05).
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Experiment 2a: Effect of Reducing the Concentration of Essential Amino Acids During Culture of Embryos from the Zygote to Day 4 pi
There was a significant inverse correlation between the concentration of the essential amino acids in culture medium during the first 72-h culture and 1) development to the 8- to 16-cell stage (r = -0.99, p < 0.01; Fig. 2a) and 2) subsequent blastocyst formation (r = -0.99, p < 0.01; Fig. 2a). There was no correlation between the concentration of essential amino acids and blastocyst cell number (r = -0.66, p > 0.05; Fig. 2b).
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Experiment 2b: Effect of Reducing the Concentration of Essential Amino Acids During Culture of Embryos from Day 4 to Day 7 pi
There was a significant inverse correlation between the concentration of the essential amino acids in culture medium during the second 72-h culture and blastocyst formation (r = -0.99, p < 0.01; Fig. 2c). There was no correlation between the concentration of essential amino acids and blastocyst cell number (r = -0.70, p > 0.05; Fig. 2d).
Experiment 3a: Role of Glutamine and Betaine During Culture of Embryos from the Zygote to Day 4 pi
Culture in NeGln resulted in significantly more embryos reaching the 8- to 16-cell stage than culture in Ne (p < 0.05; Table 5). Subsequent blastocyst development was also higher after culture in the presence of glutamine during the first 72 h (p < 0.05); however, blastocyst total cell number was not affected (p > 0.05; Table 5). Further, subsequent development to the morula stage was not affected by the absence of glutamine during the first 72-h culture as indicated by morula and blastocyst development combined (Ne vs. NeGln: p > 0.05; Table 5). In the presence of nonessential amino acids, betaine had no stimulatory effect during the first 72-h culture, with development to the 8- to 16-cell stage equivalent in Ne and NeBet (p > 0.05). However, subsequent blastocyst development from embryos cultured in NeBet was not significantly different from that of embryos cultured in the presence of glutamine (NeGln; p > 0.05). In the absence of the nonessential amino acids, culture with betaine resulted in poor development to the 8- to 16-cell stage and morula and blastocyst stages (Bet vs. Ne, NeGln, and NeBet; p < 0.05).
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Experiment 3b: Role of Glutamine and Betaine During Culture of Embryos from Day 4 to Day 7 pi
The bovine embryo had a requirement for glutamine from Day 4 to Day 7 pi. Culture in 20aa resulted in significantly more embryos reaching the blastocyst stage and the morula and blastocyst stages combined than did culture in 19aa in the presence or absence of betaine (20aa vs. 19aa and 19aaBet; p < 0.05; Table 6). Glutamine did not affect blastocyst cell number (19aa vs. 20aa; p > 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The fact that the bovine embryo exhibits a switch in its requirements for amino acids during development highlights the importance of using a two-step culture system when evaluating the role of specific media components in embryo culture. As the mammalian embryo develops in vivo, the composition of the reproductive tract changes [26] and there is a switch from maternal to embryonic genomic regulation [27]. Both of these events occur when the bovine embryo is at the 8- to 16-cell stage. In contrast to the early cleavage stages of the bovine embryo, the 8- to 16-cell stage to the blastocyst is marked by an increase in metabolic activity [28, 29], protein synthesis [30], oxygen consumption, and the uptake of carbohydrates [31]. Thus the optimization of culture medium must take into account the changing needs of the developing embryo [32–34].
In the present study, culture for the first 72 h in NeGln stimulated cleavage to the 8- to 16-cell stage and subsequent blastocyst development. During this period, EssGln and glutamine as the sole amino acid had no measurable effect on cleavage. When present with the nonessential amino acids, however, glutamine was found to be important for development of the early bovine embryo. The fact that NeGln increased cleavage is important for the optimization of culture media, as an increased rate of development of the early cleavage stage embryo has been correlated with an increase in viability [17, 35, 36]. Previous studies have illustrated the stimulatory effects of the combination of glutamine and the nonessential amino acids in development of mammalian embryos in vitro. Culture of mouse embryos from the 2-cell stage with Eagle's MEM nonessential amino acids and glutamine significantly increased blastocyst formation, cell number, hatching, and postimplantation development [13, 15]. The nonessential amino acids and glutamine were found to stimulate development of the mouse embryo by decreasing the time of the first three cleavage divisions [16]. When added during the entire culture period, the nonessential amino acids and glutamine have also been shown to stimulate development of the ruminant embryo [25, 37].
While it has been determined that the early-cleavage-stage mouse embryo has a requirement for the nonessential amino acids and glutamine [17], only one other study has addressed the effect of specific amino acids during culture of the early bovine embryo. Pinyopummintr and Bavister [38] reported that development of the bovine embryo to the 8- to 16-cell stage (72 h pi) was equivalent after culture in the presence of nonessential amino acids (media also contained glutamine), essential amino acids, or glutamine alone. The present study, however, showed that NeGln stimulated cleavage to the 8- to 16-cell stage (90 h pi) while EssGln and Gln had no effect on development. A feasible explanation for this apparent contrast is that the stimulatory effects of the nonessential amino acids and glutamine may not be evident until 90 h pi. This is supported by the fact that subsequent blastocyst development was higher after initial culture with nonessential amino acids and glutamine than after culture with the essential group [38]. Pinyopummintr and Bavister [38] found that culture with glutamine or the nonessential amino acids and glutamine to the 8- to 16-cell stage resulted in equivalent development to the blastocyst (Day 8 pi). In the present study, culture in Gln to the 8- to 16-cell stage significantly decreased subsequent blastocyst development (Day 7 pi) compared to culture in NeGln. Either culture in the presence of serum for the second culture period, or the extra day of culture in the study by Pinyopummintr and Bavister [38], could explain the observed contrariety in results. In the present study, culture of the early cleavage stages in Gln or NeGln resulted in equivalent proportions of embryos at the morula/blastocyst stages on Day 7 pi.
After Day 4 pi, the bovine embryo showed a requirement for the nonessential amino acids, the essential amino acids, and glutamine. The combination of all 20 amino acids stimulated blastocyst development, total cell number, the number of cells in the TE and ICM, and allocation of cells to the ICM. This is a significant finding for the optimization of culture medium, as the numbers of cells in the blastocyst (total) and the ICM have been positively correlated with blastocyst viability [17, 39]. Further, Iwasaki et al. [40] reported that in vitro-produced bovine blastocysts had lower total and ICM cell numbers and a smaller proportion of cells in the ICM than in vivo-produced blastocysts. While the mouse embryo showed similar requirements for amino acid groups beyond the 8-cell stage, it was found that each group had quite specific functions in the development of the mouse blastocyst [17]. The nonessential amino acids and glutamine stimulated blastocyst formation and hatching, while the essential amino acids stimulated cleavage, differentiation of cells to the ICM, and fetal development after transfer. From the present study, it is evident that the function of the individual amino acid groups is not as defined in the development of the bovine blastocyst. With respect to the function of the essential amino acids, the nonessential amino acids stimulated blastocyst formation; however, blastocyst formation was highest after culture with a combination of nonessential and essential amino acids and glutamine. Neither the nonessential nor the essential group of amino acids, on their own, affected blastocyst cell number or the differentiation of cells in the blastocyst. The essential amino acids did not stimulate development of the ICM as was found in the mouse [17]. In fact, blastocysts with the highest proportion of cells in the ICM resulted from embryos that had been cultured for the second 72 h in the presence of the nonessential amino acids and glutamine. In support of the observed differences in the role of amino acids in development of murine and bovine blastocysts, Partridge and Leese [19] and Lamb and Leese [41] reported differences between the two species in the depletion of individual amino acids from culture medium containing blastocysts. The bovine blastocyst depleted significant amounts of aspartate, glutamate, threonine, and lysine [19], while the depletion of amino acids by the mouse blastocyst included aspartate, glutamate, and a larger number of essential amino acids: tyrosine, methionine, valine, phenylalanine, isoleucine, and leucine [41].
While the mammalian embryo requires the essential amino acids beyond the third cleavage division, it is believed that they may be detrimental to development of the early cleavage stages. Culture of the mouse embryo up to the 8-cell stage with essential amino acids negated the stimulatory effect of the nonessential amino acids and glutamine, reducing subsequent blastocyst cell number and postimplantation development [13, 17]. The essential amino acids methionine, phenylalanine, and isoleucine inhibited development of the hamster 1-cell embryo [11] but were not inhibitory beyond the 8-cell stage [8, 42]. This may be due to competition for transporters between nonessential and essential amino acids. Lewis and Kaye [43] found that isoleucine, leucine, methionine, and tryptophan all reduced the uptake of glutamine in mouse 2-cell embryos. In the present study, however, the essential amino acids did not counteract the stimulatory effects of the nonessential amino acids during culture of the early cleavage stage bovine embryo. Development to the 8- to 16-cell stage, subsequent blastocyst development, cell number, and the differentiation of cells into the ICM and TE were all equivalent for embryos that had been cultured for the first 72 h in 20aa or NeGln. Previous studies in cattle [25] and sheep [37] have also shown that the presence of the essential amino acids did not negate the stimulatory effects of the nonessential amino acids and glutamine. This indicates that the transport mechanisms for nonessential and essential amino acids may differ in sheep, cattle, mice, and hamsters.
The amino acids present in Eagle's MEM essential amino acids are at higher concentrations than are found in the ruminant reproductive tract [44, 45]. Bavister and McKiernan [10] found that a reduction in the concentration of several essential amino acids for culture of the hamster embryo changed them from inhibitory to stimulatory amino acids. Liu and Foote [25] reported that culture of bovine embryos from the 4-cell stage to the blastocyst with half the concentration of essential amino acids resulted in a significant increase in the proportion of hatching blastocysts. In the present study, a reduction in the concentration of Eagle's essential amino acids (when in combination with the nonessential amino acids and glutamine) was beneficial for both the early cleavage stages and the later stages of development. When the concentration of the essential amino acids was decreased during the first 72-h culture, there was a significant inverse correlation with embryo cleavage and subsequent blastocyst development. Interestingly, this effect was not restricted to the early cleavage stages. When embryos were not exposed to the essential amino acids until Day 4 pi, there was still a significant inverse correlation with concentration and blastocyst development. Cleavage, however, was not affected by the concentration of essential amino acids, as there was no correlation between concentration and blastocyst cell number.
Glutamine has been shown to have an important role in the in vitro development of embryos from several species. Culture with glutamine helped random-bred mouse embryos through the 2-cell block [14, 46], while the hamster embryo required glutamine for development beyond the 8-cell stage [8]. In the present study, glutamine was found to be an important amino acid for development of the bovine embryo from the zygote through to the blastocyst. During the first 72-h culture, the removal of glutamine from medium containing nonessential amino acids decreased cleavage to the 8- to 16-cell stage. Similarly, Gardner and Lane [14] found that the removal of glutamine from culture medium reduced the stimulatory effects of the nonessential amino acids in helping CF1 mouse embryos through the first cleavage division. Glutamine is likely important as an energy source for the early embryo [28, 29, 47]. Rieger et al. [28, 29] found that glutamine utilization by the bovine embryo was highest at the 2- to 4-cell stages and the expanding blastocyst stage, and almost 80% of the glutamine taken up by the early cleavage stages was metabolized through the tricarboxylic acid (TCA) cycle.
As well as stimulating development of the early cleavage stages, glutamine was required by the embryo beyond Day 4 pi. Partridge and Leese [19] measured the uptake of individual amino acids by Day 7 bovine blastocysts in SOF and did not find a significant depletion of glutamine, indicating that glutamine was not required by the bovine blastocyst. Likewise, the uptake of glutamine by the mouse blastocyst was not significant [41]. The present study, however, showed that although glutamine did not affect embryo cleavage or blastocyst cell number, the removal of glutamine from medium containing nonessential and essential amino acids significantly reduced blastocyst development. Considering the findings of Partridge and Leese [19], it would appear that glutamine is required after Day 4 pi but prior to development of the blastocyst. Rieger et al. [28], however, reported that glutamine uptake was constant from the 8-cell stage to the blastocyst, increasing significantly with blastocyst expansion and hatching. Furthermore, between 43% and 73% of the glutamine taken up by the embryo from the 8-cell stage to the blastocyst was metabolized through the TCA cycle. This indicates that glutamine is used for energy production beyond Day 4 pi, particularly at the blastocyst stage, when Na+/K+ ATPase is needed for blastocyst formation and expansion. It is unknown why there are inconsistencies between the findings of Rieger et al. [28] and Partridge and Leese [19]; however, several other studies have shown that glutamine is utilized by the bovine blastocyst [29, 48, 49].
As well as being a source of energy for the embryo, glutamine can act as an osmolyte to protect the embryo from osmotic stress [50]. Regulation of osmotic pressure is a critical factor for embryo development [51–54]. Osmolytes maintain proper cell function by protecting protein structure, particularly the structure of enzymes. Betaine, a nonmetabolizable, organic osmolyte, has also been found to protect the developing mouse embryo from detrimental increases in osmolarity [50, 55]. In the present study the role of glutamine as a potential osmolyte during culture of the bovine embryo was examined by substituting betaine for glutamine. Unlike glutamine, betaine did not stimulate cleavage of the early embryo. Betaine did, however, have a beneficial effect during the first 72-h culture, but this was not evident until the blastocyst stage. These results indicate that, as well as being an energy source, glutamine may function as an osmolyte for the early-cleavage-stage bovine embryo. It is not surprising that culture with betaine in the absence of any amino acids resulted in poor embryo development. Biggers et al. [55] showed that the stimulatory effect of betaine was largely dependent on the concentration of glutamine in the culture medium. The present study indicates that the stimulatory effect of betaine during the first 72-h culture is dependent on the presence of the nonessential amino acids. While several of the nonessential amino acids (glycine, proline, and alanine) have been shown to be effective osmolytes for the mouse embryo [50], culture in Ne was inferior to culture in NeBet or NeGln. In the study by Dawson and Baltz [50], however, the nonessential amino acids were tested at 10 times the concentration present in Eagle's MEM nonessential amino acids.
Glutamine did not appear to function in the capacity of an osmolyte during development from Day 4 pi. Betaine, unlike glutamine, was not able to stimulate blastocyst formation. Liu and Foote [54] found that betaine could not protect the developing bovine embryo from an increase in the concentration of NaCl (95–122 mM). Glutamine was also present in the culture medium, indicating that glutamine was not acting as an osmolyte. However, Liu and Foote [54] looked at the effect of osmolytes from the 4-cell stage onward only. Betaine and glutamine may function as osmolytes during the first 2 cell divisions, when glutamine uptake has been reported to be high in the bovine embryo [19, 28]. In support of this, both betaine and glutamine were found to stimulate development of the early-cleavage-stage mouse embryo in vitro by reducing the time of the first 3 cleavage divisions (unpublished results). Although there was no observed effect of betaine on cleavage to the 8- to 16-cell stage in the present study, an increase in subsequent blastocyst development indicates that betaine may protect other cellular functions during development of the early cleavage stages.
Thus, while glutamine is required by the bovine embryo from the zygote through to the blastocyst, its function may change. This is not surprising, as the physiology of the embryo changes with development. In fact, Lewis and Kaye [43] found a switch in the transport mechanisms for glutamine, from systems L and gly in the mouse 2-cell embryo to system B0,+ in the blastocyst.
The presence of BSA in experiments designed to examine the role of amino acids in embryo development may be perceived as a potential source of contamination to the available amino acid pool, potentially compromising results. However, Walker et al. [56] determined that the concentration of amino acids in medium containing BSA was not significantly different from that in medium without BSA. Further, the addition of BSA to medium containing amino acids did not improve embryo viability following transfer [56].
In conclusion, this study determined the role of amino acids in the culture of the bovine embryo and showed that the embryo has a switch in its requirements for amino acids as it develops from the zygote to the blastocyst. Development of the early cleavage stages was stimulated by the nonessential amino acids and glutamine, while development beyond Day 4 pi was stimulated by a combination of the nonessential and essential amino acids and glutamine. Culture with all 20 amino acids increased blastocyst formation, cell number, and the differentiation of cells into the ICM and TE. Further, the nonessential amino acids and glutamine increased the proportion of cells allocated to the ICM. The study also revealed the embryo's requirement for glutamine from the zygote through to the blastocyst. Further, the results suggest that a reduction in the concentration of essential amino acids would be beneficial to culture of the bovine embryo. Overall, this study highlights the need to consider the species and the changing requirements of the embryo as it develops from the zygote to the blastocyst when one is optimizing culture medium.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Tracey E. Steeves, Centre for Early Human Development, Institute of Reproduction and Development, Monash University, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168, Australia. FAX: 61 3 9594 7311; tracey.steeves{at}med.monash.edu.au
3 Current address: Colorado Center for Reproductive Medicine, Englewood, CO 80110.
Accepted: April 26, 1999.
Received: February 9, 1999.
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