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Hormones, Growth and Development Program,3 Ottawa Health Research Institute,
Department of Obstetrics and Gynecology (Division of Reproductive Medicine),4
Department of Cellular and Molecular Medicine,5
Department of Biology,6 University of Ottawa, Ottawa, Ontario, Canada K1Y 4E9
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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conceptus, embryo, environment
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, media have been developed in which even blocking-type mouse embryos will develop from the fertilized egg to the blastocyst stage. The first of these were CZB, a medium developed by Chatot et al. [6] and the simplex-optimized media (SOM) [7], of which KSOM, a version with increased K+ [8], was the most successful. The development of media permitting blocking embryos to develop from the fertilized egg to the blastocyst stage in vitro has allowed the identification of several physicochemical properties that are implicated in the two-cell block. The main changes in these media that led to relief of the two-cell block were a decrease in NaCl concentration and overall osmolarity, an altered pyruvate:lactate ratio, the addition of EDTA and glutamine, and optimized concentrations of the other components, including lowered glucose and phosphate [2].
Of these, one of the best studied has been osmolarity and increased salt concentration [9]. It is now well established that the two-cell block can be alleviated by decreasing the osmolarity of the medium [9–11]. Importantly, the block can also be overcome by including compounds in the medium that function as organic osmolytes that support maintenance of cell volume without severely perturbing cell biochemistry, in contrast with osmotically equivalent amounts of inorganic ions [12]. A number of compounds can serve as organic osmolytes in embryos, protecting them against increased osmolarity [10–13], one of the most effective of which is glycine [13, 14]. Recently, it has been found that osmotically regulated glycine accumulation by embryos is mediated by the GLYT1 glycine transporter, a member of the neurotransmitter transporter family. In early preimplantation mouse embryos, GLYT1 has a previously unreported function as a cell-volume-regulatory organic osmolyte transporter, explaining how glycine exerts its osmoprotective effect and mediates release from the two-cell block [15].
It is puzzling, however, that only embryos from certain genotypes of female mice seem to be subject to the two-cell block. If the block can be induced by something as fundamental as preventing normal cell volume regulation or perturbing metabolism, it would seem that it should occur in all embryos regardless of genotype. An alternate explanation, which is that all mouse embryos are susceptible to the two-cell block but that the threshold at which it is induced varies among genotypes, has not been adequately considered. We therefore hypothesized that the two-cell block is not restricted to particular mouse genotypes but is instead a general phenomenon to which they have varying susceptibility. The experiments reported here were designed to test this hypothesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All chemicals were obtained from Sigma (St. Louis, MO) unless otherwise noted and were embryo-culture grade or cell-culture grade. Some media used were based on KSOM embryo culture medium [8], except that glutamine (which can function as an organic osmolyte in embryos [12]) was omitted, and polyvinyl alcohol (PVA; 1 mg/ml) replaced BSA as the macromolecular component of the medium. Thus, the components of the KSOM medium used here were (in mM) NaCl (95), KCl (2.5), KH2PO4 (0.35), MgSO4·7H2O (0.2), Na lactate (10), glucose (0.2), Na pyruvate (0.2), NaHCO3 (25), CaCl2 (1.7), EDTA (0.01), K penicillin G (0.16), streptomycin (0.03). Other media were based on mMTF, a medium developed by Gardner and Lane based on measured concentrations of components in mouse tubal fluid [16], again with BSA replaced by PVA. This mMTF medium contained (in mM) NaCl (103), KCl (4.8), KH2PO4 (1.2), MgSO4·7H2O (1.2), Na lactate (4.8), glucose (3.4), Na pyruvate (0.37), NaHCO3 (25), CaCl2 (1.7), EDTA (0), K penicillin G (0.16), and streptomycin (0.03). Other modifications to media were made as described in the Results. To adjust osmolarity, appropriate amounts of the trisaccharide D(+) raffinose, which is not transported nor metabolized by mammalian cells, were added as previously described [12]; osmolarity of KSOM with no added raffinose was 250 mOsM, while osmolarity of mMTF was 270 mOsM. Osmolarities were confirmed using a vapor-pressure osmometer (model 5520; Wescor, Logan, UT). For flushing zygotes from the oviduct, modified Hepes-KSOM [8] was used in which glutamine was omitted and BSA replaced by PVA, as above.
Embryos and Culture
All mice were obtained from Charles River Canada (St-Constant, PQ). Their use in these experiments was approved by the Animal Care Committee of the Ottawa Health Research Institute (protocol 2003003) and followed the guidelines of the Canadian Council on Animal Care. Blocking embryos were obtained from female outbred CF1 mice. KSOM medium has been reported to support development of CF1-derived fertilized eggs past the two-cell stage [8], while, in contrast, embryos from these outbred mice have been reported to block completely at the two-cell stage in many media including mMTF [2, 16]. Nonblocking embryos were obtained from female B6D2F1 (referred to throughout as BDF1), which are F1 hybrid offspring of inbred C57Bl/6 females and DBA/2 males. To obtain embryos, females were superovulated by intraperitoneal injection of 5 IU eCG followed 47.5 h later by 5 IU hCG. Immediately after hCG injection, the females of either type were caged with BDF1 males overnight. Female mice were killed by cervical dislocation, and fertilized eggs (obtained approximately 20–22 h post-hCG) or two-cell embryos (44–46 h post-hCG) were removed from the excised oviducts by flushing with Hepes-KSOM. For eggs, 300 µg/ml hyaluronidase was added to remove adherent cumulus. Normal fertilization of eggs was confirmed by the presence of two pronuclei. Embryos were cultured in groups of 15 according to standard techniques, in microdrop cultures under mineral oil (Sigma or Aldrich Chemical Co., Milwaukee, WI) at 37°C in 5% CO2:air.
Data Analysis
For embryo culture experiments, development from fertilized eggs was recorded each day for 6 days, with the day on which eggs were obtained designated as Day 1 (1 day post-hCG). Development of in vivo-produced two-cell embryos was recorded each day for 5 days, with the day two-cell embryos were obtained designated as Day 2 (2 days post-hCG). Data are presented as the percentage of embryos reaching the two-cell stage on Day 2, the four-cell or greater stage on Day 3, the morula stage on Day 4, and the blastocyst stage on Days 5 and 6 post-hCG. Replicates consisted of individual groups of 15 embryos obtained on separate days and cultured under the same conditions. Data are presented as the percentage ± SEM of replicates to allow visual comparisons, but this SEM was not used for statistical comparison of these response data. Instead, for statistical comparisons, data were expressed as the inverse sine of the fraction of embryos in each replicate reaching the specified stage.
For data in which each treatment group was essentially independent and could be considered categorical (data in Figs. 1, 2, 5, and 8), one-way ANOVA was used to determine if there was significant variance above that expected by chance, which, if found, was then followed by the Tukey-Kramer multiple comparisons test to perform pairwise comparisons between individual means (InStat, GraphPad Software, San Diego). Thus, the significance of differences between each pair of individual treatment groups was obtained. Data that were instead best described by two independent variables (data in Figs. 4, 6, and 7), where one variable was categorical (female genotype, i.e., CF1 or BDF1) and one continuous (i.e., osmolarity or glucose/phosphate levels), were tested by a two-factor ANOVA (Microsoft Excel and Vista Visual Statistics System, F.W. Young, L.L. Thurston Psychometric Laboratory, University of North Carolina, Chapel Hill, NC). Here, whether the main effect of each independent variable is significant can be determined, but it is not possible to test for significant differences between individual points. Finally, data in which there were three independent variables (Fig. 3; one categorical, female genotype [CF1 or BDF1], and two continuous, glycine level and osmolarity) were assessed by univariate analysis with ANOVA assuming a linear model for dependence on the variables (SPSS 12.0, SPSS Inc., Chicago, IL). Again, whether the main effect of each independent variable is significant can be determined, but it is not possible to test for significant differences between individual points. Analyses of such embryo development data have previously been described in detail by Lawitts and Biggers [17]. Graphs are presented with superscripts to indicate differences between treatment groups when each group was considered independent and post hoc tests could be used. Where the data were considered to depend on continuous variables, graphs show only the data with the significance of the main effects of each independent variable given in the text.
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RESULTS |
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To confirm that the putative blocking and nonblocking embryos that we used here behaved as expected, fertilized eggs derived from CF1 or BDF1 females were cultured in parallel in either KSOM or mMTF medium. In KSOM, virtually all embryos from either CF1 or BDF1 females developed past the two-cell stage to the four-cell stage (Fig. 1, A and B; P > 0.05 for CF1 versus BDF1). In mMTF, almost all BDF1-derived embryos also developed past the two-cell stage (Fig. 1C; P > 0.05 versus either CF1 or BDF1 in KSOM). In contrast, embryos from CF1 females were nearly all blocked at the two-cell stage in mMTF, with fewer than 10% reaching the four-cell stage (Fig. 1D; P < 0.001 versus the other three cases). This confirmed that the two-cell block occurs in CF1 embryos but not BDF1 embryos in mMTF, while both will develop past the two-cell stage in KSOM, as expected.
A high proportion of embryos continued to develop to the blastocyst stage in KSOM medium for embryos from both female genotypes (CF1 or BDF1). However, development of CF1 embryos in KSOM seemed to be somewhat slower that that of BDF1 embryos because there was a trend toward a higher proportion of BDF1 embryos reaching the blastocyst stage by Day 5 in culture than CF1 embryos (although the difference did not reach significance), even though the final proportions reaching the blastocyst stage became nearly identical by Day 6 (Fig. 1, A and B; P < 0.05).
In mMTF medium, virtually no CF1 embryos reached the blastocyst stage, as expected from their poor development past the two-cell stage (Fig. 1D). Even BDF1 embryos, however, developed relatively poorly in mMTF medium compared with KSOM (Fig. 1C; P < 0.05 for blastocysts on Day 5, P < 0.01 on Day 6), with only about half the proportion of blastocysts forming in mMTF as in KSOM, even though transit through the two-cell block was nearly complete.
Thus, the two-cell block phenomenon can be clearly demonstrated to occur in CF1 but not BDF1 embryos in the blocking medium, mMTF. However, it is also clear that subsequent development of even nonblocking BDF1 embryos is compromised in mMTF.
A Two-Cell Block Can Be Induced in CF1 Embryos by Increased Osmolarity and Rescued by Glycine
A two-cell block can be induced in CF1 embryos cultured in KSOM simply by increasing the osmolarity of this medium [9, 12]. Almost all embryos reached the two-cell stage at 250 or 310 mOsM (100% and 99%, respectively). At 310 mOsM with glycine present, there was a small decrease in development to the two-cell stage (95%). When the osmolarity of KSOM was increased here from 250 to 310 mOsM by adding raffinose, more than 80% of embryos cultured from fertilized eggs became arrested at the two-cell stage (Fig. 2, A and B; P < 0.001 for development to the four-cell stage at 310 versus 250 mOsM). As previously shown [12, 15], glycine added to 310-mOsM medium was able to rescue development past the two-cell stage (Fig. 2C; development to the four-cell stage was not significantly different from development at 250 mOsM). Development to the blastocyst stage occurred at a high proportion for CF1 embryos cultured in KSOM at 250 mOsM, but was almost completely abolished at 310 mOsM (Fig. 2, A and B; P < 0.001). As expected, glycine substantially rescued development to the blastocyst stage at 310 mOsM (P < 0.01 versus without glycine), although it did not completely restore the levels supported at 250 mOsM (Fig. 2C). Thus, we confirmed that development of CF1-derived embryos can be blocked at the two-cell stage in KSOM medium by increasing its osmolarity.
Comparison of the Response of CF1 and BDF1 Embryos Cultured from Fertilized Eggs to Increased Osmolarity and Glycine
We next compared the effects of increasing osmolarity of KSOM medium on CF1- and BDF1-derived embryo development from fertilized eggs. Development from the one- to two-cell stages occurred at nearly 100% in KSOM at osmolarities up to 370 mOsM in embryos from both female genotypes (Fig. 3A). BDF1-derived embryos were somewhat more resistant to further increases in osmolarity than CF1, as can be seen most clearly by their substantially better development at 390 mOsM. Above this level, embryos of either type arrested at the one-cell stage, so that virtually no cleavage to the two-cell stage occurred at 410 mOsM or above. In embryos from both female genotypes (CF1 or BDF1), the presence of glycine rescued development from the one- to two-cell stage, so that deleterious effects were not evident below 430 mOsM in the presence of glycine, with cleavage to the two-cell stage substantially decreased only at 450 mOsM and above (Fig. 3A). The main effects of osmolarity, female genotype (CF1 or BDF1), and glycine on development to the two-cell stage were highly significant (P < 10–5 for each).
Further development past the two-cell stage to the four-cell stage was much more sensitive to increased osmolarity. CF1-derived embryos were blocked at the two-cell stage above about 290 mOsM, while glycine was able to rescue development up to about 350 mOsM. BDF1-derived embryos became blocked at the two-cell stage at osmolarities of 310 mOsM and above (Fig. 3B). Like CF1-derived embryos, development of BDF1-derived embryos was rescued by glycine, with the range of osmolarity that permitted development past the two-cell stage being extended by 60– 70 mOsM for both embryo types in the presence of glycine (Fig. 3B). The main effects of osmolarity, female genotype (CF1 or BDF1), and glycine on development past the two-cell stage to at least the four-cell stage were highly significant (P < 10–6 for each).
Subsequent development followed a similar pattern (Fig. 3, C–E), with BDF1-derived embryos continuing to exhibit better development at higher osmolarities than CF1-derived embryos. Another effect, wherein glycine exerted a deleterious effect on development of CF1-derived embryos at low osmolarities, was also evident. The etiology of this is unknown, but it has been previously shown to be reversed by preventing glycine import into the embryo [15], and thus may be due to excessive displacement of inorganic ions from the cytoplasm by accumulated glycine. Again, the main effects of osmolarity, female genotype (CF1 or BDF1), and glycine on development to the morula or blastocyst on Day 5 or 6 were highly significant (P < 10–6 for each).
Comparison of the Response of CF1 and BDF1 In Vivo-Produced Two-Cell Embryos to Increased Osmolarity
We then performed similar experiments, but instead beginning with two-cell embryos that had been produced in vivo and removed from the female tract at the late two-cell stage (Fig. 4). Again, for each stage of development, the effect of osmolarity on development was highly significant (P < 10–8). After 1 day in culture, CF1-derived embryos developed to at least the four-cell stage at osmolarities up to 330 mOsM, with the proportion that reached the four-cell stage falling to approximately 50% at 350 mOsM and showing complete arrest at 370 mOsM (Fig. 4A). In contrast, BDF1-derived two-cell embryos were more resistant to increased osmolarity, developing optimally up to 350 mOsM and still reaching about 30% development at 370 mOsM (Fig. 4A). Development to subsequent stages exhibited a similar pattern, with BDF1-derived embryos developing to morulae and blastocysts at a slightly higher rate than CF1 at increased osmolarity (Fig. 4, B–D). The effect of female genotype was significant for development to the four-cell or greater stage (Fig. 4A; P = 0.02) and to the blastocyst stage on Day 5 (Fig. 4C; P = 0.03). The differences in development between CF1- and BDF1-derived in vivo-produced two-cell embryos were small, however, compared with the large differences seen when culturing fertilized eggs. Thus, in vivo-produced embryos from either CF1 or BDF1 females will develop to the four-cell stage and beyond at much higher osmolarities than permit the development of their fertilized eggs to this point.
Induction of the Two-Cell Block Independent of Osmolarity
The results obtained above showed that embryos from both female genotypes would develop from fertilized eggs to the two-cell stage, or from in vivo-produced two-cell embryos to the four-cell stage, at much higher osmolarities than would permit development from fertilized eggs past the two-cell stage. We next wished to determine whether the apparent two-cell block phenomenon in classically nonblocking embryos, such as those derived from BDF1 females, only arose when development was perturbed by increased osmolarity or if this was a more general phenomenon. Thus, we sought to identify conditions that induced a two-cell block independent of osmolarity.
Above, we confirmed that mMTF medium produced a two-cell block in CF1-derived embryos. We therefore tested a series of media in which components of mMTF were altered stepwise from their concentrations in mMTF to those in KSOM, but with osmolarity held constant (at the higher level of mMTF) with added raffinose. We found that altering KCl and MgSO4 from the concentrations found in mMTF to those in KSOM did not significantly improve development past the two-cell stage, and the further addition of EDTA to this formulation also did not significantly increase development, although there was a clear trend toward improvement, especially with addition of EDTA (Fig. 5). Because glucose and phosphate have been reported to act synergistically to produce the two-cell block, we altered their concentrations simultaneously. We found that lowering the concentrations of glucose and phosphate (KH2PO4) to those found in KSOM (while maintaining constant osmolarity) significantly improved the proportion of CF1-derived embryos developing past the two-cell block to the four-cell or greater stage on Day 3 and to the morula stage on Day 4. Development to the four-cell or greater stage on Day 3 was now significantly better than in mMTF or in medium with lowered KCl and MgSO4 and showed a trend toward higher development (although not significant) over medium with these components lowered and with EDTA also added (Fig. 5, B and C). This improvement occurred with or without the addition of EDTA, as there was no further improvement with EDTA present. A further improvement, to the levels of development seen in KSOM, was then obtained either if glutamine was also added or if raffinose was omitted, and this also fully rescued subsequent development to the blastocyst stage. Because glutamine (like glycine) acts as an organic osmolyte in mouse embryos, both these effects are most likely due to osmotic effects, as shown above. Thus, of the components that were tested other than those directly related to osmolarity, glucose and phosphate were found to most strongly affect the ability of CF1-derived fertilized eggs to develop past the two-cell stage in vitro. This indicated that, under the conditions used here, a simultaneous increase in glucose and phosphate could, by itself, significantly increase the proportion of CF1-derived embryos that arrest at the two-cell stage independent of osmolarity.
Comparison of the Response of CF1- and BDF1-Derived Embryos Cultured from Fertilized Eggs to Increased Glucose and Phosphate
Because higher levels of glucose and phosphate in mMTF significantly increased the proportion of CF1-derived embryos blocking at the two-cell stage, we next tested whether further increasing glucose and phosphate could also induce a two-cell block in BDF1-derived embryos (which are not blocked at the level of glucose and phosphate in mMTF; Fig. 1C). As the base medium, we used modified mMTF in which the KCl, MgSO4, glucose, and phosphate concentrations had been altered to those of KSOM, so that development of CF1-derived fertilized eggs past the two-cell stage was supported, while retaining the osmolarity of mMTF with added raffinose (Fig. 5). To determine the effect of increased glucose and phosphate on embryos from both female genotypes (CF1 or BDF1), glucose and phosphate were increased from the levels in KSOM (0.2 mM glucose, 0.35 mM KH2PO4) to 1x, 2x, and 4x those of mMTF (3.4 mM glucose, 1.2 mM KH2PO4 = 1x).
Development from fertilized eggs to the two-cell stage was unaffected for BDF1-derived embryos at any glucose and phosphate level tested, but decreased somewhat for CF1-derived embryos at the highest level (4x; Fig. 6A). The main effect of glucose and phosphate just reached significance (P = 0.047), but there was not a significant effect of female genotype (CF1 or BDF1; P = 0.07) for development to the two-cell stage. Development to the four-cell stage was much more sensitive, however, with an increasing proportion of CF1-derived embryos blocking at the two-cell stage as glucose and phosphate levels were increased over the entire range tested, and BDF1-derived embryos completely blocking at the two-cell stage with 4x glucose and phosphate (Fig. 6B). Subsequent development to morulae and blastocysts also showed a similar pattern, with BDF1-derived embryos being more resistant than CF1-derived embryos, but nonetheless exhibiting decreased development with increased glucose and phosphate (Fig. 6, C and D). For development to each stage past the two-cell stage (Fig. 6, B–E), the main effect of glucose and phosphate was highly significant (P < 0.0001), as was the effect of female genotype (CF1 or BDF1; P < 0.001).
Comparison of the Response of CF1 and BDF1 In Vivo-Produced Two-Cell Embryos to Increased Glucose and Phosphate
We then cultured in vivo-produced two-cell embryos at 2x and 4x the glucose and phosphate levels in mMTF because these had the most substantial effects on development of fertilized eggs to the four-cell stage (above). At both 2x and 4x glucose and phosphate, BDF1-derived two-cell embryos reached the four-cell stage or greater at over 90% (Fig. 7A), and subsequent development was similarly high except for a trend toward a decrease in blastocyst formation at 4x (Fig. 7, B–D). A relatively high proportion of CF1-derived two-cell embryos (
60–70%) also developed to the four-cell stage at both glucose and phosphate levels (Fig. 7A), although subsequent development to morulae showed a trend toward decreasing at 4x, and blastocyst development was low at both glucose and phosphate levels (Fig. 7, B–D). The difference in development between 2x and 4x glucose and phosphate (main effect of glucose and phosphate) was insignificant at all stages except for development to the blastocyst stage by Day 5 (P = 0.03). The main effect of female genotype (CF1 or BDF1) was significant at all stages (P < 0.01).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The data presented here indicate that a similar two-cell block occurs in embryos derived from both CF1 and BDF1 females. For embryos from both female genotypes, there exists a range of osmolarities in which the fertilized egg will arrest at the two-cell stage but in vivo-produced two-cell embryos will develop to the four-cell stage and beyond. Some media among those we tested of various osmolarities, therefore, resulted in a pattern of development that fit the definition of a two-cell block in both CF1- and BDF1-derived embryos. Examples are shown in Figure 8A, where it is evident that similar patterns of development, indicative of a two-cell block, occurred with CF1 embryos in 310 mOsM medium and in BDF1 embryos in 330 mOsM medium.
The susceptibility of these mouse embryos to arresting at the two-cell stage in vitro likely reflects the same phenomenon in embryos derived from both types of females. In each case, the two-cell block could be rescued by the presence of glycine, which extended the range of osmolarities at which development to the four-cell stage will occur by about the same amount (60–70 mOsM) in each female genotype (CF1 or BDF1). We have previously shown that this phenomenon reflects the ability of CF1-derived embryos to use glycine as an organic osmolyte [12, 14, 15], and thus a similar osmoprotective effect in BDF1-derived embryos indicates a common mechanism.
This apparent two-cell block occurring in embryos from putatively nonblocking female mice was not unique to osmotic perturbations. We also found that fertilized eggs of both female genotypes became blocked at the two-cell stage in media with increased amounts of glucose and phosphate, while, in contrast, in vivo-produced two-cells would develop in the same media (Fig. 8B). Because we increased glucose and phosphate without changing osmolarity of the media, the phenomenon of a two-cell block in both BDF1- and CF1-derived embryos appears to be general and not simply a peculiar pattern due to a single type of perturbation such as osmotic stress.
Greater Perturbations Are Required to Produce the Two-Cell Block in Nonblocking Embryos
While a phenomenon resembling a two-cell block was evident in both genotypes of embryos tested here, the level of stress required to induce the block is apparently different. CF1-derived fertilized eggs ceased to develop past the two-cell stage at lower osmolarities than those of BDF1. As can be seen in Figure 3B, the proportion that developed to the four-cell or greater stage dropped to less than 10%—an essentially complete two-cell block—between 310 and 330 mOsM for CF1-derived embryos, while the corresponding point was at about 350 mOsM for BDF1-derived embryos. Similarly, CF1-derived embryos failed to develop in media with 2x the glucose and phosphate levels of mMTF, while a similar block to development only occurred in BDF1-derived embryos in media with 4x glucose and phosphate (Fig. 6B). Thus, while a similar two-cell block phenomenon apparently occurs in embryos from both female genotypes, lower levels of stress are needed to induce it in CF1- than in BDF1-derived embryos.
In general, all stages of embryos from CF1 females were more susceptible to perturbations than were those from BDF1, even when they were past the two-cell block. Thus, development of in vivo-produced two-cell embryos from BDF1 females were somewhat more resistant to increased osmolarity than those derived from CF1 females (Fig. 4), and a similar phenomenon occurred with respect to increased glucose and phosphate (Fig. 7). Susceptibility at later stages (e.g., the morula to blastocyst transition) cannot be inferred from the data presented here, as any decrease in development may be due to damage sustained during earlier stages that is not evident until later. However, it is likely that even later stages are sensitive, as we found that four-cell embryos produced in media of any osmolarity would develop to blastocysts at a high rate if they were transferred to optimal media (KSOM supplemented with amino acids; data not shown). Thus, the appearance of a two-cell block at lower thresholds of stress in CF1- versus BDF1-derived embryos is apparently a manifestation of a greater general sensitivity of early embryos of the former to stress.
Blocking and Nonblocking Embryos
Mouse embryos have long been divided into blocking and nonblocking, based upon the genotype of the female from which they were derived. Here, however, we showed that at least one female genotype considered to yield nonblocking embryos (BDF1) exhibits a two-cell block that is indistinguishable from that of a female genotype considered strongly blocking (CF1), except that moderately higher levels of stress are required for it to appear. We therefore propose that this division of female mice was an artifact of the classic culture media that were used when these assignments were being made. Culture media such as M16 and Whitten, among others, had higher osmolarity, lacked organic osmolytes and EDTA, and had greater levels of glucose and phosphate than the current generation of culture media that support development of putatively blocking embryos, such as KSOM, CZB, and those used clinically with human embryos [2, 8, 17]. The older media also probably caused higher levels of other stresses, such as oxidative stress, which is relieved by some antioxidants (e.g., taurine) in these newer media [2]. As discussed in detail by Biggers [2], these stresses clearly interact, so that the amount of stress caused by imbalance of a given component depends strongly on the other components and physical parameters of the medium. For example, levels of glucose and phosphate, which contribute strongly to the two-cell block in CF1-derived embryos in mMTF and other media, are, in contrast, completely benign in a medium based on KSOM [18, 19]. For this reason, simultaneous optimization of all components of the medium was required to develop successful culture media for all genotypes of mouse embryos and for embryos of other mammals and humans.
This work establishes that mouse embryos that are traditionally considered blocking and those considered nonblocking are both actually susceptible to a two-cell block. While it does not answer the question of why embryos from some female genotypes, particularly hybrids between inbred strains, have a higher threshold of tolerance before their embryos become blocked, it does provide evidence that the determinants of susceptibility to the two-cell block may not be discrete differences between genotypes but rather arise from a continuum of traits that differ by degree. This should provide a basis for further investigations into the molecular and genetic basis for the two-cell block phenomenon. These observations may be important clinically as well, as the different susceptibility of embryos to stress in animals of the same species but different genotypes may extend to humans, and at least partly explain the varying success rates between patients treated for infertility using in vitro fertilization and embryo culture.
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FOOTNOTES |
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2 Correspondence: Jay M. Baltz, Moses and Rose Loeb Research Centre, Ottawa Health Research Institute, 725 Parkdale Ave., Ottawa, ON K1Y 4E9, Canada. FAX: 613 761 5327; jbaltz{at}ohri.ca
Received: 21 June 2004.
First decision: 12 July 2004.
Accepted: 30 August 2004.
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