MOLECULAR REPRODUCTION AND DEVELOPMENT 31:189-194 (1992)
Joint Effects of Sodium Chloride, Glutamine, and Glucose in Mouse Preimplantation Embryo Culture Media J.A. LAWITTS AND J.D. BIGGERS Laboratory of Human Reproduction and Reproductive Biology, Haruard Medical School, Boston, Massachusetts
ABSTRACT A new medium, SOM, produced by simplex optimization, has been used to study the joint effects of NaCI, glutamine, and glucose on the development of outbred CF1 mouse zygotes to the blastocyst stage. Contrary to previous reports, glucose has no significant inhibiting effect on development to the blastocyst stage in this medium. Even in the presence of 5 mM glucose, 70%of the embryos develop to at least four cells, and 60%reach the blastocyst stage. Raising the concentration of NaCl from 75 to 125 mM, in the absence of glutamine, progressively inhibits development. Moreover, the response to glutamine depends on the concentration of NaCl in the medium. When the NaCl concentration is low, glutamine inhibits development. In contrast,when the NaCl concentration is high, glutamine protects against the inhibitory effect of the salt. We propose that glutamine protects against high concentrations of NaCl in the medium by acting as an organic osmolyte. Key Words: Simplex optimized medium, NaCI, Osmolyte
INTRODUCTION
ten and Bavister (19831, Carney and Bavister (1987); mouse: Chatot et al. (1989); pig: Petters et al. (199011. Our experiments show that glutamine protects against elevated concentrations of NaCl in culture media, and we speculate that glutamine has this property by acting a s a n organic osmolyte. After 20 cycles of simplex optimization, a medium for the culture of mouse preimplantation embryos has now been produced that has only 85 mM NaCl (medium SOM). In this medium, described in this paper, complete development of pronuclear zygotes into blastocysts occurs in embryos from outbred CF1 mice, without any block to development at the two-cell stage. We also found the surprising fact that glucose in this medium does not inhibit development, a s has been reported in other media (Chatot e t al., 1989; Lawitts and Biggers, 1991b).
MATERIALS AND METHODS Animals Outbred female mice (Cr1:CFl BR; &8 weeks old) and hybrid male mice (BGD2Fl/CrlBR) were maintained on 14 h light and 10 h dark (lights on at 0500 h). Female mice were superovulated by intraperitoneal (i.p.) injection of 5 i.u. pregnant mare serum gonadotropin (PMSG; Sigma, St. Louis, MO) a t 1530 h, followed by a n i.p. injection of human chorionic gonadotropin (hCG; Sigma) 47 h later. After hCG injection, females were placed in cages with males and examined the following morning (day 1)for the presence of a vaginal Plug-
Most media used to culture mouse preimplantation embryos contain too much sodium chloride. This fact first became apparent from experiments using simplex optimization, which, after four cycles, generated medium aKH,PO, that maximized the yield in vitro of blastocysts from pronuclear zygotes (Lawitts and Biggers, 1991a). Additional evidence that the NaCl concentration is too high is provided by the fact that the Preparation of Culture Media two-cell block, which occurs when outbred CF1 embryos are cultured in medium M16 (Whittingham, Media were prepared from individual stock solutions 19711, disappears when the NaCl concentration is re- for each component of medium SOM (Table 1). Most of duced (Lawitts and Biggers, 1991b). A recent report the components were stored in 0.1 M solutions, with the also suggests that the concentration of NaCl in media following exceptions. NaCl and NaHCO, were stored at used to culture pig preimplantation embryos is also too 1.0 M, whereas bovine serum albumin (BSA, fraction high (Beckmann and Day, 1991). V) was stored at a concentration of 100 mg/ml, CaCl, a t High inorganic salt concentration in the extracellu- 0.171 M, EDTA at 1.0 mM, and penicillin-G and streplar microenvironment of cells may raise the intracellu- tomycin sulfate as one solution a t concentrations of lar concentrations of these ions to levels that alter the conformation of proteins, causing the disruption of many cellular processes (Yancey et al., 1982; Arakawa and Timasheff, 1985; Chamberlin and Strange, 1989). Received September 4,1991;accepted September 30,1991. Recent work on several species has focused on the need Address reprint requests to Dr. John D. Biggers, Harvard Medical for glutamine in embryo culture media [hamster: Juet- School, LHRRB, 45 Shattuck St., Boston, MA 02115.
0 1992 WILEY-LISS, INC.
190
J.A. LAWITTS AND J.D. BIGGERS
TABLE 1. Composition and Osmolarity of Medium SOM* Concentration Component mM g/liter 4.97 85.0 NaCl 0.0186 0.25 KC1 0.0476 0.35 KHsP04 0.0493 0.20 MgS04 1.87 10.0 Lactate 0.022 0.2 Pyruvate 0.036 0.2 Glucose 0.146 1.0 Glutamine 1.0 1.0 0.0038 0.01 2.10 25.0 0.251 1.71
*
*Osmolarity of SOM medium = 229 4 (S.E.), n = 6. “BSA concentration is in mg/ml. Penicillin and streptomycin were also added a t concentrations of 100 U/ml and 50 pg/ml, respectively.
10,000 U/ml and 5.0 mg/ml, respectively. All components were stored at 4”C, in 50 ml conical centrifuge tubes (USA Scientific Plastics, Ocala, FL), except for glutamine and the antibiotic mixture, which were stored a t -15°C in 5 ml polypropylene tubes (Falcon 2063, Lincoln Park, NJ). Fresh solutions of NaHCO,, pyruvate, and lactate were prepared weekly. BSA solutions were prepared every time media were made. Ten milliliters of each medium was prepared on the day prior to the start of each trial. All components, except for CaCl,, were added to culture tubes (Falcon 2051), distilled H,O was added to bring the volume of each tube to 9.0-9.5 ml, and then CaC1, was added. Finally, distilled H,O was added to bring the final volume of each medium to 10 ml. Each tube was inverted twice to mix the components, the contents were drawn into a disposable syringe (Becton-Dickinson, Rutherford, NJ), and the medium was pushed through a 0.2 pm filter (Millipore) into a second tube. The osmolarity of each medium was measured on a Wescor osmometer (Wescor, Logan, UT). E m b r y o Collection Mice were killed between 1530 and 1545 h on day 1. Pronuclear-stage zygotes were flushed from each oviduct with 0.1 ml of Dulbecco’s phosphate-buffered saline (dPBS). Pooled zygotes from several donors were washed through one drop of dPBS containing 0.65 mg/ml hyaluronidase (Sigma) to remove any cumulus cells, followed by two drops of dPBS.
37°C incubator. Each culture dish contained 4 drops. Ten zygotes were transferred from dPBS into one of the drops of culture medium. Any remaining dPBS in the transfer pipette was expelled and the pipette was refilled from another drop of culture medium. The ten zygotes were then transferred into the second drop, the medium was expelled from the pipette, and new medium was drawn up from a third drop. The zygotes were then transferred into the third drop. The culture dishes were returned to the culture chamber a t 37”C, which was gas equilibrated and then sealed. Developmental progress was recorded, a s described below (statistical methods), on days 2, 3, and 5. When the culture dishes were outside the culture chamber and not on the microscope stage, they were maintained at 37°C in a benchtop incubator (Baxter Scientific, Bedford, MA). Experimental Design and Statistical Analysis Three experiments were done. Each experiment compared three different components at three different levels, in a 33 factorial design. Thus 27 media were compared in a single trial in each experiment. During each trial, ten zygotes were transferred at random into each of the media used in each experiment. Three trials were conducted for each experiment, for a total of 30 embryos per treatment group. The responses to the media were assessed as the proportion that were four cells or more on day 3 post-hCG and the proportion that reached the blastocyst stage on day 5. In all experiments, three factors were examined, each a t three concentrations, simultaneously. The experiments were analyzed by a n analysis of variance after transformation of the data by a two-term inverse sine transformation, proposed by Laubscher (1961), given by:
t4 = n”2sin-1(x/n)1’2 + (n + 1)1/2sinp1[(x+ 3/4)/(n + 3/2)1”’, where x = the number of responders and n = the group size. The advantage of this transformation over the commonly used angular transformation is that i t is more stable for proportions cO.1 and >0.9. The theoretical variance of this distribution approaches 1as n increases. Individual glutamine concentration response lines were determined by three points. These points were fitted exactly by a quadratic equation expressed as a linear combination of the orthogonal polynomials 1, x , (3x2 - 2):
Embryo Culture Zygotes were cultured in tissue culture dishes (Costar 3060, Cambridge, MA) in 50 p1 drops of medium where b, 6 the mean, bl and b, are the regression coefunder 5 ml of silicone oil (50 centistokes; Sigma). The ficients of the linear and quadratic contributions to the dishes were made up the day prior to the start of each regression, x = t - 1 in experiments 1 and 2 and trial and were equilibrated overnight in a n atmosphere x = t - 2 in experiment 3. of 6% COZ, 5% O,, and 89% N, in a plastic culture The statistical advantage of using this form of the chamber (Billups-Rothenberg, DelMar, CA) inside a regression is that the linearity and curvature of the
191
NaCl AND GLUTAMINE INTERACTION
>
TABLE 2. Concentrations (mM)of Glutamine, NaCl, and Glucose in Experiments 1,2, and 3 Experiment Glutamine NaCl Glucose 1 0, 1, 2 75,85, 95 0.2, 0.4, 0.8 2 0, 1, 2 75, 100, 125 0.2, 0.8, 3.2 3 1, 293 85, 105, 125 0.2, 1.0, 5.0 different glutamine concentration response lines can be independently compared (Kennedy and Gentle, 1980). The computations are described by Sokal and Rohlf (1981), and the numerical values of the orthogonal polynomials used in the computations were obtained from Fisher and Yates (1963). The probability that bl and b, are 0 is based on the pooled error mean squares from the analyses of variance. Only the bl and b, that are significantly different from 0 (P < 0.05) are listed. RESULTS Three 33factorial experiments comparing the effects of all combinations of NaCl, glucose, and glutamine were done. The concentrations of the three factors varied between experiments and are summarized in Table 2. The analyses of variance of the transformed proportions of embryos that reached at least four cells on day 3 post-hCG and blastocysts on day 5 post-hCG are shown in Tables 3 and 4,respectively. The results of the experiments are summarized in Figures 1and 2. Glucose In all three experiments, the main effect of glucose and all interactions between glucose and the other components were not significant. The proportions of embryos reaching the four-cell stage (d3) and reaching the blastocyst stage (d5) in experiment 3, which examined the widest range of glucose concentrations, are shown in Figure 1. There is no suggestion that the concentration of glucose in medium SOM influences the extent of development. Even in the presence of 5 mM glucose, 70% of zygotes can develop to four cells (Fig. l a ) , and 60% can reach the blastocyst stage (Fig. le). This result contrasts with the inhibitory effect of glucose that has been reported using other media (Chatot et al., 1989; Lawitts and Biggers, 1991b).
-
4-CELLS
BLASTOCYSTS 85 rnM NaCl
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Fig. 1. Effect of concentration of glucose (0.2, 1, 5 mM) on the regression of the transformed proportion of embryos developing to at least four cells and blastocysts on the concentration of glutamine in the presence of three concentrations of NaCl(85, 105, 125 mM).
Joint Effect of NaCl and Glutamine Since the effects of glucose were not significant, the results have been presented as the two-way tables (Fig. 2) showing the joint effects of NaCl and glutamine. Four-cell stage or more. In all three experiments, development was totally blocked when the NaCl concentration was 125 mM and glutamine was absent (Fig.
TABLE 3. Analyses of Variance of Three 33 Factorial Experiments Showing the Effect of NaCl(85, 105, 125 mM), Glutamine (0, 1 , 2 mM), and Glucose (Varied)on the Transformed Proportion of Embryos Reaching at Least Four Cells by Day 3 After hCG Experiment 1 Experiment 2 Experiment 3 Source of variation d.f.* Mean sq F P Meansq F P Meansq F P 0.0005 8.81 15.51 0.016 4.49 8.23 2.85 0.07 2 4.35 Between replicates 61.27 < l o - 4 107.86 95.39
Joint Effects of Sodium Chloride, Glutamine, and Glucose in Mouse Preimplantation Embryo Culture Media J.A. LAWITTS AND J.D. BIGGERS Laboratory of Human Reproduction and Reproductive Biology, Haruard Medical School, Boston, Massachusetts
ABSTRACT A new medium, SOM, produced by simplex optimization, has been used to study the joint effects of NaCI, glutamine, and glucose on the development of outbred CF1 mouse zygotes to the blastocyst stage. Contrary to previous reports, glucose has no significant inhibiting effect on development to the blastocyst stage in this medium. Even in the presence of 5 mM glucose, 70%of the embryos develop to at least four cells, and 60%reach the blastocyst stage. Raising the concentration of NaCl from 75 to 125 mM, in the absence of glutamine, progressively inhibits development. Moreover, the response to glutamine depends on the concentration of NaCl in the medium. When the NaCl concentration is low, glutamine inhibits development. In contrast,when the NaCl concentration is high, glutamine protects against the inhibitory effect of the salt. We propose that glutamine protects against high concentrations of NaCl in the medium by acting as an organic osmolyte. Key Words: Simplex optimized medium, NaCI, Osmolyte
INTRODUCTION
ten and Bavister (19831, Carney and Bavister (1987); mouse: Chatot et al. (1989); pig: Petters et al. (199011. Our experiments show that glutamine protects against elevated concentrations of NaCl in culture media, and we speculate that glutamine has this property by acting a s a n organic osmolyte. After 20 cycles of simplex optimization, a medium for the culture of mouse preimplantation embryos has now been produced that has only 85 mM NaCl (medium SOM). In this medium, described in this paper, complete development of pronuclear zygotes into blastocysts occurs in embryos from outbred CF1 mice, without any block to development at the two-cell stage. We also found the surprising fact that glucose in this medium does not inhibit development, a s has been reported in other media (Chatot e t al., 1989; Lawitts and Biggers, 1991b).
MATERIALS AND METHODS Animals Outbred female mice (Cr1:CFl BR; &8 weeks old) and hybrid male mice (BGD2Fl/CrlBR) were maintained on 14 h light and 10 h dark (lights on at 0500 h). Female mice were superovulated by intraperitoneal (i.p.) injection of 5 i.u. pregnant mare serum gonadotropin (PMSG; Sigma, St. Louis, MO) a t 1530 h, followed by a n i.p. injection of human chorionic gonadotropin (hCG; Sigma) 47 h later. After hCG injection, females were placed in cages with males and examined the following morning (day 1)for the presence of a vaginal Plug-
Most media used to culture mouse preimplantation embryos contain too much sodium chloride. This fact first became apparent from experiments using simplex optimization, which, after four cycles, generated medium aKH,PO, that maximized the yield in vitro of blastocysts from pronuclear zygotes (Lawitts and Biggers, 1991a). Additional evidence that the NaCl concentration is too high is provided by the fact that the Preparation of Culture Media two-cell block, which occurs when outbred CF1 embryos are cultured in medium M16 (Whittingham, Media were prepared from individual stock solutions 19711, disappears when the NaCl concentration is re- for each component of medium SOM (Table 1). Most of duced (Lawitts and Biggers, 1991b). A recent report the components were stored in 0.1 M solutions, with the also suggests that the concentration of NaCl in media following exceptions. NaCl and NaHCO, were stored at used to culture pig preimplantation embryos is also too 1.0 M, whereas bovine serum albumin (BSA, fraction high (Beckmann and Day, 1991). V) was stored at a concentration of 100 mg/ml, CaCl, a t High inorganic salt concentration in the extracellu- 0.171 M, EDTA at 1.0 mM, and penicillin-G and streplar microenvironment of cells may raise the intracellu- tomycin sulfate as one solution a t concentrations of lar concentrations of these ions to levels that alter the conformation of proteins, causing the disruption of many cellular processes (Yancey et al., 1982; Arakawa and Timasheff, 1985; Chamberlin and Strange, 1989). Received September 4,1991;accepted September 30,1991. Recent work on several species has focused on the need Address reprint requests to Dr. John D. Biggers, Harvard Medical for glutamine in embryo culture media [hamster: Juet- School, LHRRB, 45 Shattuck St., Boston, MA 02115.
0 1992 WILEY-LISS, INC.
190
J.A. LAWITTS AND J.D. BIGGERS
TABLE 1. Composition and Osmolarity of Medium SOM* Concentration Component mM g/liter 4.97 85.0 NaCl 0.0186 0.25 KC1 0.0476 0.35 KHsP04 0.0493 0.20 MgS04 1.87 10.0 Lactate 0.022 0.2 Pyruvate 0.036 0.2 Glucose 0.146 1.0 Glutamine 1.0 1.0 0.0038 0.01 2.10 25.0 0.251 1.71
*
*Osmolarity of SOM medium = 229 4 (S.E.), n = 6. “BSA concentration is in mg/ml. Penicillin and streptomycin were also added a t concentrations of 100 U/ml and 50 pg/ml, respectively.
10,000 U/ml and 5.0 mg/ml, respectively. All components were stored at 4”C, in 50 ml conical centrifuge tubes (USA Scientific Plastics, Ocala, FL), except for glutamine and the antibiotic mixture, which were stored a t -15°C in 5 ml polypropylene tubes (Falcon 2063, Lincoln Park, NJ). Fresh solutions of NaHCO,, pyruvate, and lactate were prepared weekly. BSA solutions were prepared every time media were made. Ten milliliters of each medium was prepared on the day prior to the start of each trial. All components, except for CaCl,, were added to culture tubes (Falcon 2051), distilled H,O was added to bring the volume of each tube to 9.0-9.5 ml, and then CaC1, was added. Finally, distilled H,O was added to bring the final volume of each medium to 10 ml. Each tube was inverted twice to mix the components, the contents were drawn into a disposable syringe (Becton-Dickinson, Rutherford, NJ), and the medium was pushed through a 0.2 pm filter (Millipore) into a second tube. The osmolarity of each medium was measured on a Wescor osmometer (Wescor, Logan, UT). E m b r y o Collection Mice were killed between 1530 and 1545 h on day 1. Pronuclear-stage zygotes were flushed from each oviduct with 0.1 ml of Dulbecco’s phosphate-buffered saline (dPBS). Pooled zygotes from several donors were washed through one drop of dPBS containing 0.65 mg/ml hyaluronidase (Sigma) to remove any cumulus cells, followed by two drops of dPBS.
37°C incubator. Each culture dish contained 4 drops. Ten zygotes were transferred from dPBS into one of the drops of culture medium. Any remaining dPBS in the transfer pipette was expelled and the pipette was refilled from another drop of culture medium. The ten zygotes were then transferred into the second drop, the medium was expelled from the pipette, and new medium was drawn up from a third drop. The zygotes were then transferred into the third drop. The culture dishes were returned to the culture chamber a t 37”C, which was gas equilibrated and then sealed. Developmental progress was recorded, a s described below (statistical methods), on days 2, 3, and 5. When the culture dishes were outside the culture chamber and not on the microscope stage, they were maintained at 37°C in a benchtop incubator (Baxter Scientific, Bedford, MA). Experimental Design and Statistical Analysis Three experiments were done. Each experiment compared three different components at three different levels, in a 33 factorial design. Thus 27 media were compared in a single trial in each experiment. During each trial, ten zygotes were transferred at random into each of the media used in each experiment. Three trials were conducted for each experiment, for a total of 30 embryos per treatment group. The responses to the media were assessed as the proportion that were four cells or more on day 3 post-hCG and the proportion that reached the blastocyst stage on day 5. In all experiments, three factors were examined, each a t three concentrations, simultaneously. The experiments were analyzed by a n analysis of variance after transformation of the data by a two-term inverse sine transformation, proposed by Laubscher (1961), given by:
t4 = n”2sin-1(x/n)1’2 + (n + 1)1/2sinp1[(x+ 3/4)/(n + 3/2)1”’, where x = the number of responders and n = the group size. The advantage of this transformation over the commonly used angular transformation is that i t is more stable for proportions cO.1 and >0.9. The theoretical variance of this distribution approaches 1as n increases. Individual glutamine concentration response lines were determined by three points. These points were fitted exactly by a quadratic equation expressed as a linear combination of the orthogonal polynomials 1, x , (3x2 - 2):
Embryo Culture Zygotes were cultured in tissue culture dishes (Costar 3060, Cambridge, MA) in 50 p1 drops of medium where b, 6 the mean, bl and b, are the regression coefunder 5 ml of silicone oil (50 centistokes; Sigma). The ficients of the linear and quadratic contributions to the dishes were made up the day prior to the start of each regression, x = t - 1 in experiments 1 and 2 and trial and were equilibrated overnight in a n atmosphere x = t - 2 in experiment 3. of 6% COZ, 5% O,, and 89% N, in a plastic culture The statistical advantage of using this form of the chamber (Billups-Rothenberg, DelMar, CA) inside a regression is that the linearity and curvature of the
191
NaCl AND GLUTAMINE INTERACTION
>
TABLE 2. Concentrations (mM)of Glutamine, NaCl, and Glucose in Experiments 1,2, and 3 Experiment Glutamine NaCl Glucose 1 0, 1, 2 75,85, 95 0.2, 0.4, 0.8 2 0, 1, 2 75, 100, 125 0.2, 0.8, 3.2 3 1, 293 85, 105, 125 0.2, 1.0, 5.0 different glutamine concentration response lines can be independently compared (Kennedy and Gentle, 1980). The computations are described by Sokal and Rohlf (1981), and the numerical values of the orthogonal polynomials used in the computations were obtained from Fisher and Yates (1963). The probability that bl and b, are 0 is based on the pooled error mean squares from the analyses of variance. Only the bl and b, that are significantly different from 0 (P < 0.05) are listed. RESULTS Three 33factorial experiments comparing the effects of all combinations of NaCl, glucose, and glutamine were done. The concentrations of the three factors varied between experiments and are summarized in Table 2. The analyses of variance of the transformed proportions of embryos that reached at least four cells on day 3 post-hCG and blastocysts on day 5 post-hCG are shown in Tables 3 and 4,respectively. The results of the experiments are summarized in Figures 1and 2. Glucose In all three experiments, the main effect of glucose and all interactions between glucose and the other components were not significant. The proportions of embryos reaching the four-cell stage (d3) and reaching the blastocyst stage (d5) in experiment 3, which examined the widest range of glucose concentrations, are shown in Figure 1. There is no suggestion that the concentration of glucose in medium SOM influences the extent of development. Even in the presence of 5 mM glucose, 70% of zygotes can develop to four cells (Fig. l a ) , and 60% can reach the blastocyst stage (Fig. le). This result contrasts with the inhibitory effect of glucose that has been reported using other media (Chatot et al., 1989; Lawitts and Biggers, 1991b).
-
4-CELLS
BLASTOCYSTS 85 rnM NaCl
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LK
0
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0 lY
a c3
w I
[li.
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1
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100
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105 rnM NaCl
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125 rnM NaCl
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GLUTAMINE (rnM)
GLUTAMINE (rnM)
Fig. 1. Effect of concentration of glucose (0.2, 1, 5 mM) on the regression of the transformed proportion of embryos developing to at least four cells and blastocysts on the concentration of glutamine in the presence of three concentrations of NaCl(85, 105, 125 mM).
Joint Effect of NaCl and Glutamine Since the effects of glucose were not significant, the results have been presented as the two-way tables (Fig. 2) showing the joint effects of NaCl and glutamine. Four-cell stage or more. In all three experiments, development was totally blocked when the NaCl concentration was 125 mM and glutamine was absent (Fig.
TABLE 3. Analyses of Variance of Three 33 Factorial Experiments Showing the Effect of NaCl(85, 105, 125 mM), Glutamine (0, 1 , 2 mM), and Glucose (Varied)on the Transformed Proportion of Embryos Reaching at Least Four Cells by Day 3 After hCG Experiment 1 Experiment 2 Experiment 3 Source of variation d.f.* Mean sq F P Meansq F P Meansq F P 0.0005 8.81 15.51 0.016 4.49 8.23 2.85 0.07 2 4.35 Between replicates 61.27 < l o - 4 107.86 95.39
