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The beneficial effect of EDTA on development of mouse one-cell embryos in chemically defined medium
DEVELOPMENTAL
BIOLOGY
The Beneficial
61,
378-383 (1977)
Effect of EDTA on Development of Mouse One-Cell Embryos in Chemically Defined Medium’ J. ABRAMCZUK,*
The Wistar Institute
of Anatomy Received
D. SOLTER, AND H. KOPROWSKI
and Biology,
May 2,1977;
36th Street at Spruce, Philadelphia,
Pennsylvania
19104
accepted in revised form July 27,1977
An improved methology for culturing noninbred (ICR) mouse one-cell embryos is described. The successful development of one-cell embryos into blastocysts in chemically defined (Whitten’s) medium was significantly enhanced by the presence of EDTA. More than 70% of ICR one-cell embryos developed into blastocysts in Whitten’s medium in the presence of 10.8 &f EDTA, while, without EDTA, only l&30% of embryos reached blastocyst stage. A concentration of 10.8 fl EDTA also promoted the development of 65-90% of inbred C57BL/6 one-cell embryos in Whitten’s medium. This beneficial role of EDTA is probably related to the chelation of some metal ion(s) other than Ca”+ or Mgz+. INTRODUCTION
Since its introduction in 1956 (Whitten, 1956), the chemically defined medium of Whitten has been variously modified to meet the culture requirements of early mouse embryos (Biggers et al., 1971; Gwatkin, 1972; Kaufman and Sachs, 1976). Mouse embryos from the two-cell stage to the end of the preimplantation period can now be grown with high efficiency; onecell embryos, however, essentially either fail to develop or develop poorly (Biggers, 1971). The most efficient culture ever reported for one-cell embryos from noninbred mice resulted in less than half of them developing into blastocysts and required special manipulations of the concentrations of energy sources (Cross and Brinster, 1973). Only embryos derived from specific crosses between two inbred strains developed satisfactorily in culture (Biggers, 1971; Whitten and Biggers, 1968), and these, there-
fore, have been used almost exclusively in experiments with mouse one-cell embryos and parthenogenones (Dufrain and Casarett, 1975; Kaufman and Sachs, 1976). We had noticed that when mouse onecell embryos were placed on cell monolayers, they developed into blastocysts and beyond. In examining the factors that promote this development we observed that the presence of trace amounts of EDTA in the medium was necessary for successful development of the embryos. We report here that the results of culturing both inbred and noninbred mouse one-cell embryos in chemically defined medium can be improved by the addition of EDTA.
’ This work was supported by USPHS Research Grants CA-10815 and CA-17546 from the National Cancer Institute and by PDT-26 from the American Cancer Society. * Fellow of the Wistar Institute from the Department of Histology and Embryology (Institute of Biostructure), Medical Academy, Warsaw, Poland.
MATERIALS
AND
METHODS
In all experiments, except when stated otherwise, random-bred ICR female mice, housed in the dark from 7 PM to 6 AM, were used. They were caged with males and were checked each morning, and those with vaginal plugs were killed. The onecell embryos from animals killed between 10 and 11 AM were considered to be earlymiddle one-cell embryos; those from animals killed between 3 and 6 PM were considered to be middle-late one-cell embryos. This classification is based on the
378 Copyright All rights
Q 1977 by Academic Press, Inc. of reproduction
in sny form reserved.
ISSN
0012-1606
BRIEF NOTES
fact that, in our laboratory, of 7500 embryos from females killed between 3 and 6 PM, only 64 (i.e., 0.85%) were in the twocell stage. Since the range of synchronization of the first cleavage is 4-8 hr, even in superovulated mice in which the timing of ovulation can be controlled (Donahue, 1972; Abramczuk and Sawicki, in preparation), most of the eggs collected between 3 and 6 PM could be assumed to be at least 2 hr before first cleavage. Because the duration of the first mitotic cycle in noninbred mice is 12-14 hr (Abramczuk and Sawicki, 1975; Donahue, 19721, most of the eggs obtained from females killed between 10 and 11 AM can be assumed to be in the first half of the cell cycle, i.e., to be earlymiddle one-cell embryos. The oviducts were excised, and the cumulus oophorus masses were released from the ampullae into Whitten’s medium (WM) in which sodium bicarbonate was replaced with Hepes (Sigma Chemical Co.) (WM-H), and the pH of the medium was 7.2. To remove corona cells, cumulus oophorus masses were transferred to WM-H medium to which hyaluronidase (100-300 NF units/ml, Sigma) had been added. The eggs were then washed four times in WMH and twice in WM buffered with bicarbonate and then were transferred to the culture. All one-cell embryos recovered were used even if polar bodies or pronuclei were not conspicuous. The isolation was carried out at room temperature in air. One and one-half to four hours elapsed between the killing of the animals and the time the eggs were placed in culture. Superovulated mice of inbred C57BL/6 and BALB/c strains were used in some experiments. Females were injected with 5-7.5 units of pregnant mare serum (Equinex, Ayerst) and, 44-48 hr later, with 57.5 units of human chorionic gonadotrophin (HCG) (APL, Ayerst). Twenty-two or twenty-three hours after HCG injection, females with plugs were killed, and eggs were cultured as described for ICR mice. The chemically defined medium WM
379
(Whitten, 1971) was used, modified by decreasing the concentration of pyruvate to 0.25 mM (Cross and Brinster, 1973) and increasing the concentration of bovine serum albumin to 4 mg/ml (Biggers, 1971). Water used for WM was collected directly from the deionizer (Ultrapure Water System C3-44, Hydro Service and Supplies, Inc.); its conductivity was 1.5 pmho. Embryos were placed in 2 ml of WM in 35mm plastic tissue culture dishes and were incubated in an atmosphere of 5% COZ, 5% 02, and 90% N, at 37°C. EDTA (Baker Chemical Co., Phillipsburg, N. Y .) and ethylene glycol bis(@ aminoethyl acid ether)N,N’-tetracetic (EGTA) (Sigma) were mixed with deionized water and sodium hydroxide to obtain the desired concentrations of disodium (tetrasodium when stated) salts. The solutions of chelating agents were always added to the culture at a volume of 0.1 ml immediately after the embryos; 0.1 ml of deionized water or saline was added to control cultures. The pH of cultures to which EDTA was added was the same as in control cultures. Cultures of embryos were examined after 4 or 5 days. The formation of a normal-looking blastocyst was the criterion for the successful development of a one-cell embryo. If all blastomeres were not completely integrated into the blastocyst or if the cells of the inner cell mass and/or trophectoderm appeared dark, the blastocyst was considered abnormal and was not classified as successfully developed. The frequency of hatched or hatching normal blastocysts was also recorded. Since the results obtained in several repeated experiments were comparable, they were pooled and expressed as proportions with accompanying 95% confidence limits evaluated from the table of confidence belts for proportions (Dixon and Massey, 1957). Only in one case (Table 2) two separate experiments gave considerably different results and were presented separately.
380
DEVELOPMENTAL BIOLOGY RESULTS
one-cell embryo development in both these strains, although differences in the efZciency of culture (see Table 2) were observed between strains and between separate experiments. Blastocysts developed in WM with or without EDTA were transferred to Eagle’s minimal essential medium. Both groups hatched, attached, and formed outgrowths at approximately the same rate. Our preliminary data also show that blastocysts developed in the presence of EDTA and transferred to the uteri of pseudopregnant mothers can implant and develop to term. The effect of another chelating agent, EGTA, on the development of one-cell embryos in vitro was compared with the effect of EDTA. In the range of concentrations optimal for EDTA, EGTA had no significant effect on the development of
In WM alone about 30% of one-cell-stage embryos developed into blastocysts, whereas about 70% developed when EDTA was added at a final concentration of 10.8 @4 (see Table 1). At a concentration of 668.0 fl of EDTA only 4% of embryos reached blastocyst stage. EDTA also supported the development in vitro of ICR early-middle one-cell embryos (Table 1). The maximum percentage of formed blastocysts (about 60%), however, was lower than it was in the case of middle-late onecell embryos. To check if observations on the beneficial effects of EDTA could be generalized to embryos of inbred mouse strains, C57BL/6 and BALB/c one-cell embryos were cultured in various concentrations of EDTA. EDTA had supporting effects on TABLE EFFECT OF EDTA
1
ON THE DEVELOPMENT OF EARLY-MIDDLE AND MIDDLE-LATE ICR ONE-CELL EMBRYOS INTO BLASTOCYSTSIN CHEMICALLY DEFINED MEDIUM
Concentration of EDTA (Pm 0
Proportion
of developed
Early-middle 0.26
0.7 2.7
10.8
(0.19-0.34) ND’ ND
blastocysts
nb
Middle-late
n
0.32 (0.20-0.44) 0.32 (0.20-0.44)
66 65 66 66 66 66 53
(95% confidence
limits)
1
n
BALBlc
n 29
2.7 10.8 43.0 172.0 688.0
0.89 (0.71-1.00) -
0.10 (0.02-0.27) -
27
0.38 (0.20-0.60)
26
-
D In Experiment 1, animals after HCG injection.
developed
into blastocyst
Experiment
27
27 27
2
of one-cell embryos
0.52 (0.27-0.72)
0.71 (0.50-0.87)
(0.56-0.79) (0.40-0.66) (0.02-0.13)
ONE-CELL EMBRYOS IN CHEMICALLY DEFINED MEDIUM IN THE PRESENCE OF EDTA”
0
0.00 (0.00-0.14)
(0.61-0.84)
of two to four experiments.
TABLE
C57/BL/6
0.74 0.68 0.53 0.04
61 138 61
THE DEVELOPMENT OF C57BL/6 AND BALB/c
Experiment
1imits.Y
0.41 (0.29-0.54)
a Each proportion is based on the results b n = number of embryos cultured. c ND = not done.
Proportion
(95% confidence
179
96
0.48 (0.38-0.60) 0.54 (0.41-0.66) 0.59 (0.50-0.68) 0.25 (0.15-0.38)
43.0 172.0 688.0
Concentration of EDTA mf)
VOLUME 61. 1977
0.00 (0.00-0.15) 0.00 (0.00-0.15) were killed
26 26
at 23 hr after
C57BLl6 0.28 0.50 0.65 0.64 0.53
(0.15-0.46) (0.32-0.67) (0.47-0.80)
(0.47-0.79) (0.37-0.70)
0.39 (0.24-0.57) HCG injection,
2
n
BALBlc
n
36 36 34 36 36 36
0.02 (0.00-0.11) 0.00 (0.00-0.08) 0.07 (0.02-0.19) 0.02 (0.00-0.10) 0.02 (0.00-0.11) 0.04 (0.01-0.16)
45
and, in Experiment
56
45 5.5
45 45
2, at 22 hr
381
BRIEF NOTES
one-cell embryos into the blastocyst stage (Table 3). This suggests that the stimulating effect of EDTA is related to its specific action probably as a chelating agent and not to some unspecific action of acetic acid groups. To test this assumption, one-cell embryos were cultured with completely neutralized EDTA, Na, EDTA, and with a 10.8 fl mixture of acetic acid and sodium acetate. Nar EDTA stimulated development to an extent comparable to Nan EDTA; the mixture of acetic acid and sodium acetate did not (Table 3). This result again suggests that the stimulator-y effect of EDTA on the preimplantation development of one-cell embryos in culture is related to its action as a chelating agent. In order to ascertain whether the results of culturing one-cell embryos could be further improved by not exposing embryos to Hepes and by minimizing the time embryos were exposed to atmospheric oxygen, an experiment was performed in which one-cell embryos were maintained in WM saturated with 5% oxygen, 5% COP, and 90% nitrogen from within 2 min after the animals were sacrificed until the embryos were placed in culture. None of the 37 one-cell embryos manipulated in this way and cultured in WM alone developed into blastocysts, whereas 24 blastocysts TABLE
3
THE EFFECTS OF NaZ EDTA, Na, EDTA, AND Naz EGTA ON THE DEVELOPMENT OF ICR MIDDLE-LATE ONE-CELL EMBRYOS Treatment
Plain WM NaZ EDTA, Nar EDTA, Na acetate acid, 10.8 Na, EGTA, Na, EGTA, Nap EGTA, Nap EGTA,
10.8 fl 10.8 fl and acetic gM 172.0 fl 43.0 a 10.8 @ 2.7 g.M
a Each proportion experiments.
Proportion developed into blastocysts (95% confidence limitsY
n
0.13 0.67 0.64 0.18
(0.05-0.29) (0.50-0.81) (0.46-0.79) (0.08-0.35)
38 39 39 38
0.16 0.05 0.18 0.16
(0.06-0.32) (0.01-0.18) (0.08-0.35) (0.06-0.32)
38 38 38 38
is based on the results
of two
developed from 37 embryos processed this way and cultured in the presence of 10.8 fl EDTA. In a parallel experiment, onecell embryos were exposed for 2 hr to WMH in air before being placed in culture. Three of thirty such eggs cultured in WM alone developed into blastocysts versus 24 blastocysts from 30 eggs cultured in the presence of 10.8 pM EDTA. DISCUSSION
EDTA stimulated the development of mouse one-cell embryos into blastocysts when the embryos were cultured in a chemically defined medium. The optimal concentration of EDTA was 10.8 a, allowing 74% to reach the blastocyst stage, and, at this concentration, twofold more embryos developed into blastocysts in chemically defined medium than did the corresponding controls grown without EDTA. In preliminary experiments, this promoting effect of EDTA on development in vitro was also observed in mouse parthenogenones. Some interstrain differences were observed in the efficiency of the culture of one-cell embryos. The proportions of ICR and C57BL/6 one-cell embryos developing into blastocysts either in plain WM or in WM with Naz EDTA were much higher than the proportions of BALB/c embryos. One possible explanation is that BALB/c one-cell embryos were much less advanced in the first cell cycle progression at the start of culture; it was noticed during the collection of BALB/c one-cell embryos for Experiment 2 (Table 2), in fact, that very few possessed conspicuous pronuclei. It is also possible that EDTA can be beneficial to the development in vitro of one-cell embryos only after they reach a certain critical point during the one-cell stage. This is supported by the fact that the development of ICR early-middle one-cell embryos in vitro was slightly less efficient than the development of ICR middle-late one-cell embryos. Despite the published report (Swenerton
382
DEVELOPMENTAL BIOLOGY
and Hurley, 1971) on the deleterious effects on rat embryos and fetuses of EDTA in the maternal diet, our mouse blastocysts obtained from one-cell embryos cultured in the presence of EDTA subsequently developed in vitro and in viva as did blastocysts grown in plain WM. Our results show that the effect of EDTA on the development of one-cell embryos in vitro is specific and probably due to its chelating action. It is unlikely that the chelation involved Ca2+ and/or Mg2+ ions as their concentrations in WM, at least 2 mM each, both as normal ingredients and as impurities of other components, are several hundred times higher than the optimal concentrations of EDTA. It is probable, therefore, that the chelation of some other metal (e.g., heavy metal) ion or ions is critical to the beneficial action of EDTA. Many EDTA-metal ion complexes are considerably more stable than the similar complexes with Mg2+ or Ca2+ (Hodgeman, 1960); therefore, even in a large excess of the latter, other metal ions can still be chelated by EDTA. It is difficult to establish whether the described stimulatory effect of EDTA is characteristic only of the development of one-cell embryos under artificial conditions (e.g., where toxic metal ions not present in the natural environment of the egg can be introduced), or whether a chelating action of some analogous substance(s) is also necessary for one-cell embryo development in uivo. Many biogenic molecules have chelating properties (Lindenbaum, 1973), and chelation may be essential in the regulation of many biochemical systems (Eichhorn et al., 1973; Sanui and Pace, 1967; Wheeler, 1972). It is interesting to note that catecholamines, which have chelating properties (Lindenbaum, 1973), have recently been detected in unfertilized eggs and cleaving mouse embryos (R. Tasca, personal communication). Preliminary studies, in which we exposed one-cell embryos to 14C-labeled EDTA for 5 hr and found that EDTA did
VOLUME 61, 1977
not enter the embryo (data not shown) suggest that EDTA acts either in the media or on the cell surface. Studies to characterize the mechanism of action of EDTA on one-cell embryo development are in progress, but whatever the mechanism is, our findings have obvious methodological implications. Some variations in the efficiency of culture of one-cell embryos in plain WM do occur (compare Tables 1 and 3), but they are minimized when EDTA is used. Current difficulties in growing one-cell mouse embryos of noninbred or inbred parental strains are overcome by the addition of EDTA to the culture medium, making it possible to use such strains in experiments on early embryonic development and parthenogenesis. Since the quality of chemicals and water used for WM differs between laboratories, a screening experiment should be performed to find the optimal concentration of EDTA. Our results also show that mouse onecell embryos can be manipulated for a considerable length of time in the air atmosphere and in the presence of organic buffer-Hepes and then can be successfully grown in uitro. REFERENCES ABRAMCZUK, J., and SAWICKI, W. (1975). Pronuclear synthesis of DNA in fertilized and parthenogenetically activated mouse eggs. A cytophotometric study. Exp. Cell Res. 92, 361-372. BIGGERS, J. D. (1971). New observations on the nutrition of the mammalian oocyte and the preimplantation embryo. In “The Biology of the Blastocyst” (R. J. Blandau, ed.), pp. 319-327. The University of Chicago Press, Chicago. BIGGERB, J. D., WHITTEN, W. K., and WHITTINGHAM, D. G. (1971). The culture of mouse embryos in vitro. In “Methods in Mammalian Embryology” (J. C. Daniel, ed.), pp. 86-116. W. H. Freeman, San Francisco. CROSS, P. C., and BRINSTER, R. L. (19731. The sensitivity of one-cell mouse embryos to pyruvate and lactate. Exp. Cell Res. 77, 57-62. DIXON, W. J., and MASSEY, F. J. (19571. “Introduction to Statistical Analysis,” p. 415. McGrawHill, New York. DONAHUE, R. P. (1972). Fertilization of the mouse
BRIEF NOTES oocyte: Sequence and timing of nuclear progression to the two-cell stage. J. Exp. 2001. 180, 305318. DUFRAIN, R. J., and CASARETT, A. P. (1975). Response of the pronuclear mouse embryo to Xirradiation in vitro. Radiat. Res. 63, 494-500. EICHHORN, G. L., BERGER, N. A., BUTZOW, J. J., CLARK, P., HEIM, J., PITHA, J., RICHARDSON, C., RIFKIND, J. M., SHIN, Y., and TARIEN, E. (1973). Some effects of metal ions on the structure and function of nucleic acids. Advan. Exp. Med. Biol. 40, 43-66. GWATKIN, R. B. L. (1972). Chemically-defined media for mammalian eggs and early embryos. In Vitro 8, 59-67. HODGEMAN, C. D. (ed.) (1960). “Handbook of Chemistry and Physics,” pp. 1476-1491. The Chemical Rubber Pub. Co., Cleveland, Ohio. KAUFMAN, M. H., and SACHS, L. (1976). Complete preimplantation development in culture of parthenogenetic mouse embryos. J. Embryol. Exp. Morphol. 35, 179-190.
383
LINDENBAUM, A. (1973). A survey of naturally occurring chelating ligands. Advan. Exp. Med. Biol. 40, 67-77. SANUI, H., and PACE, H. (1967). Effect of ATP, EDTA and EGTA on the simultaneous binding of Na, K, Mg and Ca by rat liver microsomes. J. Cell. Physiol. 69, 11-20. SWENERTON, H., and HURLEY, L. S. (1971). Teratogenie effects of a chelating agent and their prevention by zinc. Science 173, 62-64. WHEELER, K. P. (1972). Activation of membrane adenosine triphosphatase by chelating reagents and phospholipids. Biochem. J. 125, 71~. WHITTEN, W. K. (1956). Culture of tubal mouse ova. Nature 177, 96. WHITTEN, W. K. (1971). Nutrient requirements for the culture of preimplantation embryos in vitro. Advan. Biosci. 6, 129-141. WHITTEN, W. K., and BIGGERS, J. D. (1968). Complete development in vitro of the pre-implantation stages of the mouse in a simple chemically defined medium. J. Reprod. Fert. 17, 399-401.
BIOLOGY
The Beneficial
61,
378-383 (1977)
Effect of EDTA on Development of Mouse One-Cell Embryos in Chemically Defined Medium’ J. ABRAMCZUK,*
The Wistar Institute
of Anatomy Received
D. SOLTER, AND H. KOPROWSKI
and Biology,
May 2,1977;
36th Street at Spruce, Philadelphia,
Pennsylvania
19104
accepted in revised form July 27,1977
An improved methology for culturing noninbred (ICR) mouse one-cell embryos is described. The successful development of one-cell embryos into blastocysts in chemically defined (Whitten’s) medium was significantly enhanced by the presence of EDTA. More than 70% of ICR one-cell embryos developed into blastocysts in Whitten’s medium in the presence of 10.8 &f EDTA, while, without EDTA, only l&30% of embryos reached blastocyst stage. A concentration of 10.8 fl EDTA also promoted the development of 65-90% of inbred C57BL/6 one-cell embryos in Whitten’s medium. This beneficial role of EDTA is probably related to the chelation of some metal ion(s) other than Ca”+ or Mgz+. INTRODUCTION
Since its introduction in 1956 (Whitten, 1956), the chemically defined medium of Whitten has been variously modified to meet the culture requirements of early mouse embryos (Biggers et al., 1971; Gwatkin, 1972; Kaufman and Sachs, 1976). Mouse embryos from the two-cell stage to the end of the preimplantation period can now be grown with high efficiency; onecell embryos, however, essentially either fail to develop or develop poorly (Biggers, 1971). The most efficient culture ever reported for one-cell embryos from noninbred mice resulted in less than half of them developing into blastocysts and required special manipulations of the concentrations of energy sources (Cross and Brinster, 1973). Only embryos derived from specific crosses between two inbred strains developed satisfactorily in culture (Biggers, 1971; Whitten and Biggers, 1968), and these, there-
fore, have been used almost exclusively in experiments with mouse one-cell embryos and parthenogenones (Dufrain and Casarett, 1975; Kaufman and Sachs, 1976). We had noticed that when mouse onecell embryos were placed on cell monolayers, they developed into blastocysts and beyond. In examining the factors that promote this development we observed that the presence of trace amounts of EDTA in the medium was necessary for successful development of the embryos. We report here that the results of culturing both inbred and noninbred mouse one-cell embryos in chemically defined medium can be improved by the addition of EDTA.
’ This work was supported by USPHS Research Grants CA-10815 and CA-17546 from the National Cancer Institute and by PDT-26 from the American Cancer Society. * Fellow of the Wistar Institute from the Department of Histology and Embryology (Institute of Biostructure), Medical Academy, Warsaw, Poland.
MATERIALS
AND
METHODS
In all experiments, except when stated otherwise, random-bred ICR female mice, housed in the dark from 7 PM to 6 AM, were used. They were caged with males and were checked each morning, and those with vaginal plugs were killed. The onecell embryos from animals killed between 10 and 11 AM were considered to be earlymiddle one-cell embryos; those from animals killed between 3 and 6 PM were considered to be middle-late one-cell embryos. This classification is based on the
378 Copyright All rights
Q 1977 by Academic Press, Inc. of reproduction
in sny form reserved.
ISSN
0012-1606
BRIEF NOTES
fact that, in our laboratory, of 7500 embryos from females killed between 3 and 6 PM, only 64 (i.e., 0.85%) were in the twocell stage. Since the range of synchronization of the first cleavage is 4-8 hr, even in superovulated mice in which the timing of ovulation can be controlled (Donahue, 1972; Abramczuk and Sawicki, in preparation), most of the eggs collected between 3 and 6 PM could be assumed to be at least 2 hr before first cleavage. Because the duration of the first mitotic cycle in noninbred mice is 12-14 hr (Abramczuk and Sawicki, 1975; Donahue, 19721, most of the eggs obtained from females killed between 10 and 11 AM can be assumed to be in the first half of the cell cycle, i.e., to be earlymiddle one-cell embryos. The oviducts were excised, and the cumulus oophorus masses were released from the ampullae into Whitten’s medium (WM) in which sodium bicarbonate was replaced with Hepes (Sigma Chemical Co.) (WM-H), and the pH of the medium was 7.2. To remove corona cells, cumulus oophorus masses were transferred to WM-H medium to which hyaluronidase (100-300 NF units/ml, Sigma) had been added. The eggs were then washed four times in WMH and twice in WM buffered with bicarbonate and then were transferred to the culture. All one-cell embryos recovered were used even if polar bodies or pronuclei were not conspicuous. The isolation was carried out at room temperature in air. One and one-half to four hours elapsed between the killing of the animals and the time the eggs were placed in culture. Superovulated mice of inbred C57BL/6 and BALB/c strains were used in some experiments. Females were injected with 5-7.5 units of pregnant mare serum (Equinex, Ayerst) and, 44-48 hr later, with 57.5 units of human chorionic gonadotrophin (HCG) (APL, Ayerst). Twenty-two or twenty-three hours after HCG injection, females with plugs were killed, and eggs were cultured as described for ICR mice. The chemically defined medium WM
379
(Whitten, 1971) was used, modified by decreasing the concentration of pyruvate to 0.25 mM (Cross and Brinster, 1973) and increasing the concentration of bovine serum albumin to 4 mg/ml (Biggers, 1971). Water used for WM was collected directly from the deionizer (Ultrapure Water System C3-44, Hydro Service and Supplies, Inc.); its conductivity was 1.5 pmho. Embryos were placed in 2 ml of WM in 35mm plastic tissue culture dishes and were incubated in an atmosphere of 5% COZ, 5% 02, and 90% N, at 37°C. EDTA (Baker Chemical Co., Phillipsburg, N. Y .) and ethylene glycol bis(@ aminoethyl acid ether)N,N’-tetracetic (EGTA) (Sigma) were mixed with deionized water and sodium hydroxide to obtain the desired concentrations of disodium (tetrasodium when stated) salts. The solutions of chelating agents were always added to the culture at a volume of 0.1 ml immediately after the embryos; 0.1 ml of deionized water or saline was added to control cultures. The pH of cultures to which EDTA was added was the same as in control cultures. Cultures of embryos were examined after 4 or 5 days. The formation of a normal-looking blastocyst was the criterion for the successful development of a one-cell embryo. If all blastomeres were not completely integrated into the blastocyst or if the cells of the inner cell mass and/or trophectoderm appeared dark, the blastocyst was considered abnormal and was not classified as successfully developed. The frequency of hatched or hatching normal blastocysts was also recorded. Since the results obtained in several repeated experiments were comparable, they were pooled and expressed as proportions with accompanying 95% confidence limits evaluated from the table of confidence belts for proportions (Dixon and Massey, 1957). Only in one case (Table 2) two separate experiments gave considerably different results and were presented separately.
380
DEVELOPMENTAL BIOLOGY RESULTS
one-cell embryo development in both these strains, although differences in the efZciency of culture (see Table 2) were observed between strains and between separate experiments. Blastocysts developed in WM with or without EDTA were transferred to Eagle’s minimal essential medium. Both groups hatched, attached, and formed outgrowths at approximately the same rate. Our preliminary data also show that blastocysts developed in the presence of EDTA and transferred to the uteri of pseudopregnant mothers can implant and develop to term. The effect of another chelating agent, EGTA, on the development of one-cell embryos in vitro was compared with the effect of EDTA. In the range of concentrations optimal for EDTA, EGTA had no significant effect on the development of
In WM alone about 30% of one-cell-stage embryos developed into blastocysts, whereas about 70% developed when EDTA was added at a final concentration of 10.8 @4 (see Table 1). At a concentration of 668.0 fl of EDTA only 4% of embryos reached blastocyst stage. EDTA also supported the development in vitro of ICR early-middle one-cell embryos (Table 1). The maximum percentage of formed blastocysts (about 60%), however, was lower than it was in the case of middle-late onecell embryos. To check if observations on the beneficial effects of EDTA could be generalized to embryos of inbred mouse strains, C57BL/6 and BALB/c one-cell embryos were cultured in various concentrations of EDTA. EDTA had supporting effects on TABLE EFFECT OF EDTA
1
ON THE DEVELOPMENT OF EARLY-MIDDLE AND MIDDLE-LATE ICR ONE-CELL EMBRYOS INTO BLASTOCYSTSIN CHEMICALLY DEFINED MEDIUM
Concentration of EDTA (Pm 0
Proportion
of developed
Early-middle 0.26
0.7 2.7
10.8
(0.19-0.34) ND’ ND
blastocysts
nb
Middle-late
n
0.32 (0.20-0.44) 0.32 (0.20-0.44)
66 65 66 66 66 66 53
(95% confidence
limits)
1
n
BALBlc
n 29
2.7 10.8 43.0 172.0 688.0
0.89 (0.71-1.00) -
0.10 (0.02-0.27) -
27
0.38 (0.20-0.60)
26
-
D In Experiment 1, animals after HCG injection.
developed
into blastocyst
Experiment
27
27 27
2
of one-cell embryos
0.52 (0.27-0.72)
0.71 (0.50-0.87)
(0.56-0.79) (0.40-0.66) (0.02-0.13)
ONE-CELL EMBRYOS IN CHEMICALLY DEFINED MEDIUM IN THE PRESENCE OF EDTA”
0
0.00 (0.00-0.14)
(0.61-0.84)
of two to four experiments.
TABLE
C57/BL/6
0.74 0.68 0.53 0.04
61 138 61
THE DEVELOPMENT OF C57BL/6 AND BALB/c
Experiment
1imits.Y
0.41 (0.29-0.54)
a Each proportion is based on the results b n = number of embryos cultured. c ND = not done.
Proportion
(95% confidence
179
96
0.48 (0.38-0.60) 0.54 (0.41-0.66) 0.59 (0.50-0.68) 0.25 (0.15-0.38)
43.0 172.0 688.0
Concentration of EDTA mf)
VOLUME 61. 1977
0.00 (0.00-0.15) 0.00 (0.00-0.15) were killed
26 26
at 23 hr after
C57BLl6 0.28 0.50 0.65 0.64 0.53
(0.15-0.46) (0.32-0.67) (0.47-0.80)
(0.47-0.79) (0.37-0.70)
0.39 (0.24-0.57) HCG injection,
2
n
BALBlc
n
36 36 34 36 36 36
0.02 (0.00-0.11) 0.00 (0.00-0.08) 0.07 (0.02-0.19) 0.02 (0.00-0.10) 0.02 (0.00-0.11) 0.04 (0.01-0.16)
45
and, in Experiment
56
45 5.5
45 45
2, at 22 hr
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BRIEF NOTES
one-cell embryos into the blastocyst stage (Table 3). This suggests that the stimulating effect of EDTA is related to its specific action probably as a chelating agent and not to some unspecific action of acetic acid groups. To test this assumption, one-cell embryos were cultured with completely neutralized EDTA, Na, EDTA, and with a 10.8 fl mixture of acetic acid and sodium acetate. Nar EDTA stimulated development to an extent comparable to Nan EDTA; the mixture of acetic acid and sodium acetate did not (Table 3). This result again suggests that the stimulator-y effect of EDTA on the preimplantation development of one-cell embryos in culture is related to its action as a chelating agent. In order to ascertain whether the results of culturing one-cell embryos could be further improved by not exposing embryos to Hepes and by minimizing the time embryos were exposed to atmospheric oxygen, an experiment was performed in which one-cell embryos were maintained in WM saturated with 5% oxygen, 5% COP, and 90% nitrogen from within 2 min after the animals were sacrificed until the embryos were placed in culture. None of the 37 one-cell embryos manipulated in this way and cultured in WM alone developed into blastocysts, whereas 24 blastocysts TABLE
3
THE EFFECTS OF NaZ EDTA, Na, EDTA, AND Naz EGTA ON THE DEVELOPMENT OF ICR MIDDLE-LATE ONE-CELL EMBRYOS Treatment
Plain WM NaZ EDTA, Nar EDTA, Na acetate acid, 10.8 Na, EGTA, Na, EGTA, Nap EGTA, Nap EGTA,
10.8 fl 10.8 fl and acetic gM 172.0 fl 43.0 a 10.8 @ 2.7 g.M
a Each proportion experiments.
Proportion developed into blastocysts (95% confidence limitsY
n
0.13 0.67 0.64 0.18
(0.05-0.29) (0.50-0.81) (0.46-0.79) (0.08-0.35)
38 39 39 38
0.16 0.05 0.18 0.16
(0.06-0.32) (0.01-0.18) (0.08-0.35) (0.06-0.32)
38 38 38 38
is based on the results
of two
developed from 37 embryos processed this way and cultured in the presence of 10.8 fl EDTA. In a parallel experiment, onecell embryos were exposed for 2 hr to WMH in air before being placed in culture. Three of thirty such eggs cultured in WM alone developed into blastocysts versus 24 blastocysts from 30 eggs cultured in the presence of 10.8 pM EDTA. DISCUSSION
EDTA stimulated the development of mouse one-cell embryos into blastocysts when the embryos were cultured in a chemically defined medium. The optimal concentration of EDTA was 10.8 a, allowing 74% to reach the blastocyst stage, and, at this concentration, twofold more embryos developed into blastocysts in chemically defined medium than did the corresponding controls grown without EDTA. In preliminary experiments, this promoting effect of EDTA on development in vitro was also observed in mouse parthenogenones. Some interstrain differences were observed in the efficiency of the culture of one-cell embryos. The proportions of ICR and C57BL/6 one-cell embryos developing into blastocysts either in plain WM or in WM with Naz EDTA were much higher than the proportions of BALB/c embryos. One possible explanation is that BALB/c one-cell embryos were much less advanced in the first cell cycle progression at the start of culture; it was noticed during the collection of BALB/c one-cell embryos for Experiment 2 (Table 2), in fact, that very few possessed conspicuous pronuclei. It is also possible that EDTA can be beneficial to the development in vitro of one-cell embryos only after they reach a certain critical point during the one-cell stage. This is supported by the fact that the development of ICR early-middle one-cell embryos in vitro was slightly less efficient than the development of ICR middle-late one-cell embryos. Despite the published report (Swenerton
382
DEVELOPMENTAL BIOLOGY
and Hurley, 1971) on the deleterious effects on rat embryos and fetuses of EDTA in the maternal diet, our mouse blastocysts obtained from one-cell embryos cultured in the presence of EDTA subsequently developed in vitro and in viva as did blastocysts grown in plain WM. Our results show that the effect of EDTA on the development of one-cell embryos in vitro is specific and probably due to its chelating action. It is unlikely that the chelation involved Ca2+ and/or Mg2+ ions as their concentrations in WM, at least 2 mM each, both as normal ingredients and as impurities of other components, are several hundred times higher than the optimal concentrations of EDTA. It is probable, therefore, that the chelation of some other metal (e.g., heavy metal) ion or ions is critical to the beneficial action of EDTA. Many EDTA-metal ion complexes are considerably more stable than the similar complexes with Mg2+ or Ca2+ (Hodgeman, 1960); therefore, even in a large excess of the latter, other metal ions can still be chelated by EDTA. It is difficult to establish whether the described stimulatory effect of EDTA is characteristic only of the development of one-cell embryos under artificial conditions (e.g., where toxic metal ions not present in the natural environment of the egg can be introduced), or whether a chelating action of some analogous substance(s) is also necessary for one-cell embryo development in uivo. Many biogenic molecules have chelating properties (Lindenbaum, 1973), and chelation may be essential in the regulation of many biochemical systems (Eichhorn et al., 1973; Sanui and Pace, 1967; Wheeler, 1972). It is interesting to note that catecholamines, which have chelating properties (Lindenbaum, 1973), have recently been detected in unfertilized eggs and cleaving mouse embryos (R. Tasca, personal communication). Preliminary studies, in which we exposed one-cell embryos to 14C-labeled EDTA for 5 hr and found that EDTA did
VOLUME 61, 1977
not enter the embryo (data not shown) suggest that EDTA acts either in the media or on the cell surface. Studies to characterize the mechanism of action of EDTA on one-cell embryo development are in progress, but whatever the mechanism is, our findings have obvious methodological implications. Some variations in the efficiency of culture of one-cell embryos in plain WM do occur (compare Tables 1 and 3), but they are minimized when EDTA is used. Current difficulties in growing one-cell mouse embryos of noninbred or inbred parental strains are overcome by the addition of EDTA to the culture medium, making it possible to use such strains in experiments on early embryonic development and parthenogenesis. Since the quality of chemicals and water used for WM differs between laboratories, a screening experiment should be performed to find the optimal concentration of EDTA. Our results also show that mouse onecell embryos can be manipulated for a considerable length of time in the air atmosphere and in the presence of organic buffer-Hepes and then can be successfully grown in uitro. REFERENCES ABRAMCZUK, J., and SAWICKI, W. (1975). Pronuclear synthesis of DNA in fertilized and parthenogenetically activated mouse eggs. A cytophotometric study. Exp. Cell Res. 92, 361-372. BIGGERS, J. D. (1971). New observations on the nutrition of the mammalian oocyte and the preimplantation embryo. In “The Biology of the Blastocyst” (R. J. Blandau, ed.), pp. 319-327. The University of Chicago Press, Chicago. BIGGERB, J. D., WHITTEN, W. K., and WHITTINGHAM, D. G. (1971). The culture of mouse embryos in vitro. In “Methods in Mammalian Embryology” (J. C. Daniel, ed.), pp. 86-116. W. H. Freeman, San Francisco. CROSS, P. C., and BRINSTER, R. L. (19731. The sensitivity of one-cell mouse embryos to pyruvate and lactate. Exp. Cell Res. 77, 57-62. DIXON, W. J., and MASSEY, F. J. (19571. “Introduction to Statistical Analysis,” p. 415. McGrawHill, New York. DONAHUE, R. P. (1972). Fertilization of the mouse
BRIEF NOTES oocyte: Sequence and timing of nuclear progression to the two-cell stage. J. Exp. 2001. 180, 305318. DUFRAIN, R. J., and CASARETT, A. P. (1975). Response of the pronuclear mouse embryo to Xirradiation in vitro. Radiat. Res. 63, 494-500. EICHHORN, G. L., BERGER, N. A., BUTZOW, J. J., CLARK, P., HEIM, J., PITHA, J., RICHARDSON, C., RIFKIND, J. M., SHIN, Y., and TARIEN, E. (1973). Some effects of metal ions on the structure and function of nucleic acids. Advan. Exp. Med. Biol. 40, 43-66. GWATKIN, R. B. L. (1972). Chemically-defined media for mammalian eggs and early embryos. In Vitro 8, 59-67. HODGEMAN, C. D. (ed.) (1960). “Handbook of Chemistry and Physics,” pp. 1476-1491. The Chemical Rubber Pub. Co., Cleveland, Ohio. KAUFMAN, M. H., and SACHS, L. (1976). Complete preimplantation development in culture of parthenogenetic mouse embryos. J. Embryol. Exp. Morphol. 35, 179-190.
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LINDENBAUM, A. (1973). A survey of naturally occurring chelating ligands. Advan. Exp. Med. Biol. 40, 67-77. SANUI, H., and PACE, H. (1967). Effect of ATP, EDTA and EGTA on the simultaneous binding of Na, K, Mg and Ca by rat liver microsomes. J. Cell. Physiol. 69, 11-20. SWENERTON, H., and HURLEY, L. S. (1971). Teratogenie effects of a chelating agent and their prevention by zinc. Science 173, 62-64. WHEELER, K. P. (1972). Activation of membrane adenosine triphosphatase by chelating reagents and phospholipids. Biochem. J. 125, 71~. WHITTEN, W. K. (1956). Culture of tubal mouse ova. Nature 177, 96. WHITTEN, W. K. (1971). Nutrient requirements for the culture of preimplantation embryos in vitro. Advan. Biosci. 6, 129-141. WHITTEN, W. K., and BIGGERS, J. D. (1968). Complete development in vitro of the pre-implantation stages of the mouse in a simple chemically defined medium. J. Reprod. Fert. 17, 399-401.