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Embryotropic role of hemoglobin and ethylenediaminetetraacetic acid in preimplantation development of ICR mouse 1-cell embryos
FERTILITY AND STERILITY威 VOL. 74, NO. 5, NOVEMBER 2000 Copyright ©2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Embryotropic role of hemoglobin and ethylenediaminetetraacetic acid in preimplantation development of ICR mouse 1-cell embryos Sung E. Park, M.S.,a Hyung M. Chung, Ph.D.,a Jung J. Ko, Ph.D.,a Byeong C. Lee, Ph.D.,b Kwang Y. Cha, M.D.a, and Jeong M. Lim, Ph.D.a College of Medicine, Pochon CHA University; and Seoul National University, Seoul, Korea
Received February 15, 2000; accepted May 31, 2000. Reprint requests: Jeong M. Lim, Applied Embryology Laboratory, Infertility Medical Center of CHA General Hospital, 606-5 Yeoksam 1-Dong, Kangnam-Gu, Seoul 135081, Korea (FAX: 82-2-5018704; E-mail: jmlcimc @netsgo.com). a Infertility Medical Center of CHA General Hospital, College of Medicine. b Department of Theriogenology, Seoul National University. 0015-0282/00/$20.00 PII S0015-0282(00)01536-3
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Objective: To evaluate embryotropic action of hemoglobin (Hb) and ethylenediaminetetraacetic acid (EDTA) on preimplantation embryo development. Design: In vitro model study using mouse embryos. Setting: University affiliated hospital, Pochon CHA University. Animals: Four-week-old block strain ICR mice naturally mated after superovulation. Intervention(s): One-cell embryos were cultured in serum-free, modified preimplantation-1 medium, to which 1 g/ml Hb and/or 0.1 mM EDTA were added. Main Outcome Measure(s): Preimplantation development and blastomere number. Result(s): More (P⬍.05) 1-cell embryos developed to the 4-cell (52% vs. 67%– 84%), 8-cell (48% vs. 65%– 81%), and blastocyst (40% vs. 61%–79%) stages after the addition of hemoglobin (Hb) and/or EDTA than after no addition. Highest proportion of embryos developed to each stage after the combined addition of Hb⫹EDTA. EDTA specifically stimulated the development before the 8-cell stage, which was as similar as Hb⫹EDTA. On the contrary, higher ratio of morula to blastocyst transformation was obtained after the addition of Hb or Hb⫹EDTA than after no addition (0.76 vs. 0.96 – 0.98). Significant increases in the cell number of blastocysts (46.5– 47.2 vs. 53.2 cells), inner cell mass (ICM) cells (16.7–17.5 vs. 21 cells), and the ratio of ICM cells to trophoblasts (0.3– 0.37 to 0.39) were found after the combined addition of Hb⫹EDTA, compared with no addition or with the addition of EDTA or Hb alone. Conclusions: Hb and EDTA have stage-specific effects on supporting preimplantation embryo development; Hb promotes both the development before the 8-cell stage and the morula to blastocyst transformation, whereas EDTA mainly promotes the development to the 8-cell stage. The combined exposure of embryos to Hb and EDTA improves not only preimplantation development but also the growth and quality of blastocysts. (Fertil Steril威 2000;74:996 –1000. ©2000 by American Society for Reproductive Medicine.) Key Words: Embryo, hemoglobin, EDTA, preimplantation development, blastocysts
Developing human embryo culture system is important for increasing the rates of pregnancy and implantation after in vitro fertilization with embryo transfer (IVF-ET). Tremendous efforts have been made to develop such technology, and a number of animal experiments were conducted before the clinical application of developed methods. To date, numerous substances have proved to have embryotropic actions (1–7), and these have been added to culture medium at the different stages of embryo development. On the basis of the latter studies, a sequential culture method using the “proven” embryotropins has been
developed for supporting preimplantation embryo development before transfer (8 –13). We recently developed a sequential culture system using glucose-, phosphate- and serumfree culture media, to which hemoglobin (Hb), a nitric oxide scavenger essential and nonessential amino acids BSA and glucose are supplemented to preimplantation-1 (P-1) medium at different times after culture (14, 15). To optimize our culture system, we subsequently planned to examine the embryotropic effect of ethylendiaminetetraacetic acids (EDTA), calcium and metallic ion chelators, which stimu-
late the development of mouse embryos beyond the stage of embryonic genome activation in the absence of Hb (1, 16 – 18). The combined addition of Hb and EDTA to culture medium may further promote in vitro embryo development. In this study, outbred “block” strain (ICR) mouse 1-cell embryos were cultured in P-1 medium after the supplementation of Hb and/or EDTA. In vitro development to the hatched blastocyst stage, the cell numbers of blastocysts and inner cell mass (ICM) cells, and the ratio of ICM cells to trophoblasts were examined.
MATERIALS AND METHODS Collection of Embryos Four-week-old female ICR mice were maintained under controlled lighting conditions (14L:10D) and superovulated by the injection of 5 IU PMSG (Folligon; Intervet Co., The Netherlands). At 48 h after PMSG injection, 5 IU hCG (Chorulaon; Intervet) was administered and natural mating was concomitantly initiated at the time of hCG injection. The presence of a vaginal plug was examined at 16 h post-hCG injection, and 1-cell embryos with male and female pronuclei were then collected from the oviduct of mated female mice at 18 h after hCG injection. After washing several times in culture medium, embryos were provided for each experiment.
Embryo Culture Medium P-1 medium (Irvine Scientific Co., San Diego, CA) consisting of 101.6 mM NaCl, 4.69 mM KCl, 2.04 mM CaCl2 䡠 2H2O, 0.2 mM MgSO4 䡠 7H2O, 0.33 mM sodium pyruvate, 21.4 mM sodium lactate, 25 mM NaHCO3 and 0.15 mg/ml (w/v) sodium citrate, 0.05 mM taurine, 10 g/ml gentamycin, as well as 5 mg/ml phenol red, was used as a based medium. This medium does not contain glucose, inorganic phosphate, or protein. For the culture of embryos, fatty acid–free BSA (cat. no. A-4161, 3 mg/ml, Sigma Co., St. Louis, MO), Modified Eagle medium nonessential (0.5%, v/v), and essential (1%, v/v) amino acids solutions (Gibco BRL, Grand Island, NY) were added to P-1 medium (designated as modified P-1 medium), and 5.6 mM glucose was subsequently supplemented at 66 h after hCG injection. The osmolarity and pH of modified P-1 medium were within the ranges of 295–305 mOsm and pH 7.3–7.4, respectively.
Culture of Embryos and Assessment of Preimplantation Development
A group of 15 to 20 embryos were cultured in 5 l droplet of modified P-1 medium supplemented with Hb and/or EDTA at 37°C, 5% CO2 in humidified air atmosphere, and embryos were transferred to the medium additionally supplemented with 5.6 mM glucose at 66 h post-hCG injection. Media used for embryo culture were equilibrated in such atmosphere at least for 3 h before culture, and the droplets of equilibrated medium were covered with warm mineral oil (BDH Co., Poole, Dorset, UK). The number of embryos FERTILITY & STERILITY威
developed to the 2-cell, 4-cell, 8-cell, morula, blastocyst, and hatched blastocyst stages was monitored under an inverted microscope (Eclipse TE300, Nikon, Tokyo, Japan) at 42, 66, 90, 114, 138, and 162 h after hCG injection, respectively.
Assessment of the Quality of Blastocysts To evaluate the growth and quality of blastocysts, the cell number of total blastomeres and inner cell mass (ICM) cells as well as the ratio of ICM cells to trophoblasts were counted by the method of Hardy et al. (19) with a slight modification. The zona pellucida of blastocysts obtained at 162 h after hCG injection were removed by 0.5% (v/v) protease solution (cat. no. P-8811, Sigma), and the blastocysts were placed in 15 mM trinitrobezene sulfonic acid (cat. no. P-2297, Sigma) for 15 min at 4°C. Blastocysts were then incubated for 10 min in Tyrode’s lactate solution supplemented with 25 mM Hepes and 0.1 mg/ml anti-dinitrophenol-BSA (cat no. 61007-1, ICN, Irvine, CA) at 39°C. Subsequently, they were treated with 0.01 mg/ml propidium iodide (cat. no. P-4170, Sigma) and incubated with 15% (v/v) guinea pig complement (cat. no. S-1639, Sigma) for 20 to 30 min at 39°C. The blastocysts were then placed in absolute ethanol solution supplemented with 0.05 mM fluorochrome bisbenzimide (cat. no. B-2261, Sigma) overnight at 4°C. After washing in absolute ethanol, the stained blastocysts were mounted on a glass slide, and cell numbers were examined under the inverted microscope with epifluorescent apparatus (HB10104AF, Nikon, Tokyo, Japan).
Experimental Design and Statistical Analysis In experiment 1, the effects of combined addition of Hb (1 g/ml; methemoglobin, cat no. H-7379, Sigma) and/or EDTA (0.1 mM; cell culture–tested, cat no. E-6758, Sigma) on the development of mouse 1-cell embryos to the 2-cell, 4-cell, 8-cell, and blastocyst stages were evaluated. The dosage of tested substances was determined by our preliminary study (data not shown). In experiment 2, embryos were allotted in the same experimental group of experiment 1 and sequentially cultured up to the morula stage (114 h after hCG injection) without any observation. Morula to blastocyst transformation and the development of blastocysts to the hatched blastocyst stage were then monitored. In experiment 3, the number of total blastomeres and ICM cells, and the ratio of ICM cells to trophoblasts in blastocysts obtained in the same groups of experiment 1 were evaluated at 162 h post-hCG injection. Each experiment was replicated 3 (experiments 2 and 3) or 4 (experiment 1) times. Embryos developed to the 4-cell, 8-cell, morula, blastocyst, and hatched blastocyst stages were individually scored as 1 (developed). Oocytes that did not develop to the appropriate stages were scored as 0 (not developed). The mean percentage of development, the mean cell number of total blastomeres and ICM cells, and the mean ratio of ICM cells to trophoblasts in each experiment were calculated. Those scores were then subjected to analysis of variance using the general linear model (PROC997
TABLE 1 Effects of the combined addition of hemoglobin (Hb, 1 g/ml) and/or ethylenediaminetetraacetic acid (EDTA, 0.1 mM) to modified preimplantation-1 medium supplemented with 5.6 mM glucose at 66 h after hCG injection on the development of ICR mouse 1-cell embryos to the blastocyst stage. Mean percentage (⫾SE) of 1-cell embryos developed to Supplements
No. of embryos cultured
2-cell [42]a
4-cell [66]a
8-cell [90]a
Blastocyst [138]a
No addition ⫹Hb ⫹EDTA ⫹Hb⫹EDTA
75 82 80 79
100 100 100 100
52.0 ⫾ 5.1b 67.1 ⫾ 4.9c 80.0 ⫾ 4.9d 83.5 ⫾ 5.0d
48.0 ⫾ 5.3b 64.6 ⫾ 5.0c 75.0 ⫾ 5.1c,d 81.0 ⫾ 5.1d
40.0 ⫾ 5.4b 61.0 ⫾ 5.2c 63.8 ⫾ 5.3c 78.5 ⫾ 5.3d
Note: Model effects (P value) in the development to the 2-cell, 4-cell, 8-cell, and blastocyst stages were 1, .0001, .0001, and .0001, respectively. a Hours after hCG injection. b,c,d Different superscripts in each parameter were significantly different, P⬍.05. Park. Embryotropic action of hemoglobin and EDTA. Fertil Steril 2000.
GLM) in the SAS software program (20). When the significance of the main effects was detected in each experimental parameter, the treatment effects were compared by the least square method. A statistical significance was determined where P value was less than .05.
RESULTS Experiment 1 A total of 316 1-cell embryos were provided for this experiment, and all embryos developed to the 2-cell stage until 42 h after hCG injection, regardless of the treatments. As shown in Table 1, a significant treatment effect (P⫽ .0001) was found in the development to the 4-cell, 8-cell, and blastocyst stages. The addition of Hb or EDTA to modified P-1 medium significantly promoted the developments compared with no addition (52% vs. 67%– 80%, P⬍.0329 in the 4-cell; 48% vs. 65%–75%, P⬍.0228 in the 8-cell; and 40% vs. 61%– 64%, P⬍.0057 in the blastocyst stages). The promoting effects of EDTA on embryo development to the 4-cell and 8-cell stages were as similar as those of EDTA⫹Hb and similar proportion of embryos developed to the 8-cell stage in these 2 treatments. However, more embryos developed to blastocysts after the addition of Hb⫹ EDTA than after the addition of either Hb or EDTA alone.
Experiment 2 Of those 360 1-cell embryos cultured after each treatment, 243 (67%) embryos developed to the morula stage were used for analysis. As shown in Table 2, a significant treatment effect was found in the morula to blastocyst transformation (P⫽.0002). Higher transformation rate was obtained after the addition of Hb alone than after no addition (0.76 vs. 0.98; P⬍.0001). However, no significant effect was found after the addition of EDTA, regardless of the presence (0.96 vs. 0.98) or the absence (0.76 vs. 0.89) of Hb in the medium. No significant main effect (P⫽.41) was detected in 998
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Embryotropic action of hemoglobin and EDTA
the development of blastocysts to the hatched blastocyst stage.
Experiment 3 A significant treatment effect was observed in the cell numbers of blastocysts (P⫽.0438) and ICM cells (P⫽.0031) and in the ratio of ICM cell to trophoblast (P⫽.0338). As shown in Table 3, higher cell numbers of blastomeres (46.5– 47.2 cells vs. 53.2 cells per blastocyst, P⬍.019) and ICM cells (16.7–17.5 cells vs. 21 cells per blastocyst, P⬍.002) in blastocysts were obtained after the addition of Hb⫹EDTA to P-1 medium than after no addition or after the addition of EDTA or Hb alone. The ratio of ICM cells to trophoblasts was also significantly increased after the combined addition than any other combinations (0.36 – 0.37 vs. 0.39, P⬍.05).
DISCUSSION The results of this study demonstrated that Hb and EDTA have different roles in supporting in vitro preimplantation development of outbred block strain ICR mouse 1-cell embryos. Hb significantly promoted the development to the 8-cell stage and the morula to blastocyst transformation; EDTA mainly strongly stimulated early cleavage up to the 8-cell stage. Combined exposure of 1-cell embryos to Hb and EDTA during their preimplantation development improved not only development to the blastocyst stage but also the growth and quality of blastocysts; 78% of 1-cell embryos sequentially cultured in modified P-1 medium developed to the blastocyst stage after the supplementation of Hb and EDTA. In the case of bovine embryos, Hb exerts its stimulatory action only in the presence of cumulus cells (21), and this result suggests that Hb stimulates blastocyst formation by scavenging nitric oxide secreted from co-cultured cumulus cells. In our recent (14) and present studies, Hb improved in Vol. 74, No. 5, November 2000
TABLE 2 Morula to blastocyst transformation and development of blastocyst to the hatched blastocyst stage in embryos cultured after the addition of hemoglobin (Hb, 1 g/ml) and/or ethylenediaminetetraacetic acid (EDTA, 0.1 mM) to modified preimplantation-1 medium supplemented with 5.6 mM glucose at 66 h after hCG injection. Morula/blastocyst transformation
Hatched blastocyst
Supplements
Number of blastocysts/ Number of morulae
Ratio (⫾ SE)
Number examined
Number developed (Mean % ⫾ SE)
No addition ⫹Hb ⫹EDTA ⫹Hb⫹EDTA
42/55 50/52 63/71 64/65
0.76 ⫾ 0.04a 0.96 ⫾ 0.04b 0.89 ⫾ 0.03a,b 0.98 ⫾ 0.04b
42 50 63 64
28.6 ⫾ 7.4 38.0 ⫾ 6.8 33.3 ⫾ 6.1 43.8 ⫾ 6.0
Note: Model effect (P value) in the ratio of morula/blastocyst transformation and development to the hatched blastocyst was .0002 and .41, respectively. Different superscripts in each parameter were significantly different, P⬍.05.
a,b,c
Park. Embryotropic action of hemoglobin and EDTA. Fertil Steril 2000.
vitro preimplantation development of mouse embryos even in the absence of cumulus cells. From these data, it appears that (i) Hb has an embryotropic action in both mouse and bovine embryos and that (ii) the detailed action of Hb on embryo development of the mouse and the bovine species was different. Additional results of our previous study using bovine embryos (22) showed that several nitric oxide synthase inhibitors, such as L-nitroarginine-methylester did not stimulate preimplantation development. Research is currently under way to clarify how Hb promotes mouse embryo development. From the results of our study, further information on Hb action during in vitro development was obtained: Hb added to culture medium promotes all stages of mouse preimplantation development. Hb improves embryo development during their own genome activation before the second cleavage, the development to the 8-cell stage, and morula to blastocyst transformation. These results were comparable to those of our bovine embryo study that Hb significantly and specifically enhances the morula to blastocyst transformation. Probably, species specificity (bovine vs. mouse) and/or culture milieu (coculture vs. cell-free culture) affect the embryotropic action of Hb. In contrast to the action of Hb, EDTA mainly exerts its promoting effect on supporting the development during early cleavage, and such effect was stronger than that of Hb (Table 1). These results raised the possibility that nitric oxide and metallic ion contaminants of culture media independently inhibit different stages of preimplantation development with different embryotoxicities. This hypothesis was strongly supported by our result showing the different embryotropic roles of Hb and EDTA during in vitro development to the blastocyst stage. Considering the previous reports on the action of EDTA on embryo development (1, 16 –18), EDTA assists mouse 1-cell embryos to overcome in vitro developmental block phenomenon during their 1st cleavage, regardless of the presence of nitric oxide in culture systems. FERTILITY & STERILITY威
ICM cell and total blastomere numbers in blastocyst are the indicators of embryo growth, whereas ICM cell to trophoblast ratio represents the quality of the blastocyst. Synergism of Hb and EDTA appeared not only as an improved development of 1-cell embryos to the blastocyst stage but also as increased ICM cell and trophoblast numbers yielding high ICM cell to trophoblast ratio. Accordingly, a combined addition of these substances stimulates both proliferation and differentiation of mouse embryos during in vitro culture, which enhances the quality and growth of blastocysts. In the basis of our present results, we renovated our sequential culture system for preimplantation development of mouse embryos. This system uses small volume of me-
TABLE 3 Effects of the combined addition of hemoglobin (Hb, 1 g/mL) and/or ethylenediaminetetraacetic acid (EDTA; 0.1 mM) to modified preimplantation (P)-1 medium supplemented with 5.56 mM glucose at 66 h after hCG injection on the cell number of blastomeres and inner cell mass (ICM) cells in blastocysts derived from ICR mouse 1-cell embryos. Cell numbers (mean ⫾ SE) of blastocysts Supplements
Total
ICM cells
ICM cell to trophoblast ratio
No addition ⫹Hb ⫹EDTA ⫹Hb⫹EDTA
46.5 ⫾ 2.2a 50.4 ⫾ 1.9a 47.2 ⫾ 1.8a 53.2 ⫾ 1.7b
16.7 ⫾ 1.0a 18.8 ⫾ 0.9a 17.5 ⫾ 0.8a 21.0 ⫾ 0.8b
0.36 ⫾ 0.01a 0.37 ⫾ 0.01a 0.37 ⫾ 0.01a 0.39 ⫾ 0.01b
Note: Model effect (P value) in the cell numbers of total blastomere and ICM cells and the ratio of ICM to trophoblast was .0438, .0031, and .0338, respectively. a,b Different superscripts in each parameter were significantly different, P⬍.05. Park. Embryotropic action of hemoglobin and EDTA. Fertil Steril 2000.
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dium (5 l medium for culturing 15–20 embryos), which supports paracrine action of embryos during in vitro culture. To intoxicate the embryotoxic factors present in our system, Hb, EDTA, and taurine are present in culture media. Thus, P-1 medium was supplemented with amino acids, BSA, EDTA and Hb for the first culture phase during the development to the 4-cell stage, and this modified P-1 medium was additionally supplemented with glucose at the 4-cell stage for later preimplantation development. Attempts have been made to apply this culture system for human IVF-ET program after further modification of our system for developing chemically defined system.
Acknowledgments: This work was supported by a grant from the Interdisciplinary Research Program of the KOSEF (grant 1999-2-205-002-5) and by a grant from the Korea Research Foundation (KRF-99-041-G00065).
REFERENCES 1. Gardner DK, Lane M. Alleviation of the “2-cell block” and development to the blastocyst of CF1 mouse embryos: role of amino acids, EDTA and physical parameters. Hum Reprod 1996;11:2703–12. 2. Gardner DK, Lane M. Amino acids and ammonium regulate mouse embryo development in culture. Biol Reprod 1993;48:377– 85. 3. Lane M, Gardner DK. Differential regulation of mouse embryo development and viability by amino acids. J Reprod Fertil 1997;109:153– 64. 4. Sakkas D, Urner F, Menezo Y, Leppens G. Effects of glucose and fructose on fertilization, cleavage, and viability of mouse embryos in vitro. Biol Reprod 1993;49:1288 –92. 5. Harvey MB, Kaye PL. IGF-2 stimulates growth and metabolism of early mouse embryos. Mech Dev 1992;38:169 –73. 6. Drakakis P, Loutradis D, Milingos S, Bletsa R, Kallianidis K, Michalas S. The in vitro development of mouse embryos beyond the blastocyst stage into the hatching and outgrowth stage using different energy sources. J Assist Reprod Genet 1996;13:786 –92. 7. Lavranos TC, Rathjen PD, Seamark RF. Tropic effects of myeloid leukaemia inhibitory factor (LIF) on mouse embryos. J Reprod Fertil 1995;105:331– 8.
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8. Gardner DK, Leese HJ. Concentrations of nutrients in mouse oviduct fluid and their effects on embryo development and metabolism in vitro. J Reprod Fertil 1990;88:361– 8. 9. Gardner DK, Lane M. Culture of viable human blastocysts in defined sequential serum-free media. Hum Reprod 1998;13:148 –59. 10. Menezo YL, Hamamah S, Hazout A, Dale B. Time to switch from co-culture to sequential defined media for transfer at the blastocyst stage. Hum Reprod 1998;13:2043– 44. 11. Menezo Y, Chouteau J, Guyader-Joly C, Veiga A. [Sequential Media: why and how]? Contracept Fertil Sex 1999;27:449 –51. 12. Fong CY, Bongso A. Comparison of human blastulation rates and total cell number in sequential culture media with and without co-culture. Hum Reprod 1999;14:774 – 81. 13. Schoolcraft WB, Gardner DK, Lane M, Schlenker T, Hamiliton F, Meldrum DR. Blastocyst culture and transfer: analysis of results and parameters affecting outcome in two in vitro fertilization programs. Fertil Steril 1999;72:604 –9. 14. Park SE, Lim JM, Chung HM, Lee BC, Lee ES, Hwang WS, Cha KY. Embryotropins in a glucose-, phosphate- and serum-free culture, and development of a chemically defined, protein-free medium for mouse 1-cell embryos derived from in vivo and in vitro. Fertil Steril. Submitted. 15. Park SE, Lim JM, Chung HM, Shim H, Ko JJ, Lee BC, et al. Improved development of embryogenome activated mouse embryos by the addition of amino acids to a serum-, phosphate- and glucose-free medium. Theriogenology. Submitted. 16. Mehta TS, Kiessling AA. Development potential of mouse embryos conceived in vitro and cultured in ethylenediaminetetraacetic acid with or without amino acids or serum. Biol Reprod 1990;43:600 – 6. 17. Mehta TS, Kiessling AA. The developmental potential of mouse embryos conceived in Ham’s F-10 medium containing ethylenediaminetetraacetic acid. Fertil Steril 1993;60:1088 –93. 18. Nasr-Esfahani MH, Winston NJ, Johnson MH. Effects of glucose, glutamine, ethylenediaminetetraacetic acid and oxygen tension on the concentration of reactive oxygen species and on development of the mouse preimplantation embryo in vitro. J Reprod Fertil 1992;96:219 – 31. 19. Hardy K, Handyside AH, Winston RML. The human blastocyst: cell number, death and allocation during late preimplantation development in vitro. Development 1989;107:597– 604. 20. Statistical Analysis Systems Institute. SAS Users Guide. Statistics. Cary, NC: Statistical Analysis Systems Institute, 1992. 21. Lim JM, Hansel W. Improved development of in vitro-derived bovine embryos by use of a nitric oxide scavenger in a cumulus-granulosa cell coculture system. Mol Reprod Dev 1998;50:45–53. 22. Lim JM, Mei Y, Chen B, Godke RA, Hansel W. Effect of nitric oxide removal from an embryo culture system and use of different type of nitric oxide inhibitors. Theriogenology 1999;51:941–9.
Vol. 74, No. 5, November 2000
Embryotropic role of hemoglobin and ethylenediaminetetraacetic acid in preimplantation development of ICR mouse 1-cell embryos Sung E. Park, M.S.,a Hyung M. Chung, Ph.D.,a Jung J. Ko, Ph.D.,a Byeong C. Lee, Ph.D.,b Kwang Y. Cha, M.D.a, and Jeong M. Lim, Ph.D.a College of Medicine, Pochon CHA University; and Seoul National University, Seoul, Korea
Received February 15, 2000; accepted May 31, 2000. Reprint requests: Jeong M. Lim, Applied Embryology Laboratory, Infertility Medical Center of CHA General Hospital, 606-5 Yeoksam 1-Dong, Kangnam-Gu, Seoul 135081, Korea (FAX: 82-2-5018704; E-mail: jmlcimc @netsgo.com). a Infertility Medical Center of CHA General Hospital, College of Medicine. b Department of Theriogenology, Seoul National University. 0015-0282/00/$20.00 PII S0015-0282(00)01536-3
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Objective: To evaluate embryotropic action of hemoglobin (Hb) and ethylenediaminetetraacetic acid (EDTA) on preimplantation embryo development. Design: In vitro model study using mouse embryos. Setting: University affiliated hospital, Pochon CHA University. Animals: Four-week-old block strain ICR mice naturally mated after superovulation. Intervention(s): One-cell embryos were cultured in serum-free, modified preimplantation-1 medium, to which 1 g/ml Hb and/or 0.1 mM EDTA were added. Main Outcome Measure(s): Preimplantation development and blastomere number. Result(s): More (P⬍.05) 1-cell embryos developed to the 4-cell (52% vs. 67%– 84%), 8-cell (48% vs. 65%– 81%), and blastocyst (40% vs. 61%–79%) stages after the addition of hemoglobin (Hb) and/or EDTA than after no addition. Highest proportion of embryos developed to each stage after the combined addition of Hb⫹EDTA. EDTA specifically stimulated the development before the 8-cell stage, which was as similar as Hb⫹EDTA. On the contrary, higher ratio of morula to blastocyst transformation was obtained after the addition of Hb or Hb⫹EDTA than after no addition (0.76 vs. 0.96 – 0.98). Significant increases in the cell number of blastocysts (46.5– 47.2 vs. 53.2 cells), inner cell mass (ICM) cells (16.7–17.5 vs. 21 cells), and the ratio of ICM cells to trophoblasts (0.3– 0.37 to 0.39) were found after the combined addition of Hb⫹EDTA, compared with no addition or with the addition of EDTA or Hb alone. Conclusions: Hb and EDTA have stage-specific effects on supporting preimplantation embryo development; Hb promotes both the development before the 8-cell stage and the morula to blastocyst transformation, whereas EDTA mainly promotes the development to the 8-cell stage. The combined exposure of embryos to Hb and EDTA improves not only preimplantation development but also the growth and quality of blastocysts. (Fertil Steril威 2000;74:996 –1000. ©2000 by American Society for Reproductive Medicine.) Key Words: Embryo, hemoglobin, EDTA, preimplantation development, blastocysts
Developing human embryo culture system is important for increasing the rates of pregnancy and implantation after in vitro fertilization with embryo transfer (IVF-ET). Tremendous efforts have been made to develop such technology, and a number of animal experiments were conducted before the clinical application of developed methods. To date, numerous substances have proved to have embryotropic actions (1–7), and these have been added to culture medium at the different stages of embryo development. On the basis of the latter studies, a sequential culture method using the “proven” embryotropins has been
developed for supporting preimplantation embryo development before transfer (8 –13). We recently developed a sequential culture system using glucose-, phosphate- and serumfree culture media, to which hemoglobin (Hb), a nitric oxide scavenger essential and nonessential amino acids BSA and glucose are supplemented to preimplantation-1 (P-1) medium at different times after culture (14, 15). To optimize our culture system, we subsequently planned to examine the embryotropic effect of ethylendiaminetetraacetic acids (EDTA), calcium and metallic ion chelators, which stimu-
late the development of mouse embryos beyond the stage of embryonic genome activation in the absence of Hb (1, 16 – 18). The combined addition of Hb and EDTA to culture medium may further promote in vitro embryo development. In this study, outbred “block” strain (ICR) mouse 1-cell embryos were cultured in P-1 medium after the supplementation of Hb and/or EDTA. In vitro development to the hatched blastocyst stage, the cell numbers of blastocysts and inner cell mass (ICM) cells, and the ratio of ICM cells to trophoblasts were examined.
MATERIALS AND METHODS Collection of Embryos Four-week-old female ICR mice were maintained under controlled lighting conditions (14L:10D) and superovulated by the injection of 5 IU PMSG (Folligon; Intervet Co., The Netherlands). At 48 h after PMSG injection, 5 IU hCG (Chorulaon; Intervet) was administered and natural mating was concomitantly initiated at the time of hCG injection. The presence of a vaginal plug was examined at 16 h post-hCG injection, and 1-cell embryos with male and female pronuclei were then collected from the oviduct of mated female mice at 18 h after hCG injection. After washing several times in culture medium, embryos were provided for each experiment.
Embryo Culture Medium P-1 medium (Irvine Scientific Co., San Diego, CA) consisting of 101.6 mM NaCl, 4.69 mM KCl, 2.04 mM CaCl2 䡠 2H2O, 0.2 mM MgSO4 䡠 7H2O, 0.33 mM sodium pyruvate, 21.4 mM sodium lactate, 25 mM NaHCO3 and 0.15 mg/ml (w/v) sodium citrate, 0.05 mM taurine, 10 g/ml gentamycin, as well as 5 mg/ml phenol red, was used as a based medium. This medium does not contain glucose, inorganic phosphate, or protein. For the culture of embryos, fatty acid–free BSA (cat. no. A-4161, 3 mg/ml, Sigma Co., St. Louis, MO), Modified Eagle medium nonessential (0.5%, v/v), and essential (1%, v/v) amino acids solutions (Gibco BRL, Grand Island, NY) were added to P-1 medium (designated as modified P-1 medium), and 5.6 mM glucose was subsequently supplemented at 66 h after hCG injection. The osmolarity and pH of modified P-1 medium were within the ranges of 295–305 mOsm and pH 7.3–7.4, respectively.
Culture of Embryos and Assessment of Preimplantation Development
A group of 15 to 20 embryos were cultured in 5 l droplet of modified P-1 medium supplemented with Hb and/or EDTA at 37°C, 5% CO2 in humidified air atmosphere, and embryos were transferred to the medium additionally supplemented with 5.6 mM glucose at 66 h post-hCG injection. Media used for embryo culture were equilibrated in such atmosphere at least for 3 h before culture, and the droplets of equilibrated medium were covered with warm mineral oil (BDH Co., Poole, Dorset, UK). The number of embryos FERTILITY & STERILITY威
developed to the 2-cell, 4-cell, 8-cell, morula, blastocyst, and hatched blastocyst stages was monitored under an inverted microscope (Eclipse TE300, Nikon, Tokyo, Japan) at 42, 66, 90, 114, 138, and 162 h after hCG injection, respectively.
Assessment of the Quality of Blastocysts To evaluate the growth and quality of blastocysts, the cell number of total blastomeres and inner cell mass (ICM) cells as well as the ratio of ICM cells to trophoblasts were counted by the method of Hardy et al. (19) with a slight modification. The zona pellucida of blastocysts obtained at 162 h after hCG injection were removed by 0.5% (v/v) protease solution (cat. no. P-8811, Sigma), and the blastocysts were placed in 15 mM trinitrobezene sulfonic acid (cat. no. P-2297, Sigma) for 15 min at 4°C. Blastocysts were then incubated for 10 min in Tyrode’s lactate solution supplemented with 25 mM Hepes and 0.1 mg/ml anti-dinitrophenol-BSA (cat no. 61007-1, ICN, Irvine, CA) at 39°C. Subsequently, they were treated with 0.01 mg/ml propidium iodide (cat. no. P-4170, Sigma) and incubated with 15% (v/v) guinea pig complement (cat. no. S-1639, Sigma) for 20 to 30 min at 39°C. The blastocysts were then placed in absolute ethanol solution supplemented with 0.05 mM fluorochrome bisbenzimide (cat. no. B-2261, Sigma) overnight at 4°C. After washing in absolute ethanol, the stained blastocysts were mounted on a glass slide, and cell numbers were examined under the inverted microscope with epifluorescent apparatus (HB10104AF, Nikon, Tokyo, Japan).
Experimental Design and Statistical Analysis In experiment 1, the effects of combined addition of Hb (1 g/ml; methemoglobin, cat no. H-7379, Sigma) and/or EDTA (0.1 mM; cell culture–tested, cat no. E-6758, Sigma) on the development of mouse 1-cell embryos to the 2-cell, 4-cell, 8-cell, and blastocyst stages were evaluated. The dosage of tested substances was determined by our preliminary study (data not shown). In experiment 2, embryos were allotted in the same experimental group of experiment 1 and sequentially cultured up to the morula stage (114 h after hCG injection) without any observation. Morula to blastocyst transformation and the development of blastocysts to the hatched blastocyst stage were then monitored. In experiment 3, the number of total blastomeres and ICM cells, and the ratio of ICM cells to trophoblasts in blastocysts obtained in the same groups of experiment 1 were evaluated at 162 h post-hCG injection. Each experiment was replicated 3 (experiments 2 and 3) or 4 (experiment 1) times. Embryos developed to the 4-cell, 8-cell, morula, blastocyst, and hatched blastocyst stages were individually scored as 1 (developed). Oocytes that did not develop to the appropriate stages were scored as 0 (not developed). The mean percentage of development, the mean cell number of total blastomeres and ICM cells, and the mean ratio of ICM cells to trophoblasts in each experiment were calculated. Those scores were then subjected to analysis of variance using the general linear model (PROC997
TABLE 1 Effects of the combined addition of hemoglobin (Hb, 1 g/ml) and/or ethylenediaminetetraacetic acid (EDTA, 0.1 mM) to modified preimplantation-1 medium supplemented with 5.6 mM glucose at 66 h after hCG injection on the development of ICR mouse 1-cell embryos to the blastocyst stage. Mean percentage (⫾SE) of 1-cell embryos developed to Supplements
No. of embryos cultured
2-cell [42]a
4-cell [66]a
8-cell [90]a
Blastocyst [138]a
No addition ⫹Hb ⫹EDTA ⫹Hb⫹EDTA
75 82 80 79
100 100 100 100
52.0 ⫾ 5.1b 67.1 ⫾ 4.9c 80.0 ⫾ 4.9d 83.5 ⫾ 5.0d
48.0 ⫾ 5.3b 64.6 ⫾ 5.0c 75.0 ⫾ 5.1c,d 81.0 ⫾ 5.1d
40.0 ⫾ 5.4b 61.0 ⫾ 5.2c 63.8 ⫾ 5.3c 78.5 ⫾ 5.3d
Note: Model effects (P value) in the development to the 2-cell, 4-cell, 8-cell, and blastocyst stages were 1, .0001, .0001, and .0001, respectively. a Hours after hCG injection. b,c,d Different superscripts in each parameter were significantly different, P⬍.05. Park. Embryotropic action of hemoglobin and EDTA. Fertil Steril 2000.
GLM) in the SAS software program (20). When the significance of the main effects was detected in each experimental parameter, the treatment effects were compared by the least square method. A statistical significance was determined where P value was less than .05.
RESULTS Experiment 1 A total of 316 1-cell embryos were provided for this experiment, and all embryos developed to the 2-cell stage until 42 h after hCG injection, regardless of the treatments. As shown in Table 1, a significant treatment effect (P⫽ .0001) was found in the development to the 4-cell, 8-cell, and blastocyst stages. The addition of Hb or EDTA to modified P-1 medium significantly promoted the developments compared with no addition (52% vs. 67%– 80%, P⬍.0329 in the 4-cell; 48% vs. 65%–75%, P⬍.0228 in the 8-cell; and 40% vs. 61%– 64%, P⬍.0057 in the blastocyst stages). The promoting effects of EDTA on embryo development to the 4-cell and 8-cell stages were as similar as those of EDTA⫹Hb and similar proportion of embryos developed to the 8-cell stage in these 2 treatments. However, more embryos developed to blastocysts after the addition of Hb⫹ EDTA than after the addition of either Hb or EDTA alone.
Experiment 2 Of those 360 1-cell embryos cultured after each treatment, 243 (67%) embryos developed to the morula stage were used for analysis. As shown in Table 2, a significant treatment effect was found in the morula to blastocyst transformation (P⫽.0002). Higher transformation rate was obtained after the addition of Hb alone than after no addition (0.76 vs. 0.98; P⬍.0001). However, no significant effect was found after the addition of EDTA, regardless of the presence (0.96 vs. 0.98) or the absence (0.76 vs. 0.89) of Hb in the medium. No significant main effect (P⫽.41) was detected in 998
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the development of blastocysts to the hatched blastocyst stage.
Experiment 3 A significant treatment effect was observed in the cell numbers of blastocysts (P⫽.0438) and ICM cells (P⫽.0031) and in the ratio of ICM cell to trophoblast (P⫽.0338). As shown in Table 3, higher cell numbers of blastomeres (46.5– 47.2 cells vs. 53.2 cells per blastocyst, P⬍.019) and ICM cells (16.7–17.5 cells vs. 21 cells per blastocyst, P⬍.002) in blastocysts were obtained after the addition of Hb⫹EDTA to P-1 medium than after no addition or after the addition of EDTA or Hb alone. The ratio of ICM cells to trophoblasts was also significantly increased after the combined addition than any other combinations (0.36 – 0.37 vs. 0.39, P⬍.05).
DISCUSSION The results of this study demonstrated that Hb and EDTA have different roles in supporting in vitro preimplantation development of outbred block strain ICR mouse 1-cell embryos. Hb significantly promoted the development to the 8-cell stage and the morula to blastocyst transformation; EDTA mainly strongly stimulated early cleavage up to the 8-cell stage. Combined exposure of 1-cell embryos to Hb and EDTA during their preimplantation development improved not only development to the blastocyst stage but also the growth and quality of blastocysts; 78% of 1-cell embryos sequentially cultured in modified P-1 medium developed to the blastocyst stage after the supplementation of Hb and EDTA. In the case of bovine embryos, Hb exerts its stimulatory action only in the presence of cumulus cells (21), and this result suggests that Hb stimulates blastocyst formation by scavenging nitric oxide secreted from co-cultured cumulus cells. In our recent (14) and present studies, Hb improved in Vol. 74, No. 5, November 2000
TABLE 2 Morula to blastocyst transformation and development of blastocyst to the hatched blastocyst stage in embryos cultured after the addition of hemoglobin (Hb, 1 g/ml) and/or ethylenediaminetetraacetic acid (EDTA, 0.1 mM) to modified preimplantation-1 medium supplemented with 5.6 mM glucose at 66 h after hCG injection. Morula/blastocyst transformation
Hatched blastocyst
Supplements
Number of blastocysts/ Number of morulae
Ratio (⫾ SE)
Number examined
Number developed (Mean % ⫾ SE)
No addition ⫹Hb ⫹EDTA ⫹Hb⫹EDTA
42/55 50/52 63/71 64/65
0.76 ⫾ 0.04a 0.96 ⫾ 0.04b 0.89 ⫾ 0.03a,b 0.98 ⫾ 0.04b
42 50 63 64
28.6 ⫾ 7.4 38.0 ⫾ 6.8 33.3 ⫾ 6.1 43.8 ⫾ 6.0
Note: Model effect (P value) in the ratio of morula/blastocyst transformation and development to the hatched blastocyst was .0002 and .41, respectively. Different superscripts in each parameter were significantly different, P⬍.05.
a,b,c
Park. Embryotropic action of hemoglobin and EDTA. Fertil Steril 2000.
vitro preimplantation development of mouse embryos even in the absence of cumulus cells. From these data, it appears that (i) Hb has an embryotropic action in both mouse and bovine embryos and that (ii) the detailed action of Hb on embryo development of the mouse and the bovine species was different. Additional results of our previous study using bovine embryos (22) showed that several nitric oxide synthase inhibitors, such as L-nitroarginine-methylester did not stimulate preimplantation development. Research is currently under way to clarify how Hb promotes mouse embryo development. From the results of our study, further information on Hb action during in vitro development was obtained: Hb added to culture medium promotes all stages of mouse preimplantation development. Hb improves embryo development during their own genome activation before the second cleavage, the development to the 8-cell stage, and morula to blastocyst transformation. These results were comparable to those of our bovine embryo study that Hb significantly and specifically enhances the morula to blastocyst transformation. Probably, species specificity (bovine vs. mouse) and/or culture milieu (coculture vs. cell-free culture) affect the embryotropic action of Hb. In contrast to the action of Hb, EDTA mainly exerts its promoting effect on supporting the development during early cleavage, and such effect was stronger than that of Hb (Table 1). These results raised the possibility that nitric oxide and metallic ion contaminants of culture media independently inhibit different stages of preimplantation development with different embryotoxicities. This hypothesis was strongly supported by our result showing the different embryotropic roles of Hb and EDTA during in vitro development to the blastocyst stage. Considering the previous reports on the action of EDTA on embryo development (1, 16 –18), EDTA assists mouse 1-cell embryos to overcome in vitro developmental block phenomenon during their 1st cleavage, regardless of the presence of nitric oxide in culture systems. FERTILITY & STERILITY威
ICM cell and total blastomere numbers in blastocyst are the indicators of embryo growth, whereas ICM cell to trophoblast ratio represents the quality of the blastocyst. Synergism of Hb and EDTA appeared not only as an improved development of 1-cell embryos to the blastocyst stage but also as increased ICM cell and trophoblast numbers yielding high ICM cell to trophoblast ratio. Accordingly, a combined addition of these substances stimulates both proliferation and differentiation of mouse embryos during in vitro culture, which enhances the quality and growth of blastocysts. In the basis of our present results, we renovated our sequential culture system for preimplantation development of mouse embryos. This system uses small volume of me-
TABLE 3 Effects of the combined addition of hemoglobin (Hb, 1 g/mL) and/or ethylenediaminetetraacetic acid (EDTA; 0.1 mM) to modified preimplantation (P)-1 medium supplemented with 5.56 mM glucose at 66 h after hCG injection on the cell number of blastomeres and inner cell mass (ICM) cells in blastocysts derived from ICR mouse 1-cell embryos. Cell numbers (mean ⫾ SE) of blastocysts Supplements
Total
ICM cells
ICM cell to trophoblast ratio
No addition ⫹Hb ⫹EDTA ⫹Hb⫹EDTA
46.5 ⫾ 2.2a 50.4 ⫾ 1.9a 47.2 ⫾ 1.8a 53.2 ⫾ 1.7b
16.7 ⫾ 1.0a 18.8 ⫾ 0.9a 17.5 ⫾ 0.8a 21.0 ⫾ 0.8b
0.36 ⫾ 0.01a 0.37 ⫾ 0.01a 0.37 ⫾ 0.01a 0.39 ⫾ 0.01b
Note: Model effect (P value) in the cell numbers of total blastomere and ICM cells and the ratio of ICM to trophoblast was .0438, .0031, and .0338, respectively. a,b Different superscripts in each parameter were significantly different, P⬍.05. Park. Embryotropic action of hemoglobin and EDTA. Fertil Steril 2000.
999
dium (5 l medium for culturing 15–20 embryos), which supports paracrine action of embryos during in vitro culture. To intoxicate the embryotoxic factors present in our system, Hb, EDTA, and taurine are present in culture media. Thus, P-1 medium was supplemented with amino acids, BSA, EDTA and Hb for the first culture phase during the development to the 4-cell stage, and this modified P-1 medium was additionally supplemented with glucose at the 4-cell stage for later preimplantation development. Attempts have been made to apply this culture system for human IVF-ET program after further modification of our system for developing chemically defined system.
Acknowledgments: This work was supported by a grant from the Interdisciplinary Research Program of the KOSEF (grant 1999-2-205-002-5) and by a grant from the Korea Research Foundation (KRF-99-041-G00065).
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