to the Solution at Various Temperatures1

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1 BIOLOGY OF REPRODUCTION 47, (1992) Survival of Mouse Morulae Vitrified in an Ethylene Glycol-Based Solution after Exposure to the Solution at Various Temperatures1 M. KASAI,2 M. NISHIMORI, S.E. ZHU, T. SAKURAI, and T. MACHIDA Laboratoiy of Animal Science, College of Agriculture, Kochi University, Nankoku, Kochi 783, Japan ABSTRACT Mouse morulae were exposed in one step to a vitrification solution (EFS, a modified PBS containing 40% ethylene glycol, 18% Ficoll, and 0.3-M sucrose) at various temperatures, then cooled rapidly in liquid nitrogen, and then warmed rapidly. All of the embryos exposed to the EFS solution for 0.5 mm at 25#{176}Cbefore vitrification developed in culture. However, survival rates were lower if the duration of exposure was prolonged to 2, 5, or 10 mm. At lower ambient temperatures (20, 10, and 5#{176}C), high survival rates were associated with longer exposure to the EFS solution. The toxicity of the EFS solution was also lower at lower temperatures. The toxic injury of morulae was manifested as decompaction of the blastomeres. Among the three additives in the EFS solution, ethylene glycol, which can cross cell membranes, was responsible for the toxicity. The results show that the optimum time for exposure of the embryos to the EFS solution before rapid cooling varies with the ambient temperature, i.e., 0.5 mm at 25#{176}C, mm at 20#{176}C, 2-5 mitt at 10#{176}C, and 2-10 mitt at 5#{176}C. If they are exposed for an optimum period, almost all mouse morulac can survive vitrification (94-100%). INTRODUCTION In 1985, RaIl and Fahy [1] reported the first successful cryopreservation of mouse embryos by vitrification. The vitrification solution they used (VS 1) was composed of dimethylsulfoxide (DMSO), acetamide, propylene glycol, and polyethylene glycol as additives, and the total concentration of the additives was 6.54 mol/l. One obstacle to successful vitrification is the toxicity caused by high concentrations of additives. One way to avoid this toxicity is to equilibrate embryos in a diluted solution at room temperature, and then transfer them to more concentrated solutions at a low temperature (4#{176}C) before cooling rapidly with liquid nitrogen [1]. However, this procedure is not convenient and takes about 35 mm. There have been several attempts to improve the vitrification solution and the procedure for embryo treatment [2-5]. Very recently, Kasai et al. [6] reported that mouse morulae can be vitrified by a simple method, without appreciable loss of viability. They used a minimally toxic vitrification solution called EFS, which consists of 40% ethylene glycol, 18% Ficoll, and 0.3 M sucrose dissolved in modified PBS (PB1) [7]. They exposed mouse embryos directly to the solution for only 2 mm at 20#{176}C, and then plunged them into liquid nitrogen. The embryos were rapidly warmed at 20#{176}C, and nearly all of them developed in culture (97-98%). Although this method is simple and efficient, the ambient temperature during exposure to the solution was strictly controlled (20 ± 0.5#{176}C).However, temperature must be critical to survival, because the permeability of embryos to an additive is closely related to the temperature [8-10], and Accepted August 12, Received May 22, This work was supported by a grant-in-aid for scientific research from the Mmiso) of Education, Science and Culture, Japan. 2Correspondence: FAX: permeation of additives strongly affects both cryoprotection and toxic injury. In the present study, mouse morulae were exposed to EFS solution in one step at various temperatures and then vitrified; survival was assessed with reference to the toxicity of the additives. Enthyos MATERIALS AND METHODS Female ICR mice (6-12 wk old, CLEAJapan, Inc., Tokyo, Japan) were induced to superovulate by i.p.injections of 5 IU ecg (Serotropin, Teikokuzoki, Tokyo, Japan) and 5 IU hcg (Puberogen, Sankyozoki, Tokyo, Japan) given 48 h apart. They were mated with adult male ICR or BDF1 mice. Embryos were flushed from the upper half of excised uteri and part of the oviducts with PB1 medium h after the injection of hcg. The embryos were washed in fresh PB1 and only morphologically normal compacted morulae were used. Toxicity Test of the Vitrification Solution and Individual Additives In the first series of experiments, the toxicity of the vitrification solution (EFS) [6] was tested. The EFS solution was 40% (v/v) ethylene glycol and 60% PB1 medium containing 30% (w/v) Ficoll (average molecular weight 70000; Pharmacia, Uppsala, Sweden) and 0.5 M sucrose; thus the final concentrations of Ficoll and sucrose were 18% and 0.3 mol/l, respectively. About 100 pa EFS solution was placed in a culture dish (35 x 10 mm; Nunc Inc., Naperville, IL) under paraffin oil (Nacalai Tesque, Inc., Kyoto, Japan), and kept at 5 ± 1, 10 ± 1, 20 ± 0.5, 25 ± 0.5, or 30 ± 0.5#{176}C for at least 3 h. Experiments at 5 and 10#{176}C were conducted in a temperature-controlled cooling chamber (1.8 X 1.8 X 1.9 m) located in a corner of a laboratory room, and ex- 1134

2 VITRIFICATION OF MOUSE EMBRYOS 1135 periments at 20#{176}C or above were done in another laboratory room where an adjustable air conditioner was located. The ambient temperature was monitored on the table and set to be within the range. Nine to 10 ICR embryos were suspended in the EFS solution. They were left for 2, 5, or 10 mm at each temperature. Then they were transferred to PB1 medium containing 0.5 M sucrose (S-PB1) and finally to fresh PB1 medium 5 mm later at each temperature, except for embryos suspended at 5#{176} and 10#{176}C, which recovered at 20#{176}C. In the second series of experiments, the toxicity of individual additives in the EFS solution was tested. Embryos (9-11) were suspended in 100 pa of PB1 medium containing either ethylene glycol (30 or 40% [v/v]), Ficoll (50% [w/v]), or sucrose (0.5, 0.75, or 1.0 M) under paraffin oil in a culture dish at 25#{176} or were 30#{176}C. After 20 mm, the embryos in ethylene glycol were transferred to PB1 medium containing the same concentration of ethylene glycol and 0.5 M sucrose, then to S-PB1, and then to fresh PB1 medium at 5-mm intervals. Embryos in Ficoll and sucrose were directly transferred to fresh PB1 medium. The experiments were done four times, and a total of embryos were tested in each group. Vitrification of Embryos The embryos were exposed to EFS solution in a room where the temperature was controlled at 5 ± 1, 10 ± 1, 20 ± 0.5, or 25 ± 0.5#{176}C, as described above. EFS solution was prepared in a 0.25-ml plastic straw (I.M.V., L Aigle, France) as described previously [6]. Briefly, a large column of S-PB1 medium (-100 pa) and two columns of EFS solution (-6 and -40 il) were aspirated, separated by air, and the straw was held horizontally. When the embryos were vitrified after 0.5 mm of exposure, they were directly transferred to the larger column of EFS solution via a pipette with a small volume ( pa) of PB1 medium. When the embryos were exposed to EFS solution for 2 mm or more before vitrification, they were first suspended in EFS solution in a watch glass, washed in the solution twice, and transferred to the EFS column. Then air (-6 pa), EFS solution (-6 pa), air (-15 pa), and S-PB1 medium (-20 pa) were aspirated successively before being sealed with straw powder. The configuration of the straw has been described elsewhere [6]. After 0.5, 2, 5, or 10 mm of exposure to EFS solution, the straws were immersed in liquid nitrogen vertically in two steps to prevent bursting: first, about half the straw (including the EFS solution) was immersed rapidly (2400#{176}C! mm, between 0#{176}C and - 150#{176}C),and then the rest of the straw (including the larger column of S-PB1) was immersed slowly. The straws were stored for at least 1 day, and then warmed rapidly in 20#{176}C water (1600#{176}C/mm, between -150#{176}C and 0#{176}C). As soon as the crystallized S-PB1 medium in the straw began to melt (about 5 sec), the embryos in the vitrification solution were expelled onto a watch glass by flushing the straw with -0.8 ml of S-PB1 medium, agitated gently, and then transferred into fresh S-FBi medium at 20#{176}C. About 5 mm after the embryos were flushed out, they were transferred to fresh PB1 medium. The experiments were done five times, and a total of 50 embryos were tested in each group. Assessment of Suivival Recovered embryos were washed in a modified Krebs- Ringer-bicarbonate medium [11] and were cultured in -0.3 ml of the medium under paraffin oil in 5% CO2 in air at 37#{176}C. As a control, fresh morulae were cultured after being held in PB1 medium at 5 ± 1, 10 ± 1, 20 ± 0.5, 25 ± 0.5, or 30 ± 0.5#{176}C for at least 30 mm. The survival of the embryos was assessed by the development of expanded biastocysts during 48 h in culture. In one experiment, embryos from pigmented mice (ICR X BDF1) were vitrified after 0.5 mm of exposure to EFS solution at 25#{176}C. After recovery, 5-6 embryos were transferred to each uterine horn of ICR mice (10-12 embryos per animal) on Day 3 of pseudopregnancy. The recipients were allowed to deliver their litters. Stattstics In vitro survival rate after each treatment was compared with that in the temperature control group, with the x2 test. Temperature Control RESULTS In the temperature control where 50 mouse morulae were held in PB1 medium for 20 mm at 5, 10, 20, 25, or 30#{176}C, 100% of the embryos developed to expanded blastocysts in culture, irrespective of the temperature. Toxicity Test As shown in Figure 1 (closed circles), when embryos were exposed to EFS solution for 2, 5, and 10 mm at 5 or 10#{176}C, 100% of them developed into expanded blastocysts in culture. At 20 or 25#{176}C, almost all of them (95-100%) also survived after exposure to the solution for 2 and 5 mm; however, survival rates after 10 mm of exposure were reduced signifirantly (78% at 20#{176}C, and 0% at 25#{176}C; P < 0.001). At 30#{176}C, after exposure to the solution for only 2 mm, only 18% of embryos survived (P < 0.001); the rates exposed for 5 and 10 mm were 5% and 0%, respectively. Figure 2 shows the morphological observation of the morulae exposed to EFS solution for 10 mm at 30#{176}C. When compact morulae (Fig. 2A) were suspended in EFS solution, they were considerably shrunken (Fig. 2B). Just after recovery in PB1 medium, they were apparently normal compacted morulae (Fig. 2C). After only 2 h of culture, however, they decompacted into -16 cell embryos (Fig. 2D), which arrested development.

3 1136 KASAI ET AL w I- 100#{149} 0#{149} 100#{149} 0-100#{149} 0- B C - I I I I D EXPOSURE TIME (MIN) Since toxicity of EFS solution was manifested at higher temperatures, the toxicity of individual additives was tested at 25 and 30#{176}C(Table 1 ). All of the embryos exposed to 30% ethylene glycol at 25#{176}C developed in the culture. However, the survival rates were very low when the concentration of ethylene glycol was 40% (3% ), and also when the temperature was 30#{176}C (24%). Ficoll was not toxic; 98% of the embryos survived suspension in 50% Ficoll, even at 30#{176}C. Sucrose was not harmful at 25#{176}C even when the concentration was 1.0 mol/l (100% survival). At 30#{176}C, 0.5 M sucrose was not harmful (100% survival), but survival rates of embryos exposed to 0.75 and 1.0-M sucrose were significantly different from control (50 and 10%, respectively; p < 0.001). Vitrification of Embryos In total, 800 morulae of ICR mice were vitrified in EFS solution, and 774 (97%) were recovered after warming. As shown in Figure 1 (open circles), of the embryos recovered after exposure to the solution at 25#{176}C and rapid cooling in liquid nitrogen, all developed in culture after exposure to EFS solution for only 0.5 mm. However, survival rate was inversely related to the duration: the rates being 92% after 2 mm of exposure (p < 0.05), and 38% and 4% after 5 and 10 mm of exposure, respectively (p < 0.001). With embryos exposed to the solution at 20#{176}C before cooling, survival rates were quite high (98-100%) over a wide range of exposure times (0.5, 2, and 5 mm), but the rate decreased significantly (p < 0.001) after 10 mm of exposure (72%). At 10#{176}C, survival rates were also high with embryos exposed to the EFS solution for 2 or 5 mm (94-100%), but not 0.5 mm (79%;p < 0.001) and 10 mm (84%; p < 0.01). When the ambient temperature during the exposure was 5#{176}C, the survival rate after 0.5 mm of exposure was lower (23%; p < 0.001), but most embryos survived after 2, 5, or 10 mm of exposure (96-98%). In the transfer experiment, a total of 79 embryos were transferred to 7 recipient mice, 6 of which got pregnant and delivered 50 live young and 1 stillborn fetus in total (29 females and 22 males) on Days The percentage of young was 65% when based on the number of transferred embryos (51/79), and was 74% when based on embryos transferred to pregnant recipients (51/69). DISCUSSION Inclusion of one or more cryoprotective additives in the freezing medium is essential for successful cryopreserva- FIG. 1. Effect of the temperature at which embryos were exposed to tion of mammalian embryos in liquid nitrogen. In vitrifi- EFS solution on the survival of mouse morulae stored at -196#{176}Cby vitri- cation, toxicity of the additive(s) limits embryo survival, befication. Embryos were held in EFS solution at 25 (A), 20 (B), 10 (C), or 5#{176}C cause the concentration of additives required for vitrification (D) for various times, and then they were either recovered without vitrifi-. cation (toxicity test; closed circles) or vitrified in liquid nitrogen (open cir- is high. There are at least two strategies for reducing the des). Forty embryos were treated for each toxicity test group, and toxicity of any given solution: one is to lower the temperembryos were examined for each vitrification group. Survival rates were ature, and the other is to shorten the exposure period [3]. assessed by the proportions of embryos developed to expanded blasto-. - cysts in culture, based on the number of recovered embryos after vitrifi- The temperature of the solution to which embryos were cation. exposed significantly affected embryo survival, and the tox-

4 VITRIFICATION OF MOUSE EMBRYOS 1137 icity of the solution was higher at higher temperatures (Fig. 1, closed circles). When the EFS solution was first made, ethylene glycol was used because it is less toxic to mouse embryos than other permeating additives [6, 12]. However, it is clear that the harmful substance in the EFS solution is ethylene glycol (Table 1). Since the permeability of mouse embryos to an additive is higher at higher temperatures [8-10], the harmful effect of ethylene glycol may be exerted mntracellularly after permeation. At higher temperatures, biochemical toxicity of the additive may be higher even if the intracellular concentration of the additive is the same. There may be a possibility that the ethylene glycol-mnduced injury was caused by osmotic events, because the intracellular concentration of ethylene glycol, and its toxicity, is time-dependent. When embryos are exposed to injurious hypotonic stress, they overswell and presumably the plasma membrane is damaged [13]. We have observed that such cells shrink into dead cell mass in culture. However, the embryos injured by exposure to EFS solution appeared to be morphologically normal compacted morulae just after recovery, but they decompacted into clear blastomeres with arrested development in the following culture (Fig. 2). This TABLE 1. Survival of mouse morulae exposed to various cryoprotectants in PB1 for 20 mm at 25#{176}C and 30#{176}C. Cryoprotectants Ethylene Ficoll 70 Sucrose Control glycol Solution Concentration 30% (v/v) 40% (v/v) 50% (w/v) 0.5 M 0.75 M 1.0 M - No. of embryos developed/treated (%) 25#{176}C Temperature 30#{176}C 40/40 (100) 10/42 (24) 1/40 (3) 0/40 (0) 40/40 (100) 39/40 (98) 40/40 (100) 39/39 (100) 40/40 (100) 20/40 (50) 40/40 (100) 4/40 (10) 50/50 (100) 50/50 (100) Embryos were held in PB1 medium at each temperature for 20 mm. p < 0.001; significantly different from control. observation strongly suggests that the injury was biochemical, not osmotic. If so, the compacted morula seems to be a good material for toxicity test, because this event does not occur in precompaction embryos, which have clear blastomeres. The following circumstances support that the embryos were less sensitive to osmotic stress: 1) morulae are probably highly permeable to ethylene glycol, because FIG. 2. When fresh mouse morulae in PB1 (A) were suspended in EFS for 10 mm at 30#{176}C. they were apparently normal compacted morulae just after recovery in PB1 medium (C). However, after 2 h of culture, they decompacted into -16 cell embryos (D). Embryos suspended in EFS solution (at 25#{176}C) remained shrunken and did not reexpand (B). x186.

5 1138 KASAI ET AL. the permeability of embryos to an additive increases as development proceeds [8]; 2) ethylene glycol has a lower molecular mass than other commonly used additives; and 3) the amount of intracellular ethylene glycol must be limited, because sucrose caused embryos in the EFS solution to shrink (Fig. 2B). In contrast, the toxicity of nonpermeating agents, especially Ficoll, was nonexistent, probably due to its high molecular mass, which had little or no osmotic effect. Although sucrose is known to protect refrigerated embryos [12, 14], it had harmful effects at 30#{176}C at higher concentrations (Table 1), and also at 20#{176}C if the embryos were exposed for longer periods [12]. Prolonged shrinkage or dehydration, or both, caused by osmosis may be injurious at high temperatures. In general, embryos are not always manipulated at a strictly controlled ambient temperature before vitrification, and the effects of the temperature during exposure to the additive have not been extensively studied. The present results show that temperature is of critical importance for the survival of vitrified embryos, even at normal room temperatures, e.g., 20#{176}C and 25#{176}C. The effect of additives is probably increased by their very high concentrations in the vitrification solution. In conventional embryo freezing, however, the concentration of the additive is relatively low, and embryos are resistant to longer exposure to the storage solution over a wide range of temperatures; survival of mouse morulae was not lowered after suspension in 1.5 M ethylene glycol, DMSO, or glycerol at 20#{176}Cfor 6 h [12], and mouse and rabbit embryos were resistant to equilibration for mm at 37#{176}C [15-17]. Permeation of cell membranes by an additive is considered a necessity for successful embryo preservation by vitrification [3], but if the internal concentration of additives is too high, there can be toxic injury. The low survival rates of vitrified embryos exposed for longer times to the EFS solution at higher temperatures (Fig. 1, open circles) are mainly attributable to the toxicity of the EFS solution, because these embryos were liable to injury without vitrification (Fig. 1, closed circles). In some cases, however, vitrification caused more damage than can be accounted for by the toxicity of the EFS solution; e.g., when embryos were exposed to the EFS solution at 25#{176}C for 5 mm, the survival rate was 95% without cooling, but it was only 38% after vitrification (Fig. 1A). The lower survival rate would not be attributed to intracellular freezing, because the embryos survived vitrification after a much shorter period of exposure at this temperature. For the same reason, vitrification per se or thermal shock alone is not harmful. Therefore, the embryos stressed by a low level of additive toxicity which is not detectable by in vitro culture may be more susceptible to vitrification. The low survival rates of vitrified embryos exposed for short periods at low temperatures (Fig. 1, open circles) strongly suggest that permeation of ethylene glycol is not a sufficient condition for survival; in those embryos, intracellular ice may have formed. However, even at 5#{176}C, enough ethylene glycol seems to have penetrated during 2 mm of exposure (96%; Fig. 1D). Ax this temperature, in addition, embryos can be resistant to 10 mm of exposure to EFS solution before vitrification, probably because the ethylene glycol permeated more slowly or because the additives were less toxic at that temperature, or both. From a practical point of view, embryo manipulation at 20 or 25#{176}Cwould be very convenient, because the room temperature falls between these values in most laboratories. At 20 or 2 5#{176}C, only 0.5 mm of exposure to EFS solution was enough for a high post-vitrification survival rate, which indicates that enough ethylene glycol permeates the embryos during this short period. Thirty seconds would be just enough time to load the embryos in one step into a straw and seal it. The results also show that embryos survived vitrification even when they were directly suspended in EFS solution in a straw, without being washed in the solution before cooling. Thus, a small amount of PB1 introduced into the EFS column does not have a detrimental effect. Vitrification after a much shorter exposure at room temperature was reported by Nakagata [5,18]. He vitrified mouse oocytes and embryos after only 5-10 sec of exposure to a solution modified from VS1. In his procedure, however, oocytes/embryos in a sample tube had to be transferred to liquid nitrogen within 5-10 sec, which would make the procedure less practical. Therefore, our ethylene glycol-based EFS solution must be much less toxic than the DMSO-based modified VS1, which is a significant advantage. The present results show that the optimum time for exposure of embryos to EFS solution before cooling varies with the ambient temperature. If embryos are exposed within the optimum period for a given temperature, that is, 0.5 mm at 25#{176}C, mm at 20#{176}C, 2-S mm at 10#{176}C, or 2-10 mm at 5#{176}C, mouse morulae can be vitrified after a short period of exposure to EFS solution over a wide range of temperatures, with no appreciable effect on survival. REFERENCES 1. RaIl WF, Fahy GM. Ice-free cryopreservation of mouse embryos at - 196#{176}Cby vitrification. Nature 1985; 313: Scheffen B, Van Ocr Zwalmen P. Massip A. A simple and efficient procedure for preservation of mouse embryos by vitrification. Cryo Leu 1986; 7: RaIl WF. Factors affecting the survival of mouse embryos cryopreserved by vitofication. Cryobiology 1987; 24: Rail WF, Wood MJ, Kirby C, Whittingham 0G. Development of mouse embryos cryopreserved by vitrification. J Reprod Fertil 1987; 80: Nakagata N. Survival of 4-cell mouse embryos derived from in vitro fertilization after ultrarapid freezing and thawing. Exp Anim 1989; 38: , Kasai M, Komi J1-l, Takakamo A, Tsudera H, Sakurai T, Machida T. A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution without appreciable loss of viability. J Reprod Fertil 1990; 89: Whittingham 0G. Survival of mouse embryos after freezing and thawing. Nature 1971; 233: Mazur F, Rigopoulos N, Jackowski SC, Leibo SF. Preliminary estimates of the permeability of mouse ova and early embryos to glycerol. Biophys J 1976; 16:232a.

6 VITRIFICATION OF MOUSE EMBRYOS Leibo SP. Fundamental cryobiology of mouse ova and embryos. In: Elliott K, Whelan J (eds.), The Freezing of Mammalian Embryos. Amsterdam: Elsevler; 1977: Jackowski S, Leibo SF, Mazur P. Glycerol permeabilities of fertilized and unfertilized mouse ova.j Exp Zool 1980; 212: Toyoda Y, Chang MC. Fertilization of rat eggs in vitro by epididymal spermatozoa and the development of eggs following transfer. J Reprod Fertil 1974; 36: Kasai M, Niwa K, Iritani A. Effects of various cryoprotective agents on the survival of unfrozen and frozen mouse embryos. J Reprod Fertil 1981; 63: Mazur F, Schneider U. Osmotic responses of preimplantation mouse and bovine embryos and their cryobiological implications. Cell Biophys 1986; 8: Kasal M, Niwa K, Iritani A. Protective effect of sucrose on the survival of mouse and i-at embryos stored at 0#{176}C. J Reprod Fertil 1983; 68: Whittingham 0G. Viability of frozen-thawed mouse blastocysts. J Reprod Fertil 1974; 37: Tsunoda Y, Sugie T. Survival of rabbit eggs preserved in plastic straws in liquid nitrogen. J Reprod Fertil 1977; 49: Kojima 1, Soma T, Oguri N. Effect of rapid addition and dilution of dimethyl sulfoxide and 37#{176}Cequilibration on viability of rabbit morulae thawed rapidly. Cryobiology 1987; 24: Nakagata N. High survival rate of unfertilized mouse oocytes after vitrification. J Reprod Fertil 1989; 87: