Cryopreservation of Mammalian Embryos by Vitrification

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1 - Review- Cryopreservation of Mammalian Embryos by Vitrification Shigeki OHBOSHI Faculty of Veterinary and Animal Science, Nippon Veterinary and Animal Science University, Musashino-shi Key words: Mammalian embryos, Cryopreservation, Vitrification, Cellular injury Cryopreservation of mammalian embryos, coupled with embryo transfer techniques, have made important contributions to recent advances in reproductive technology. The discovery that glycerol is an effective cryoprotectant for spermatozoa41) led to research on cryopreservation of mammalian embryos. In the past quarter of a century since the first successful cryopreservation of mouse embryos58,60), basic and applied research has resulted in the cryopreservation of embryos of many mammalian species. Improvements have been made in nearly all steps of the cryopreservation process, resulting in considerable simplification of the original slow freezing and thawing procedures. Recently, the first successful cryopreservation of murine embryos by vitrification was reported by Rall and Fahy43). Using highly concentrated solutions of cryoprotectants, they obtained high survival rates of mouse min. This approach to cryopreservation has some advantages over conventional freezing methods, such as reduction of the cost of embryo freezing due to the simplified process and elimination of the need for costly computerized freezing units (programmed freezer). Another advantage is that the solution forms no extracellular ice, which is one of the major causes of cell injury27). A higher survival rate of embryos can therefore be expected with vitrification than with conventional freezing. However, highly concentrated solutions of cryoprotectants have extremely harmful effects on embryos. If the disadvantages of high concentrations of cryoprotectant mixtures can be overcome, good embryo recovery after warming will be obtained, and vitrification of embryos might become commercially practical. Characteristics of vitrification During the treating and cooling of cells, and during the restoration of the cryopreserved cells into a physiological state, the cells are exposed to the risk of injury. The major causes of cellular injuries are as follows: toxicity of chemicals, chilling injury, physical injury by extracellular ice, toxicity of electrolytes concentrated with extracellular ice, formation and growth of intracellular ice, fracture damage, and osmotic stress21). Vitrification is a physical process in which a highly concentrated solution of cryoprotectants solidifies during cooling without formation of ice crystals. The solution becomes Anim. Sci. Technol. (Jpn.) 69 (12):

2 OHBOSHI increasingly viscous and turns into an amorphous state called "glass", which retains the normal molecular and ionic distributions of the liquid state44). The glassy form is devoid of all ice crystals, and embryonic cells are not subjected to the physical damage that is associated with ice crystal formation43). Therefore, the major advantage of vitrification is that mechanical damage to the embryo due to intraand extracellular ice formation does not occur. An additional advantage is that freezing is accomplished by plunging the embryos into liquid nitrogen. Although the presence of cryoprotectants in the vitrification solution decreases the probability of ice formation when very rapid cooling speed is employed, the highly concentrated cryoprotectant is toxic and causes osmotic injury to the embryonic cells. Therefore, several methods have been used to reduce the harmful effects of chemicals : short time of exposure to cryoprotectants9), use of low-toxicity cryoprotectants44) or mixtures of relatively dilute cryoprotectants25), addition of nonpermeating cryoprotectants9), reduction of the cryoprotectant concentration44), and exposure to the vitrification solution at low temperatures44). The embryos at certain stages in some species, especially pig, can be severely altered or irreversibly damaged by being cooled below physiological temperatures. This hypothermic sensitivity appears to be dependent on the developmental stage of the embryo and on the conditions under which it develops42). Vitrification reduces the possibility of both intracellular and extracellular ice formation, and rapid cooling of embryos reduces the detrimental hypothermic-induced cellular changes that take place during slow cooling28). Fracture damage is thought to be caused by a nonuniform change in the volume of the solution during the rapid phase change between liquid and solid45). The incidence of this physical injury in vitrification also seems to be less than in conventional freezing19,20). Kasai et al.20) showed that fracture damage occurs both during cooling and warming but more damage occurs during warming, so that the fracture damage can be prevented completely by controlling the cooling and warming velocities during passage through the tempera- change occurs in vitrification. Other factors such as cooling rate, type of cryoprotectant and sample volume also seem to affect the survival rate of embryos following vitrification1). Cryopreservation can be extremely disruptive to the cellular organization of embryos. Ice crystal formation can disrupt plasma membranes, storage in liquid nitrogen can denature critical intracellular functions and organelles, and the central cytoarchitecture of a cell can be destroyed. As mentioned above, it is easier to deduce the optimal conditions for vitrification than for the conventional freezing method because of the lack of extracellular ice formation. However, the causes of injury in cryopreserved cells are complex and remain only partially understood. Further information about cellular damage during or after vitrification would lead to improved protocols for embryo cryopreservation and a better understanding of mammalian embryos. Cellular injury of embryos by cryopreservation Morphological features Whittingham and Anderson59) first reported the ultrastructure of frozen-thawed mouse 8- cell embryos, in which they found no disorganization of any membranous or membraneassociated structures. Mohr and Trounson29) described the ultrastructure of cryopreserved cow embryos. In that study, cellular damage was observed, but the extent of the damage varied between and within embryos. The Day 5 or younger embryos appeared to sustain the most damage to the plasma membrane, although the junctional complexes appeared to be intact, and the trophectoderm cells in Day 13 blastocysts sustained the most damage, while

3 Cryopreservation of Mammalian Embryos by Vitrification the undifferentiated embryonic cells appeared to survive the freezing and thawing process. Wilson et al.61) showed that mitochondrial damage was the major defect found in the cells of the frozen-thawed Day 6 horse embryos. Although there is a substantial amount of information about cell morphology after conventional freezing procedures, only a few studies have been made of morphological changes in vitrified embryos, and most of them were made in bovine oocytes or embryos produced in vitro. Kuwayama et al.22) reported that small vesicles and distinct intramembrane particle aggregation were frequently observed in the plasma membranes of vitrified embryos equilibrated by the two-step method. They further suggested that the extent of injury depends on the procedure used for equilibration in the vitrification solution, and that increasing the number of steps of equilibration in the vitrification solution may minimize ultrastructural damage to the plasma membrane due to the osmotic stress. Our previous ultrastructural observations of vitrified bovine embryos produced in vitro (Fig.1)37) are consistent with this suggestion, and also showed other cellular changes in microvilli, mitochondria and the endoplasmic reticulum. Darvelid et al.4) observed ultrastructural changes in microvilli, nuclei and mitochondria of in vivo-developed embryos warmed after vitrification. Vajta et al.54) investigated morphological signs of injury and regeneration following vitrification and warming of bovine embryos. Their investigation showed embryos with extensive ultrastructural injury im-

4 OHBOSHI mediately following warming, including distension or shrinkage of mitochondria, loss of cell adhesion, and disruption of plasma membrane of some cells. Although the majority of irreversibly damaged cells and cellular debris were expelled to the perivitelline space, most of the severe damage was progressively repaired during a short period of in vitro culture. The ultrastructure of bovine embryos after vitrification, warming and 24h in culture did not appear to be different from that of fresh embryos (not vitrified), irrespective of the vitrification solution39). In theory, vitrification should be a practical technique for cryopreservation of mammalian oocytes30,31). However, it has not been very successful when applied to bovine oocytes and has resulted in very low rates of fertilization and cleavage2,10,14). The nature of the damage caused by vitrification was elucidated by using transmission electron microscopy11,12). Vitrification induced profound ultrastructural modifications in microvilli and mitochondria, resulted in vesicle formation, and caused the premature release of the cortical granules. These examinations also showed that germinal vesicle stage oocytes were more sensitive to vitrification than in vitro-matured oocytes, and further suggested that differences of sensitivity to cryopreservation between species may be due to differences in cell volume, membrane permeability, tolerance to cryoprotectants, lipid content, and other as yet unrecognized factors that differ between species. In horse oocytes, vitrification also induced ultrastructural changes, such as the dilatation of mitochondria with reduced matrix density, the destruction of cumulus cell projections surrounding the oocytes, and production of large vacuoles located peripherally in the ooplasm16). Cryopreservation treatments of embryos increased glutamine metabolism, possibly as a result of disturbances in mitochondrial function46), which could be interpreted as reflecting a decrease in mitochondrial ATP production26). Mitochondrial damage results in cell death61) and injury of microvilli and the endoplasmic reticulum seriously affect the subsequent development of the embryos37). Cytoskeleton Permeating cryoprotectants, such as DMSO, glycerol and propylene glycol, intracellularly influence microfilament and microtubule dynamics17,40,57) and act to depolymerize microfilaments and microtubules. Although depolymerization is beneficial in protecting these cytoskeletal components during osmotic stresses induced by exposure to or removal of cryoprotectants, highly concentrated cryoprotectants may lead to long-lasting disruption of the microfilaments or microtubules, resulting in irreversible and lethal damage to embryos8). Dobrinsky7) showed that cytoplasmic microfilaments and microtubules of bovine embryos were influenced by the vitrification treatment, but that these changes were reversible, since the post-warming embryos displayed progressive increases in both cytoskeletal components, and this recovery seemed to be dependent on the methods used to dilute or remove the cryoprotectants from the post-warming embryos. Cytoskeletal components reacted quite differently to cryoprotectants depending on the embryonic stage of development and species of embryo6,8). Dobrinsky8) also suggested that the disruption of the cellular organization of porcine embryos during cryopreservation caused irreversible damage of membrane lipids, intracellular lipids, and the embryonic cytoskeleton, which led to denaturation of critical intracellular structures and organelles. Metabolic activity Very little work has been done on measuring the metabolic activity of the embryos before or after cryopreservation because of the difficulty of the measurements themselves and insufficient understanding of embryonic metabolism. Furthermore, no information is available on the metabolism of vitrified embryos, but some results from cryopreserved embryos subjected 1072

5 to slow freezing might be relevant to the metabolism of the vitrified embryos, Gardner et al.13) demonstrated that glucose and pyruvate uptake and utilization by bovine blastocysts are significantly reduced after freezing, suggesting that energy metabolism of the embryos is affected by freezing, and they also proposed that the assay of glucose uptake and glucose utilization via glycolysis would be useful for selecting viable blastocysts immediately after thawing, since glycolysis is a major energy-generating pathway for the thawed blastocysts. Takagi et al.50) investigated DNA synthesis in the inner cell mass of bovine cryopreserved embryos by using monoclonal bromodeoxyuridine-specific antibody. Their results demonstrated that the number of immunoreactive cells in frozen-thawed embryos was significantly lower than that in unfrozen embryos, suggesting that DNA synthesis in the inner cell mass cells of the blastocysts is affected by cryopreservation. There is a delay in the resumption of normal development in embryos after freezing or vitrification, and there is a significant reduction in metabolic activities even of the viable embryos. It has been thought that these phenomena might be due to a slow and gradual restoration of normal metabolic and synthetic activity of the cryopreserved embryos, and that the metabolic changes might cause the reduced rate of subsequent development of cryopreserved compared to fresh embryos. Recently, Uechi et al.52) showed that the normally developed in vitro blastocysts derived from embryos cryopreserved at the two-cell stage had low viability, and that the 2-deoxyglucose uptake of blastocysts developed in vitro from frozenthawed two-cell embryos was also significantly lower than that of blastocysts developed in vitro from fresh two-cell embryos, although there was no significant difference in cell number, and the two types of blastocysts were morphologically indistinguishable. Moreover, they also demonstrated that the expression of glucose transporter (GLUT) protein in embryos developed from frozen-thawed two-cell embryos was reduced, paralleling the reduction of 2-deoxyglucose uptake. These results suggested that cryopreservation of embryos at an early developmental stage influences the gene expression during subsequent embryonic development, including implantation. However, since chromosome damage may be related to the freezing properties of the cryoprotectant solution49), it is still unknown whether critical damage of the embryonic genome is caused by vitrification. Improvement of survival rate Most recently, methods for in vitro production of embryos have been established, leading to pregnancies after transfer of the embryos to recipients, and various new approaches using vitrification have tried for achieving higher embryonic viability, based on theoretical or empirical considerations. In general, in vitroproduced embryos are considered to be more sensitive to freezing than their in vivo-derived counterparts23,42). It is suggested that cryopreservation methods appropriate for in vivoderived embryos might not be completely satisfactory for in vitro-produced embryos, because there are many differences between in vivo and in vitro embryos (for reviews, see Massip et al.26) and Thompson51)). Recently reported improvements in the survival rates of vitrified embryos are attributed to improved culture conditions and changes in the cryopreservation technology itself. The culture system affects the quality of the resulting embryos and consequently their cryosurvival potential. The presence of serum during culture of embryos might have an effect on cryoresistance of the resulting embryos (Table 1)5,36,48) and co-culture with buffalo rat liver cells and leukemia inhibitory factor appear to be beneficial for cryopreservation of bovine embryos (Table 1)15,18,26). Dobrinsky7,8) showed that cytoskeletal stabilizers could enhance the survival rates of the 1073

6 OHBOSHI Table 1. In vitro survival of bovine vitrified-warmed blastocysts cultured in vitro in media with various supplements1 1 Bovine in vitro-produced zygotes were cultured for up to 8 days in modified synthetic oviduct fluid medium supplemented with 5% calf serum, 3mg/ml BSA (fraction V or fatty acid free), or 1mg/ml polyvinyl alcohol (PVA) in a humidified atmosphere of 5% CO2, 5% O2 and 90% N2. Embryos were then vitrified and warmed according to the method of Ohboshi et al.35). Percentage 2 of the post-warming embryos. abvalues within the same column with different superscripts are significantly different (P< 0.05; x2-test). vitrified bovine and porcine embryos, and suggested that microfilament depolymerization prior to cryopreservation significantly improves the embryonic development. As lipid content is thought to have a major effect on plasma membrane composition and consequently on the freezability of the embryos, manipulation of the protein and/or lipid content of embryos affects their sensitivity to freezing. Removal of lipid from embryos reduced the chilling sensitivity and greatly improved the survival of the embryos32-34). Although the addition of heat-stable plant proteins did not improve the in vitro survival of warmed embryos39), antifreeze glycoproteins isolated from antarctic fishes could protect cell membranes from the damage caused by low temperature and improve the viability of oocytes and embryos when added to the vitrification solution3,38,41). Very recently, efficient procedures to cryopreserve bovine oocytes and embryos have been reported by Martino et al.24) and Vajta et al.53,55). They successfully achieved much higher cooling and warming rates and shorter exposure of the oocytes and embryos to the vitrification solution by devising new cryopreservation equipment. However, there seem to be discrepancies between the rates of in vitro survival and in vivo development to fetus8,26), and these problems remain under investigation, along with the problem of the system for producing embryos in vitro. In conclusion, cryopreservation by vitrification has been developed as a simple and alternative technique for preservation of various mammalian oocytes and embryos. The higher survival of vitrified embryos suggests that vitrification is more appropriate than conventional slow freezing for cryopreservation of mammalian oocytes and embryos, and the viability of the oocytes and embryos has also been confirmed by embryo transfer on a large scale56,62). Further research on the optimal conditions for embryonic viability and the reduction of cellular and molecular damage during and after vitrification of oocytes and embryos will lead to wider practical application of vitrification and a better understanding of animal reproduction. Acknowledgements The author wishes to thank Drs. Noboru Fujihara (Kyushu Univ.) and Hiroshi Tomogane (Nippon Vet. and Anim. Sci. Univ.) for advice and encouragement throughout the study. 1074

7 Cryopreservation of Mammalian Embryos by Vitrification References 1) Arav A. Vitrification of oocytes and embryos. In: Embryonic Development and Manipulation in Animal Production. (Lauria S, Gandolti F eds.) Portland Press. London and Chapel Hill ) Arav A, Shehu D, Mattioli M. Osmotic and cytotoxic study of vitrification of immature bovine oocytes. J. Reprod. Fert., 99: ) Arav A, Rubinsky B, Seren E, Roche JF, Boland MP. The role of thermal hysteresis proteins during cryopreservation of oocytes and embryos. Theriogenology, 41: ) Darvelid U, Gustafsson H, Shamsuddin M, Larsson B, Rodrigues Martinez H. Survival rate and ultrastructure of vitrified bovine in vitro andd in vivo developed embryos. Acta Vet. Scand., 35: ) Dinnyes A, Carolan C, Lonergan P, Massip A, Mermillod P. Survival of frozen or vitrified bovine blastocysts produced in vitro in synthetic oviduct fluid. Theriogenology, 46: ) Dobrinsky JR, Johnson LA. Cryopreservation of porcine embryos by vitrification: a study of in vitro development. Theriogenology, 42: ) Dobrinsky JR. Cellular approach to cryopreservation of embryos. Theriogenology, 45: ) Dobrinsky JR. Cryopreservation of pig embryos. J. Reprod. Fert. Suppl., 52: ) Fahy GM, MacFarlane DR, Angell CA, Meryman HT. Vitrification as an approach to cryopreservation. Cryobiology, 21: ) Fuku E, Kojima T, Shioya Y, Marcus GJ, Downey BR. In vitro fertilization and development of frozen-thawed bovine oocytes. Cryobiology, 29: ) Fuku E, Xia L, Downey BR. Ultrastructural changes in bovine oocytes cryopreserved by vitrification. Cryobiology, 32: ) Fuku E, Liu J, Downey BR. In vitro viability and ultrastructural changes in bovine oocytes treated with a vitrification solution. Mol. Reprod. Dev., 40: ) Gardner DK, Pawelczynski M, Trounson AO. Nutrient uptake and utilization can be used to select viable day 7 bovine blastocysts after cryopreservation. Mol. Reprod. Dev., 44: ) Hamano S, Koikeda A, Kuwayama M, Nagai T. Full-term development of in vitro matured, vitrified and fertilized bovine oocytes. Theriogenology, 38: ) Han YM, Lee ES, Mogoe T, Lee KK, Fukui Y. Effect of human leukemia inhibitory factor on in vitro development of IVF-derived bovine morulae and blastocysts. Theriogenology, 44: ) Hochi S, Kozawa M, Fujimoto T, Hondo E, Yamada J, Oguri N. In vitro maturation and transmission electron microscopic observation of horse oocytes after vitrification. Cryobiology, 33: ) Johnson MH, Pickering SJ. The effect of dimethyl sulfoxide on the microtubular system of the mouse oocyte. Development, 100: ) Kaidi S, Donnay I, Van Langendonckt A, Dessy F, Massip A. Comparison of two co-culture systems to assess the survival of in vitro produced bovine blastocysts after vitrification. Anim. Reprod. Sci., 52: ) Kasai M, Hamaguchi Y, Zhu SE, Miyake T, Sakurai T, Machida T. High survival of rabbit morulae after vitrification in an ethylene glycol-based solution by a simple method. Biol. Reprod., 46: ) Kasai M, Zhu SE, Pedro PB, Nakamura K, Sakurai T, Edashige K. Fracture damage of embryos and its prevention during vitrification and warming. Cryobiology, 33: ) Kasai M. Vitrification: refined strategy for cryopreservation of mammalian embryos. J. Mamm. Ova Res., 14: ) Kuwayama M, Fujikawa S, Nagai T. Ultrastructure of IVM-IVF bovine blastocysts vitrified after equilibration in glycerol 1, 2- propanediol using 2-step and 16-step procedures. Cryobiology, 31: ) Leibo SP, Loskutoff NM. Cryobiology of in vitro-derived bovine embryos. Theriogenology, 39: ) Martino A, Songsasen N, Leibo SP. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol. Reprod., 54: ) Massip A, van der Zwalmen P, Scheffen B, Ectors F. Pregnancies following transfer of cattle embryos preserved by vitrification. 1075

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