Cryopreservation of Murine Zygotes for Use in Testing Culture Environments

Thomas K. Meyer,1 Melissa R. Yurchak,2 Jose F. Pliego,1 Thomas J. Wincek,1 W. Richard Dukelow,3 and Thomas J. Kuehl1,4*

Abstract Developing one-cell mouse zygotes are more sensitive to in vitro environmental conditions than are cleavage-stage embryos. However, for convenience and reproducibility, cryopreserved two-cell zygotes are routinely used for such assays. Concern over the possibility of inducing damage by exposing one-cell zygotes to cryoprotective agents and freeze-thaw procedures during syngamy led us to examine one-cell zygotes, with and without visible pronuclei, in an effort to minimize or avoid these effects and obtain the highest possible developmental rate. In vivo fertilized mouse zygotes were collected 21 to 43 h after administration of human chorionic gonadotropin (hCG). Suspensions of zygotes in 2M ethylene glycol were aspirated into 0.25-ml plastic insemination straws and slowly cooled at -0.5°C/min to -40°C before being plunged into liquid nitrogen for storage. Zygotes were thawed, rinsed, and placed in culture. Zygotes were examined initially for damage from the freeze-thaw procedure. Daily in vitro development was recorded. In this group of zygotes, no damage was apparent immediately after thawing, and a high degree of development in vitro was observed. Thus, usefulness of a cryopreservation method for one-cell murine zygotes has been confirmed.

Human in vitro fertilization (IVF) environments require pronuclear status, using the cryopreservation protocol. For strict quality control to ensure a program’s success. Protein the latter comparison, we used noncontemporary and con- sources, water quality, lot variation of media, and incubator temporary experimental designs. conditions all provide opportunities for the introduction of toxic effects that necessitate routine, systematic testing for Materials and Methods potential embryotoxicity (1–7). For this purpose, the devel- Oocyte source and classification: Six- to seven-week- opment in vitro of murine preimplantation embryos is often old female B6C3F1/HSD mice were injected intraperito- the method of choice (1, 3, 7–9). This method allows the early neally with 7 IU of pregnant mare serum gonadotropin detection of suboptimal factors and the opportunity to rede- (PMSG; Sigma Chemical Co., St. Louis, Mo.), followed 47 h fine culture conditions to avoid problems. later with 7 IU of human chorionic gonadotropin (hCG; Many investigators have shown one-cell mouse zygotes to Sigma Chemical Co.) to induce superovulation. Female mice be more sensitive to in vitro environmental conditions than were then placed individually with B6D2F1/HSD male mice are cleavage-stage embryos (2, 5, 10). However, due to con- for breeding. All husbandry and veterinary care, as well as venience, reproducibility, and commercial availability, experimental procedures, were conducted in accordance cryopreserved two-cell mouse zygotes are routinely used for with a protocol approved by the Scott and White IACUC such assays. Similar approaches to the storage of one-cell and AALAS guidelines. Zygotes were retrieved from the zygotes have not been as successful as those used for the excised oviducts after hCG administration at 23 h for the two-cell zygote. In addition, the possibility of inducing dam- pronuclear stage, 29 h for the syngamy stage, and 43 h for age by exposing one-cell zygotes to cryoprotective agents and the two-cell stage. All zygotes were collected in modified freeze-thaw procedures during syngamy has been suggested human tubal fluid (mHTF; Irvine Scientific, Irvine, Calif.) (10, 11). The purpose of the study reported here was to refine with bovine serum albumin (BSA; Sigma Chemical Co., our freezing technique and to provide easy access to one-cell 5 mg/ml) and were examined microscopically. zygotes for in vitro testing of the IVF environment. In addi- Freezing and thawing procedure: The modified cul- tion, we examined the effect of freezing, with respect to ture methods used in this study were derived from those of earlier culture studies (3). A modified one-step cryopreservation procedure based on several published reports (12, 13) was used. The first of those reports involved cryo- Department of Obstetrics and Gynecology,1 Pathology,2 and Medical Bio- chemistry and Genetics,4 Texas A & M University Health Science Center, preserved zygotes for the production of five types of College of Medicine, Temple, Texas, and Endocrine Research Center, Michi- transgenic mice. Those research workers, using albino ICR gan State University, East Lansing, Michigan3 female mice mated with albino FVB males and with the *Address reprint requests and correspondence to Dr. Thomas J. Kuehl, Department of Obstetrics and Gynecology, Texas A & M University Health zygotes cryopreserved in CZB medium with 3M ethylene Science Center, College of Medicine, Temple, TX 76508. glycol, grew 225 of 269 (83.6% ± 5.1%) to the blastocyst

496 Mouse Zygote Cryopreservation

Figure 1. Design of contemporary trials of mouse zygote cryopreservation. Cryopreserved zygotes were frozen at the time of recovery and thawed together for subsequent evaluation and culture. stage after 5 days of culture following thawing. Others (13) of the cryoprotectant with 0.5M sucrose in M2 medium used vitrification as a cryopreservation technique with within the straw; the contents were then warmed in a 37°C pronucleate-stage mouse zygotes that were subjected to water bath for 3 min. This suspension was expelled from injection of DNA constructs. The outcome measure was the straw; zygotes were recovered, rinsed in fresh medium, development to the two-cell stage. Vitrified oocytes devel- and equilibrated for 10 min in mHTF containing 5 mg of oped at a 64% rate, compared with 83% of those under- BSA/ml. Embryos were examined for freeze-thaw damage going vitrified treatment. at this stage, and all were placed into culture. The study reported here differed from the earlier study Noncontemporary trial of development versus stage (12) in mouse strain, recovery and culture media, freezing at time of cryopreservation: Pronuclear and syngamic rates, and the amount of cryoprotectant. Briefly, in vivo one-cell zygotes were compared with two-cell zygotes on the fertilized mouse zygotes were collected into M2 medium basis of freeze-thaw survival and in vitro development. (14) containing 5 mg of BSA/ml, washed, and examined for Zygotes at each stage were collected from different groups evidence of fertilization. All zygotes were placed in M2 of mice. Groups of cryopreserved zygotes at various stages medium containing 30 mg of BSA/ml and were allowed to were thawed on different days. Therefore, the collection and equilibrate for 5 min. Zygotes were then allotted to groups freezing processes and the thawing and culturing processes and were transferred into M2 medium with 2M ethylene were performed in a manner similar to that of usual embry- glycol (Mallinckrodt, Paris, Ky.) and 30 mg of BSA/ml for ologic laboratory practice (noncontemporary). 10 min. Suspensions of equilibrating zygotes in this cryo- After thawing, zygotes were cultured in mHTF with protective solution were loaded into 0.25-ml plastic insemi- 5 mg of BSA/ml (ICN ImmunoBiologicals, Costa Mesa, nation straws (IMV Corp., Minneapolis, Minn.) containing Calif.) in a humidified environment of 5% CO2/5% O2/90% a 0.015-ml volume of the cryoprotectant solution together N2 at 37°C. Development was observed microscopically with a 0.15-ml volume of 1M sucrose in M2 medium, the every 24 h. The cumulative number of developing embryos two solutions being separated by a 1-cm-long air bubble. was expressed as a percentage of the total number of all The tubes were heat sealed at both ends. All zygote manip- zygotes recovered at the time of thaw without exclusions ulation was performed at room temperature (26 to 28°C). for damaged, degenerated, or noncleaving zygotes. After equilibration in cryoprotectant for 10 min at room Contemporary controlled trial of development ver- temperature, the straws were cooled to -7°C in the ethanol sus stage at time of cryopreservation: Groups of five bath of a FTS Bio-cool freezer (FTS Systems Inc., Stone to six female mice underwent procedures in contemporary Ridge, N.Y.), seeded to induce ice formation with a cold probe manner (i.e., freezing and thawing for all treatments touching the plugged end of straws distal from the zygotes, occurred at the same time [Figure 1]). At the time of collec- and kept for 10 min. Straws were then frozen slowly at tion, a representative proportion of fertilized zygotes was -0.5°C/min to -40°C and kept at -40°C for 10 min prior to placed directly into culture medium as fresh in vitro con- being plunged directly into liquid nitrogen for storage. trols. The remaining preimplantation embryos were then Straws were stored for a minimum of 5 days prior to being cryopreserved as described previously, stored, and thawed thawed. Thawing was accomplished by allowing the con- by use of a contemporary, one-step procedure with repre- tents of each straw to equilibrate for 2 min at room tem- sentative straws thawed from each of the three develop- perature followed by vigorous shaking as a one-step dilution mental stages. Zygotes were cultured in mHTF with 5 mg

497 Vol 47, No 5 Laboratory Animal Science October 1997

Table 1. Noncontemporary trial comparing stage developmental Table 2. Contemporary controlled trial comparing fresh differences after cryopreservation and frozen-thawed zygotes Percentage of embryos developing to Percentage of embryos developing to Furthest Furthest Stage N 1-cell 2-cell Morula Blastocyst stage Stage N 1-cell 2-cell Morulab Blastocystc stage 1-cell 78 100 96 90a 78a HB Fresha Syngamy 115 100 95 60b 31b EB 1-cell 87 100 93 84 84 HB 2-cell 161 – 100 98c 93c HB Syngamy 33 100 79 79 79 EB HB = hatching blastocyst; EB = expanded blastocyst 2-cell 21 – 100 100 90 HB a,b,cProportions with differenc superscripts are significantly different, using Frozen- chi-square analysis, at P < 0.0037 for blastocytes and P < 0.0007 for morula. thawed 1-cell 70 100 89 86 81 HB Syngamy 60 100 80 78 78 EB 2-cell 27 – 100 93 85 HB of BSA/ml in a humidified environment of 5% CO2/95% air at 37°C. Development was observed and scored as described aNo significant differences between development of fresh and frozen-thawed embryos of like stages, chi-square analysis (P < 0.75, 0.96, and 0.20 for previously. 1-cell, syngamy, and 2-cell stages respectively). Minimal standard of development and statistical bDevelopment to morula stage was significantly different between 2-cell analysis: Previous investigators have suggested the need and syngamy-stage embryos, using chi-square analysis (P < 0.007), but neither 2-cell nor syngamy-stage embryos were different from 1-cell-stage for minimal standards of embryo development, such as 80% embryos. development to the blastocyst stage (3) or development to cNo significant differences in development of embryos to blastocyst stae, expanded or hatching blastocysts (6). In this study, these using chi-square analysis. Development expressed as a percentage of the total in human tubal fluid relationships have been provided (Tables 1 and 2). Further- medium with 10 mg of BSA/ml. more, statistical analysis has been used to determine the See Table 1 for key. significance of differences between treatments and controls. The overall significance of treatments and development in scope of this study. these experiments was determined using chi-square analy- Contemporary trial: In vitro development was compa- sis, with significance determined at P < 0.017. This level rable in all groups with that of the corresponding fresh con- was chosen to account for multiple comparisons between trol groups (Table 2). The viability of the syngamy group three groups. after freeze-thawing is demonstrated by the ability of the zygotes to develop to the blastocyst stage. However, none Results of these was observed to develop beyond this expanded stage Classification of embryos: Groups of seven female mice in either the control or the frozen-thawed group. were simultaneously stimulated at each trial. Each group was separated into three subgroups, and embryos were Discussion collected at various time intervals for the desired develop- Results of this study confirm success of a cryopreserva- mental stage (Figure 1). A total of 43 female mice were tion method for pronuclear zygotes using ethylene glycol. used in this project, and a total of 1,723 embryos were This method results in development equal to that of fresh obtained, from which 1,209 were cryopreserved at either zygotes. From other observations, one-cell zygotes may be the one- or two-cell zygotic stage in various lots. After col- more sensitive to toxic contaminants than are two-cell lection, the integrity of the embryos was assessed by light zygotes (1–4, 15). Thus, cryopreserved pronuclear zygotes microscopy to exclude those with previous damage, such offer a more sensitive model for quality control testing of as cracked zona pellucida, cytoplasmic fragmentation, or in vitro culture environments than do cryopreserved two- degeneration, which were then culled from the experimen- cell embryos, the current detection model. tal group. Furthermore, developmental stages were con- Although a higher survival rate of pronuclear versus firmed at that time prior to cryopreservation. syngamic one-cell zygotes, based on development, was docu- Survival and cryoinjury: Of the 511 frozen mouse mented in the noncontemporary trial, significant difference zygotes thawed for this experiment, none had obvious dam- was not observed in the contemporary trial. The exact cause age at the time of thawing. Recovered zygotes were included of this is unknown, but it indicates that adverse effects on in the culture results (Tables 1 and 2). the zygotes in syngamy were not sustained due to the Noncontemporary trial: Initial in vitro development freeze-thaw process. The difference between the trials may was found to be comparable in all groups (>95%); however, be caused by a unique sensitivity to chilling to room tem- a significant reduction in morulae and blastocyst forma- perature, washing or culture in buffered media during pri- tion was seen in the syngamy group, compared with the mary recovery, and in vitro manipulation. If the embryos frozen-thawed one-cell pronuclear and two-cell zygotes of other species are susceptible to this phenomenon, it could (Table 1). Additionally, hatching was not observed in zygotes be important to the successful use of in vitro manipula- of the syngamy group, but was prevalent in those of the tions at this developmental stage. Additionally, the devel- pronuclear stage one-cell and two-cell zygotes. Only the opmental rate in the contemporary controlled trial was no highest degree of development was evident. The longer significantly different from that of the pronuclear phenomenon of blastocyst expanding and hatching is diffi- stage or two-cell zygotes treated in a similar manner. These cult to gauge for individual embryos with variable cell num- differences in survival between the noncontemporary and bers, and cell counting of the blastocysts was beyond the contemporary trials may be due to improved culture condi-

498 Mouse Zygote Cryopreservation tions after BSA concentration was increased to 10 mg/ml 4. Nag, R. K., J. T. Janousek, T. Moody, et al. 1986. Factors (which may have acted as a buffer to factors damaging to influencing murine embryo bioassay: effects of proteins, aging of the medium and surgical glove coating. Fertil. Steril. syngamic one-cell development) (16). 46:914–991. Common freeze-thaw damage in the form of fractured 5. Davidson, A., M. Vermesh, R. A. Lobo, et al. 1988. Mouse zonae pellucidae and blastomeres was avoided by use of embryo culture as quality control for human in vitro fertiliza- plastic straws and optimization of freezing and warming tion: the one-cell versus the two-cell model. Fertil. Steril. conditions (17). These factors became apparent in the con- 49:516–521. 6. Byrd, W. 1992. Quality assurance in the reproductive biol- temporary trial because development was found to be simi- ogy laboratory. Arch. Pathol. Lab. Med. 116:418–422. lar to that of the fresh control zygotes in all instances. 7. Beyler, S. A., and A. P. Chong. 1992. In vitro fertilization Morphologic damage was not obvious in any zygotes thawed culture medium surveys. Arch. Pathol. Lab. Med. 116:425–429. from either trial (n = 511). 8. Ackerman, S. B., G. L. Stokes, R. J. Swanson, et al. 1985. Toxicity testing for human in vitro fertilization programs. Studies exploring zygote age and the relationship to J. IVF ET 2:132–134. cryopreservation have been only sparsely addressed in lit- 9. Cheung, S. W., R. C. Strickler, V. C. Yang, et al. 1985. A erature. This is an area in which studies using human mouse embryo culture system for quality control testing of zygotes have preceded studies using animal models. Stan- human in vitro fertilization and embryo transfer media and dard freezing protocols for eight-cell preimplantation fetal cord sera. Gamete Res. 11:411–419. 10. John, D. P., and A. A. Klessling. 1988. Improved pronuclear embryos with slight modifications have been applied to mouse embryo development over an extended pH range in these early zygotes, with potential benefit at the patient’s Ham’s F-10 medium without protein. Fertil. Steril. 49:150–155. expense (18–20). Dimethyl sulfoxide and propanediol have 11. Cohen, J., G. W. DeVane, C. W. Elsner, et al. 1988. Cryo- been the cryoprotectants of choice for these procedures. preservation of zygotes and early cleaved human embryos. Fertil. Steril. 49:283–289. Mandelbaum et al. (21) observed that dimethyl sulfoxide 12. Leibo, S. P., F. J. DeMayo, and B. O’Malley. 1991. Produc- had direct interference with the mitotic spindle of tion of transgenic mice from cryopreserved fertilized ova. Mol. metaphase-I mouse oocytes, causing aneuploidy. This cre- Reprod. Dev. 30:313–319. ated the concern that the morphologically similar sperm 13. Tada, N., M. Sato, K. Kasai, et al. 1995. Production of trans- aster could be destroyed. To the authors’ knowledge, these genic mice by microinjection of DNA into vitrified pronucleate stage eggs. Transgenic Res. 4:208–213. topics have not yet been fully investigated. However, in 14. Quinn, P., C. Barros, and D. G. Whittingham. 1983. Pres- our experiments, significant difference was not apparent ervation of hamster oocytes to assay the fertilizing capacity in the ability of a zygote to survive the cryopreservation of human spermatozoa. J. Reprod. Fertil. 66:161–168. process using this protocol, on the basis of age or stage of 15. Davidson, A., M. Vermesh, R. A. Lobo, et al. 1988. The tem- poral effects of changes in in vitro fertilization culture media development. A difference was seen in the ability of syn- on the one-cell mouse embryo system. J. IVF ET 5:149–152. gamy zygotes to develop past the expanded blastocyst stage 16. Ogawa, T., and R. P. Marrs. 1987. The effect of protein under our culture conditions. supplementation on single-cell mouse embryos in vitro. Fertil. There are a large number of applications of successful Steril. 47:156–161. cryopreservation methods for one-cell mouse embryos. For 17. Rall, W. F., and T. K. Meyer. 1986. Fracture damage to the zonae of mammalian embryos during cryopreservation and example, the procedure provides material for in vitro its avoidance. Theriogenology 25:186–188. manipulations, such as production of transgenic clones for 18. Tesart, J., B. Lassalle, J. Belaisch-Allart, et al. 1986. High basic and applied research (12) and for embryo banking of pregnancy rate after early embryo freezing. Fertil. Steril. mouse stocks. Because of the importance of mice as a mam- 64:268–276. 19. Todorow, S. J., E. R. Siebzehhnrubl, R. Koch, et al. 1989. malian genetic model, the procedures provide a rapid way Comparative results on survival of human and animal eggs of storing transgenic and mutant lines of mice efficiently. using different cryoprotectants and freeze-thawing regimens. I. Mouse and hamster embryos. Human Reprod. 4:805–811. 20. Todorow, S. J., E. R. Siebzehhnrubl, R. Koch, et al. 1989. Comparative results on survival of human and animal eggs Acknowledgement using different cryoprotectants and freeze-thawing regimens. This study was supported in part by grant no. P42 ES04911 of II. Human. Human Reprod. 4:812–816. the National Institutes of Health Sciences and by the Scott, 21. Mandelbaum, J., A. M. Junca, C. Tibi, et al. 1987. La Sherwood and Brindley Foundation. congelation de l’ovocyte. Contracept. Fertil. Sex 15:792–795.

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