Cryopreservation of nuclear material as a potential method of fertility preservation

FERTILITY AND STERILITY VOL. 79, NO. 2, FEBRUARY 2003 Copyright 2003 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Cryopreservation of nuclear material as a potential method of fertility preservation Zhiying He, M.S., Hung-Ching Liu, Ph.D., and Zev Rosenwaks, M.D. Institute for Reproductive Medicine, The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, New York, New York Objective: To establish a cryopreservation method for female nuclear materials with the aim of creating viable embryos or offspring by nuclear transfer. Design: A randomized controlled study. Setting: Clinical and academic research facility. Animal(s): B6D2F1 mice. Intervention(s): Female pronuclei (FPNs) and second polar bodies (2PBs) isolated from superovulated mouse zygotes were cryopreserved, thawed, and transferred into donor zygotes. Main Outcome Measure(s): Survival rates after freeze thaw and blastocyst formation rates were evaluated. Result(s): Female pronuclei and 2PBs preserved with three tested methods resulted in survival rates ranging from 11.5% to 85% for FPNs vs. 46.9% to 95.0% for 2PBs and blastocyst formation rates ranging from 0% to 35.5% for FPNs vs. 9.1% to 47.4% for 2PBs. Live birth offspring also resulted from both FPNs and 2PBs preserved with vitrification. Conclusion(s): We have established a new system to effectively revive frozen nuclei into viable embryos by a combination of nuclei preservation, zygote reconstruction, and coculturing of reconstructed zygotes with mouse embryonic fibroblast cells. Our data suggest that the preserved female genomic DNA material can be potentially used for future nuclear transfer to preserve female fertility. (Fertil Steril 2003;79:347 54. 2003 by American Society for Reproductive Medicine.) Key Words: Nuclear transfer, cryopreservation, second polar bodies, female pronuclei, vitrification Received November 6, 2001; revised and accepted August 2, 2002. Reprint requests: Hung- Ching Liu, Ph.D., Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, 515 East 71 Street, S500, New York, New York 10021 (FAX: 212-746-8996; E-mail: hcliu@mail.med.cornell.edu). 0015-0282/03/$30.00 dio:10.1016/s0015-0282(02) 04674-5 Various attempts to cryopreserve human oocytes have been unsuccessful, as demonstrated by the low survival rates after thawing and the rare incidence of pregnancies following IVF (1 8). Cryopreservation imparts detrimental effects on oocytes (1), which include hardening of the zona pellucida (1, 2, 9, 10), spindle microtubular disruption, and injuries to the oolemma (1, 2, 11). It has also been proposed that cryopreservation of ova may result in excessive mutagenicity caused by sister chromatid exchange errors during meiosis and early development (1, 2, 12 16). The search for an effective protocol of oocyte preservation or other alternative methods for preserving female fertility has thus far remained elusive. The final stages of oocyte maturation in mammals are characterized by the first and second meiotic chromosomal divisions. The primary oocyte undergoes germinal vesicle breakdown and releases the first polar body in the process of becoming mature metaphase II oocytes, the stage that is preserved until oocyte activation is triggered by a fusing spermatozoon. The second polar body (2PB) and female pronuclei (FPNs) are formed after fertilization. Both PB and FPN are subcellular components of the zygote. They are membrane-bound nuclear materials, free of meiotic microtubings or ooplasmic organelles, and more importantly, contain a full haploid complement of female chromosomes. In addition, they are smaller in volume and less complex in composition than oocytes, and could be less sensitive to cryoinjury. Hence, we theorized that these components could be safely cryopreserved and serve as an alternative method to preserve female fertility. The potential in this new approach for female fertility preservation still relies heavily on the efficiency of both cryopreservation and nuclear transfer of the thawed 2PBs or FPNs. 347

Pronuclei have been successfully cryopreserved within pronuclear stage embryos and in fact, pronuclear stage freezing has been routinely used in IVF institutes worldwide (2, 17). However, both 2PBs and FPNs undergo cellular reorganization during isolation by micromanipulation. In addition, the survival rate of these isolated 2PBs and FPNs after thawing had, until now, not been evaluated. Nuclear transfer of freshly isolated pronuclei (1, 18) and the first polar body (19) has been successfully used to generate offspring. Whether freezing and thawing of FPNs and 2PBs have a negative impact on the success of nuclear transfer also requires further investigation. Therefore, the objective of this study is to establish a cryopreservation method for mouse female nuclear materials (namely, the 2PBs and FPNs) with the aim of creating viable embryos or offspring by nuclear transfer. The methods used were: [1] isolation of 2PBs and FPNs from mouse zygotes by micromanipulation; [2] cryopreservation of 2PBs and FPNs; [3] nuclear transfer of the thawed 2PBs and FPNs to form the reconstructed zygotes; [4] coculturing the reconstructed zygotes with murine embryonic fibroblasts (MEFs); then [5] transferring the cultured embryos to a recipient to create viable offspring. In this study, we have proven that FPNs or 2PBs removed from a mouse two-pronuclei zygote can be cryopreserved thawed and later used to generate embryos leading to live births. Whether this nuclear cryopreservation technique can be applied clinically requires further study. When applied to humans, this method may potentially be used to conserve fertility and may be beneficial for [1] young cancer patients who require treatment with chemotherapy or radiotherapy; [2] patients who are at risk for premature ovarian failure; [3] patients who undergo hysterectomy or ovarectomy; and [4] patients who wish to prolong or delay their fertility. Chemotherapy and radiotherapy are known to cause infertility by destroying ovarian cells, and oocytes can be deteriorated with age or with medical reasons (e.g., patients with polycystic ovaries [PCO]). For those patients, their female nuclear materials can be preserved before medical treatment or when they are young and later used after treatment or when they deem ready. Therefore, our nuclear cryopreservation method may potentially provide an alternative method for fertility in the future. MATERIALS AND METHODS Superovulation of Mice and Collection of Zygotes The use of mice for this study has been approved by our Institutional Animal Use and Care Committee under protocol #9803-494A. B6D2F1 (C57BL/6female DBA/2male) female mice (6 8 weeks old) were superovulated with 10 IU of pregnant mare serum gonadotropin (PMSG) (Sigma, St. Louis, MO) and 10 IU hcg (Sigma) at 46-hour intervals and caged with male mice overnight. Zygotes were then collected from the females oviducts 18 hours after hcg injection. Collected zygotes were treated with 0.1% hyaluronidase (type V from sheep testes) (Sigma) in phosphatebuffered saline (PBS) (GIBCO, Grand Island, NY) for 2 3 min to remove its surrounding cumulus cells, and then thoroughly rinsed in the M2 medium. Isolation of 2PBs and FPNs Partial zona dissection and isolation of membrane-bound FPNs and 2PBs were performed by micromanipulation using a Nikon Diaphot inverted microscope with a pair of Narishige hydraulic remote control micromanipulators (MO- 188NE; Narishige, Tokyo, Japan) (20). The enucleation micropipettes were made from standard-wall borosilicate glass capillaries (Clark Electromedical Instruments, Fircroft Way, Edenbridge, Kent, UK), which were flame-pulled by a horizontal micropuller (model 753, Camiden Instrument, Ltd., Loughborough, UK) and cut, fine-polished on a Narishige MF-9 microforge (New York/New Jersey Scientific Inc., Middlebush, NJ) to create inner diameters of 20 m. Second polar bodies and FPNs were isolated according to published methods (20). Zygotes were partially dissected (10%) on zona pellucida with a fine dissection needle, then incubated in M2 medium containing cytochalasin B (Sigma) and colcemid (Sigma) at 37 C. After incubation for 15 35 min, FPNs and 2PBs were removed from the zygote through the zona pellucida by a micromanipulator with an enucleation pipette. In mice, FPNs can be accurately selected based on their proximal location to the 2PBs and its small size relative to the male pronuclei. Unfortunately, in human zygotes, both female and male pronuclei are not distinguishable and can easily be confused. Clinically, a mistaken transfer of the wrong pronuclei could lead to major problems because it has been shown in mice that a zygote requires both a male and female pronuclei to develop to full term (21). A zygote formed by two female pronuclei and an absence of a male pronucleus will generate an embryo with a negligible placenta. On the other hand, a zygote formed by two male pronuclei and an absence of female pronuclei will generate a nonviable embryo with a large placenta. To confirm the success of selecting the female pronucleus in human zygotes, the remaining pronuclei can be used to detect the presence of sperm-specific nuclear proteins by preimplantation genetic diagnosis techniques. Preparation of Donor Zona Pellucida The donor zona pellucida used for packing FPNs and 2PBs were prepared from deselected oocytes, which were usually atretic. These oocytes were incubated in M2 medium containing cytochalasin B and colcemid at 37 C with 5% CO 2 for 15 minutes. All contents of the oocytes were then removed by the enucleation pipette through the partially dissected hole. After flushing with the new M2 medium, 348 He et al. Embryogenesis from preserved female pronuclei Vol. 79, No. 2, February 2003

empty zona pellucidas were transferred to the new droplet of the medium. The isolated 2PBs and FPNs were grouped (four to six per group) and then packed into a donor zona pellucida by an enucleation pipette. The packed nuclei were then washed thoroughly with M2 medium before cryopreservation. Cryopreservation of the Packed Nuclear Materials The packed zona pellucida containing either 2PBs or FPNs was cryopreserved using either published slow-rate freezing protocol (22), ultra-rapid rate freezing method (23), or by open pulled straw vitrification (24) for comparison. Cryosolutions containing Dulbecco s PBS (Sigma) with 10% fetal calf serum (FCS) (GIBCO), 1.5 M ethylene glycol (Sigma), and 0.1 M sucrose (Sigma), 10% egg yolk, and 1 mg/ml polyethylene glycol were used in both the slow-rate freezing and the ultra-rapid rate freezing protocols. The packed 2PBs or FPNs were first equilibrated with cryosolution in vials at 37 C. For slow-rate freezing, the vials were frozen at an initial temperature drop of 2 C/min to 7 C, changing from a rate of 0.3 C/min to 35 C, then to a rate of 50 C/min to 180 C. The frozen 2PBs and FPNs can then be stored in liquid nitrogen until usage. For ultra-rapid rate freezing, the vials were plunged directly into liquid nitrogen for freezing and storage. Open-pulled straw vitrification was performed according to a previously published method (24) with modification. The packed 2PBs or FPNs were first incubated in 7.5% ethylene glycol and 7.5% dimethylsulfoxide dissolved in holding medium (TCM-HEPES) supplemented with 20% FCS for 3 minutes and then transferred in approximately 1 2 ml of solution into a 20-mL droplet of 16.5% ethylene glycol and 16.5% dimethylsulfoxide dissolved in holding medium and 0.5 M sucrose. Microtubings with an inner diameter of 0.15 mm were used for study. The packed 2PBs and FPNs were loaded into the narrow end of the microtubings from a droplet with the packed 2PBs or FPNs by a capillary effect. These loaded microtubings were then immediately submerged into frozen vials with liquid nitrogen and stored for a designated time. Warming was performed by placing the open end of the microtubing directly into the holding medium. The vitrified medium became liquefied within 1 2 seconds, releasing the packed 2PBs or FPNs out of the microtubes by sedimentation. All frozen FPNs and 2PBs were stored in liquid nitrogen for 2 weeks before thawing for further study. Assessment of the Viability of Thawed FPNs and 2PBs After incubation, the thawed FPNs or 2PBs were isolated with a solution containing propidium iodide (Sigma) and Hoechst 33342 (5 mg/ml) (Sigma) for 20 minutes. The viability of the thawed FPNs and 2PBs was assessed by a fluorescence microscope with UV light and DAP1/Fluorescein/Texas red mono-triple-filter. Propidium iodide (red stain) stains positive only for nonviable nuclear materials, whereas Hoechst 33342 (blue stain) stains positive for both viable and nonviable nuclear materials. Preparation of Male Karyoplasts and Donor Zona Pellucida Another group of zygotes were used for preparation of male karyoplasts. These zygotes were also exposed to M2 medium containing cytochalasin B and colcemid for 30 minutes at 37 C. Both FPNs and 2PBs were removed, except for the male pronuclei in the cytoplast of donor zygotes. The prepared male karyoplasts were then washed five times in M2 medium and left to recover in M16 medium in 5% CO 2 at 37 C for approximately 30 minutes before fusion. Nuclear Transfer Nuclear transfer was undertaken in a small drop of M2 medium containing cytochalasin (5 mg/ml) (18). The intact 2PBs and FPNs were transferred into the perivitelline space of male karyoplasts prepared as mentioned previously. The grafted zygotes were washed in the M2 medium for 10 minutes and transferred to a fusion chamber containing 700 ml of 0.3 M mannitol, 0.1 M MgSO 4 and 0.1 mm CaCl 2 in deionized water. Fusion and activation were induced by application of a direct current pulse of 1.5 kev/cm for 60 microseconds using an ECM2001 electrocell manipulator (BTX Inc., San Diego, CA). The reconstructed zygotes were washed five times in M2 medium and were cocultured with the feeder cell layer of MEF cells. Mouse Fetus and Culture of Mouse Embryonic Fibroblast Cells Mouse embryonic fibroblast cells were prepared from mouse fetus recovered at autopsy on day 10 of pregnancy. The head was removed before tissues were cut into small pieces and the cells dispersed by exposure to trypsin. Culture was supplemented with 10% FCS. At 90% confluence, the cells were passaged with a 1:2 division. The fibroblast-like cells between passages two and four were used for culture with the reconstructed embryos for early embryo development. Transfer of Reconstructed Embryos to Surrogate Recipients In the first experiment, reconstructed embryos were cocultured with MEF for 1 day and then transferred to the oviducts of pseudopregnant CD1 female mice that had been mated with vasectomized males of the same strain. In the second experiment, the reconstructed embryos were cocultured with MEF for 4 days until they reached the blastocyst stage and then transferred to the uterus of pseudopregnant CD1 female mice at days 3 4 after the reconstruction. Implantation rates or living pups were checked at day 19 after transfer or after natural delivery. FERTILITY & STERILITY 349

FIGURE 1 Isolation and packaging of female pronuclei for cryopreservation. (A), Female pronuclei were isolated from zygotes by micromanipulation. (B), The isolated pronuclei were packed into an empty donor zona pellucida by an enucleation pipette. Statistics Category variables were assessed by calculating 2 with Yate s correction or Fisher s exact test in the case of small samples. P.05 was considered as statistically significant. RESULTS After isolation from mouse zygotes by micromanipulation, the FPNs and 2PBs were packed into a donor zona pellucida (Fig. 1) for easy handling and preventing loss during cryopreservation, as well as for the ease of location after thawing. The packed FPNs and 2PBs were then cryopreserved. The FPNs (n 109) and 2PBs (n 120) were used to check the efficiency of cryopreservation. The viability of the thawed FPNs and 2PBs was assessed by propidium iodide and Hoechst 33342 dual staining (Fig. 2). Comparison of three cryoprotocols was conducted in the first tryout (76 FPNs and 72 2PBs). Our data showed that all three tested protocols could be used to preserve these nuclear materials and the ultra-rapid method demonstrated significantly lower survival rates in both FPNs and 2PBs groups (Table 1). Vitrification, being both effective and easy to operate, was performed in the second tryout (21 FPNs and 28 2PBs) and the third tryout (21 FPNs and 20 2PBs) to confirm the consistently high survival rate (Table 1). Another group of FPNs (n 97) and 2PBs (n 103) was used to test the potential of embryogenesis after cryopreservation and zygote reconstruction. After thawing, the viable FPNs and 2PBs were inserted into the perivitelline space of the male karyoplasts to form reconstructed zygotes after electrical fusion. Reconstructed zygotes reached the twocell, four-cell, hatching, and hatched stages after coculture with mouse embryonic fibroblasts for 1, 2, 4, and 5 days. Comparing the tested protocols, the first tryout (41 FPNs and 67 2PBs) demonstrated that the ultra-rapid method exhibited a significantly lower blastocyst formation rate in the 2PBs group (Table 2). Furthermore, the second (37 FPNs and 45 2PBs) and third (19 FPNs and 10 2PBs) tryouts confirmed that vitrification consistently resulted in high fusion and blastocyst formation rates (Table 2). In addition to this high success rate for blastocyst formation, the liveborn rate was further evaluated after ET to the recipient. In this ET study, all reconstructed embryos were frozen thawed with the vitrification method. These reconstructed embryos were either transferred at the blastocyst stage to the uterus cavity or at the two-cell stage to the oviduct of the recipients. The first tryout was performed by transfer through the uterus. Unfortunately, after 20 days of gestation, no pups TABLE 1 Survival rates of frozen-thawed female pronuclei (FPNs) and second polar bodies (2PBs) No. tryout Cryopreserved method No. frozen No. survived (%) FPNs 2PBs FPNs 2PBs 1 Slow-rate 21 19 11 (52.4) 16 (84.2) Ultra-rapid 26 32 3 (11.5) 15 (46.9) Vitrification 20 21 17 (85.0) 16 (76.2) P.001 a P.001 a 2 Vitrification 21 28 12 (57.1) 24 (85.7) 3 Vitrification 21 20 14 (66.7) 19 (95.0) a Ultra-rapid group has significantly lower blastocyst formation rates than the other groups. 350 He et al. Embryogenesis from preserved female pronuclei Vol. 79, No. 2, February 2003

FIGURE 2 Assessment of nuclei viability. The viability of thawed nuclear materials (i.e., female pronuclei or second polar bodies) was evaluated by propidium iodide and Hoescht 33342 dual staining. After staining, pictures were taken under a light microscope (A), fluorescence microscope with Texas red filter (B), under fluorescence microscope with DAP1 filter (C), or under fluorescence microscope with DAP1/fluorescein/Texas red triple-filter (D). were born. At sacrifice, we found that all fetuses were normally formed except one with placenta but no fetus. The implantation rates were 17.6% and 37.3% in the 2PB and FPN groups (P.201), respectively. In the second tryout, embryos were transferred through the oviduct. This time, three live offspring resulted from each FPN and 2PB transfer and an additional stillborn resulted from the FPN group (Table 3). TABLE 2 Outcome of thawed female pronuclei (FPNs) and second polar bodies (2PBs) after transplantation. No. tryout Cryopreserved method No. frozen No. fused (%) No. blastocysts (%) a FPNs 2PBs FPNs 2PBs FPNs 2PBs 1 Slow-rate 15 26 5 (33.3) 18 (69.2) 3 (20.0) 8 (30.8) Ultra-rapid 9 22 5 (55.6) 17 (77.3) 0 (0.0) 2 (9.1) Vitrification 17 19 12 (70.6) 17 (89.6) 6 (35.3) 9 (47.4) P.107 P.274 P.114 P.001 b 2 Vitrification 37 45 19 (51.4) 41 (91.1) 8 (21.6) 13 (28.8) 3 Vitrification 19 10 13 (68.4) 8 (80.0) 2 (10.5) 4 (40.0) a Percentage per number frozen. b Ultra-rapid group has significantly lower blastocyst formation rates than the other groups. FERTILITY & STERILITY 351

FIGURE 3 Reconstructed mice, recipients, and their offspring. (A), Reconstructed B6D2F1 mice (ebony) after nuclear transfer of cryopreserved second polar bodies and their CD1 recipient (white). (B), Three generations of mice. The first generation CD1 recipient (white), second generation reconstructed mice (big ebony), and the third generation offspring (4 small ebony). To distinguish the reconstructed pronuclei from the offspring of the surrogate mother, the transferred nuclei was obtained from mice of ebony color (i.e., B6D2F1), as compared to the stark white color of the surrogate mother (i.e., CD1). All of the offspring were ebony-colored showing that they were indeed derived from the reconstructed embryos (Fig. 3A). After 20 weeks, reconstructed mice that were products of both FPN and 2PBs were mated naturally, and the second generation of mice was born, demonstrating that these mice were anatomically normal in terms of fertility (Fig. 3B) and displayed all the morphologic characteristics (such as color of coat) resembling that of their reconstructed mothers. DISCUSSION Whereas cryopreservation of sperm has long been established for banking and has been applied successfully in IVF clinics worldwide, the freeze-banking of oocytes has remained substandard. Cryoinjury to meiotic microtubings and ooplasmic organelles (i.e., mitochondria and intracellular lipid droplets) often results in abnormal maturation, fertilization, and embryonic development. However, after fertilization of the oocyte, the 2PB and FPN are formed, both serving as subcellular components of zygotes. Both polar bodies and pronuclei are membranebound nuclear materials that are free of microtubings or ooplasmic organelles. More important, they contain a full haploid complement of female chromosomes. They can be safely cryopreserved to circumvent cryoinjury and used later for reconstruction of the embryo, thereby preserving female fertility potential. We have established a novel method to generate viable offspring through nuclei packaging in donor zona pellucida, cryopreservation, zygote reconstruction, coculturing of re- TABLE 3 Development of reconstructed embryos after transplantation. No. tryout Embryo transfer location Type of transplant No. reconstructed zygotes No. blastocysts formed (%) No. ETs No. implant sites (%) Post-preimplantation develoment No. placenta alone (%) No. fetuses (%) No. offspring (%) 1 Uterus 2PBs 67 25 (37.3) 17 3 (17.6) 0 3 (17.6) 0 FPNs 71 22 (31.0) 16 6 (37.5) 1 (6.3) 5 (31.3) 0 P.200 P.362 2 Oviduct 2PBs 41 26 1 (3.9) 3 (11.5) FPNs 36 27 0 3 (11.1) P.961 352 He et al. Embryogenesis from preserved female pronuclei Vol. 79, No. 2, February 2003

constructed zygotes with murine embryonic fibroblasts, and subsequent ET. This is a report of the successful cryopreservation of female nuclear materials and nuclear transplantation that resulted in apparently normal liveborn offspring with normal fertility, as evidenced by the delivery of healthy second-generation mice. Our results clearly demonstrate that both 2PBs and FPNs can be preserved in all three tested protocols, with ultra-rapid method exhibiting a significantly lower survival rate. The open-pulled straw vitrification method, which renders very high cooling ( 20,000 C/min) and short contact with concentrated cryoprotectant additives ( 30 seconds over 180 C) offers a possibility to circumvent cryoinjury and to decrease toxic and osmotic damage (24). These advantages may also explain why open-pulled straw vitrification is an effective protocol for cryopreservation of the packed 2PBs and FPNs. Our data also showed that the conventional slowrate freezing method may be as effective as open-pulled straw vitrification. However, to have a conclusive outcome, this experiment needs to be repeated with larger sample numbers in all three tested groups. Competent oocyte/zygote cytoplasm has been demonstrated to support the development of embryos produced by nuclear transfer of freshly isolated somatic cell nuclei (25 27), first polar bodies (19), second polar bodies (18), and female pronuclei (1). Our data show that frozen thawed FPNs and 2PBs had similar embryogenesis potential after transfer to donor cytoplasts. As determined by the survival, blastocyst formation and live birth rates, our novel strategy of nuclei vitrification, zygote reconstruction, and coculturing of reconstructed zygotes with MEFs is far superior to all other published protocols for oocyte cryopreservation. Our novel approach can serve as an alternative method for oocyte banking. It can preserve female nuclear material before oocyte damage, which may result from chemotherapy or age-related oocyte deterioration. The FPNs or 2PBs could be preserved at a young age or preceding medical treatment deemed toxic to oocytes to preserve a woman s fertility. At the designated time, a healthy zygote could be obtained after transferring the thawed FPNs or 2PBs to a donor cytoplast and a liveborn baby might be resulted after ET. This new approach can also be clinically used to avoid the confusion of maternal identity in donor egg patients. Normally, oocytes from donors are fertilized with sperm of the recipient s husband to form zygotes that are later transferred to the patient s uterus. These zygotes contain genetic constitution of both the donor and the patient s husband. Using female pronuclear transfer technique, the donor s female pronucleus is replaced by the patients own female pronucleus at the zygote stage. Although it cannot avoid the creation of a triparental embryo composed of normal biparental origin with mitochondrial DNA from the donor (2, 28), our data showed that these disposable 2PBs can be used to successfully generate viable embryos and offspring. It is therefore conceivable that two viable embryos or offspring from one zygote can be created, offering patients an additional hope of generating viable embryos and offspring from their disposable 2PBs. In our laboratory, we have successfully applied openpulled straw vitrification to cryopreserve other subcellular components such as germinal vesicle cytoplast, metaphase II oocyte cytoplasts, zygote cytoplasts, germinal vesicles, first polar bodies, and male pronuclei (data not shown). Cryopreservation of subcellular components makes possible a concept to constitute a subcellular parts workshop. All of the components of oocytes or zygotes preserved separately can be assembled later to reconstruct oocytes or zygotes. A particular hindrance to nuclear transfer in the past, for example, has been ensuring that the cytoplasm was at the correct developmental stage and under the optimal activation stage for the male and female nuclear material to fuse (1, 26). Cytoplasm presumably contains unidentified factors that allow for the fusing of male and female pronuclei. Although electrofusion will trigger activation, the cytoplast and karyoplast must be synchronized or the procedure will fail. Cryopreservation of subcellular components allows the selection of well-synchronized cellular components for effective oocyte or zygote reconstruction. It also provides an opportunity to study the synchronization and mechanism of nuclei and karyoplast interaction during nuclear transfer. In conclusion, we have established a new system to effectively generate live offspring by a combination of nuclei manipulation and preservation, zygote reconstruction, and coculturing of reconstructed zygotes with MEF. Our data suggest that preserved female genomic DNA materials can be potentially used for future nuclear transfer to preserve female fertility and that the otherwise disposable 2PBs can be used to successfully generate viable, healthy embryos. References 1. Levron J, Willadsen SM, Shimmel T, Cohen J. Cryopreservation of activated mouse oocytes and zygote reconstitution after thaw. Hum Reprod 1998;13:109 16. 2. Alikani M, Cohen J. Human oocyte and embryo cryopreservation. Curr Opin Obstet Gynecol 1990;2:714 7. 3. Fugger EF. Clinical status of human embryos in the USA until 1989. Fertil Steril 1989;52:986 90. 4. Whittingham DG. Fertilization in vitro and development to term of unfertilized mouse oocytes previously stored at 196 C. J Reprod Fertil 1977;49:89 94. 5. Schroeder AC, Charplin AK, Mobraaten LE, Eppig J. Developmental capacity of mouse oocytes cryopreserved before and after maturation in vitro. J Reprod Fertil 1990;39:43 50. 6. Gook DA, Osborn SM, Jonnston VI. Cryopreservation of the human oocytes using 1.2-propandiol and the configuration of the meitotic spindle. Hum Reprod 1993;8:1101 9. 7. Van der Elst J, Nerincks S, Van Steirteghem AC. Association of ultrarapid freezing of mouse oocytes with increased polyploidy at the pronucleate stage, reduced cell numbers in blastocysts and impaired fetal development. J Reprod Fertil 1993;99:2 32. 8. Van der Elst J, Nerincks S, Van Steirteghem AC. Slow and ultrapid freezing of fully grown GV stage mouse oocytes: optimization of survival rate outweighed by defective blastocyst formation. J Assist Reprod Genet 1993;10:202 12. 9. Carrol J, Depypere H, Matthews. Freeze thaw-induced damage of the zona pellucida explains decreased rates of fertilization in frozen thawed mouse oocytes. J Reprod Fertil 1990;90:547 53. FERTILITY & STERILITY 353

10. Vincent C, Pickering SJ, Johnson MH. The hardening effect of DMSO on the mouse zona pellucida requires the presence of an oocyte and is associated with a reduction in the number of cortical granules present. J Reprod Fertil 1990;89:253 9. 11. Younis AI, Toner M, Biggers JD. Cryobiology of non-human primate oocytes. Hum Reprod 1996;11:156 65. 12. Bouquet M, Selva J, Auroux M. The incidence of chromosomal abnormalities in frozen-thawed mouse oocytes after in-vitro fertilization. Hum Reprod 1992;7:76 80. 13. Bouquet M, Selva J, Auroux M. Cryopreservation of mouse oocytes: mutagenic effects in the embryo. Biol Reprod 1993;49:746 9. 14. Sathanantan AH, Kirby C, Trounson A, Philipatos D, Shaw J. The effects of cooling on mouse oocytes. J Assist Reprod Genet 1992;9: 139 48. 15. Aigner S, Van der Elst J, Siebzehnrubl E, Wildt L, Lang N, Van Steirteghem AC. The influence of slow and ultra-rapid freezing on the organization of the meiotic spindle of the mouse oocyte. Hum Reprod 1992;7:857 64. 16. Sterzik K, Rosenbusch B, Grab D, Wahl A, Beier HM, Lauritzen C. Numerical chromosome anomalies after fertilization of freeze-thawed mouse oocytes. Arch Gynecol Obstet 1992;251:133 8. 17. Veeck LL, Amundson CH, Brothman LJ, DeScisciolo C, Maloney MK, Muasher SJ, et al. Significantly enhanced pregnancy rates per cycle through cryopreservation and thaw of pronuclear stage oocytes. Fertil Steril 1993;59:1202 7. 18. Wakayama T, Hayashi Y, Ogura A. Participation of the female pronucleus derived from the second polar body in full embryonic development of mice. J Reprod Fertil 1997;110:263 6. 19. Wakayama T, Yanagimachi R. The first polar body can be used for the production of normal offspring in mice. Biol Reprod 1998;59:100 4. 20. Tsunoda Y, Yasui T, Nakamura K, Uchida T, Sugie T. Effect of cutting the zona-pellucida on the pronuclear transplantation in the mouse. J Exper Zoo 1986;240:119 25. 21. Surani MAH, Barton BC, Norris ML. Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis. Nature 1984;308:548. 22. Gosden RG, Baird DT, Wade JC, Webb R. Restoration of fertility to oophorectomized sheep by ovarian autografts at 196 degrees C. Hum Reprod 1994;9:597 603. 23. Isachenko VV, Isachenko EF, Ostashko FI, Grishchenko VI. Ultrarapid freezing of rat embryos with rapid dilution of permeable cryoprotectants. Cryobiology 1997;34:157 64. 24. Vajta V, Holm P, Kuwayama M, Booth PJ, Jacobsen H, et al. Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Develop 1998;51:53 8. 25. Dominko T, Mitalipova M, Haley B, Beyhan Z, Erdogan M, McKusick B, First NL. Bovine oocyte cytoplasm supports development of embryos produced by nuclear transfer of somatic cell nuclei from various mammalian species. Biol Reprod 1999;60:1496 502. 26. Wolf DP, Meng L, Ouhibi N, Zelinski-Wooten M. Nuclear transfer in the rhesus monkey: practical and basic implications. Biol Reprod 1999; 60:199 204. 27. Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KHS. Viable offspring derived from fetal and adult mammalian cells. Nature 1997; 385:810 3. 28. Cohen J, Levron J, Palermo GD, Munne S, Adler A, Alikani M, et al. Atypical activation and fertilization patterns in humans. Theriogenology 1995;43:129 40. 354 He et al. Embryogenesis from preserved female pronuclei Vol. 79, No. 2, February 2003