Generation of Healthy Cloned Mice Using Enucleated Cryopreserved Oocytes

Abstract

The successful generation of cloned animals and the establishment of embryonic stem (ES) cell lines from somatic cells suggest that these techniques may be used in human regenerative medicine. However, the fact that oocytes must be donated by women undergoing infertility treatment remains a fundamental ethical objection, as they might be concerned about the potential exploitation of their genome. Here, we investigated the reprogramming potential of enucleated and cryopreserved oocytes for the development of full-term cloned mice. BDF1 strain mouse oocytes were cryopreserved at metaphase II, before and after enucleation. After thawing, cumulus cell nuclei were microinjected to generate clones. Although the rate of development of cloned embryos to the blastocyst stage using the treated oocytes was lower than that obtained using fresh oocytes, three live pups were delivered after embryo transfer into pseudopregnant females (0.4% of the oocytes used). Thus, although cryopreservation reduces the potential of oocytes, these cells retain the ability to support the full-term development of cloned embryos. In addition, the removal of DNA from human oocytes may alleviate the ethical and psychological problems for women who are undergoing infertility treatment and are considering oocyte donation for research or therapeutic purposes.

Introduction

Nuclear-transfer-derived embryonic stem (ntES) cells can be established from cloned embryos (Munsie et al., 2000; Wakayama et al., 2001) generated by somatic cell nuclear transfer (SCNT) into an enucleated oocyte (Wakayama et al., 1998; Wilmut et al., 1997). Such ntES cells have the same level of pluripotency as conventionally derived ES cells (Wakayama et al., 2006). Although the development of human ntES cell has not been successful, even when using fresh oocytes, “personalized” ntES cells are expected to have future applications in human regenerative medicine, without the problems associated with tissue rejection by the host (Rideout et al., 2002; Tabar et al., 2008). However, the source of oocytes remains a major ethical and practical obstacle to the application of this technology in humans (Spar, 2007), as SCNT is inefficient in all species studied to date (Thuan et al., 2010). The safe collection of hundreds of oocytes is prohibitively expensive, time consuming, and risky for donors (Covington and Gibbons, 2007; Daley et al., 2007).

Several approaches have been developed to overcome this problem. Previously, we reported the possibility of using aged fertilization-failed (AFF) oocytes for SCNT; these AFF oocytes are routinely discarded in clinical practice, as they lose developmental potential with age. Although cloned mouse embryos derived from AFF oocytes did not develop to full term, ntES cell lines were established with the same success rate as that obtained using fresh oocytes (Wakayama et al., 2007).

Sung et al. (2010) reported the potential use of “excess” cryopreserved oocytes for the treatment of human infertility. These authors used the mouse as a model to demonstrate that cryopreserved oocytes retain the potential for reprogramming the somatic cell genome and succeeded in establishing ntES cell lines. Although they generated chimeric mice from ntES cell lines using tetraploid complementation, these researchers did not examine the full-term development of cloned embryos.

In addition, women are faced with psychological problems associated with the decision to donate excess oocytes. Here, we propose that the fact that the oocyte contains genomic information is one of the obstacles to human oocyte donation; we suggest that asking a woman to donate oocytes lacking a genome, even if the procedure of oocyte collection remains the same, may reduce the difficulties inherent in oocyte donation. Although it is not clear yet whether enucleated and cryopreserved oocytes retain their full genomic reprogramming potential, this approach may help overcome such ethical problems. However, Endoh et al. (2007) reported that frozen–thawed oocytes often show lower full-term developmental potential after intracytoplasmic sperm injection in mice. Moreover, to our knowledge, no cloned mice have been born from SCNT using cryopreserved oocytes. If cryodamaged oocytes lose their potential to support full embryogenesis, we must determine whether ntES cell lines established from such oocytes also exhibit abnormalities. It is important to clarify this point before making any attempts to apply this technology to human medicine. Here, we attempted to produce cloned mice from enucleated cryopreserved oocytes, as the generation of healthy offspring represents the strongest evidence of the quality of the oocytes after treatment.

Materials and Methods

Animals

Six-week-old female B6D2F1 mice (C57BL/6 × DBA/2, registered as Jcl:BDF1) and 6-week-old female and male ICR mice (registered as Jcl:ICR) were obtained from CLEA Japan Inc. (Higashiyama, Tokyo, Japan). The mice were handled according to the Guidelines of the Center for Life Science Research, University of Yamanashi, and the Animal Experiment Handbook at the Riken Center for Developmental Biology. The experimental protocol of this study was approved by the Animal Care and Use Committee, University of Yamanashi. The mice were fed food and water ad libitum and were maintained under a 12/12 h light/dark cycle.

Chemicals and laboratory equipment

Unless otherwise indicated, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Preparation of oocytes and enucleation

Female B6D2F1 mice were superovulated via subcutaneous administration of 5 IU PMSG, followed by the administration of 5 IU hCG 48 h later. Cumulus–oocyte complexes (COCs) were collected from oviducts 14 h after hCG administration. Oocytes were denuded using 0.05% hyaluronidase in modified HEPES-buffered CZB medium (Kimura and Yanagimachi, 1995), transferred into fresh CZB medium (Chatot et al., 1990), and stored at 37°C under a humidified mixture of 5% CO₂ and 95% air.

Enucleation was performed as described previously (Wakayama et al., 1998), either before or after cryopreservation. Briefly, oocytes at the metaphase II (MII) stage were treated with 5 μg/mL cytochalasin B in HEPES-buffered CZB before enucleation. Subsequently, the MII chromosome–spindle complex was removed using a micropipette. Enucleated oocytes were incubated for at least 1 h at 37°C in 5% CO₂ in air before vitrification.

Vitrification and thawing of oocytes

We adopted the ultrarapid vitrification method for oocyte cryopreservation (Gomes et al., 2008; Kuwayama, 2007; Kuwayama et al., 2005). Briefly, oocytes were suspended in an equilibration solution containing 7.5% ethylene glycol (EG), 7.5% dimethylsulfoxide (DMSO), and 20% serum substitute supplement (SSS; Irvine Scientific, Santa Ana, CA, USA) at room temperature for 60 sec. The oocytes were subsequently transferred into a vitrification solution (VS) containing 15% EG, 15% DMSO, 0.5 M sucrose, and 20% SSS, and were held at room temperature for 30 sec. The original protocol indicated that a maximum of three oocytes could be vitrified simultaneously in a droplet held in a Cryotop (Kitazato BioPharma Co., Ltd., Shizuoka, Japan). However, because hundreds of oocytes were needed daily to perform the experiments, we decided to treat more oocytes simultaneously using a dish instead of the Cryotop (we termed this the Schale method). In this method, approximately 40 oocytes were loaded onto a polystyrene dish 3.5 cm in diameter (Falcon #35-3801, Becton, Dickinson and Co., Franklin Lakes, NJ, USA) containing a minimum amount of VS (about 3 μL). The oocytes on the dish were immediately plunged into liquid nitrogen at −196°C and cryopreserved for at least 24 h before thawing.

The cryopreserved oocytes were warmed using the thawing media set supplied (Code # VT102; Kitazato BioPharma) (Kuwayama, 2007). Oocytes were thawed in 1 M sucrose plus 20% SSS for 60 sec at 37°C, followed by incubation in a diluent solution containing 0.5 M sucrose plus 20% SSS for 3 min at room temperature. Then, oocytes were rinsed twice in a washing solution containing 20% SSS, to dilute out the cryoprotectants. After thawing, the oocytes were incubated for recovery in CZB for at least 1 h at 37°C in 5% CO₂ in air. Finally, the surviving oocytes—which were assessed by color and membrane integrity—were selected and used for SCNT.

SCNT procedure

SCNT was carried out as described previously (Thuan et al., 2010; Wakayama, 1998, 2007). Briefly, cumulus cells isolated from the COCs at the time of oocyte retrieval were used as nuclear donor cells. The nucleus was prepared by breaking the plasma membrane of the donor cell in the injection pipette using piezo pulses (Prime Tech, Ltd., Ibaraki, Japan), followed by injection into the oocytes to generate reconstructed embryos. The reconstructed embryos were placed in activation medium (Kishigami and Wakayama, 2007) containing 5 nM trichostatin A (TSA) for 6 h (Kishigami et al., 2006; Ono et al., 2008). After activation, the embryos were transferred to fresh CZB medium containing 5 nM TSA for 12 h, followed by incubation in CZB at 37°C under humidified 5% CO₂ and 95% air until blastocyst formation. The number of embryos that reached the pronuclear, two-cell, four-cell, and blastocyst stages was counted.

Generation of cloned mice

Female ICR mice to be used as surrogate mothers were mated with vasectomized male ICR mice. Two-cell-stage cloned embryos generated as described above were transferred into the oviducts at day 1 of pseudopregnancy. The offspring were delivered by cesarean section 18.5 to 19.5 days after SCNT.

Statistics

The data were compared using chi-squared tests and significance was set at p < 0.05.

Results and Discussion

In this study, we attempted to generate cloned mice from cryopreserved oocytes. For this purpose, we started by investigating the effects of vitrification on the developmental potential of mouse oocytes before and after enucleation. The recovery and survival rates of the cryopreserved oocytes are shown in Table 1. The survival rate was decreased significantly in enucleated and cryopreserved oocytes compared with intact cryopreserved oocytes (p < 0.01), although their recovery rates were similar, indicating that the enucleated oocytes were more sensitive to cryopreservation. It is known that the hole made in the zona pellucida of zygotes has no negative effects on vitrification (Macas et al., 2008). However, in this study, oocytes were enucleated under cytochalasin B treatment, which disrupts actin filaments. Enucleation damage to the oolemma or disruption of the cytoskeleton may have caused this disruption.

Table 1.

Oocyte Collection and Survival Rate

Experimental group	No. of vitrified oocytes	No. of oocytes recovered from dish	No. of oocytes that survived after warming
Enucleated–cryopreserved	200	197	166^a
Intact–cryopreserved	204	200	196^b

a vs. b: p < 0.01.

Next, we analyzed the early embryonic development of the embryos cloned using cryopreserved oocytes. As shown in Table 2, the rates of pronuclear embryo development and first cleavage were not different between groups. However, after 4 days of culture, although the rate of blastocyst formation in the enucleated and cryopreserved oocytes was higher than that observed in the cryopreserved and enucleated group (16 vs. 7%; p < 0.05), these rates were significantly lower in both compared with the control group (47%; p < 0.01). Thus, the developmental potential of the ooplasm was clearly impaired by cryopreservation. However, Sung et al. (2010) reported that the rate of cloned blastocyst development using cryopreserved mouse oocytes was decreased by only ∼30% compared with fresh oocytes. The reason for this discrepancy is not clear, as we used a similar vitrification protocol. One possibility is the different SCNT methods used; for example, Sung et al. (2010) fused fibroblasts with oocytes, whereas we injected cumulus cell nuclei directly into the oocyte. Possibly, cryopreserved oocytes are more sensitive to the membrane damage caused by microinjection.

Table 2.

In vitro Development of Cloned Embryos

	No. of nuclear-transferred oocytes		No. of embryos showing in vitro development
Experimental group	Survived after injection	Pronuclear formation (%)	Two-cell stage (%)	Four- to eight-cell stage (%)	Blastocyst stage (%)
Fresh control	240	207 (86.3)	195 (94.2)	170 (82.1)^a	97 (46.8)^c
Enucleated–cryopreserved	204	172 (84.3)	155 (90.1)	76 (44.2)^b	27 (15.7)^d
Cryopreserved–enucleated	247	210 (85.0)	186 (88.6)	81 (38.6)^b	11 (6.7)^e

a vs. b: p < 0.01; c vs. d. e: p < 0.01: d vs. e: p < 0.01.

Based on these initial results, we used enucleated and then cryopreserved oocytes mainly as the recipients in subsequent experiments, aiming to achieve full-term clone development. As shown in Table 3, more than 1000 attempted SCNT procedures using enucleated–cryopreserved oocytes yielded three live offspring. The body and placental weights were similar among experimental and control groups, and all three clones grew to adulthood. Although the success rate of this experiment was only one-tenth of that obtained using fresh controls (0.4 vs. 4%), this study proves the principle that live offspring can be produced by cloning from enucleated–cryopreserved oocytes. To our knowledge, this is the first report of this achievement in mice.

Table 3.

Generation of Cloned Mice using Enucleated–Frozen–Thawed (E–F) Oocytes

		No. of nuclear-transferred oocytes			No. of oocytes exhibiting in vitro development			Average weight (g)
Experimental group	No. of oocytes used	Survived after injection	Survived after activation	Pronuclear formation	Fragment and one-cell stage	Two-cell stage^*	No. of live clones (%)	Pups	Placenta
Fresh control	126	95	93	75	20	73	3 (4)^a	1.53	0.24
Enucleated–cryopreserved	1102	961	941	834	170	771	3 (0.4)^b	1.71	0.27
Cryopreserved–enucleated	68	63	58	58	10	48	0	—	—

All two-cell-stage embryos were transferred into a recipient female.

a vs. b: p < 0.01.

Several reports exist on the development of cloned embryos reconstructed using cryopreserved oocytes in bovines (Atabay et al., 2004; Dinnyes et al., 2000; Hou et al., 2005; Yang et al., 2008). In these studies, intact oocytes were enucleated after freeze–thawing. All of these reports concluded that cryopreserved oocytes exhibited decreased developmental potential for embryos cloned in vitro, and only one cloned calf was carried to a live birth (Hou et al, 2005). Taken together, these findings suggest that the deterioration of the developmental potential of the ooplasm after cryopreservation is similar between mice and cattle.

In conclusion, although the developmental potential of the ooplasm was poor after cryopreservation, some enucleated oocytes maintained sufficient potential for genomic reprogramming of the somatic cell nucleus to permit full-term development. This study established that, in principle, SCNT using enucleation followed by cryopreservation of oocytes may help alleviate the psychological and ethical problems associated with oocyte donation in the setting of human regenerative medicine.

Footnotes

Acknowledgments

The authors gratefully acknowledge the technical support of Ms Ayano Shimoji. We are grateful to the Center for Life Science Research of the University of Yamanashi and the Laboratory for Animal Resources and Genetic Engineering of the RIKEN-KOBE Center for the housing of the mice. This study was supported by grants from the Ministry of Education, Science, Sports, Culture and Technology of Japan (20591910 to S. H.; 20791140 to H. F.; and 15080211 to T. W.).

Author Disclosure Statement

The authors declare that no conflicting financial interests exist.

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