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Laboratory of Developmental Engineering, Department of Life Science, School of Agriculture, Meiji University, Tama, Kawasaki 214-8571, Japan
ABSTRACT
We have successfully produced healthy piglets following cryopreservation of embryos derived from oocytes matured and fertilized in vitro. The appropriate timing of cryopreservation pretreatment (removal of cytoplasmic lipid droplets [delipation] and vitrification) was initially determined using parthenogenetic embryos derived from in vitro-matured (IVM) oocytes. Viable embryos were obtained at the highest rate when embryos were delipated at the four- to eight-cell stages (Day 2 of embryo culture) and were vitrified approximately 15 h later (Day 3) by means of the minimum volume cooling method. After cryopreservation of embryos derived from oocytes matured and fertilized in vitro under the most appropriate conditions, 401 embryos were transferred to five recipient gilts, and the recipients all became pregnant. At autopsy of one of the recipients, which had received 47 embryos, eight fetuses (17.0%) were found. Three recipients each gave birth to two to four piglets (1.4%–6.0%). These results demonstrate that normal offspring can be produced from vitrified porcine embryos derived from IVM oocytes by a strategic combination of delipation and vitrification at the early cleavage stages. This approach has great potential in the reproduction of micromanipulated porcine embryos, such as cloned and sperm-injected embryos, produced from IVM oocytes.
developmental biology,, embryo, in vitro fertilization
Pigs are a valuable resource not only as livestock but also as experimental animals in biomedical research [1]. Developmental engineering has greatly influenced the use of pigs for scientific purposes, leading to the production of designed pigs, including genetically modified and cloned pigs. These pigs currently are used as human disease models with important applications in advanced medical research, such as organ transplantation and regenerative medicine [2–4].
Oocytes that have been in vitro matured (IVM) are used commonly to produce specifically designed pigs [4–7]. The production cost of designed pigs can be considerably reduced by using IVM oocytes instead of in vivo-derived oocytes. One of the current limitations in the production of designed pigs is the requirement of a large number of embryos to obtain the offspring. This problem arises from the low developmental competence of embryos that are produced using IVM oocytes by current reproduction technology [4–7].
Against this background, if proven feasible, the cryopreservation of porcine embryos derived from IVM oocytes would be of great practical value, because in vitro-produced (IVP) embryos, including those obtained by somatic cell nuclear transfer, can be stored in preparation for transfer to recipients. However, reproductive research of the cryopreservation of porcine embryos has lagged behind that of other experimental and livestock animals [8]. It has been demonstrated recently that cryopreserved porcine embryos possess practical levels of viability, but only when in vivo-derived embryos are used [9, 10]. Cryopreservation of embryos derived from IVM oocytes currently remains at the experimental stage [11]. This is in sharp contrast to bovine reproduction, in which the cryopreservation of in vitro maturation-derived embryos is performed routinely [12–15].
This study examined the possibility of cryopreservation of porcine embryos derived from IVM oocytes at early cleavage stages, with the goal of applying the technology to cloned and sperm-injected embryos. We have shown previously that through a combination of delipation (decreasing the amount of cytoplasmic lipid droplets) [16, 17] and vitrification by means of the minimum volume cooling (MVC) procedure [18], cryopreservation of parthenogenetic porcine embryos derived from IVM oocytes can produce viable embryos in vitro [11]. Encouraged by the results of this study, we first determined the appropriate conditions for delipation and vitrification for early cleavage-stage embryos derived from IVM oocytes. Live piglets were obtained following vitrification of porcine embryos produced by in vitro maturation and fertilization of oocytes.
All of the animal experiments in this study were approved by Institutional Animal Care and Use Committee of Meiji University (IACUC-02–002).
Chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated.
In Vitro Maturation of Oocytes
Ovaries were collected at a local abattoir and were transported to the laboratory in Dulbecco phosphate-buffered saline (PBS) containing 75 µg/ml potassium penicillin G, 50 µg/ml streptomycin sulfate, and 0.1% (w/v) polyvinyl alcohol (PVA). Cumulus-oocyte complexes (COCs) were collected by aspiration from ovarian antral follicles with a diameter of 3.0–6.0 mm. COCs having at least three layers of compacted cumulus cells were selected and cultured in NCSU23 medium [19] supplemented with 0.6 mM cysteine, 10 ng/ml epidermal growth factor (EGF), 10% (v/v) porcine follicular fluid, 75 µg/ml potassium penicillin G, 50 µg/ml streptomycin sulfate, 10 IU/ml eCG (Teikoku Zouki Co., Tokyo, Japan), and 10 IU/ml hCG (Teikoku Zouki). The COCs were cultured for 22 h with eCG and hCG in a humidified atmosphere of 5% CO2 and 95% air at 38.5°C, followed by culture for 22 h without eCG and hCG in an atmosphere of 5% CO2, 5% O2, and 90% N2 [20].
Electrical Activation of IVM Oocytes
IVM oocytes with expanded cumulus cells were treated with 1 mg/ml hyaluronidase dissolved in Tyrode lactose medium supplemented with 10 mM Hepes and 0.3% (w/v) polyvinyl-pyrrolidone (Hepes-TL-PVP), and were denuded of cumulus cells by gentle pipetting. Oocytes having evenly granulated ooplasm and an extruded first polar body were selected for the experiments. Oocytes were washed twice in an activation solution consisting of 0.28 M mannitol (Nacalai Tesque Inc., Kyoto, Japan), 50 µM CaCl2, 100 µM MgSO4, and 0.01% PVA. Oocytes were lined up between two wire electrodes (1.0 mm apart) in a fusion chamber overlaid with 0.2 ml activation solution. A single DC pulse of 150 V/mm was applied for a duration of 100 µsec using an electric pulsing machine (ET-1; Fujihira Industries, Tokyo, Japan). To prevent extrusion of the second polar body, activated oocytes were cultured for 3 h in modified NCSU23, as described below, in the presence of 5 µg/ml cytochalasin B (CB).
In Vitro Fertilization of IVM Oocytes
IVM oocytes were fertilized in vitro in accordance with Yoshioka et al. [21] and using epididymal sperm that had been cryopreserved as described by Kikuchi et al. [22]. Briefly, frozen sperm (1.7 x 109/ml) were thawed in a 38.5°C water bath and were centrifuged three times in Dulbecco PBS supplemented with 0.1% BSA at 970 x g for 4 min to remove cryoprotective agents. After discarding the supernatant, the sperm pellet was suspended in an insemination medium (PGM-tac) [21], and IVM oocytes (15–20) with expanded cumulus cells were placed in a 100-µl drop of PGM-tac containing sperm (5 x 106/ml) and then incubated for 20 h at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2. After the insemination period, cumulus cells were removed by pipetting. Fertilized oocytes with both an extruded second polar body and uniformly granulated ooplasm were selected and used for embryo culture.
In vitro culture of the embryos was performed in 20-µl droplets of modified NCSU23 supplemented with 4 mg/ml BSA and 0.5 mg/ml hyaluronic acid [23] under paraffin oil in a plastic Petri dish, which was maintained in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 at 38.5°C. The NCSU23 medium was modified by the addition of 0.17 mM sodium pyruvate and 2.73 mM sodium lactate instead of D-glucose for the first 48 h of culture of one-cell stage embryos [24]. Osmolality of the medium was adjusted to 290 mOsm for the first 48 h and to 256 mOsm for the rest of the culture period [25]. For culturing embryos beyond the morula stage, 10% (v/v) fetal calf serum (FCS; catalog no. 12303–500M, lot no. 4C0702; JRH Biosciences Inc., Kansas City, KS) was added to the medium.
Embryos to be transferred to recipients were cultured in Porcine Zygote Medium-5 (PZM-5; Functional Peptide Inc., Yamagata, Japan).
Removal of Cytoplasmic Lipid Droplets from Embryos (Delipation)
Removal of cytoplasmic lipid droplets from the embryos was carried out by a previously described method [16, 17]. Briefly, to polarize lipid granules in the cytoplasm, embryos were centrifuged (12,000 x g, 23 min) at 38°C in Hepes-TL-PVP containing 7.5 µg/ml CB using a 1.5-ml microcentrifuge tube (Fukae Kasei Co., Hyogo, Japan). The resultant lipid layer was removed (Fig. 1) by micromanipulation using a beveled suction pipette (diameter: 28–32 µm) attached to the micromanipulator (MO-202U; Narishige, Tokyo, Japan) under an inverted microscope (TE300; Nikon, Tokyo, Japan).
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Cryopreservation of embryos was carried out by vitrification using the MVC method [18]. All solutions used during vitrification and thawing were prepared with a basal medium composed of TCM199 containing 20 mM Hepes, 4.2 mM NaHCO3, 75 µg/ml potassium penicillin G, and 50 µg/ml streptomycin sulfate. Embryos were equilibrated with an equilibration solution containing 7.5% (v/v) ethylene glycol (EG; Nacalai Tesque), 7.5% (v/v) dimethyl sulfoxide (DMSO; Wako Pure Chemical Industries Co., Osaka, Japan), and 20% (v/v) calf serum (CS; catalog no. 12133–500M, lot no. 4J0214; JRH Biosciences) for 4 min followed by exposure to a vitrification solution containing 15% EG, 15% DMSO, 0.5 M sucrose (Nacalai Tesque), and 20% CS. Embryos then were loaded onto an MVC plate (Cryotop; Kitazato Supply, Shizuoka, Japan) and immediately plunged into liquid nitrogen. The process, beginning with embryo exposure to a vitrification solution and ending with the plunging, was completed within 1 min. Embryos were thawed by immersing the MVC plate directly in a thawing solution containing 1 M sucrose and 20% CS at 37°C for 1 min. Recovered embryos were transferred to a diluent solution containing 0.5 M sucrose and 20% CS, where they were kept for 3 min, after which they were kept for 10 min in a washing solution containing 20% CS in order to remove the cryoprotectant. All solutions except for the thawing solution were maintained at room temperature.
The recovered embryos that met all of the following criteria were assessed as live embryos: 1) all blastomeres were present without damage, 2) no damage was observed on the blastomere membranes, and 3) no discoloration or granulation of the blastomere cytoplasm was observed, and the blastomere cytoplasm was transparent.
Fixation and Staining of Embryos
To determine the normal fertilization rate for in vitro-fertilized (IVF) embryos, some embryos were fixed with acetoalcohol (1:3) and stained with 1% acetoorcein for 20 h following insemination. Cryopreserved and control embryos also were fixed and stained 8 days after activation or insemination to determine cell number.
Transfer of Cryopreserved Embryos
Crossbred (Large White/Landrace x Duroc) prepubertal gilts weighing 100–105 kg were used as recipients of the cryopreserved embryos. In order to induce estrus, 1000 IU eCG was injected intramuscularly, followed by an injection of 1500 IU hCG 72 h later.
The cryopreserved embryos were cultured for 0.5–5.0 h prior to transfer. Embryos that maintained normal morphology were selected for transfer. Embryos were transferred into the oviducts of recipient gilts approximately 72 h after hCG injection. Some of the recipients were kept until parturition, and others were autopsied on Day 22 or 23 of gestation to collect fetuses.
Determining the appropriate timings of delipation and vitrification. The appropriate conditions for cryopreservation were determined in parthenogenetic embryos at 2 days (Day 2) and 3 days (Day 3) after activation. Parthenogenetically activated embryos were delipated and vitrified at various time points ranging from 44 to 67 h after activation (Fig. 2), and their in vitro survival rates were compared after culture (experiments 1–3). In addition, the ability of vitrified embryos to develop into blastocysts after thawing was compared to that of the parthenogenetic control (nondelipated/nonvitrified embryos).
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Each experiment was conducted at least three times, and at least 13 embryos were randomly assigned to each group in each replicate.
Experiment 1. This experiment was carried out to determine the appropriate timing of delipation, and embryos at early cleavage stages were used. As shown in Figure 2, delipation and subsequent vitrification were performed early on the second day following activation (early Day 2, approximately 44 h after activation; the two- to four-cell stage) or late on the second day following activation (late Day 2, approximately 52 h after activation; the four- to eight-cell stage), and then the postthaw viability of embryos was compared. The interval between vitrification and delipation was approximately 1 h.
Experiment 2. In experiment 1 we determined the appropriate timing of delipation. In experiment 2 we examined the interval (culture period) between delipation and vitrification (Fig. 2). Delipation was performed on late Day 2, and the interval between delipation and vitrification was changed to examine its effect on postthaw embryonic viability. Vitrification was performed approximately 3 or 15 h (early on Day 3) after delipation, and the embryo survival rate was compared after thawing.
Experiment 3. In experiment 2 it was found that a high embryo survival rate was obtained when the embryos were delipated late on Day 2, cultured for 15 h, and vitrified early on Day 3. In experiment 3 we examined the effects of different culture periods (between delipation and vitrification) on embryonic viability by fixing the time of vitrification at early on Day 3 (Fig. 2). Delipation was performed approximately 15 h before vitrification (late on Day 2; the test conditions in this group were the same as those in one group in experiment 2) or 3 h before vitrification (Day 3), and the postthawing embryo survival rates were compared.
Evaluation of Developmental Competence of Cryopreserved Embryos Derived from IVM Oocytes
Experiment 4. The conditions that gave the highest embryonic viability (delipation on late Day 2 and vitrification on early Day 3, 15 h after delipation) were used for cryopreservation of parthenogenetic and IVF embryos, and the developmental competence of these embryos was examined after transfer to recipient gilts. Postthaw survival of the IVF embryos was assessed by either in vitro culture or transfer. IVF embryos were delipated approximately 52 h after insemination. Embryos were all cryopreserved for 6–35 days in liquid nitrogen before examination of postthaw survival.
The rate of embryo development was compared using the chi-square test. Student t-test was used to compare the mean cell numbers of the embryos.
Appropriate Timings for Delipation and Vitrification
In experiments 1–3, the appropriate timing for delipation and vitrification was determined by examination of parthenogenetic embryos. The mean in vitro maturation rate of the oocytes provided for these experiments (n = 1606) was 75.3%. A total of 1209 IVM oocytes were activated, and 791 cleaved embryos with normal morphology (65.4%) were obtained. These embryos were used in the experiments described below, and the results are summarized in Table 1.
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In experiment 1, the timing of delipation was compared between early and late Day 2. There was no significant difference in the blastocyst formation rates between embryos delipated late and early on Day 2, and these rates were significantly lower (P < 0.05) than those of the nonvitrified control. Cell number in the embryos delipated on early Day 2 also was significantly lower (P < 0.05) than that of nonvitrified control; however, the cell number of the embryos delipated on late Day 2 was comparable to that of the control. Thus, late Day 2 was selected as the better delipation timing option.
In experiment 2, vitrification was performed after approximately 3 or 15 h of culture following delipation on late Day 2. The embryo viability was significantly higher when embryos were vitrified on early Day 3; that is, after 15 h of culture (36 [46.8%] of 77, vs. 24 [29.6%] of 81; P < 0.05). Most notably, the percentage of vitrified embryos in the 15-h culture group that developed into blastocysts was as high as that of the control nondelipated/nonvitrified embryos (51.7%).
In experiment 3, all embryos were vitrified on early Day 3, and the timing of the preceding delipation was changed (15 or 3 h before vitrification). The effect of these conditions on embryonic viability was examined. There was no significant difference in blastocyst formation rate between embryos delipated at 3 h versus 15 h before vitrification; however, the cell number was significantly higher (P < 0.05) in the embryos vitrified 15 h after delipation compared with that of the 3-h culture group. In the 15-h culture group, the percentage of vitrified embryos that developed into blastocysts and the number of cells were as high as those of the nondelipated/nonvitrified control embryos (blastocysts: 28 [57.1%] of 49; number of cells: 56.0 ± 4.8). Thus, the appropriate timing with the highest embryonic viability was delipation on late Day 2 and vitrification on early Day 3.
Developmental Ability of Porcine Embryos Derived from IVM Oocytes
In experiment 4 a total of 1978 COCs were provided for in vitro maturation and subsequent insemination procedures. Oocytes that extruded one to two polar bodies after insemination (1585 oocytes, 80.1%) were regarded as fertilized and were provided for in vitro culture.
Embryos were cultured for 2 days, and 737 (46.5%) of 1585 embryos reached the four- to eight-cell stages. Of these, 623 embryos were delipated, and cytoplasmic lipid droplets were removed successfully without damaging blastomeres in 588 embryos (94.4%). These embryos were used in the vitrification experiment. Some IVF embryos were fixed and stained (n = 75) in order to examine the fertilization rate, which was 45.3% (polyspermy rate: 32.4% of the fertilized embryos). IVF embryos were delipated and vitrified under the conditions that had been determined in experiment 3.
The in vitro development of IVF embryos after vitrification is presented in Table 2. A high yield (71 [94.7%] of 75) of vitrified embryos recovered after thawing had retained their previtrification morphology, and 30 embryos (40.0%) developed to the blastocyst stage (Fig. 1). This blastocyst formation rate was comparable to that of the nonvitrified controls (nondelipated/nonvitrified embryos 35 [44.3%] of 79) and delipated/nonvitrified embryos (23 [43.4%] of 53). There was no difference between the vitrified (49.4 ± 4.7) and nonvitrified controls (nondelipated: 54.7 ± 5.4; delipated: 55.9 ± 5.4) with respect to blastocyst cell number.
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Table 3 shows the developmental competence of the vitrified IVF and parthenogenetic embryos after transfer to recipient gilts.
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Transfer of a total of 401 vitrified IVF embryos to five gilts (47–142 embryos per recipient) resulted in pregnancy in all of the recipients. When one gilt that had received 47 embryos was autopsied at 23 days of pregnancy, eight fetuses (17.0%) were collected (Fig. 3). Three other recipients farrowed two, three, and four (including one stillbirth) piglets (Fig. 3). One recipient that had been judged pregnant on Day 27 by ultrasonography returned to estrus on Day 41.
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When a total of 251 parthenogenetic embryos were transferred to four gilts (50–80 embryos per recipient), three recipients became pregnant. When these gilts were autopsied at 22–23 days of pregnancy, they were carrying 2 (2.9%) to 11 (21.6%) somite-stage fetuses (Fig. 3).
To the best of our knowledge, this is the first report of live piglets produced from cryopreserved porcine embryos derived from oocytes matured and fertilized in vitro. Previously, cryopreservation of porcine embryos produced by in vitro maturation and fertilization of oocytes had proven unsuccessful, primarily due to the intrinsic sensitivity of porcine embryos to low temperatures. In addition, embryos derived from IVM oocytes are generally less cryoresistant than in vivo-derived embryos [26–28]. Together, these factors have constituted a barrier to successful cryopreservation of porcine embryos derived from oocytes matured and fertilized in vitro.
Results of the present study demonstrate that the cryosensitivity of porcine embryos derived from IVM oocytes can be overcome by a combination of delipation and vitrification. The fact that the postthaw developmental ability of vitrified IVF embryos was similar to the nonvitrified controls indicated that the method used in this study for cryopreservation did not negatively affect the viability of porcine embryos derived from IVM oocytes. Viable embryos could be obtained at the highest rate when embryos were delipated at the four- to eight-cell stage and vitrified approximately 15 h later by means of the MVC method. In addition, the survival of in vivo-derived embryos at the four-cell stage was 100% when they were vitrified by the same delipation and MVC procedures in a separate experiment (Nagashima et al., unpublished results). Together, these results indicate that the method in this report has attained a highly reproducible level for the cryopreservation of porcine embryos derived from IVM oocytes.
It has also been suggested that the duration of the culture period between delipation and vitrification might be one of the determinants for embryonic survival after cryopreservation. Interestingly, a longer culture period (15 h) between delipation and vitrification increased embryonic survival after vitirification compared with a shorter culture (3 h). The physiologic changes that embryos undergo during culture are not clear, but delipation-associated damage to the cell membrane and other qualitative changes may take a period of time to be restored. The next investigative step is to precisely determine the culture conditions that influence cryoresistance in porcine embryos.
In cattle, cryopreservation of embryos produced by in vitro maturation and fertilization of oocytes and subsequent in vitro culture is performed routinely, and it is well established that the culture conditions employed greatly influence the cryoresistance of the embryo [29, 30]. In this study, recent advances in porcine embryonic culture media no doubt contributed to the success of the cryopreservation. In fact, the modified NCSU23 [23–25] and PZM-5 [31] media used in the present study markedly improved the in vitro developmental competence of porcine embryos derived from IVM oocytes compared with our previous data (Nagashima et al., unpublished results). In particular, we confirmed that the number of embryos that develop to the hatching blastocyst stage and the number of cells in the blastocyst stage are significantly higher when porcine parthenogenetic embryos are cultured in PZM-5 medium rather than modified NCSU (Nagashima et al., unpublished data). As such, PZM-5 is considered an excellent culture medium for porcine embryos, and it is for this reason that PZM-5 was used in the embryo transfer experiments in this study.
In this study, we first determined the appropriate timing of delipation and vitrification using parthenogenetic embryos. There is room for discussion as to whether parthenogenetic embryos are the optimal model for IVP embryos, such as cloned and transgenic embryos. However, many recent studies have reported the production of IVP embryos from IVM oocytes. Since the parthenogenetic embryos that we used in this study also were derived from IVM oocytes, we therefore consider many of the findings in this study to be fundamentally true for IVP embryos derived from IVM oocytes.
Although the pregnancy rate after transfer of the vitrified IVF embryos was 100% in this study, the overall efficiency of the vitrified embryos developing into piglets was low. The fact that approximately 30% of the IVF embryos in this study were polyspermic acted as a constraint on the low efficiency. In addition, transfer of an unnecessarily large number of vitrified IVF embryos to ensure conception also lowered the rate of piglet production. In contrast to the IVF embryos, the developmental rate to fetus (>20%) of the vitrified parthenogenetic embryos is comparable to the development of the nonvitrified, parthenogenetic embryos examined in our previous study [32]. As such, qualitative improvements in IVF embryos possess great potential to increase the production efficiency of live piglets from vitrified embryos derived from IVM/IVF oocytes. Furthermore, the piglets obtained from the vitrified IVF embryos all were healthy and showed favorable growth until weaning (they were culled after weaning). Therefore, there is a strong likelihood of applying this cryopreservation technology to micromanipulated embryos, such as those that have been cloned or sperm injected.
In a number of studies [2–4, 6, 7, 33], IVP embryos, such as cloned and sperm-injected embryos, were transferred at early cleavage stages (two- to eight-cell stages), which we used as the basis for determining the appropriate conditions for cryopreservation of embryos derived from IVM oocytes. However, porcine embryos are generally more cryotolerant at later embryonic stages [34–36]. It is possible that embryos derived from IVM/IVF oocytes would show a higher viability if they were to be vitrified later than the timing employed in this study. Conversely, it is known that prolonged culture before cryopreservation also lowers the developmental potency after transfer [37]. Thus, considering the percentage of embryos that develop normally to the late stage in vitro, it is unclear at present whether cryopreserving embryos is more effective at the early cleavage stage or at a later stage. For cryopreservation of later-stage embryos, the first step must be the optimization of culture conditions.
In conclusion, this study demonstrates that porcine embryos derived from oocytes matured and fertilized in vitro can be effectively cryopreserved at early cleavage stages, and healthy piglets can be produced by the transfer of these embryos.
FOOTNOTES
1Supported by the 21st Century Green Frontier Research Project, National Institute of Agrobiological Science, Japan; the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN); and Biotechnology Venture Research and Development Base Maintenance Enterprise Project for Private Universities, Ministry of Education, Culture, Sports, Science and Technology, Japan, 2002–2006. 
Correspondence: 2Hiroshi Nagashima, Laboratory of Developmental Engineering, Department of Life Science, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama, Kawasaki, Kanagawa 214-8571, Japan. FAX: 81 44 934 7824; e-mail: hnagas{at}isc.meiji.ac.jp
Received: 2 April 2006.
First decision: 17 April 2006.
Accepted: 22 January 2007.
REFERENCES
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