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Embryo |
Laboratory of Developmental Engineering,5 Department of Life Science, School of Agriculture, Meiji University, Kawasaki 214-8571, Japan
Chiba Prefectural Animal Experimental Station,6 Chiba, 289-1113, Japan
Kato Ladies' Clinic,7 Shinjuku, Tokyo, 160-0023, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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embryo, gamete biology, developmental biology, early development, ovum
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has only fairly recently become feasible to produce progeny from frozen porcine embryos [2–5], although the highly variable cryotolerance of pig embryos over the developmental stages has limited applications for embryos at certain developmental stages [2, 6]. Porcine embryos at the perihatching stage are known to be most resistant to freezing [6, 7], and their cryopreservation by the conventional freezing method has successfully led to offspring production [2, 8]. Conventional freezing, however, has been plagued by a low survival rate in that after thawing, even perihatching-stage embryos showed poor survival, at about 30% [2].
Vitrification has improved the survival rate of perihatching-stage porcine embryos subsequent to cryopreservation (see [9] for a review). Moreover, technological improvements have led to higher survival rates in porcine morulae and early blastocysts, which are notoriously sensitive to damage by conventional freezing [10]. This technique has been further improved to achieve more stable vitrification using the open pulled straw [11] and minimum volume cooling (MVC) [12] methods such that higher embryo viability is provided [13].
In an effort to improve survival after cryopreservation, alternative approaches have been directed at increasing cryotolerance of porcine embryos. Nagashima et al. [14, 15] showed that delipation, which removes cytoplasmic lipid droplets in embryos, remarkably improves embryo viability following cryopreservation. And since then, the transfer of delipated porcine morulae and blastocysts was demonstrated to provide a practically feasible birth rate after cryopreservation [16, 17].
These advancements in cryopreservation of porcine embryos have been successful only for in vivo-derived embryos, whereas in recent pig cloning research, an in vitro-matured (IVM) oocyte is usually employed [18–22], as is also the case with transgenic pig production [23] and in vitro fertilization [24]. Because improvements in cryopreservation technology of in vitro-produced (IVP) embryos will further facilitate studies on pig cloning and the establishment of a gene bank of transgenic pigs, it is necessary to establish cryopreservation technology for embryos derived from IVM oocytes. Successful cryopreservation of IVP embryos, however, remains elusive.
These considerations led to the present study, which describes a cryopreservation method for IVP porcine embryos. As a model, we used embryos that were parthenogenetically developed from IVM oocytes. Postvitrification viability of embryos at the perihatching stage was first investigated due to their high resistance to cryopreservation, after which embryos were vitrified at the early cleavage stages with the future intent to transfer IVP embryos to the recipient's oviduct. We also report on the effect of delipation on cryotolerance of IVP porcine embryos, and on the successful vitrification of porcine IVP embryos, which were delipated by a newly developed noninvasive method.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Unless otherwise indicated chemicals were obtained from Sigma Chemical Company (St. Louis, MO).
In Vitro Maturation of Oocytes
Ovaries were collected at a local abattoir and transported to the laboratory in 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 from antral follicles (3.0–6.0 mm in diameter) of ovaries by aspiration. COCs having at least three layers of compacted cumulus cells were selected and cultured in NCSU23 medium [25] 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, and 10 IU/ml of eCG and hCG (Teikoku Zouki Co., Tokyo, Japan). They were cultured for 22 h with hormones and then 22 h without hormones in a humidified atmosphere of 5% CO2 and 95% air at 38.5°C.
Electric 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) polyvinylpyrrolidone (PVP) (Hepes-TL-PVP), then denuded of cumulus cells by gentle pipetting. Oocytes having extruded the first polar body were selected and washed twice in an activation solution consisting of 0.3 M mannitol (Nacalai Tesque Inc., Tokyo, Japan), 50 µM CaCl2, 100 µM MgCl2, and 0.01% PVA. Next, they were lined up between two wire electrodes (1.0 mm apart) of a fusion chamber overlaid with 0.2 ml of activation solution, and a single 150-V/mm DC pulse was applied for 100 µsec using an electrical pulsing machine (ET-1; Fujihira, Tokyo, Japan). Activated oocytes were finally treated with 5 µg/ml cytochalasin B (CB) for 3 h.
In Vitro Culture of Embryos
In vitro culture of IVP embryos except for those in experiment 3 was performed in 20-µl droplets of NCSU23 supplemented with 4 mg/ml BSA under paraffin oil in a plastic Petri dish in humidified air with 5% CO2 at 38.5°C. For culturing embryos that developed beyond the morula stage, 10% (v/v) fetal calf serum (FCS) was added to the medium.
For culturing the IVP embryos for the first 2 days in experiment 3, NCSU medium was modified to contain 0.17 mM sodium-pyruvate and 0.17 mM sodium-lactate instead of glucose. The gas atmosphere used in experiment 3 was 5% CO2, 5% O2, and 90% N2.
Removal of Cytoplasmic Lipid Droplets from Embryos (Delipation)
Removal of cytoplasmic lipid droplets was carried out by a method described previously [14, 15]. Briefly, to polarize lipid granules in the cytoplasm, embryos were centrifuged (12 000 x g, 23 min) at room temperature in Hepes-TL-PVP containing 7.5 µg/ml CB using a 1.5-ml microcentrifuge tube (Treff Lab, Schweiz, Switzerland). The resultant lipid layer was removed by micromanipulation using a beveled suction pipette (diameter, 28–32 µm) attached to micromanipulators (MO-108; Narishige, Tokyo, Japan) under an inverted microscope (TE300; Nikon, Tokyo, Japan).
Noninvasive delipation of embryos was also carried out. IVP morulae at 4 days after activation were treated with 4% trypsin (in PBS) for 3.5 min, so that the zona pellucidae were swollen due to slight digestion. Embryos were then washed twice by PBS + 10% FCS. Embryos with swollen zona were centrifuged in the presence of CB as described above to polarize the cytoplasmic lipid droplets in the perivitelline space. After centrifugation embryos were subjected to vitrification within 2 min.
Vitrification of Embryos
Cryopreservation of embryos was carried out by vitrification using the MVC method [12]. All solutions used during vitrification and thawing were prepared with TCM-199 containing 20 mM Hepes, 4.2 mM NaHCO3, 75 µg/ml potassium penicillin G, and 50 µg/ml streptomycin sulfate as the basal medium. Embryos were equilibrated with equilibration solution containing 7.5% (v/v) ethylene glycol (Nacalai Tesque), 7.5% (v/ v) dimethylsulfoxide (DMSO; Wako Pure Chemical Industries Co., Osaka, Japan), and 20% (v/v) calf serum (JRH Biosciences, Inc., Lenexa, KS) for 4 min followed by exposure to vitrification solution containing 15% ethylene glycol, 15% DMSO, 0.5 M sucrose (Nacalai Tesque), and 20% calf serum. Embryos were then loaded onto an MVC plate (Cryotop; Kitazato Supply, Tokyo, Japan) and immediately plunged into liquid nitrogen. The process from exposure of embryos to plunging was completed within 1 min. Embryos were thawed by immersing the MVC plate directly into thawing solution containing 1 M sucrose and 20% calf serum at 37°C for 1 min. Recovered embryos were transferred to a diluent solution containing 0.5 M sucrose and 20% calf serum and kept for 3 min, after which they were kept for 10 min in a washing solution containing 20% calf serum in order to remove cryoprotectant. All solutions except for the thawing solution, were used at room temperature.
Fixation and Staining of Embryos
Cryopreserved and control IVP embryos were fixed with aceto-alcohol (1:3) 8 days after activation. Embryos were stained with 1% aceto-orcein to determine cell number.
Experiment 1
This experiment was performed to examine survival of IVP porcine blastocysts after cryopreservation. As the cryotolerance of in vivo-derived porcine blastocysts is known to reach maximum at the perihatching stage, including the expanded blastocyst stage [6, 7], expanded blastocysts 6 days after parthenogenetic activation were subjected to vitrification.
We also tested whether survival of IVP porcine blastocysts after vitrification can be improved by delipation, which is known to be effective in rendering in vivo-derived porcine embryos cryotolerant. IVP embryos at the morula stage were delipated at 4 days after activation and further cultured for 2 days. Delipation at the morula stage was determined by our preliminary data that delipation of embryos at the expanded blastocyst stage turned out to be technically difficult. Embryos developing to expanded blastocysts were vitrified. Vitrified embryos and control IVP embryos were cultured up to Day 8 to examine their survival and cell number.
Experiment 2
This experiment was conducted to examine the possibility of cryopreserving IVP porcine embryos at an early cleavage stage, assuming that IVP porcine embryos such as transgenic or cloned embryos are transferred to recipients at an early developmental stage.
At 2 days after activation, IVP embryos at the 4-cell stage were delipated, further cultured for 15 h, and vitrified. As a control, nondelipated embryos at the 4-cell stage were also vitrified. Vitrified embryos and nonvitrified IVP embryos were cultured up to Day 8 to examine their development to the blastocyst stage.
Experiment 3
The aim of this experiment was to investigate the possibility of cryopreserving IVP embryos without using micromanipulation for delipation.
Survival of embryos vitrified after delipation by the noninvasive method (tryp/centr–morula; Fig. 1a) was compared with other embryos, including delipated morulae, morulae centrifuged without trypsin treatment (centr–morula; Fig. 1b), and intact control morulae.
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Vitrified embryos and nonvitrified IVP embryos were cultured up to Day 8 to examine their development to the blastocyst stage.
Statistical Analysis
Survival rates of embryos were compared using 
2-test. The Student t-test was used to compare mean cell numbers of embryos.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Table 1 summarizes the results of cryopreserving IVP embryos vitrified at the expanded blastocyst stage, where about 40% of the IVP blastocysts survived vitrification (Fig. 2a), although their average cell number was lower compared to that of the nonvitrified blastocysts (38.4 ± 5.1 vs. 61.9 ± 5.8, P < 0.05). Note that survival rate and embryo quality of IVP embryos was significantly improved (41.2% vs. 83.3%; 38.4 ± 5.1 vs. 51.8 ± 4.2, P < 0.05) when cryopreserved after delipation. In fact, the survival rate of vitrified delipated blastocysts was nearly the same as that of nonvitrified controls (intact blastocysts, 88.0%; delipated nonvitrified blastocysts, 92.9%). Cell number of the delipated blastocysts after vitrification was also not significantly different compared to that of nonvitrified controls.
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As shown in Figure 2b, many of the delipated blastocysts tended to herniate from the zona pellcidae in culture after thawing, indicating that decline of embryo quality was minimized when IVP blastocysts were cryopreserved after delipation.
Experiment 2
Table 2 summarizes the results of cryopreserving IVP embryos at the 4-cell stage. Most embryos vitrified without delipation degenerated within 24 h after thawing (Fig. 3a), having a very low developmental rate to blastocysts (6/62, 9.7%). By contrast, delipation of embryos prior to vitrification significantly improved post-thaw development (32/ 89, 36.0%, P < 0.05). Most embryos vitrified after delipation herniated from the zona pellucidae as shown in Figure 3b in culture after thawing.
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Experiment 3
As shown in Table 3, survival of IVP morulae vitrified after trypsin treatment and centrifugation (tryp/centr; 52/ 63, 82.5%; Fig. 4, b and c) was equal to that of the delipated morulae (46/56, 82.1%; Fig. 4e). These survival rates were as high as those of the nonvitrified control embryos (Fig. 4a).
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Centrifugation without trypsin treatment was also shown to render the IVP morulae cryotolerant (survival, 24/44, 54.5%; Fig. 4d), although their survival rate was significantly lower than that of the tryp/centr and delipated groups (P < 0.05). In contrast, only a few (3/35, 8.6%) of the intact morulae survived vitrification (Fig. 4f).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The porcine embryo is intrinsically quite sensitive to hypothermic exposure such that cryopreservation of IVP embryos has been thus far unobtainable. The key to their successful cryopreservation as shown here is the use of the MVC method (i.e., vitrification of embryos using a trace amount of vitrification solution allows for a rapid drop in temperature in the vitrification solution and stable vitrification in the presence of a low concentration of cryoprotective agent [13, 31, 32]). In our preliminary study, we confirmed that porcine IVP blastocysts could survive cryopreservation better with the MVC method than the conventional vitrification methods using high concentrations of ethylene glycol, DMSO, or propylene glycol (unpublished data). Furthermore, the MVC method is reported by other workers to provide higher survival rates of embryos compared to conventional vitrification in cattle [31], humans [12], pigs [33], and rabbits [34].
In addition, we used embryos at the expanded blastocyst stage, which also contributed to success (i.e., embryos at the perihatching stage including the expanded blastocyst stage are most cryotolerant [6, 7]). In a preliminary study, we confirmed that IVP embryos at earlier stages had lower viability following cryopreservation (unpublished data). On the other hand, because IVP embryos at the expanded blastocyst stage require longer culture, its effect on cryotolerance becomes another concern. It has been reported that some components in media, especially fetal calf serum, remarkably disturb cryotolerance of bovine IVP embryos [35, 36], thus, based on this, it is important to develop culture conditions suitable for cryopreservation of porcine IVP embryos.
Our results show that use of the MVC method in conjunction with delipation (removal of cytoplasmic lipid droplets) provides for effective cryopreservation of porcine IVP embryos. It was found that delipation of expanded blastocyst-stage embryos significantly improved the viability after cryopreservation (P < 0.05). Although the MVC method alone did not produce viable 4-cell and morula-stage embryos after cryopreservation, delipation increased the survival rate to 36.0% and 82.1%, respectively. These data suggest that delipation is effective not only for improving post-thaw survival of embryos with some cryotolerance, but for cryopreservation of highly cryosensitive embryos as well. In general, the presence of lipid droplets in the cytoplasm is considered an important aspect for embryonic development in terms of energy metabolism [37], yet delipation of porcine IVP embryos does not appear to pose a problem because delipated embryos have produced progeny [15].
We previously demonstrated that cryopreservation of delipated morulae is feasible using in vivo-derived embryos [16, 38]. Results of the present study further demonstrated that cryopreservation of delipated IVP morulae is also successful. In particular, development of a cryopreservation protocol for IVP morulae in conjunction with the noninvasive delipation method should have conspicuous practical value, because use of micromanipulation for delipation can be avoided.
Although our results demonstrated that conjunction of the MVC method with delipation dramatically increases postvitrification survival of IVP embryos, this protocol requires micromanipulation of embryos. In addition, our data indicated that embryos delipated by micromanipulation tended to have fewer cell numbers at the blastocyst stage, and this could be a drawback of the protocol, including delipation by the invasive method. In contrast, the embryos that had been vitrified after delipation by the noninvasive method had equal cell numbers as the intact control embryos.
By centrifuging IVP morulae with swollen zona, cytoplasmic lipid droplets could be well separated from the blastomeres (Fig. 1a). Therefore, these embryos were believed to be rendered as cryotolerant as the embryos delipated using micromanipulation. This was a notable difference from centrifuging embryos with intact zona pellucida (Fig. 1b), in which separation of the lipid droplets from the blastomeres tended to be less complete, hence embryos were not rendered highly cryotolerant [38].
When an embryo with an intact zona was centrifuged in the presence of CB, the mass of the cytoplasmic lipid droplets that were polarized in the perivitelline space tended to remain connected with blastomeres with string-like structures [38]. This bridge-like structure may cause redistribution of the lipid droplets into some blastomeres during the process of vitrification and thawing [38]. In contrast, when an embryo with swollen zona was centrifuged, its larger perivitelline space might allow complete cutoff of the bridges, so that separation of lipid droplets and blastomeres could be completed.
Cloned embryos generally have very low developmental potency, and hundreds of cloned embryos are required to obtain a single offspring [18, 22]. In addition, IVM oocytes are typically used to produce cloned embryos [18–22]. Taken together, this indicates that if cryopreservation technology for IVM-derived cloned embryos is established, efficient production of cloned pigs might be possible by storing embryos necessary for embryo transfer. Cloned embryos are in general transferred at the early cleavage stages [20, 22, 39], with cryopreservation of IVP embryos at the 4-cell stage being demonstrated here such that application to embryo cloning appears feasible. On the other hand, effective pig cloning has been achieved by transfer of cloned embryos developed to the blastocyst stage in a temporal recipient [40]. This indicates, together with the data of our present study, an alternative path for cryopreservation of cloned pig embryos at the later developmental stages.
In conclusion, we have confirmed that porcine IVP embryos derived from IVM oocytes can be effectively cryopreserved using the MVC method in conjunction with delipation. To our knowledge, this is the first report on the successful cryopreservation of IVP embryos in the pig. It is expected that our study will open the doors for cryopreservation of highly manipulated IVP embryos such as cloned embryos.
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FOOTNOTES |
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2 Correspondence: Hiroshi Nagashima, Laboratory of Developmental Engineering, Department of Life Science, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama, Kawasaki 214-8571, Japan. FAX: 81 44 934 7824; hnagas{at}isc.meiji.ac.jp
3 Current address: Laboratory of Anatomy and Embryology, Institute of Basic Medical Science, Tsukuba University, 1-1-1, Tennoudai, Tsukuba 305-8577, Japan
4 Current address: National Institute of Genetics, 1111 Yata, Mishima, Shizuoka, 411-8540, Japan
Received: 13 December 2003.
First decision: 26 December 2003.
Accepted: 16 March 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 2002 295:1089-1092This article has been cited by other articles:
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  T. Somfai, M. Ozawa, J. Noguchi, H. Kaneko, M. Nakai, N. Maedomari, J. Ito, N. Kashiwazaki, T. Nagai, and K. Kikuchi Live Piglets Derived from In Vitro-Produced Zygotes Vitrified at the Pronuclear Stage Biol Reprod, January 1, 2009; 80(1): 42 - 49. [Abstract] [Full Text] [PDF]
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 Y Du, C S Pribenszky, M Molnar, X Zhang, H Yang, M Kuwayama, A M Pedersen, K Villemoes, L Bolund, and G Vajta High hydrostatic pressure: a new way to improve in vitro developmental competence of porcine matured oocytes after vitrification Reproduction, January 1, 2008; 135(1): 13 - 17. [Abstract] [Full Text] [PDF]
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H. Nagashima, K. Hiruma, H. Saito, R. Tomii, S. Ueno, N. Nakayama, H. Matsunari, and M. Kurome Production of Live Piglets Following Cryopreservation of Embryos Derived from In Vitro-Matured Oocytes Biol Reprod, May 1, 2007; 76(5): 900 - 905. [Abstract] [Full Text] [PDF]
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