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ARTICLES |
a Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon 305-600, Korea
b National Livestock Research Institute, Chonan 330-800,
c Suwon 441-350, Korea
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IVF/ART
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several combined chemical treatments, such as Ca-ionophore/6-DMAP [6, 15], ionomycin/6-DMAP [8], or cycloheximide/cytochalasin B [16], have been used to activate reconstructed bovine oocytes injected with somatic cells. In the pig, some of reconstructed eggs activated by an exposure to thimerosal/dithiothreitol could develop to blastocysts [12]. When porcine NT eggs using fibroblasts were activated and fused simultaneously by electrical stimulation, a few embryos developed to blastocyst stage [11]. The electrical stimulation was also effective in parthenogenetic activation of pig oocytes [17, 18].
In NT using embryonic or somatic cells, a part of the oocyte cytoplasm is removed during the enucleation procedure. This reduced cytoplasmic volume could decrease the developmental potential and cell number of NT embryos in cattle [19]. Bovine NT embryos injected with fibroblasts showed a lower developmental rate (12%) to the blastocyst stage [6] than those injected with mural or cumulus granulosa cells (approximately 50%) [7, 8]. This difference reflects that cell types as donor nuclei may have different developmental potential in NT embryos. However, little information has been gained about the developmental competence of NT porcine embryos.
The aim of this study was to determine the optimal activation conditions for porcine NT eggs using somatic cells. The developmental abilities of porcine NT eggs induced by different electrical voltages or activation times after electrofusion were examined. Thereafter, developmental competence of NT embryos was compared to parthenogenetic and in vitro-fertilized (IVF) embryos.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Unless otherwise mentioned, all chemicals used in this study were purchased from Sigma Chemical Co. (St. Louis, MO).
Prepubertal porcine ovaries were collected from a local slaughterhouse and transported to the laboratory at 25–30°C in 0.9% (w/v) saline supplemented with 75 µg/ml potassium penicillin G and 50 µg/ml streptomycin sulfate. Cumulus-oocyte complexes (COC) were obtained from follicles with a diameter of 3 to 6 mm using an 18-gauge needle connected to a 10-ml disposable syringe [20]. The COC were washed three times with Tyrode's lactate (TL)-Hepes medium [21]. Approximately 50 COC were cultured in 500 µl of maturation medium in each well of a 4-well multidish (Nunc, Roskilde, Denmark) at 39°C, 5% CO2 in air. After 20 to 22 h of culture, oocytes were washed three times with TL-Hepes medium and further cultured in the maturation medium without hormone supplements (eCG and hCG) for 22 h. The medium was covered with warm paraffin oil (light mineral oil) and equilibrated at 39°C, 5% CO2 in air for at least 2 h prior to use. The medium used for oocyte maturation was BSA-free North Carolina State University (NCSU) 23 medium [22] supplemented with 10% (v/v) porcine follicular fluid, 0.57 mM cysteine, 25 µg/ml gentamycin, 10 ng/ml epidermal growth factor, 10 IU/ml eCG, and 10 IU/ml hCG. Porcine follicular fluid was collected from follicles of 3–6 mm in diameter, centrifuged at 1600 x g for 30 min at 4°C, filtered through 1.2-µm syringe filters (Sartorius AG, Goettingen, Germany), and stored in aliquots at -20°C until use.
In Vitro Fertilization
The basic medium used for IVF was essentially the same as described by Abeydeera and Day [23]. This IVF medium, designated modified Tris-buffered medium (mTBM), consists of 113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl2·2H2O, 20 mM Tris (crystallized free base; Fisher Scientific, Fair Lawn, NJ), 11 mM glucose, 5 mM sodium pyruvate, and no antibiotics. Fresh semen was kindly supplied from Darby Pig AI Center (Anseong, Korea) once a week and kept at 17°C for 5 days. Semen was washed three times by centrifugation with Dulbecco's PBS (DPBS) supplemented with 1 mg/ml BSA (Fraction V), 100 µg/ml penicillin, and 75 µg/ml streptomycin. At the end of washing, the spermatozoa were resuspended in Tris-buffered medium (pH 7.8). After the completion of IVM, cumulus cells of oocytes were removed by treatment with 0.1% (w/v) hyaluronidase in NCSU 23 medium. The denuded oocytes were washed three times with the mTBM supplemented with 2.5 mM caffeine and 4 mg/ml BSA and placed into 50 µl of mTBM under paraffin oil. Fifty microliters of diluted spermatozoa was added to 50 µl of the fertilization medium containing oocytes to give a final sperm concentration of 5 x 105 cells/ml. The oocytes were coincubated with spermatozoa for 6 h at 39°C in an atmosphere of 5% CO2 in air.
Parthenogenetic Activation of Oocytes
Electrical activation of pig oocytes was carried out as described previously [18]. Briefly, cumulus-free oocytes were equilibrated for 1 min in 0.3 M mannitol solution supplemented with 0.5 mM Hepes, 0.01% BSA (fatty acid free), 0.01 mM CaCl2, and 0.01 mM MgCl2, and transferred to a chamber consisting of two electrodes 1 mm apart that was overlaid with activation solution. Oocytes were exposed to a single AC pulse of 3 V/mm for 5 µsec followed by a single DC pulse of 180 V/mm for 30 µsec using a BTX Electro-cell Manipulator 2001 (BTX, San Diego, CA).
Preparation of Fetal Fibroblasts
Experiments were conducted according to The Animal Care and Use Committee guidelines of the National Livestock Research Institute of Korea. Fetal fibroblasts were isolated from fetuses at Day 40 of gestation in Korean native swine (Sus scrofa domesticus L.). Fetal fibroblasts were prepared as described previously [24]. The same procedure for the isolation of porcine fetal fibroblasts has been used to produce the embryos from somatic cell NT [12, 25]. Briefly, porcine fetuses were washed three times with Ca2+- and Mg2+-free PBS (DPBS-). The heads of fetuses were removed using iris scissors, and soft tissues such as liver and intestine were discarded by scooping out with two watchmaker's forceps. After washing twice with DPBS-, the carcass was minced with a surgical blade on a 100-mm culture dish. The minced tissues were incubated in 10 ml of 0.25% (w/v) trypsin/3.65 mM EDTA solution at 39°C for 30 min. Trypsin activity was inhibited by adding an equal volume of cell culture medium supplemented with 15% fetal bovine serum (FBS; Gibco, Life Technologies Inc., Grand Island, NY). The cell culture medium was composed of Dulbecco's modified Eagle's medium (DMEM; Gibco), 15% FBS, 1000 units of penicillin, and 1000 µg/ml of streptomycin (Gibco) [24]. After vigorous pipetting, the supernatant was centrifuged at 150 x g for 5 min. The cells were suspended, adjusted to a final concentration of 2 x 106 cells/ml, and then cultured in 10 ml of the culture medium into 175-cm2 tissue culture flask (Nunc) at 39°C, 5% CO2 in air. The fetal fibroblasts were passaged three times before used as a source of donor nuclei.
Enucleation of Porcine Oocytes
After 44 to 46 h of IVM, the oocytes were transferred to 500 µl of TL-Hepes supplemented with 0.1% hyaluronidase and free of cumulus cells by vortexing for 3 min. The zonae pellucidae of oocytes were partially dissected using a fine glass needle as described by Tsunoda et al. [26]. Oocyte manipulation, including enucleation and cell injection, was performed by using a micromanipulator equipped with an inverted microscope (Leitz, Ernst Leitz Wetzlar GmbH, Germany). The medium used for oocyte manipulation was DPBS- containing 7.5 µg/ml cytochalasin B. The first polar body and partial cytoplasm presumptively containing metaphase II chromosomes were removed together by using a micropipette with an inner diameter of 20 µm. After the enucleation, oocytes were stained with 5 µM bisbenzimide (Hoechst 33342) for 5 min and observed on an epifluorescent microscope (Olympus, Tokyo, Japan). Oocytes having DNA materials were excluded in subsequent experiments. The proportion of oocytes completely enucleated was approximately 90.3% (280/310) in this study.
Cell Injection, Electrofusion, and Activation
Single fibroblasts were individually transferred into enucleated oocytes. Small cells with a smooth surface were used as donor nuclei. Reconstructed embryos were equilibrated in a 50-µl drop of cell fusion medium for 10 to 20 sec and transferred to a fusion chamber with two electrodes 1 mm apart overlaid with cell fusion medium. The cell fusion medium consisted of 0.3 M mannitol, 0.5 mM Hepes, 0.01% BSA, 0.1 mM CaCl2, and 0.1 mM MgCl2. Cell fusion was induced with a single DC pulse of 160 V/mm for 30 µsec. After the electrofusion, embryos were activated using a 5-sec pulse of 3 V/mm AC followed by a 30-µsec pulse of 120 V/mm DC.
Experimental Designs and In Vitro Culture
In experiment 1, the effect of activation voltage on in vitro development of NT embryos to the blastocyst stage was examined. At 2 to 3 h after electrofusion, reconstructed embryos were activated at a pulse of 3 V/mm AC for 5 sec followed by a pulse of 120 or 150 V/mm DC for 30 µsec.
In experiment 2, whether the timing of activation after electrofusion affects the developmental ability of NT embryos was investigated. Approximately one-third of fused eggs were activated at a pulse of 3 V/mm AC for 5 sec followed by a single pulse of 120 V/mm DC for 30 µsec at different times (2, 4, or 6 h) after electrofusion.
In experiment 3, the developmental competence of NT embryos with fetal fibroblasts was compared to parthenogenetic and IVF-derived porcine embryos.
In all experiments, the activated eggs were cultured in 50-µl drops of NCSU 23 medium supplemented with 4 mg/ml BSA at 39°C, 5% CO2 in air. After 72 h of culture, cleavage-stage embryos were selected. Forty to fifty cleaved embryos were cultured together in a 50-µl drop of NCSU 23 medium supplemented with 10% FBS at 39°C, 5% CO2 in air for 3 days. After 6 days of culture, blastocyst formation was observed, and cell numbers of blastocysts were counted on a fluorescent microscope following Hoechst 33342 (2.5 µg/ml) staining.
Cell Number of NT Embryos
All blastocysts developed from NT embryos using somatic cells were counted for nuclei after Hoechst staining. Briefly, the embryos were fixed with 1% formaldehyde in PBS for 10 min at room temperature and then placed in a drop of mounting medium on a slide. The mounting medium consisted of 25% (v/v) glycerol in PBS containing 2.5 mg/ml sodium azide and 2.5 µg/ml Hoechst 33342. A coverslip was placed on the top of the embryos and the edge of the slide was sealed with fingernail polish. The nuclei numbers were counted on an epifluorescent microscope (Olympus, Japan).
Statistical Analysis
Four or five replicates for each experiment were conducted. All percentage data were subjected to arcsine transformation. The data for cleavage and developmental rates were expressed as mean ± SD. All percentage data and data sets obtained from the study were presented as (mean ± SD, data set) throughout the text. Developmental rate and cell numbers of NT embryos between experimental groups were analyzed by Student's t-test and Duncan's multiple range test using the General Linear Models procedure in the Statistical Analysis System. Probability of P < 0.05 was considered to be significant statistically.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To evaluate if electrical fields affect in vitro development of NT embryos, the fused embryos were activated at different voltages (120 and 150 V/mm DC) after electrofusion and then cultured in vitro for 6 days. As shown in Table 1, there was no difference in the cleavage rate between groups. The rate of blastocyst development (11.6 ± 1.6%, 14/121) of NT embryos in a field strength of 120 V/mm was significantly high as compared to 150 V/mm group (6.5 ± 2.3%, 8/121) (P < 0.05). However, mean cell numbers of blastocysts were similar between the two groups (29.5 ± 11.3 and 27.6 ± 10.5, respectively) (P > 0.05). For subsequent experiments, the activation was performed with a single 120-V/mm DC pulse.
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Effect of Activation of NT Embryos at Different Times after Electrofusion (Experiment 2)
This experiment was carried out to determine the optimal activation timing after fusion (Table 2). The cleavage rates of NT embryos in 2-h and 4-h groups were higher than those of the 6-h group. A higher proportion of NT embryos (11.6 ± 2.9%, 20/187) activated 2 h after electrofusion developed to the blastocyst stage, as compared to the 4-h (6.6 ± 2.3%, 11/190) and 6-h (8.1 ± 3.3%, 14/184) groups. Mean cell numbers of NT embryos in 2-, 4-, and 6-h groups were 30.4 ± 10.4, 24.6 ± 10.1, and 16.5 ± 7.4, respectively. A significant difference was observed between the 2- and 6-h groups (P < 0.05). These results suggest that activation timing after electrofusion can affect developmental potential of porcine NT eggs.
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Comparison of Developmental Ability of Porcine NT Embryos to IVF-Derived or Parthenogenetic Embryos (Experiment 3)
The NT embryos using fibroblasts showed a lower developmental rate (9.4 ± 0.9%, 20/289) to the blastocyst stage than IVF-derived (21.4 ± 1.9%, 43/201) or parthenogenetic embryos (22.4 ± 7.2%, 43/189) (P < 0.01), although there was no difference in the cleavage rate of the embryos among experimental groups (Table 3). After 6 days of culture, most blastocysts (16/20) from NT embryo were hatched (Fig. 1), although the mean number of nuclei was smaller than that of IVF-derived blastocysts (Fig. 2). The reason for early hatching of NT blastocysts seems to be due to the partial dissection of the zona pellucida during the enucleation procedure. The mean cell number of NT embryos (28.9 ± 11.5, ranging from 14 to 61, n = 18) was smaller than that of IVF-derived blastocysts (36.2 ± 9.7, ranging from 18 to 67, n = 22) (P < 0.05), while similar to that of parthenogenetic embryos (29.9 ± 12.1, ranging from 13 to 50, n = 24) (Table 3). These results represent that the porcine NT embryos using fibroblasts could develop to blastocysts in vitro, although having lower developmental competence when compared to IVF-derived embryos. However, it remains to be examined whether the porcine NT embryos using nuclei of somatic cells can normally develop to term.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In cattle, NT embryos activated at 4 to 8 h after fusion of donor cells with MII cytoplasts showed improved embryonic development [33, 34]. A prolonged exposure of transferred nuclei to oocyte cytoplasmic factors facilitated nuclear remodeling and reprogramming [35, 36]. Hence, several hours may be required to facilitate nuclear remodeling or reprogramming of the fused embryos prior to activation, although reliable evidence for this has not been reported. In the present study the porcine NT embryos activated at 2 h after fusion showed improved development as compared with the longer periods (4 and 6 h) (Table 2). This difference may be due to the timing of genomic activation between species. Activation of the embryonic genome occurs at the 4-cell stage in the pig, while during the 8- to 16-cell stage in sheep and cattle [37]. In subsequent experiments, rates of blastocyst formation in reconstructed embryos were 2.1 (2/97), 3.2 (2/85), and 10.4% (11/106), respectively, when they were activated at 0, 30 min, and 1 h after electrofusion. Thus, this study demonstrated that the electrical activation of reconstructed porcine oocytes is most effective 1 to 2 h after electrofusion. These results also suggest that the time required for the remodeling or reprogramming of donor nuclei in recipient cytoplasts after fusion may differ among species.
Approximately 70% of reconstructed embryos using pig fibroblasts failed to divide when activated with thimerosal/dithiothreitol treatment [12]. As shown in Table 3, the cleavage rate of porcine NT eggs activated by a single electric stimulus was similar to those of parthenogenetic and IVF-derived embryos. This result indicates that the activation procedure applied in our experiments was appropriate. As shown in Table 3, porcine NT eggs with somatic cells have a lower blastocyst development than parthenogenetic or IVF-derived embryos. Porcine NT embryos reconstituted with 8- to 16-cell nuclei have developed to the morula stage [38], and recently, it has been reported that a few blastocysts could develop from porcine NT eggs using fibroblasts [11, 12]. Thus, the developmental competence of porcine NT embryos using nuclei of somatic cells is still low, although improved in this study. Many factors involved in the NT procedure could result in the low developmental competence of porcine NT embryos. First, the lower developmental potential of porcine NT eggs may come from the inappropriate cell cycle of donor nuclei, as they could have received cells that were not in the G0 or G1 phase of the cell cycle [6–8, 32] and a consequence of inadequate nuclear reprogramming from using nonquiescent cells. Second, it may be essential to control the ploidy of the reconstructed embryo after the activation stimulus is applied. In order to encourage normal development, bovine NT embryos have been incubated in medium supplemented with 6-DMAP following exposure to ionomycin or ionophore [6, 15, 26]. It has been known that 6-DMAP, a protein kinase inhibitor, may inhibit the phosphorylation necessary for the spindle apparatus [39] and therefore prevent micronuclei formation known to occur when fusion precedes activation [40]. In this study the porcine NT eggs were activated by only a single electric stimulation without any treatment. The lack of any treatment to prevent micronuclei formation may explain in part the poor development. Third, inadequate recipient cytoplasts may result in low development of NT embryos. If recipient cytoplasts were activated, and cytoplasmic kinase (maturation promoting factor) activity declined prior to transfer of the donor nucleus, no nuclear envelope breakdown and/or premature chromosome condensation occur. Thereafter, DNA synthesis occurs in relation to the cell cycle stage of the nucleus at the time of transfer [41]. In vitro-matured porcine oocytes were used as sources of recipient cytoplasts in this study. Therefore, synchronization of recipient oocytes prior to the enucleation may be an important factor in the subsequent development of NT embryos.
Nuclear transfer blastocysts had a smaller cell number than IVF-derived blastocysts (Table 3). This difference might arise from the reduced cytoplasmic volume removed during the enucleation procedure [19]. The low pregnancy rates and high abortion rates in NT embryos may be attributable simply to reduced cell numbers [42]. Tao et al. [12] reported that two porcine NT blastocysts using nuclei of fibroblasts contained 14 and 11 nuclei, respectively. Our data showed the increased nuclei number of NT blastocysts, although numbers were still small compared to IVF-derived blastocysts (Fig. 2). Even for IVF-derived porcine embryos, blastocysts that developed in vitro had lower cell numbers than the embryos grown in vivo [43]. Culture of NT embryos in vivo rather than in vitro may result in blastocyst-stage embryos that have at least twice the number of nuclei [44]. Further studies should be carried out to examine the developmental potential of porcine NT embryos using somatic cells at term.
It has been reported that using the donor nucleus in the G1 or G0 phase (pre-S phase) of the cell cycle is beneficial for NT [45, 46]. However, the phase of the cell cycle resulting in better development after NT is still controversial. Wilmut et al. [3] suggested that the donor cell for NT needs to be in the G0 phase of the cell cycle, a quiescent state. Nuclei of serum-starved fibroblasts supported the development of reconstructed embryos to the blastocyst stage significantly better than those of nonstarved fibroblasts [16]. Alternatively, cycling cells could be successfully used for NT in cattle [6]. In the present study, porcine fibroblasts used as donor nuclei were normally cultured until confluent without serum starvation. The proportion of G1/G0 phases of porcine fetal fibroblasts at confluency was as high as the cells serum-starved for 5 or 10 days [24].
The results from the present study suggest that a single electrical stimulation 1 to 2 h after electrofusion was sufficient to activate NT oocytes in pigs and that fibroblasts used as donor nuclei support the development of porcine NT embryos to the blastocyst stage in vitro. Development of somatic cell NT procedures in the pig will be useful for breeding high-valued pigs and for producing transgenic pigs for xenotransplantation or to serve as disease models. However, the efficacy of the techniques in the production of NT embryos in pigs remains to be improved in future studies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported by grants (HS2705 and HS2550) of Ministry of Science and Technology. 
2 Correspondence: Yong-Mahn Han, Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yusong, Taejon 305-600, Korea. FAX: 82 42 860 4608; ymhan{at}kribb4680.kribb.re.kr
Accepted: May 8, 2000.
Received: December 16, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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