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Regular Article |
a Department of Animal Sciences, University of Missouri-Columbia, Columbia, Missouri 65211
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, early development, embryo, gene regulation, ovum
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The medium used for maturation of oocytes was tissue culture medium (TCM) 199 (no. 31100-035; Gibco, Grand Island, NY) supplemented with 0.1% (w/v) polyvinyl alcohol, 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 0.5 µg/ml of LH (L-5269; Sigma Chemical Co, St. Louis, MO), 0.5 µg/ml of FSH (F-2293; Sigma), 10 ng/ml of epidermal growth factor (E-4127; Sigma), 75 µg/ml of penicillin G, and 50 µg/ml of streptomycin.
The medium used for enucleation was TCM 199 supplemented with 0.3% BSA and 7.5 µg/ml of CB, and the medium for injection was the same medium (with or without CB). The medium used for activation consisted of 0.3 M mannitol, 1.0 mM CaCl2·H2O, 0.1 mM MgCl2·6H2O, and 0.5 mM HEPES.
The medium used for the culture of reconstructed embryos was North Carolina State University-23 medium [11] supplemented with 0.4% BSA.
Collection and Culture of Cumulus-Oocyte Complexes
Ovaries were collected from prepubertal gilts at a local abattoir and transported to the laboratory in 0.9% NaCl solution at 35–39°C. Cumulus-oocyte complexes (COCs) were aspirated from 2- to 6-mm diameter antral follicles with an 18-gauge needle fixed to a 10-ml disposable syringe. The COCs were washed three times in maturation medium, and 50–60 COCs were transferred to 500 µl of the same medium that had been covered with mineral oil in a four-well multidish (Nunc, Roskilde, Denmark) and equilibrated at 39°C in an atmosphere of 5% CO2 in air overnight.
Preparation of Fetal Fibroblasts
A porcine fetus was obtained from pregnant gilt at Day 35 after insemination, and the tissue was cut into small pieces with fine scissors. The cells were incubated for 30 min at 37°C in PBS containing 0.05% trypsin and 0.5 mM EDTA, and this suspension was centrifuged [6]. The cell pellet was resuspended and cultured in Dulbecco modified Eagle medium supplemented with 75 µg/ml of penicillin G, 50 µg/ml of streptomycin, and 15% (v:v) fetal calf serum. The cells were passaged up to seven times.
EGFP Gene Infection into Fetal Fibroblasts
To generate transgenic cells, a replication-defective vector based on Moloney murine leukemia virus, pseudotyped with the envelope glycoprotein of vesicular stomatitis virus (VSV-G), was used. Retroviral vector pseudotyped with VSV-G was carrying an EGFP gene under the control of the CMV promoter (LNCE-[VSV-G]), which was kindly provided by Dr. A.W.S. Chan [12]. The LNCE had long terminal repeat (L), neomycin-resistant gene (N), cytomeyalovirus (CMV) promoter (C), and EGFP gene (E). A 0.75-kilobase fragment containing the entire coding region of EGFP gene was recovered by HpaI and HindIII digestion of the EGFP expression vector, pEGFP-N1 (Clontech Laboratories, Inc., Palo Alto, CA). The EGFP gene fragment was inserted into the HpaI and HindIII sites of the multiple cloning site in the retrovirus expression vector, pLNCX (Clontech). The EGFP gene was regulated by a CMV promoter; the final vector was named pLNC-EGFP [12]. The pLNC-EGFP was stably transduced into a 293 GP packaging cell line. The EGFP-expressing cell line was sorted by flow cytometry and selected by using neomycin (G-418). The packaging cell was infected with VSV-G. The supernatant was collected and concentrated by ultracentrifugation [12]. The viral titer was determined, and the aliquoted solution was stored at -80°C.
The cells were infected with the retroviral vector by the following procedures: Polybrene (0.1%) was diluted 1:30 (v/v) with 0.1x HBS medium (22.9 mM Hepes, 140.3 mM NaCl, and 0.7 mM NaH2PO4·H2O). Three hundred microliters of the diluted medium were added to 4 µl of vector solution (108 cfu/ml). The solution was diluted in 5 ml of culture medium and incubated overnight. The G-418 selection was started the following day and continued for 13 days, and then the cells were frozen. Cells were a pool of infected cells and not from a single colony. Cells were thawed, cultured, and then serum-starved (0.5% serum) for 3–5 days before NT (Fig. 1).
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Micromanipulation
After 42–44 h of culture, oocytes were freed from cumulus cells by vigorous vortexing for 4 min in TL-Hepes [11] supplemented with 0.1% polyvinyl alcohol and 0.1% hyaluronidase. Cumulus-free (denuded) oocytes were enucleated by aspirating the first polar body and adjacent cytoplasm in enucleation medium with a glass pipette (diameter, 30 µm). A single donor cell was placed in the perivitelline space of the oocyte to contact the oocyte membrane.
Fusion/Activation of Oocytes
Injected oocytes were placed between 0.2-mm-diameter platinum electrodes 1 mm apart in activation medium. Fusion/activation was induced with DC pulses on a BTX Elector-Cell Manipulator 200 (BTX, San Diego, CA) according to the experimental design.
Culture of Embryos
After FA, 20–30 reconstructed embryos were transferred to a 50-µl drop of culture medium covered with mineral oil in a 35-mm dish, and the dishes were held in 5% CO2 in air at 39°C. Nonmanipulated oocytes were electrically activated and cultured as controls. Some reconstructed embryos were stained with 5 µg/ml of bisbenzimide (Hoechst 33342) to identify nuclei by using an epifluorescent microscope (Nikon, Melville, NY). After 6 days of culture, all embryos were stained with Hoechst 33342 to determine the number of nuclei by using an epifluorescent microscope, and embryos with two or more nuclei were determined to have cleaved. For detection of EGFP expression, embryos were examined on an epifluorescent microscope using a standard fluorescein isothiocyanate filter set.
Experimental Design
In experiment 1, we compared two different FA conditions: two successive DC pulses of 1.2 kV/cm for 30 µsec (group A), or one DC pulse of 1.6 kV/cm for 30 µsec followed in 30 min by one DC pulse of 1.2 kV/cm for 30 µsec (group B). Parthenogenetic controls were created by using the group A parameter. The reconstructed embryos were cultured for 6 days, examined, and stained to count the number of nuclei.
In experiment 2, we compared two successive DC pulses of 1.2 kV/cm for 30 µsec (group C) versus two successive DC pulses of 1.3 kV/cm for 30 µsec (group D). For two parthenogenetic controls, the same pulses as those given to group C or D were supplied, respectively. The reconstructed embryos were cultured for 6 days.
In experiment 3, we examined whether the presence of CB during donor cell injection affects the development of NT embryos. Cells were placed in the perivitelline space of enucleated oocytes in injection medium with CB (+CB) or without CB (-CB). The NT and parthenogenetic controls were created by using the group A parameter of experiment 1. The reconstructed embryos were cultured for 6 days.
Statistical Analysis
Data were analyzed by analysis of variance and Duncan multiple-range test by using the general linear models in the Statistical Analysis System software to determine treatment differences. All percentage data were subjected to arcsine transformation before statistical analysis. Data are expressed as the mean ± SEM. A probability of P < 0.05 was considered to be statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In experiment 1, the fusion rate in group A (68.4% ± 3.9%) was higher (P < 0.05) than in group B (59.4% ± 2.3%). The rates of cleavage (50.1% ± 4.6% to 62.8% ± 5.5%) were not different among control and treatment groups. However, the rate of embryos developing to the blastocyst stage in the control (18.1% ± 3.1%) was higher (P < 0.05) than in treatments (5.9% ± 1.7% and 4.9% ± 2.5%) (Table 1).
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In experiment 2, the fusion rate in group D (70.6% ± 4.2%) was higher (P < 0.05) than in group C (58.9% ± 2.7%). The cleavage rates were not different among control and treatment groups (58.1% ± 8.1% to 73.6% ± 6.0%). However, the rate of embryos developing to the blastocyst stage in group D (3.5% ± 1.7%) was lower (P < 0.05) than in controls and group C (11.4% ± 2.0% to 16.4% ± 1.1%) (Table 2).
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In experiment 3, the fusion rate of oocytes of the +CB group (78.4% ± 1.4%) was higher (P < 0.05) than in the -CB group (70.9% ± 0.2%). The cleavage rate in the control group (85.5% ± 4.9%) was higher (P < 0.05) than in the treatment groups (61.6% ± 2.7% and 63.9% ± 4.3% in +CB and -CB, respectively). However, the rates of embryos developing to the blastocyst stage (8.1% ± 2.5% to 19.1% ± 6.0%) and the mean cell number of blastocysts (29.4 ± 5.2 to 45.7 ± 6.4) were not different among control and treatment groups, respectively (Table 3).
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Figure 2 shows the EGFP expression in several stages of NT embryos. Before fusion, EFGP was observed only in the donor cell. However, EGFP was observed from 1 h postfusion to the blastocyst stage in all embryos. Most fused embryos had pronuclei at 18 h (fusion time was considered to be 0 h) (Fig. 2C'). Cleavage (Fig. 2D) was observed at 26 h, and embryos had developed to the 4-cell stage by 50 h (Fig. 2E) and to the 8-cell stage by 74 h (Fig. 2F). Compaction and blastocoele formation (Fig. 2G) occurred between the 8- and 16-cell stage (98 h), and a hatching blastocyst (Figs. 2H and 3A) was observed at 146 h.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The blastocyst formation rate was 8%, and the mean cell number of blastocysts was 34–46 (range, 11–100) (Table 3). Blastocyst formation and the cell number of blastocysts were examined at Day 6 of culture. Betthauser et al. [9], who reported the first piglets produced from an in vitro maturation system, showed 7% blastocyst formation and a mean cell number of 66 (range, 16–125) when cultured for 7 days. Because those authors cultured embryos one more day compared to our system, the results cannot be compared directly. However, assuming a cleavage division per day, our results are quite comparable. Koo et al. [4] reported 10% blastocysts and a mean cell number of 30 (range, 23–65) when cultured for 6 days. Both groups applied activation stimuli after fusion. However, the developmental potential [4, 9] to the blastocyst stage is similar to our simultaneous FA system. This indicates that their activation stimuli [4, 9] might not be necessary, or that the other stimuli might be necessary for normal activation if fusion stimuli are not sufficient to activate NT embryos. Both groups used a single pulse of 1.6 kV/cm [4] or 1.9 kV/cm [9] for fusion. These fusion pulses are higher than ours (1.2 kV/cm), but we supplied two pulses. However, direct comparison is difficult because of several differences in the fusion and culture system.
Cytochalasin B is commonly used during micromanipulation of oocytes in vitro in several species, including Sus scrofa (pig). However, when donor cells were placed in CB-containing medium, the cell membranes blebed out and were broken more easily when aspirated into the pipette than when placed in non-CB-containing medium (Fig. 1, C and D). Therefore, the population of smooth surface cells from which to select is reduced in CB-containing medium. Nuclei of smooth-surface cells are more capable of early morphological reprogramming of nuclei (nuclear envelope breakdown, chromosome condensation, and pronuclear formation) than nuclei of rough-surface cells [1]. It is easier to find smooth cells in non-CB-containing medium than in CB-containing medium. Moreover, the in vitro developmental potential is not different in either medium (Table 3). Therefore, CB is not necessary during donor cell injection.
To our knowledge, this is the first report in which the developmental process of transgenic NT embryos is recorded from fusion to the blastocyst stage. Uhm et al. [3] showed some 2- and 4-cell stage and blastocyst stage embryos, but other stages were not described. Koo et al. [5] showed just the blastocyst stage. In the present study, the time of fusion was considered to be 0 h, and we recorded embryo development every day (Fig. 2). Fusion occurred within 1 h, and embryos cleaved by Day 2. Four-, 8-, and 16-cell stage embryos were observed at Days 2, 3, and 4, respectively. Compaction occurred between the 8- to 16-cell stage. Blastocoele formation began from the 16-cell stage. Blastocysts were observed from Day 5, and some hatching occurred at Day 6. From Day 7, blastocysts became degenerated and collapsed. The mean cell number of blastocysts was 34–46 (Table 3). Wang et al. [14] reported fewer cells within in vitro-produced, Day 6 blastocysts (37.3) compared to in vivo-derived, Day 6 embryos (164.5).
Uhm et al. [3] and Koo et al. [5] reported in vitro development of porcine NT embryos using EGFP-infected fetal fibroblasts. However, they reported EGFP expression in embryos without a negative control. In this study, we showed the lack of fluorescence in parthenogenetic controls to confirm EGFP expression of NT blastocysts (Fig. 3). Koo et al. [5] showed mean cell number, but Uhm [3] did not describe the quality of embryos. In the present study, we found that EGFP was detected from at least 1 h postfusion to the blastocyst stage. Theoretically, EGFP expression in just-fused embryos resulted from protein in the cytoplasm of the donor cells. Some protein and mRNA [15] from the donor cells are introduced into the cytoplasm of oocytes during cell fusion. After fusion, the EGFP protein and mRNA would be dispersed into the cytoplasm of oocytes and be detected, and EGFP mRNA could produce EGFP in the cytoplasm. Moreover, the timing of the transition from maternal to zygotic control of embryonic development (maternal to zygotic transcript transition) is the 4-cell stage in the pig [16]. Therefore, if the donor nucleus is remodeled and reprogrammed, detectable EGFP during the early embryo stage (before the 4-cell stage) should result from the donor cell cytoplasm, but detection of EGFP at later stages (especially the blastocyst stage) should result from new transcription from the NT nucleus.
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In conclusion, we simplified the FA protocol in porcine NT. This system is viable for porcine NT, because cloned piglets have been produced by using this system [10].
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FOOTNOTES |
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1 Supported by Food for the 21st Century and the NCRR13438. 
2 Correspondence: Randall Prather, University of Missouri, 162 ASRC, 920 East Campus Drive, Columbia, MO 65211. FAX: 573 884 7827;pratherr{at}missouri.edu
Accepted: July 16, 2001.
Received: May 3, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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