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Biology of Reproduction 65, 1558-1564 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Feasibility of Producing Porcine Nuclear Transfer Embryos by Using G2/M-Stage Fetal Fibroblasts as Donors1

Liangxue Laia, Tao Taoa, Zoltan Machátya, Birgit Kühholzera, Qing-Yuan Suna, Kwang-Wook Parka, Bill N. Daya, and Randall S. Prather2,a

a Department of Animal Sciences, University of Missouri, Columbia, Missouri 65211

ABSTRACT

The type of donor cell most suitable for producing cloned animals is one of the topics under debate in the field of nuclear transfer. To provide useful information to answer this question, G2/M- and G0/G1-stage fetal fibroblasts were used as donor cells for nuclear transfer. In vitro-matured oocytes derived from abattoir ovaries were used as recipient cytoplasts. In both groups, nuclear envelope breakdown and premature chromosome condensation were completed within 1–2 h after donor cells were injected into the cytoplasm of oocytes. Microtubules were organized around condensed chromosomes and formed a spindle within 1–1.5 h after activation. Decondensation of chromosomes could be seen within 2–4 h after activation. Reformation of the new nuclear envelope occurred 4–6 h after activation and was followed by nuclear swelling and formation of a pronucleus-like structure (PN) 8–12 h after activation. Most (80.6%) of the reconstructed oocytes derived from G2/M cells extruded polar body-like structures (PB). However, a much lower frequency of PB (21.7%) was observed in the reconstructed oocytes derived from G0/G1 donors. A variety of PN and PB combinations were observed in reconstructed oocytes derived from G2/M-stage donors, including 1PN+0PB, 1PN+1PB, 1PN+2PB, 2PN+0PB, 2PN+1PB, 2PN+2PB, and 3PN+1PB. Chromosomes of most embryos (10/13) derived from G2/M stage were diploid. The percentage of cleavage and blastocysts and the average nuclear number of blastocysts in the G2/M and G0/G1 groups were not different. These results demonstrate that the G2/M stage can be morphologically remodeled by cytoplasm of MII oocytes in pigs. To maintain normal ploidy, the extra chromosomes derived from G2/M-stage cells could be expelled by oocytes as a second polar body. G2/M-stage fibroblast nuclei could direct reconstructed embryos to develop to the blastocyst stage.

developmental biology, early development, embryo, oocyte development, reproductive technology

INTRODUCTION

Successful production of cloned animals derived from somatic cells was first demonstrated in sheep [1]. Somatic cell-derived cloned animals were later obtained in cattle [2–4], goats [5], mice [6, 7], and most recently in pigs [8, 9]. Transgenic calves [10] and gene-targeted sheep [11] have also been obtained by nuclear transfer from somatic cells, but the efficiency of nuclear transfer in all species is very low.

There are many factors affecting efficiency of nuclear transfer. One of them is the cell cycle phase of the donor cells at the time of nuclear transfer. It is generally agreed that donor cells for nuclear transfer must be in pre-S phase of the cell cycle if the recipient oocyte is not activated [12, 13]. Wilmut et al. [14], in a report on sheep mammary cells, stated that the donor cells for nuclear transfer must be in G0 of the cell cycle (quiescent phase). But Cibelli et al. [15] showed that cycling cells, which may contain cells in different cycle stages, could be successfully used for nuclear transfer in cattle.

G2/M-stage synchronized donor nuclei might represent an advantage, compared with other cell cycle stages used in nuclear transfer, when considering nucleus-cytoplasm synchronization of karyoplasts and cytoplasts. Attempts to use the G2/M-stage cell as a nuclear donor to produce nuclear transfer animals were successful in mice [16, 17] and sheep [18]. Alberio et al. [19] studied the behavior of M-phase synchronized blastomeres after nuclear transfer in cattle, but no embryonic development was obtained after 7 days of culture. In all of these studies, blastomeres were used as donor cells. So far, there have been no reports on transfer of G2/M-stage fibroblast nuclei, which are the most promising somatic cells being used to produce genetically modified cloned animals, into MII oocytes in cattle, sheep, or pigs. To explore the feasibility of producing a nuclear transfer pig by using G2/M-stage fetal fibroblasts as donors, we examined the nuclear remodeling events and time table within 12 h after the donor cell had been transferred into the in vitro-matured porcine oocyte. The in vitro developmental capacity of the nuclear transfer embryos and their ploidy were also evaluated.

MATERIALS AND METHODS

Preparation of Recipient Oocytes

Porcine ovaries were obtained from an abattoir and transported to the laboratory in a thermos filled with saline maintained at 30–35°C. Follicular fluid from 3- to 6-mm antral follicles was aspirated with an 18-gauge needle attached to a 10-ml disposable syringe. Cumulus-oocyte complexes (COCs) with uniform cytoplasm and several layers of cumulus cells were selected and rinsed three times in Tyrode lactate-Hepes plus polyvinyl alchohol (PVA). Approximately 50–70 COCs were put into each well of a four-well multidish containing TCM-199 medium supplemented with PVA (0.1%), D-glucose (3.05 mM), sodium pyruvate (0.91 mM), penicillin (75 µg/ml), streptomycin (50 µg/ml), cysteine (0.57 mM), LH (0.5 µg/ml), FSH (0.5 µg/ml), and epidermal growth factor (10 µg/ml) and were covered with mineral oil. The oocytes were cultured for 42–44 h at 39°C in 5% CO2 in air. After maturation, expanded cumulus cells were removed by vigorous pipetting in the presence of 0.3 mg/ml hyaluronidase. All chemicals were obtained from Sigma Chemical Company (St. Louis, MO).

Preparation of Donor Cells

Fetal fibroblasts were obtained from a fetus on Day 30 of pregnancy. The head of the fetus was removed before tissues were minced into small pieces and cells were dispersed by digestion in Hanks balanced salt solution (HBSS) with 0.1% trypsin and 0.02% EDTA. Dissociated cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 15% fetal calf serum (FCS), 2 mM L-glutamine, 1 mM sodium pyruvate, and antibiotics. To obtain the G2/M-stage donor cells, medium was removed and replaced with medium containing colchicine (1.0 µM), and cells were cultured for 24 h. For preparation of G0/G1-stage donors, fibroblasts were serum starved with DMEM containing 0.1% serum for 48 h. The synchronized cells were harvested using HBSS supplemented with 0.1% trypsin and 0.02% EDTA and were frozen in 15- to 20-µl aliquots containing 1500–3000 cells. Before microinjection, donor cells were thawed at 37°C, 200 µl of FCS was added, and cells were cultured for 30 min. Dulbecco modified Eagle medium (800 µl) supplemented with 15% FCS was added, and the sample was centrifuged at 500 x g for 5 min. The supernatant was removed, and 50 µl of TCM-199 supplemented with Hepes was added to resuspend the cells.

Parthenogenetic Activation of Oocytes

For electrical activation, 1–2 h after microinjection reconstructed oocytes were equilibrated for 5 min in a 0.3 M mannitol solution supplemented with 0.5 mM Hepes, 0.01% BSA, 0.1 mM CaCl2, and 0.1 mM MgCl2 and transferred to a chamber consisting of two electrodes 1 mm apart that was overlaid with the same activation solution. Oocytes were exposed to a single pulse of 1.2 kV/cm for 30 µsec on an Electro-cell manipulator 200 (BTX, San Diego, CA).

Micromanipulation

Oocytes were stained with 5 µM Hoechst 33342 for 30 min, and fluorescent metaphase II chromosomes and the first polar body were removed under ultraviolet microscopy with a 25- to 30-µm (inside diameter) glass pipette in TCM-199-Hepes plus 0.3% BSA and 7.5 µg/ml cytochalasin-B. The largest cells from the colchicine-treated population and the smallest cells from the serum-starved population, which were supposed to be in G2/M stage [20–22] and G0/G1 stage [23], respectively, were chosen as the donor cells to be injected directly into enucleated oocytes in TL-Hepes plus 0.3% BSA with a 14- to 17-µm (for G2/M cells) or 10- to 12-µm (for G0/G1 cells) glass pipette.

Experimental Design

Some oocytes were fixed to observe the condensation of donor cell chromosomes at 1–2 h after microinjection, just prior to activation. To examine the progression of nuclear remodeling, some of the reconstructed oocytes were fixed every 2 h after activation for 12 h. Oocytes were mounted on glass slides under coverslips, fixed in ethanol:acetic acid (3:1) for 24 h, stained with 1% (w/v) aceto-orcein, and evaluated by using Hoffman modulation contrast optics (400x). Some oocytes were fixed with 3.7% paraformaldehyde 1–1.5 h after activation and stained with fluorescein isothiocyanate (FITC)-conjugated anti-{alpha}-tubulin antibody diluted 1:50. After two washes in PBS, oocytes were stained with 4',6'-diamidino-2-phenylindole (DAPI) and observed with an E600 epifluorescence microscope (Nikon, Melville, NY). The remaining embryos were cultured in NCSU-23 with 4 mg/ml BSA for 7 days. G0/G1-stage fibroblasts obtained from the serum starvation treatment were also used as donor cells to compare the developmental potential with G2/M donors. Nonmanipulated MII oocytes were electrically activated and cultured as controls. The percentage of cleavage and blastocyst formation was examined at 48 h and on Day 7, respectively. Blastocysts were stained with Hoechst 33342 to count the number of nuclei. Some morulae on Day 5 were treated with 0.08 µg colcemid for ploidy analysis using the procedure described by Burgoyne [24].

Statistical Analysis

All dependent variables were analyzed for normality using the Wilk-Shapiro test (SAS, Cary, NC). Percentages of formation of polar body-like structures (PB), pronucleus-like structures (PN), cleavage, and blastocysts were arcsine transformed to achieve normal distribution. Treatment effects were analyzed with the GLM procedure of SAS. A P value <0.05 was considered significant.

RESULTS

The selection of the cells at the appropriate stage of cell cycle was critical to the success of the experiments. To obtain G2/M-stage cells, two measures were taken. First, we treated the cells with the microtubule-disrupting agent colchicine, which arrested the population of cells at G2/M (with a 4C DNA complement) before nuclear transfer. In a previous study, flow cytometric cell cycle analysis revealed that after treated with colchicine, porcine fibroblasts could be efficiently synchronized at the G2/M stage [23]. In that study, some of the cells were still in G1 stage even after being treated with colchicine. To reduce the risk of using the G1-stage cells for the G2/M-stage experiment, the largest cells from the colchicine-treated population, which are almost twice the size (20–25 µm in diameter) of the smallest cells (10–12 µm in diameter), were chosen as G2/M-stage donors (Fig. 1A). According to previous studies, cells with such a relatively large cytoplasmic volume are typical of those that have advanced beyond the G1 phase in preparation for division [20, 21]. Results of a previous study also showed that after treated with serum starvation, 95% of the fibroblasts of the smallest size were in G0/G1 stage [23]. Thus, the smallest cells (10–12 µm in diameter) in the serum-starved population (Fig. 1B), which were most likely to be in cycle stage G0/G1, were chosen for the experiment.



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  		FIG. 1. Selection of different pig cell cycle stage donors. Bars = 10 µm. A) After treatment with colchicine, large cells are dominant. The largest cells (arrow) were selected as G2/M-stage donors. B) After serum starvation, small cells are dominant. The smallest cells (arrow) were selected as G0/G1-stage donors

After microinjection, survival percentage of reconstructed oocytes from the G2/M group (47.5%, 688/1449) was lower than that for the G0/G1 group (69.5%, 503/724) (P < 0.05). Nuclear envelope breakdown and premature chromosome condensation were completed within 1–2 h after donor cells were injected into the cytoplasm of oocytes in both groups. G0/G1 nuclei condensed to form a single chromosome cluster (Fig. 2A), and G2/M nuclei condensed to form single or double clusters (Fig. 2B). Scattered chromosomes could be seen frequently in one or both of the double clusters (Fig. 2C). Sixty-one percent (47/77) of chromosomes from reconstructed oocytes derived from G2/M-stage cells could condense, a percentage lower than that of the G0/G1 group (71.0%, 44/62) (P < 0.05). Decondensation of chromosomes could be seen within 2–4 h after activation (Fig. 2D). Reformation of the new nuclear envelope occurred 4–6 h after activation (Fig. 2E), followed by nuclear swelling and PN formation 8–12 h after activation (Fig. 2F). Within 1–1.5 h after activation, microtubule assembly occurred around condensed donor nuclei, regardless of whether the reconstructed oocytes were derived from G2/M stage or G0/G1-stage donors and of whether the donor nuclei were located in a central or a peripheral position. Within 1.5–2 h after activation, extrusion of a PB could be seen in the reconstructed oocytes derived from G2/M-stage donors (Fig. 3A), and spindle formation occurred during the process of PB extrusion (Fig. 3, B–D). However, these events were rarely observed in G0/G1 donor-derived oocytes. The rate of PN formation from G2/M cells (53.7%, 103/192) was lower than that from G0/G1 donors (65.4%, 83/127) (P < 0.05; Table 1).



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  		FIG. 2. Nuclear remodelling in reconstructed pig oocytes. A) G0/G1 nuclei condense to form a single chromosome cluster (arrow). B) G2/M nuclei condense to form double chromosome clusters (arrow). C) Scattered chromosomes in one of the double clusters (arrow). D) Decondensation of chromosomes 2–4 h after activation (arrow). E) Reformation of the new nuclear envelope 4–6 h after activation (arrow). F) Reconstructed oocytes with 1PN and no PB



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  		FIG. 3. Extrusion of PB and formation of spindle. A) 1.2 h after activation, a reconstructed pig oocyte starts to extrude a PB (arrow). B) Same oocyte as A. Two sets of chromosomes have been separated. C) Same oocyte as A, showing a telophase spindle between the two sets of chromosomes. D) Merged B and C


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  		TABLE 1. Number (%) embryos showing PCC, PN formation, and PB formation from reconstructed pig oocytes derived from different donor nuclei

Of the reconstructed embryos that formed PN, most (80.6%, 83/103) of those derived from G2/M cells extruded PB (Table 1). More than half of them (58.3%, 60/103) had one PB; the others had two (22.3%, 23/103) or none (19.4%, 20/103) (Table 2). The number of PN also varied greatly among reconstructed oocytes derived from G2/M-stage cells. Most of these cells had one PN (72.8%, 75/103), some of them had two (15.5%, 16/103), and some had three (11.5%, 12/103). More oocytes had one PN than had two or three (P < 0.05), but there was no significant difference in the number of oocytes with two or three PN (P > 0.05). A variety of PN and PB combinations were found in reconstructed oocytes, including 1PN+0PB, 1PN+1PB (Fig. 4A), 1PN+2PB (Fig. 4B), 2PN+0PB (Fig. 4C), 2PN+1PB (Fig. 4D), 2PN+2PB (Fig. 4E), and 3PN+1PB (Fig. 4F). Among oocytes with one PN, the number of 1PN+1PB oocytes was higher than that of oocytes with either 1PN+0PB or 1PN+2PB. Among the oocytes with two PN, the number of 2PN+1PB oocytes was also higher than that of oocytes with either 2PN+0PB or 2PN+2PB (P < 0.05). For those oocytes with three PN, only one PB was found (Table 2). In the reconstructed oocytes derived from G0/G1 donors, only one PN was found. Oocytes from these donors had a significantly lower frequency of PB (P < 0.01) than oocytes from the G2/M donor group (Table 1). Only two kinds of PN and PB combinations, 1PN+0PB (78.3%, 65/83) and 1PN+1PB (21.7%, 18/63), were found in the G0/G1 group.


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  		TABLE 2. Number (%) of reconstructed one-cell pig embryos derived from G2/M-stage fetal fibroblasts showing different combinations of PN and PB



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  		FIG. 4. One-cell-stage pig embryo with different PN and PB combinations. A) Reconstructed oocytes with 1PN and 1PB (arrow). B) Reconstructed oocytes with 1PN and 2PB (arrows). C) Reconstructed oocytes with 2PN and no PB. D) Reconstructed oocytes with 2PN and 1PB (arrow). E) Reconstructed oocytes with 2PN and 2PB (arrows). F) Reconstructed oocytes with 3PN and 1PB (arrow)

After culture for 24–48 h, reconstructed oocytes could cleave to form two-cell (Fig. 5A) and four-cell embryos (Fig. 5B). The percentage of cleavage was not different between G0/G1 and G2/M groups (P > 0.05), but cleavage percentage in both groups was lower than that in the parthenogenetic group (P < 0.05). After culture to Day 7, blastocysts were found from both G2/M- and G0/G1-stage donor groups (Fig. 5C). The percentage of blastocysts from the G2/M donor group was not significantly different from that of the G0/G1 group (P > 0.05), but the blastocyst percentage in both groups was lower than that in the parthenogenetic group (P < 0.05). The nuclear numbers of the eight blastocysts derived from G2/M donors were 8, 12, 13, 17, 21, 18, 24, and 28. The nuclear numbers of the eight blastocysts from G0/G1 donors were 10, 11, 14, 15, 18, 22, 25, and 30. Average nuclear number of blastocysts in the G2/M and G0/G1 groups was not significantly different (P > 0.05), but both groups had fewer blastocysts than did the parthenogenetic group (Table 3). Of 13 morulae on Day 5, 10 were diploid (Fig. 6A), two were tetraploid (Fig. 6B), and one was a chimeric embryo containing both diploid and tetraploid cells.



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  		FIG. 5. Pig embryos in different developmental stages after in vitro culture of reconstructed oocytes derived from G2/M-stage donors. A) A two-cell embryo with PB (arrow). B) A four-cell stage embryo. C) A blastocyst on Day 7


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  		TABLE 3. Development of reconstructed pig embryos cultured for 7 days in vitro



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  		FIG. 6. Karyotypes of pig embryos derived from G2/M-stage donors. A) Diploid embryos. B) Tetraploid embryos

DISCUSSION

The events that occur in the donor nucleus after transfer into an enucleated metaphase II oocyte using early embryonic cells as donors have been studied in a number of species, including mouse [25], rabbit [26], pig [27], and cow [28]. Immediately at fusion, donor nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) take place. These effects are catalyzed by a cytoplasmic activity termed maturation-promoting factor (MPF). This activity is found in all mitotic and meiotic cells reaching maximal activity at metaphase. Matured mammalian oocytes are arrested at metaphase of the second meiotic division (metaphase II) and have high MPF activity [29]. In our nuclear transfer experiments, donor nuclei were injected directly into nonactivated matured oocytes in which MPF levels were high, so NEBD and PCC also were found in both G2/M and G0/G1 groups. G0/G1 nuclei condensed to form a single chromosome cluster, whereas G2/M nuclei formed either a single or two independent chromosome clusters.

As a result of NEBD having allowed access of licensing factors to the DNA, nuclei, regardless of their cell cycle state, would undergo DNA replication before the first cleavage. These morphologic remodeling events could occur when using either G0/G1- or G2/M-stage cells as donors. Therefore, it is generally thought that during embryo reconstruction, correct ploidy can be maintained by transferring G0/G1 nuclei into enucleated metaphase II oocytes at the time of or before activation. However, for the G2/M-stage donors, which contain replicated chromosomes, elimination of extra chromosomes is required to restore a normal ploidy prior to nuclear formation. In our experiment, staining with FITC-conjugated anti-{alpha}-tubulin antibody and with DAPI revealed evidence of polar body extrusion events characterized by microtubule assembly around the condensed chromosome and formation of the spindle in the reconstructed oocytes derived from G2/M-stage donors following electrical activation. Also, 80.6% of the reconstructed oocytes, which formed PN, extruded PB. The formation of spindle might be triggered by extra chromosomes in the reconstructed oocytes derived from G2/M-stage donors; spindles were rarely found in oocytes derived from G0/G1-stage donors. Furthermore, donor cells in M stage may make use of their own spindle and more easily complete their pseudomeiosis.

In a previous study, the reconstructed one-cell embryos with a variety of PN and PB combinations, such as 1PN+1PB, 1PN+0PB, and 2PN+0PB, were found after activation in mice by using the late two-cell embryos as donors, which were likely in or close to G2 phase [30]. Cheong et al. [17] also found one-cell embryos with 1PN+2PB after nuclear transfer in mice. However, the present results indicate that PN and PB formation after injection of G2/M-stage fibroblasts and electrical activation is more complicated and irregular. Because G2/M-stage donor nuclei have two sets of chromosomes after activation, if a normal bipolar spindle formed, one set may remain in the cytoplasm and decondense to form a PN and the other set may be extruded to form a PB. In this way, the reconstructed embryo may maintain normal ploidy. So the 1PN+1PB combination is expected in G2/M-cell nuclear transfer. In our experiment, 37.9% of reconstructed oocytes were classified in this category. The various other types of reconstructed oocytes observed after activation in this study may have arisen for one of two reasons. First, abnormalities of the spindle-like structures occur during PCC. Spindle-like structures in the hybrids formed from one interphase blastomere and one MII oocyte have shown numerous abnormalities, which were often apolar, monopolar, or tripolar structures [31]. These abnormal spindle-like structures might contribute to the formation of 2PN+0PB, 2PN+1PB, 1PN+2PB, 2PN+2PB, or 3PN+1PB one-cell embryos. Second, the donor cells are not purified; although many of the techniques were successful in changing the percentage of the cells in one phase or another, none of the techniques were 100% effective. Colchicine can be used to acheive approximately 40% synchronization in G2/M [23]. Although selection of the largest cells would highly increase the number of G2/M-stage donors during microinjection [22], cells in G1 and S phase cannot be completely avoided and might contribute to formation of 1PN+0PB embryos.

The extrusion of a PB was not observed in early nuclear transfer experiments with embryonic donor cells in rabbits [26, 32], sheep [33, 34], pigs [27], and cattle [35]. All of these experiments used the fusion method, and the reconstructed oocytes were activated at the same time or immediately after fusion. In those cases, MPF was reduced and the PCC might not be completed before formation of the PN. Thus, PB extrusion could not occur. Alberio et al. [19] reported that in cattle, in which M-phase-synchronized blastomeres were used as donors to perform nuclear transfer, when activation was conducted 3–4 h and 6–7 h after fusion, 50% of the embryos could extrude PB, but no extrusion was observed in embryos activated immediately after fusion. These results are consistent with those of the present study. However, normal embryonic development did not occur after 7 days of culture in cattle. In the present study, activation was initiated 1–2 h after microinjection. During this time, PCC occurred in 61.0% of reconstructed oocytes, which is one of the prerequisites for extrusion of PB, resulting in a high percentage of PB formation in our experiment.

The high percentage of PB formation leads to the hypothesis that some G2/M-stage donors allow reconstructed oocytes to progress through normal early embryo development, especially those with 1PB+1PN. This hypothesis was supported by results of two other experiments. First, after further in vitro culture, some reconstructed oocytes did undergo normal cleavage and could develop to blastocyst stage. The percentage of cleavage and blastocyst formation and the nuclear number of blastocysts in the G2/M-stage donor group were similar to those in the G0/G1-stage donor group and to the results of other recent studies in which serum-starved fibroblast cells were used as donor cells [36–38]. The percentage of blastocyst formation and the average nuclear number of the blastocysts obtained with either type of donors were not high, possibly because of poor in vitro culture conditions. To overcome this problem when trying to establish a pregnancy, the in vitro culture time could be limited to less than 24 h before the reconstructed embryos are transferred to a surrogate recipient. Onishi et al. [9] reported only 2.4% blastocyst formation, but a recipient pig established pregnancy and a cloned fetus developed to term. Second, karyotype analysis provided some insight into the formation of PB. Most of the embryos (10/13) on Day 5 were diploid. The tetraploid embryos and chimeric embryo containing diploid and tetraploid cells, which occurred in low frequency, might be derived from reconstructed embryos that failed to extrude PB or formed abnormal spindle-like structures.

The results of this study demonstrate that G2/M-stage donor nuclei can be morphologically remodeled by cytoplasm of MII oocytes in pigs. The extra chromosomes derived from G2/M-stage cells can be expelled by oocytes as a second PB. G2/M-stage fibroblasts can direct the reconstructed embryo to develop to the blastocyst with normal ploidy. Future experiments are needed to establish whether the use of G2/M-stage cells as donors can result in production of viable cloned piglets with normal ploidy.

FOOTNOTES

First decision: 26 December 2000.

1 This study was supported by Food for the 21st Century and the National Institutes of Health (NCRR 13438) and is a contribution from the Missouri Agricultural Experiment Station journal series 13 091. Back

2 Correspondence: Randall S. Prather, Department of Animal Science, Lab 162, University of Missouri, Columbia, MO 65211. FAX: 573 882 6827;pratherr{at}missouri.edu Back

Accepted: June 28, 2001.

Received: November 8, 2000.

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