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Regular Article |
a Departments of Gene Expression and Development
b Genomics and Bioinformatics, Roslin Institute, Roslin, Midlothian EH25 9PS, United Kingdom
c Germplasm and Gamete Physiology Laboratory, U.S. Department of Agriculture, Beltsville, Maryland 20705
d Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 02111
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, early development, embryo, reproductive technology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, several research groups have succeeded in cloning piglets from somatic cells [5–7]. Despite differences in approach, at least 3 key similarities are evident. First, all groups relied on electrical stimulation to either activate and/or fuse reconstructed eggs. Second, despite improvements in embryo culture methodologies all groups transferred cloned embryos prior to the blastocyst stage. Third and perhaps most significant, all groups recognized the need to surmount pregnancy maintenance requirements.
Implicit to all recent NT experiments has been an appreciation of the importance of cell cycle coordination between donated nuclei and recipient egg cytoplasm to maintain DNA integrity and ploidy (reviewed in [8]). The DNA content of NT eggs can be regulated by controlling polar body extrusion after activation. This control has been achieved by the use of cytochalasin to depolymerize cortical microfilaments [9]. Thus, as exemplified in the mouse, the DNA content of an unreplicated diploid nucleus (2n, 2C) in G0/G1 can be preserved after its transfer by suppression of polar body extrusion. Conversely, the DNA content of a replicated diploid nucleus (2n, 4C) in G2 of the cell cycle or arrested in M-phase can be returned to an unreplicated state if polar body extrusion is permitted after transfer [10–13]. In cases in which the DNA content of nuclear reconstructed embryos cannot be controlled via cytochalasin treatment, an alternative strategy might involve interference with microtubule spindle function using depolymerizing agents such as nocodozole or demecolcine.
In the present study, our objective was to clone a pig by somatic cell NT. Our approach involved integration of improvements in oocyte activation [14] and embryo transfer and pregnancy maintenance with NT experiments to evaluate reconstruction strategies, cell types, and control of nuclear formation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In Vitro Oocyte and Embryo Culture
Unless otherwise noted, all chemical reagents used during oocyte/egg and embryo culture and micromanipulation were purchased from Sigma (Poole, Dorset, U.K.). Cumulus-oocyte complexes were collected from slaughterhouse ovaries and matured as described previously [14]. The base medium for embryo culture in an atmosphere of 5% CO2 in air at 39°C was NCSU 23 [15]. This medium was comprised of 108.73 mM NaCl, 4.78 mM KCl, 1.7 mM CaCl2, 1.19 MgSO4, 25.07 mM NaHCO3, 1.19 mM KH2PO4, 5.55 mM glucose, 1 mM glutamine, 7 mM taurine, 5 mM hypotaurine, 0.4% BSA-V, 100 U/L penicillin-G, and 50 mg/L streptomycin. Embryos were cultured in NCSU 23 in groups of 20–40 per 500 µl of medium. The base medium for handling mature oocytes (eggs) and embryos under atmospheric conditions was Hepes-buffered NCSU 23 (hb-NCSU 23). This medium was composed of 131.7 mM NaCl, 4.78 mM KCl, 1.7 mM CaCl2, 1.19 MgSO4, 2 mM NaHCO3, 10 mM Hepes, 1.19 mM KH2PO4, 5.55 mM glucose, 1 mM glutamine, 12 mM taurine, 0.4% BSA-V, 100 U/L penicillin G, and 50 mg/L streptomycin. For some procedures, Ca2+-free hb-NCSU 23 or NCSU 23 was used. Egg and embryo handling under atmospheric conditions was on surfaces heated to 34–37°C
In Vivo Oocyte Production
Ovulated oocytes for this study were produced from Large White gilts that were approximately 9 mo of age or older and weighed at least 120 kg at time of use. Donor gilts were superovulated after exhibiting a natural heat that may have been synchronized by feeding of 20 mg altrenogest (Regumate; Hoechst Roussel Vet Ltd., Milton Keynes, U.K.) daily for 18 days. Superovulation was achieved by 1 of 2 methods. In experiments I and II, midluteal gilts (11–16 days postestrus) were injected with 175 µg of cloprostenol (Planate; Schering-Plough Animal Health, Munich, Germany) and 1500 IU of eCG (PMSG; Intervet U.K. Ltd., Milton Keynes, U.K.) at 1900 h. Eighty-eight hours later, these gilts were injected with 750 IU hCG (Chorulon; Intervet U.K.). In experiments III–V between Day 11 and Day 15 following an observed estrus, gilts were fed 20 mg altrenogest once daily for 4 days and 20 mg altrenogest twice on the 5th day. On the 6th day, these gilts were injected with 1500 IU of eCG at 2000 h and with 750 IU hCG 83 h later.
Ovulation was monitored by transcutaneous ultrasound, using an Aloka SSD-500 machine with a 5-MHz convex linear probe. The probe was placed in the right inguinal area and directed dorsally in a sweeping fashion to locate an ovary. Gilts were first examined prior to 34 h post-hCG and again by 42 h post-hCG to identify the presence of follicles and to identify gilts that had ovulated prior to the intensive scanning. From 42 to 48 h post-hCG, gilts were scanned every 2 h. Ovulation was deemed to have occurred when all follicles were no longer visible or approximately 80% of follicles could not be detected, with ovulation time taken as the midpoint between successive positive and negative scans. Gilts that ovulated between 42 and 48 h post-hCG were used as oocyte donors for NT studies.
Nuclear Donor Cell Preparation
Fibroblast cells isolated from two Day 25 pig fetuses (A and B) were used as nuclear donors. Results in experiments II–IV were obtained using fibroblasts from fetus A, although similar results were observed using fibroblasts from fetus B (data not shown). Both cell populations were used in experiment V. Fibroblasts were isolated, frozen at low passage (passages 2–4), and cultured essentially as described by Campbell et al. [16]. After thawing, cells were cultured for 2 days at 37°C in 5% CO2 in air in Glasgow minimum essential medium (GMEM, G-5154; Sigma) modified by addition of 2 mM glutamine, 1 mM sodium pyruvate, 1x nonessential amino acids, and 10% fetal calf serum (FCS). Cells were then cultured for 5–6 days in the modified GMEM with FCS reduced to 0.5%. Confirmation that cells had left the cell cycle was obtained by immunostaining for proliferating cell nuclear antigen [17], which was normally reduced to 
1% of all cells examined. Following serum deprivation or resumption of growth in 10% FCS, >95% of cells examined (n = 50–100) retained a normal karyotype (2n = 38). In experiment III, cumulus cells were prepared by rapid dispersal of expanded cumulus complexes from either ovulated or in vitro-matured (IVM) oocytes in Ca2+-free hb-NCSU 23 using a Gilson P100 pipette.
Nuclear Transfer
Nuclear transfer was carried out as described by Campbell et al. [16] with the exceptions noted. Oocytes were stripped of cumulus by pipetting in 300 IU/ml hyluronidase in Ca2+-free hb-NCSU 23. Denuded oocytes were treated for 15 min in Ca2+-free hb-NCSU 23 containing 7.5 µg/ml cytochalasin B (CB) and 5 µg/ml Hoechst 33342 at 39°C. Oocytes were manually enucleated in Ca2+-free hb-NCSU 23 containing 7.5 µg/ml CB. Cytoplasts derived from enucleated oocytes were held and reconstructed in Ca2+-free hb-NCSU 23 at 39°C. In all experiments, the delay between fusion and activation was in Ca2+-free NCSU 23 at 39°C in 5% CO2 in air. Depending on the experiment this medium was supplemented with CB, nocodozole, or demecolcine.
Electrical pulses were used throughout the study for fusion and activation. The fusion medium was 0.3 M mannitol, 100 µM MgCl2, with the same medium supplemented with 50 µM CaCl2 for activation. For experiment I, oocytes were activated with three 80-µsec pulses of 1.25 kV/cm. This stimulation was also used in experiment II, for both fusion and activation in either delayed activation (DA) or simultaneous activation (SA) treatment groups. In all remaining experiments (III–V), this treatment was used for fusion, whereas activation of NT cytoplasts and IVM oocytes (to make parthenotes) was achieved using three 80-µsec pulses of 1 kV/cm. This transition coincided with our determination in concurrent experiments that activation by multiple pulses of the lower voltage yielded better parthenogenetic development to the blastocyst stage [14]. However, the higher voltage was still preferred for fusion. In all experiments, fusion was assessed by brightfield microscopy 2 h after administration of the first electrical stimulation. Only fused karyoplast-cytoplast complexes were activated when activation was delayed.
To make diploid parthenote embryos, activated ovulated (experiment I) or IVM (experiments III–V) oocytes were treated for 6 h with 7.5 µg/ml CB in NCSU 23 at 39°C in 5% CO2 in air. In experiment II, both DA and SA groups were cultured in the presence of 7.5 µg/ml CB for 2 h postfusion. However, DA embryos were not treated with CB after activation. This DA strategy (CB postfusion for 2 h and drug-free postactivation) was the standard against which all other postfusion or postactivation treatments were compared. In experiment III, this standard was compared against a 2-h postfusion and 6-h postactivation culture with 7.5 µg/ml CB, the latter in NCSU 23. In experiments IV and V, the standard was compared against treatment with 7.5 µg/ml CB combined with 2 µg/ml nocodozole or 0.4 µg/ml demecolcine, postfusion or postfusion and activation in NCSU 23.
In experiment II, ovulated oocytes in each replicate trial were equally allocated between both groups. Following NT, DA and SA clones were transferred into contralateral horns of recipient gilts within 2 h of activation. Clones were recovered 3–4 days later as 2- to 8-cell embryos and cultured to Day 6 to avoid reduced recovery encountered when flushing uteri to collect hatched blastocysts. In experiments III–IV, cloned embryos were cultured in vitro for 7 days. In experiment V, cloned embryos were transferred to recipients within 1 h of activation.
Confirmation of Fibroblast-Cytoplast Fusion by Membrane Stain Transfer
In experiment IV, transfer of octadecyl rhodamine B dye (R18, O-246; Molecular Probes, Eugene, OR) from fibroblast to cytoplast membranes was used to confirm cell fusion. R18 was stocked at a concentration of 1 mg/ml in ethanol and stored at -20°C for up to 1 mo. Within 1 h of being used as nuclear donors, fibroblasts in suspension (5 x 104 cells/ml) were stained with 0.1 µg/ml R18 in Dulbecco PBS (BR14; Oxoid Ltd., Basingstoke, U.K.) for 15 min at room temperature on a rocker platform. Excess stain in solution was then removed by centrifugation (5 min at 750 x g) and resuspension 3 times in 10 ml of modified GMEM before final resuspension in 1 ml of the same for NT. Although R18 dye transfer was occasionally assessed vitally under ultraviolet irradiation (excitation/emission: 546/580
), all data were collected following fixation in 4% paraformaldehyde in Dulbecco PBS.
Oocyte Recovery, Embryo Transfer, and Pregnancy Maintenance
Oocyte collection and embryo transfer were conducted under general anesthesia. Following a midline laparotomy, oocytes and embryos were recovered or transferred, respectively, in hb-NCSU 23 at 39°C. To assist with the establishment and maintenance of pregnancies in experiment V, 1 of 3 strategies was implemented. The first involved injection with 5 mg (1 ml) of estradiol benzoate on Day 11 and Day 15 following estrus. The second involved an attempt to induce an accessory set of corpora lutei by injection with 1200 IU eCG at 900 h on Day 9 following estrus and with 500 IU of hCG at 1300 h on Day 12 following estrus. The third strategy involved cotransfer of 60–65 parthenote embryos prepared using IVM oocytes activated and made diploid the day before transfer [14].
Microsatellite Analysis
Genomic DNA was extracted from whole blood of the surrogate sow, fibroblast cell cultures, and piglet ear punches according to the method of Miller et al. [17]. The genotypes were determined for 23 microsatellite loci selected from the panel recommended by the Food and Agriculture Organization-International Society for Animal Genetics for studies of genetic diversity in pigs [18] (http://www.toulouse.inra.fr/lgc/pig/panel.htm). Polymorphism for each marker was based on allelic variations in length arising from the number of simple tandem repeats at each locus. Length variations were assayed by polymerase chain reaction (PCR) amplification with fluorescently labeled locus-specific primers and PAGE on an automated DNA sequencer (ABI 373; Applied Biosystems, Cheshire, U.K.). Proprietary software (GeneScan and Genotyper; Applied Biosystems) was used to estimate PCR fragment sizes in nucleotides.
Statistical Analysis
In experiment II, the effect of activation strategy was estimated using the method of Breslow and Clayton [19] to allow for binomial variation among embryos and extra variation among trials and between uterine sides. The significance of effects in the logistic scale was determined approximately using 2-tailed Student t-tests with degrees of freedom based on the numbers of trials available for the paired comparison of strategies. In all remaining experiments, data were analyzed by a 1-way or a repeated-measures ANOVA, followed by pairwise t-test comparisons of means.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A total of 249 ovulated oocytes were recovered from 18 gilts over 6 days, with the number of oocytes recovered per gilt ranging from 7 to 23. Oocytes were activated and cultured separately according to donor identity, and the proportion of oocytes forming parthenote blastocysts after 7 days was plotted with respect to donor time of ovulation (Fig. 1). Half of the gilts ovulated between 45 and 47.5 h post-hCG, and the remaining gilts were roughly equally distributed between earlier (42–43.5 h) and later (49–50.5 h) ovulation times. The proportion of activated oocytes forming parthenote blastocysts from gilts ovulating earlier or later than the midperiod did not exceed 30%, and 4 of 9 gilts ovulating in these periods yielded no blastocysts at all. In contrast, of 9 gilts ovulating during the midperiod (45–47.5 h post-hCG), 6 yielded parthenote blastocyst rates greater than 50%, and 1 gilt produced no blastocysts at all. Regardless of ovulation time, there was no significant difference in mean (±SEM) parthenote blastocyst nuclear counts (early: 22 ± 4, n = 9 mid: 26 ± 1, n = 54 late: 23 ± 3, n = 5).
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Experiment II: Delayed Versus Simultaneous Fusion and Activation
Using the same electrical stimulation as in experiment I, development of embryos cloned from fetal fibroblast nuclei and ovulated oocytes was compared in 4 trials following delayed versus simultaneous fusion and activation. All fused karyoplast-cytoplast complexes were activated when fusion was delayed. Across replicates, cell fusion was normally higher for the DA group, with the difference approaching significance (Table 1, P = 0.08). Following in vivo development for 3–4 days, there were no apparent differences between treatment groups in embryo recovery, which was 50–70% of transferred embryos in each trial (data not shown). The average cleavage and blastocyst rates were 33% and 7% for DA and 16% and 1% for SA, respectively. Although there was no significant difference in corresponding values between these groups (P 
0.25), DA was more consistent in providing at least 1 blastocyst in every trial, with blastocyst nuclear counts ranging from 18 to 49. In contrast, only 1 trial yielded an SA blastocyst, with a low nuclear count. Based on these results, SA was abandoned as an NT strategy in the pig.
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Experiment III: Evaluating the Need for CB Following DA
We next considered whether cloned embryo development could be improved by treatment with CB for 6 h after DA, using ovulated or IVM oocytes as cytoplasts and fetal fibroblasts or adult cumulus cells as karyoplasts. For all cytoplast/karyoplast combinations, postactivation treatment with CB had no appreciable effect on the rates of cleavage, the production of blastocysts, or blastocyst nuclear counts, which remained essentially the same as in experiment II. Rates of cell fusion were consistently higher with fibroblast than with cumulus cells using either ovulated (60% vs. 40%) or IVM (80% vs. 40%) cytoplasts (Table 2). In experiments with IVM oocytes, parthenote controls were produced using the same electrical activation treatment and 6 h of CB treatment (Table 2, None = no karyoplast). Parthenote development was highly consistent across trials. After 7 days in culture, average parthenote blastocyst nuclear count was 30, with nuclear counts in cloned blastocysts approaching this value. Cloned embryo cleavage rates were comparable to those of parthenotes; however, developmentally arrested cloned embryos differed by having fewer or aberrantly structured nuclei as revealed by Hoechst staining (data not shown). Because cumulus cells were not superior karyoplasts to fibroblasts they were abandoned in subsequent experiments. In addition, because the use of CB postactivation did not confer improved development, the standard DA strategy continued to restrict CB to the postfusion period.
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Experiment IV: Control of Nuclear Retention in Cloned Embryos
We considered whether poor cloned embryo development could be related to aberrant control of DNA content during the first cell cycle and whether this problem could be corrected by combining CB with nocodozole (N; 2 µg/ml) after activation to interfere with microtubule spindle function. In preliminary experiments, we reconstructed IVM oocyte cytoplasts with fetal fibroblast cells whose membranes had been prestained with the red fluorescent dye R18. In 2 independent trials, examination 18 h postactivation consistently revealed that chromatin was completely internalized in only 4/10, 6/10, and 5/10 fused (R18 positive) cytoplasts when postfusion treatment with CB was followed by no treatment, or a 6-h postactivation culture with either CB or CB/N, respectively. Postactivation treatment with CB/N also resulted in all fused cytoplasts containing single pronuclei, in contrast to a mixture of single and multiple pronuclei (no treatment) or only multiple pronuclei (CB for 6 h).
To improve nuclear retention, we used ovulated cytoplasts with R18 stained fibroblasts and only compared our standard DA treatment (CB postfusion only) with CB/N beginning postfusion and for 6 h postactivation. Nuclear fate was followed at 2 h postfusion (hpf) and 18 h postactivation (hpa). Cell-cytoplast fusion was confirmed by dye transfer in approximately 80% of cytoplasts assessed by brightfield as fused (F) and fixed 2 hpf (Table 3). Conversely, only 5% of cytoplasts assessed as nonfused (NF) at the same time point were stained with R18, likely depicting the error of assessment by brightfield microscopy. Following activation and culture, the proportion of NF cytoplasts showing R18 membrane staining increased to 25–30%, probably because of further fusion during activation. At 18 hpa, the proportion of F cytoplasts positive for membrane staining was 66% and 90% following postactivation culture in medium alone or in medium containing CB/N, respectively, although this difference was not significant (ANOVA, P = 0.2).
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The presence of Hoechst-stained chromatin in cytoplasts was correlated with R18 staining of cytoplast membranes (Table 3). At 2 hpf, almost all Hoechst-stained chromatin in cytoplasts was condensed. At 18 hpa, the incidence of Hoechst staining in F cytoplasts was higher for the CB/N group (P < 0.05). Single and multiple pronuclei, in addition to condensed mitotic configurations of chromatin, were observed in both treatment groups at this time point. However, treatment with CB/N resulted in an increase in the prevalence of single pronuclei in F and NF cytoplast groups, approaching significance in the former (ANOVA, P = 0.06).
In light of the enhanced nuclear retention observed with CB/N begun at 2 hpf and continued up to 6 hpa, we tested this combination for development to the blastocyst stage following DA of ovulated cytoplasts receiving fibroblast nuclei. No improvement in cleavage, blastocyst rates, or blastocyst nuclear counts was observed (Table 4, treatment group 1). In addition, in 3 trials involving reconstruction of fibroblast karyoplasts with a total of 88 IVM cytoplasts, no blastocysts were produced. However, in those IVM experiments there were no differences in the development of parthenote controls treated with either CB (n = 119) or CB/N (n = 119) for 6 hpa, in cleavage rates (71% vs. 76%), blastocyst rates (39% vs. 29%), or blastocyst nuclear counts (27 ± 2, n = 19 vs. 29 ± 3, n = 22). Thus, treatment with CB/N for 6 hpa was not overtly detrimental to parthenotes.
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To strike a balance between improved nuclear retention and the possibility that prolonged treatment with microtubule and microfilament inhibitors was more detrimental to cloned embryos, a final series of experiments were conducted using N or demecolcine (D; 0.4 µg/ml) with CB after fusion alone (Table 4, treatment groups 2–4). Again, there was no evidence of enhanced development to the blastocyst stage. In these experiments, nuclear retention was retrospectively estimated by Hoechst staining of all cloned embryos, including those that had not cleaved or had failed to form blastocysts. This analysis suggested that 72%, 78%, and 67% of reconstructed cytoplasts retained nuclei following CB (n = 92), CB/N (n = 91), and CB/D (n = 90) postfusion treatments, respectively. Although imprecise, this analysis suggested that combining microtubule-depolymerizing agents with CB during the postfusion period alone did not improve nuclear retention.
Experiment V: In Vivo Competence of Cloned Embryos
Following experiments to optimize enabling methodologies and development of cloned embryos to the blastocyst stage, we assessed in vivo development with the assistance of various pregnancy maintenance strategies. Two fibroblast populations (A and B) were fused to ovulated oocyte cytoplasts and cultured with CB or CB/D for 2 h before activation and immediate transfer. Both F and NF eggs assessed by brightfield were activated and transferred. The NF eggs were expected to contribute a small proportion of embryos that were effectively simultaneously fused and activated, based on our results in experiment IV (see Table 3). Nine transfers into recipients were carried out (Table 5). Two pregnancies were established using clones treated with CB/D postfusion. Of these 2, one was established with each fibroblast cell line and was maintained with either estradiol or cotransfer of parthenotes derived from IVM oocytes (see Materials and Methods). Both were lost by 40 days of gestation. One pregnancy was established from clones produced by the standard DA strategy (CB postfusion only) and was maintained by cotransfer of parthenotes. This pregnancy resulted in a single healthy piglet delivered naturally at term (121 days; Fig. 2). Genetic analysis confirmed that the piglet was identical to the cell line (B) used for NT at all 23 polymorphic microsatellite loci examined. In addition, at 5 loci the piglet lacked any of the alleles detected in the surrogate mother, confirming that she made no genetic contribution to the piglet (Table 6). The genotype of the piglet differed from the genotype of cell line A at 13 of the 23 loci tested. At the time of submission of this manuscript, the piglet was more than 4 mo old and in good health.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oocytes used as cytoplasts in this study were electrically activated using an optimized protocol [14]. Previously, we determined that activation of oocytes by this method was capable of yielding pregnancies, which by ultrasound could be detected until at least 50 days of gestation (unpublished results). An integral part of that protocol developed using IVM oocytes involved activation during an optimal window at approximately 44 h of maturation [14]. In the present study, an increase in parthenote blastocyst potential was correlated with oocytes ovulated between 45 and 47.5 h post-hCG and activated 3–4 h later. Our results contrast with previous findings by Polejaeva et al. [5], who concluded that pig parthenote development to the blastocyst stage was not age dependent and did not surpass 30% of activated oocytes. This difference may be explained by their activation of oocytes from 51.5 to 60 h post-hCG, the former time point representing the very end of the window suggested by our results. To increase the pool of oocytes serving as cytoplasts in our study, we accepted as donors gilts that ovulated from 42 to 48 h post-hCG. However, our protocol for inducing ovulation resulted in approximately half of stimulated gilts ovulating during our optimum window. In mice, optimum parthenogenetic development is restricted to oocyte activation over a 4-h period [20]. In contrast, the developmental competence of cow oocytes extends over 20 h [21]. The capacity to predict oocyte developmental performance on the basis of ovulation time would substantially reduce the cost and effort associated with using ovulated pig oocytes in future studies.
With reliance on electrical activation to initiate cloned embryo development, we observed more consistent development to the blastocyst stage following DA than following SA. This difference may have been related in part to a reduction in the efficiency of fusion associated with the latter, resulting in fewer reconstructed cytoplasts. In the rabbit, fusion of adult fibroblasts with preactivated cytoplasts derived from ovulated oocytes is less successful than when fusion precedes activation [22]. Cortical granule exocytosis that occurs during egg activation may interfere with the apposition of membranes required for karyoplast-cytoplast fusion. Treatment with CB, which disrupts cortical microfilaments known to control granule distribution [23], may compound such an effect. A simultaneous fusion and activation strategy was an integral part of one group's failed attempt to clone pigs from fetal fibroblasts [24]. Both blastocyst yields and cell numbers in that study were comparable to our results with this method. However, Polejaeva et al. [5] successfully cloned piglets from adult granulosa cells after combining simultaneous fusion and activation with serial NT into pronuclear zygotes.
Our study joins 2 other reported implementations of a DA strategy to clone pigs by somatic NT. Onishi et al. [6] accomplished NT by microinjection and a 3- to 4-h delay in electrical activation. Using IVM oocytes, Betthauser et al. [7] also reported a 4-h delay between fusion and activation, the latter accomplished by treatment with ionomycin and then the serine-threonine kinase inhibitor 6-dimethylaminopurine (6-DMAP). Prolonged exposure of incoming nuclei to a cytoplasm rich in metaphase-promoting factor (MPF) causes chromosome condensation, as we observed after 2 h. This condensation is believed to facilitate undefined nuclear changes that are essential for development [25, 26]. Diminished development to the blastocyst stage has been reported after DA up to 6 h (vs. 2 and 4 h) following NT with pig fibroblast cells [27]. This reduced development may have been due in part to differences in the timing of activation relative to oocyte age. In the study by Betthauser et al. [7], fusion was done in double the concentration of calcium, which in our hands results in egg activation [14]. After electrical activation of oocytes or cytoplasts into which nuclei have already been introduced, pronuclear formation normally only begins after 4–6 h [28, 29]. Treatment of activated cytoplasts with ionomycin and 6-DMAP in the study by Betthauser et al. [7] would have provided an additional activation stimulus that would have accelerated the decline in MPF activity brought on by activation [30]. This decline would have reduced the period over which the cytoplast environment could influence nuclear structure.
To our knowledge, we are the first researchers to explore the inefficiency of nuclear retention in pig NT cytoplasts and the benefit of combining N with CB treatment. Others have reported poor rates of pronuclear formation [5, 31], which could be accounted for by reduced nuclear retention. When applied to parthenogenetically activated pig oocytes, CB effectively controls polar body formation and egg ploidy [14, 32]. However, in our study, postactivation treatment of cloned embryos with CB did not enhance development to the blastocyst stage, and observations on NT cytoplasts prepared from IVM oocytes confirmed that nuclear retention was poor. Pig oocytes appear to be distinct from those of the mouse, in which CB is sufficient to manipulate polar body emission in either circumstance [10–13, 33]. These differences between species may relate to differences in microtubule spindle organization. Pig meiotic spindles differ at their poles from both mitotic and meiotic spindles in mice by lacking 
-tubulin [34]. Because -tubulin normally serves to seed microtubule organization in centrosomes, its absence in pig oocyte spindle poles would make spindle dynamics even more sensitive to microtubule depolymerization [35, 36]. Associations between the spindle and the oocyte cortex are also likely to differ between pig and mouse oocytes, as suggested by differences in the direction of spindle rotation with respect to the egg cortex following activation [34].
Treatment of NT sheep and cow cytoplasts with 6-DMAP after activation has previously been shown to promote nuclear retention and the formation of single pronuclei, as described for CB/N [36, 37]. In our study, there was no improvement in development despite improved nuclear retention. In mouse oocytes, spindle assembly checkpoints prevent meiotic cell cycle progression when the spindle is disrupted with microtubule inhibitors [38, 39]. To our knowledge, this question has not been examined in pig oocytes. If a parallel exists, then persistent treatment with N after activation in the pig could have delayed cell cycle-associated changes in cytoplasmic activities until withdrawal of this agent. Such a delay could have offset any benefit incurred by improved nuclear retention.
In our study, NT embryos were transferred to recipients within a few hours of activation, earlier than those reported by other groups who have recently reported success. We were compelled to transfer prior to embryo compaction after determining that unprotected oviductal transfer of zona pellucida-compromised precompaction pig embryos permits development to term (unpublished results). Recent somatic pig cloning studies have also described transfer of cleaved NT embryos 20–48 h postactivation [6, 7]. Polejaeva et al. [5] also transferred embryos within hours of completion of serial NT. However karyoplasts in their study would have been in an oocyte cytoplasmic environment in vitro for longer, with chromatin having a greater potential exposure to damaging oxygen free radicals [40–42]. In general, our interest in transferring cloned embryos in vivo as soon as possible was driven by the perception that pig oocyte and embryo culture environments remain suboptimal. Despite this our culture system was still comparable to that reported for the production of fertilized embryos by in vitro maturation, in vitro fertilization, and in vitro culture [43, 44].
Cloned embryo pregnancies in our study were established using timed administration of estradiol and parthenote cotransfer, with the latter yielding a piglet. Of 2 of the 3 pregnancies assisted with parthenotes, 1 was lost between Day 30 and Day 40, although the fetuses that survived to this stage may have all been parthenotes, which are capable of being detected by ultrasound up to Day 50 of gestation (unpublished results). Previously, somatic NT pregnancies have been maintained to term by transfer of large numbers of NT embryos (100) per recipient [6], gonadotropin injections [5], or cotransfer with mated embryos [7]. In our study, gonadotropin injections were unsuccessful, although we only attempted this strategy with 2 recipients. We did not attempt cotransfer with mated embryos out of concern that these embryos might outcompete developmentally inferior clones [6, 24]. However, transferred and mated embryos can be reliably distinguished using microsatellite genetic analyses (unpublished results). Prior to transferring cloned embryos, we exemplified the utility of parthenote cotransfer for sustaining pregnancies with 3 fertilized embryos, less than the 4 or 5 required for an unassisted pregnancy (unpublished results). We conclude, parthenote cotransfer contributed to the maintenance of the cloned embryo pregnancy to term because the number of fused and activated karyoplast-cytoplast complexes transferred (46) coupled with our cloned blastocyst production rate in vitro (2–5%) would not have yielded more than 2 viable clones to establish a pregnancy.
Our successful cloning of a piglet by somatic cell NT represents the cumulative reward of systematic research on several fronts. This research has included studies of the control and monitoring of ovulation, pig oocyte activation [14], embryo transfer, pregnancy maintenance, and NT biology. Clearly, improvements can still be made, most significantly with respect to enhancing early development in culture [7]. A better appreciation of nuclear changes within the first cell cycle will be instrumental to future success.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was funded by Geron Bio-Med. 
2 Correspondence. FAX: 44 0131 527 4493; ian.wilmut{at}bbsrc.ac.uk
Accepted: October 22, 2001.
Received: May 31, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 M. Kawahara, T. Wakai, K.-I. Yamanaka, J. Kobayashi, S. Sugimura, T. Shimizu, H. Matsumoto, J.-H. Kim, H. Sasada, and E. Sato Caffeine promotes premature chromosome condensation formation and in vitro development in porcine reconstructed embryos via a high level of maturation promoting factor activity during nuclear transfer Reproduction, September 1, 2005; 130(3): 351 - 357. [Abstract] [Full Text] [PDF]
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S J Martins da Silva, J O Gardner, J E Taylor, A Springbett, P A De Sousa, and R A Anderson Brain-derived neurotrophic factor promotes bovine oocyte cytoplasmic competence for embryo development Reproduction, April 1, 2005; 129(4): 423 - 434. [Abstract] [Full Text] [PDF]
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L. Jiang, D. B. Carter, J. Xu, X. Yang, R. S. Prather, and X. C. Tian Telomere Lengths in Cloned Transgenic Pigs Biol Reprod, June 1, 2004; 70(6): 1589 - 1593. [Abstract] [Full Text] [PDF]
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 C. J. Koh and A. Atala Tissue Engineering, Stem Cells, and Cloning: Opportunities for Regenerative Medicine J. Am. Soc. Nephrol., May 1, 2004; 15(5): 1113 - 1125. [Full Text] [PDF]
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Y.-H. Choi, L. B. Love, M. E. Westhusin, and K. Hinrichs Activation of Equine Nuclear Transfer Oocytes: Methods and Timing of Treatment in Relation to Nuclear Remodeling Biol Reprod, January 1, 2004; 70(1): 46 - 53. [Abstract] [Full Text] [PDF]
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J.-W. Lee, S.-C. Wu, X. C. Tian, M. Barber, T. Hoagland, J. Riesen, K.-H. Lee, C.-F. Tu, W. T.K. Cheng, and X. Yang Production of Cloned Pigs by Whole-Cell Intracytoplasmic Microinjection Biol Reprod, September 1, 2003; 69(3): 995 - 1001. [Abstract] [Full Text] [PDF]
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 C. R. Cogle, S. M. Guthrie, R. C. Sanders, W. L. Allen, E. W. Scott, and B. E. Petersen An Overview of Stem Cell Research and Regulatory Issues Mayo Clin. Proc., August 1, 2003; 78(8): 993 - 1003. [Abstract] [PDF]
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Y. Hao, L. Lai, J. Mao, G.-S. Im, A. Bonk, and R. S. Prather Apoptosis and In Vitro Development of Preimplantation Porcine Embryos Derived In Vitro or by Nuclear Transfer Biol Reprod, August 1, 2003; 69(2): 501 - 507. [Abstract] [Full Text] [PDF]
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B. Gasparrini, S. Gao, A. Ainslie, J. Fletcher, M. McGarry, W.A. Ritchie, A.J. Springbett, E.W. Overstrom, I. Wilmut, and P.A. De Sousa Cloned Mice Derived from Embryonic Stem Cell Karyoplasts and Activated Cytoplasts Prepared by Induced Enucleation Biol Reprod, April 1, 2003; 68(4): 1259 - 1266. [Abstract] [Full Text] [PDF]
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K. Miyoshi, S. J. Rzucidlo, S. L. Pratt, and S. L. Stice Improvements in Cloning Efficiencies May Be Possible by Increasing Uniformity in Recipient Oocytes and Donor Cells Biol Reprod, April 1, 2003; 68(4): 1079 - 1086. [Abstract] [Full Text] [PDF]
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J. Zhu, E. E. Telfer, J. Fletcher, A. Springbett, J. R. Dobrinsky, P. A. De Sousa, and I. Wilmut Improvement of an Electrical Activation Protocol for Porcine Oocytes Biol Reprod, March 1, 2002; 66(3): 635 - 641. [Abstract] [Full Text] [PDF]
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