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research-article |
Department of Animal Science/Center for Regenerative Biology,4 University of Connecticut, Storrs, Connecticut 06269
Department of Animal Science,5 National Pingtung University of Science and Technology, Pingtung 91201, Taiwan
Evergen Biotechnologies, Inc.,6 Storrs, Connecticut 06269
Department of Animal Science,7 National Taiwan University, Taipei 10617, Taiwan
Taiwan Livestock Research Institute,8 Hsin-hua, Tainan 71210, Taiwan
Trans Ova Genetics,9 Sioux Center, Iowa 51250
ABSTRACT
Premature chromosome condensation (PCC) was believed to promote nuclear reprogramming and to facilitate cloning by somatic cell nuclear transfer (NT) in mammalian species. However, it is still uncertain whether PCC is necessary for the successful reprogramming of an introduced donor nucleus in cattle. In the present study, fused NT embryos were subjected to immediate activation (IA, simultaneous fusion and activation), delayed activation (DA, activation applied 4 h postfusion), and IA with aged oocytes (IAA, activation at the same oocyte age as group DA). The morphologic changes, such as nuclear swelling, the occurrence of PCC, and microtubule/aster formation, were analyzed in detail by laser-scanning confocal microscopy. When embryos were subjected to IA in both IA and IAA groups, the introduced nucleus gradually became swollen, and a pronuclear-like structure formed within the oocyte, but PCC was not observed. In contrast, delaying embryo activation resulted in 46.5%–91.2% of NT embryos exhibiting PCC. This PCC was observed beginning at 4 h postcell fusion and was shown as one, two, or multiple chromosomal complexes. Subsequently, a diversity of pronuclear-like structures existed in NT embryos, characterized as single, double, and multiple nuclei. In the oocytes exhibiting PCC, the assembled spindle structure was observed to be an interactive mass, closely associated with condensed chromosomes, but no aster had formed. Regardless of whether they were subjected to IA, IAA, or DA treatments, if the oocytes contained pronuclear-like structures, either one or two asters were observed in proximity to the nuclei. A significantly higher rate of development to blastocysts was achieved in embryos that were immediately activated (IA, 59.1%; IAA, 40.7%) than in those for which activation was delayed (14.2%). The development rate was higher in group IA than in group IAA, but it was not significant (P = 0.089). Following embryo transfer, there was no statistically significant difference in the pregnancy rates (Day 70) between two of the groups (group IA, 11.7%, n = 94 vs. group DA, 12.3%, n = 130; P > 0.05) or live term development (group IA, 4.3% vs. group DA, 4.6%; P > 0.05). Our study has demonstrated that the IA of bovine NT embryos results in embryos with increased competence for preimplantational development. Moreover, PCC was shown to be unnecessary for the reprogramming of a transplanted somatic genome in a cattle oocyte.
conceptus, embryo, gamete biology, implantation, pregnancy
Somatic cell nuclear transfer (NT) has successfully produced live clones in several mammalian species. In most NT studies, a highly differentiated somatic nucleus is transferred into a recipient oocyte at metaphase II (MII), when nuclear modification and reprogramming take place [1–4]. During the several hours of exposure to the MII cytoplast, prior to parthenogenetic activation, the introduced somatic nucleus (at the G0 and/or G1 phase) usually undergoes nuclear envelope breakdown (NEBD) and subsequent premature chromosome condensation (PCC) [5], likely due to the high concentrations of maturation-promoting factor (MPF) present in the oocyte [6, 7].
The degree of PCC varies, depending on the MPF activity and the duration that a transplanted nucleus is exposed to the MII cytoplast [2, 5]. In most cloning studies in mice [8, 9] and cattle [10–12], parthenogenetic activation was delayed for 1–4 h after nuclear transplantation. This was believed to allow extensive nuclear-oocyte interaction, under the hypothesis that a longer exposure of the somatic donor nucleus to the oocyte cytoplast would induce PCC and facilitate nuclear reprogramming. The mechanism for the molecular basis of nuclear reprogramming is still unknown and appears to be highly complicated [13]. In mice [8] and pig [14] NT, a higher embryo development rate was achieved by inducing PCC, suggesting that PCC promotes effective nuclear reprogramming of the donor nucleus and developmentally competent gene expression in cloned embryos [7]. However, it is not known whether PCC is essential for efficient reprogramming of a somatic cell in cattle NT. We found that a 30%–40% blastocyst development rate was achieved when reconstructed oocytes were activated immediately after cell fusion [15, 16]. Meanwhile, we demonstrated that the use of an MII oocyte was required to reprogram a somatic nucleus [15]; furthermore, the reprogramming factors present in the MII oocyte became inactive within hours of oocyte activation [2, 3, 15]. Choi et al. [17] reported that a higher rate of development was achieved in restructured oocytes activated within 2 h after fusion than in those with a prolonged exposure (3–5 h) to MII oocytes. Therefore, we believe it is important to explore a series of cellular events taking place in the oocytes, as well as the morphology of an introduced somatic nucleus, when they are subjected to a brief exposure to MPF. We further wish to evaluate whether PCC is required to promote cloning efficiency.
In the present study, we determined the effect of exposing a bovine donor nucleus to MII oocyte cytoplasm by examining the morphologic progression of nuclear structures. This study, in particular, focused on the occurrence of PCC and the changes in the intracellular microtubule cytoskeleton; subsequently, the developmental potential of cloned embryos was determined in vitro and in vivo.
Unless otherwise indicated, all chemicals were purchased from Sigma (St. Louis, MO). The basic cell culture medium was Dulbecco minimum Eagle medium (DMEM; Gibco, Grand Island, NY); the basic oocyte culture medium was Medium 199 (M199) with Earle salts, L-glutamine, sodium bicarbonate at 2.2 g/L, and 25 mM Hepes (12340–014; Gibco). Dulbecco PBS (D-PBS, 15240–013; Gibco) containing 20% fetal bovine serum (FBS) (SH0070.03; Hyclone, Logan, UT) was used as the standard manipulation medium. Oocyte and embryo cultures were maintained at 39°C in 5% CO2 and humidified air, unless otherwise specified.
All animal care-related procedures described within were reviewed and approved by the University of Connecticut Institutional Animal Care and Use Committee and the Livestock Research Institute, Council of Agriculture of Taiwan, according to The Guide for the Care and Use of Agricultural Animals in Agricultural Research. The procedures were performed in accordance with the "Guiding Principles for the Care and Use of Laboratory Animals."
Donor Cell Collection, Culture, and Cell Cycle Analysis
For the in vitro portion of the present study, cultured bovine cumulus cells were used as the source of nuclear donors. Bovine cumulus oocyte complexes (COCs) were collected from a 4-yr-old Holstein dairy cow of high merit, obtained from the University of Connecticut's Kellogg Dairy Center, by standard oocyte ultrasound-guided retrieval (ovum pickup). Briefly, the COCs were recovered with an Aloka 5005 scanner fitted with a human vaginal probe (5 MHz) and a sterile hypodermic needle. With the aid of vacuum pressure, follicular fluid was aspirated along with the COCs. These COCs, in groups of 3–5, were collected and cultured in DMEM containing 20% FBS and antibiotics in Falcon 35- x 10-mm culture dishes (3001; Becton Dickinson, Franklin Lakes, NJ). Cumulus cells were expanded to different passages by a brief washing with D-PBS and subsequently subjected to a 3-min digestion by 0.05% trypsin (103140; ICN, Aurora, OH) and 0.5 mM EDTA (8991; Baker, Phillipsburg, NJ) at 37°C. Cumulus donor cells at passages 5–10 were used for NT. The cell cycle stage of quiescent cumulus cells was determined by flow cytometry [16], which showed 95% ± 2% of the cultured cumulus cells at the G0/G1 stage, as previously published by our laboratory [18].
For embryo transfer (ET) to test the in vivo viability of cloned embryos, both cumulus cells collected from the above cow and skin fibroblasts from several cow donors in the United States and Taiwan were used for NT. Skin tissues were collected by ear notching. Skin explants were subsequently cultured in Falcon 35- x 10-mm culture dishes (3001; Becton Dickinson) with 10% FBS DMEM at 37°C in 5% CO2 humidified air. Fibroblast monolayers formed around the tissue explants in about 2 wk. The explants were then removed and placed into new culture dishes. For passaging, cells were washed with 1 ml of D-PBS and then gently digested by a 3-min incubation in 250 µl of 0.05% trypsin (103140; ICN) and 0.5 mM EDTA (8991; Baker) at 37°C. The reaction was terminated by adding 5% FBS in DMEM. Subsequently, the collected cells were resuspended, divided into three new dishes, and maintained for 6–7 days. For cell storage, cells cultured to various numbers of passages were collected and frozen in 7% dimethyl sulfoxide (D-5879) and 7% glycerol (G–2025) at –80°C for 1 day and then stored in liquid nitrogen.
Nuclear donor cells were disassociated by 2–3 min of trypsinization at 37°C and resuspended in 1 ml of 5% FBS in DMEM. Prior to NT, cell suspensions were allowed to recover for approximately 30 min at 37°C.
Oocyte Maturation, NT, Parthenogenetic Activation, and Embryo Culture
Bovine COCs from slaughterhouse ovaries with at least four intact, tight layers of cumulus cells were selected, washed three times in D-PBS containing 0.1% polyvinyl alcohol (PVA; P-8136) (D-PBS + PVA), placed in CO2 gas balanced maturation medium in 1.0-ml vials, and shipped overnight to the laboratory in a portable incubator at 39°C. Maturation medium was M199 containing 7.5% (v/v) FBS and supplemented with ovine FSH at 0.5 µg/ml (NIDDK), ovine LH at 5.0 µg/ml (NIDDK), estradiol at 1.0 µg/ml (E-8875), and antibiotics. After 20–22 h postmaturation (hpm) (average, 21 hpm), matured oocytes with well-expanded cumulus layers were selected. The cumulus cells were then denuded according to the method of Du et al. [19]; the COCs were placed into 0.1% hyaluronidase in PBS, vortexed for 3 min, and repeatedly pipetted until the cumulus cells were removed completely. Oocytes with a polar body were selected for enucleation and NT.
All micromanipulations were carried out by our standard procedure [16, 19]. Enucleation was performed by making a slit in the zona pellucida with a glass needle and applying pressure until the polar body, along with the surrounding cytoplasm (estimated at approximately one-eighth total cytoplasm), was extruded. Successful enucleation was confirmed by fluorescent microscopy after staining with Hoechst 33342 (10 µg/ml). A donor cell with a diameter of around 20 µm was selected and transferred into the perivitelline space of an enucleated oocyte. Donor cell-cytoplasm pairs were fused by applying two direct current pulses of 1.67 kV/cm for a duration of 10 µsec each with a BTX 200 Electro Cell Manipulator (Biotechnologies & Experimental Research Inc., San Diego, CA) [19]. Following the completion of electric fusion, reconstructed oocytes were incubated at room temperature for 15 min to facilitate cell fusion. According to our observations, most cell fusion was completed within 15 min following the electric pulses. All fused oocytes were randomly assigned to three groups, according to the experimental design. The parthenogenetic activation of cloned embryos was accomplished by a 1-h incubation in M199 + 7.5% FBS (M199-FBS) containing 10 µg/ml cycloheximide (CHX; C-6255) and 2.5 µg/ml cytochalasin D (CD; C-8273) and then a culture in M199-FBS containing 10 µg/ml CHX for an additional 4 h.
Following activation, reconstructed oocytes were cultured for 44 h in CR1aa containing BSA at 6 mg/ml in a mixed gas environment of 5% CO2, 5% O2, and 90% N2 at 39°C. Cleavage rates were recorded, and four- to eight-celled embryos were cultured further in CR1aa containing 10% FBS on a cumulus cell monolayer for an additional 5 days. NT embryo development to cleavage (two- to eight-cell stage), morula, and blastocyst stages was evaluated on Days 2, 4, and 7, respectively, according to the standard of the International Embryo Transfer Society.
Immunohistochemistry and Laser-Scanning Confocal Microscopy
The reconstructed oocytes were collected at different time periods, according to the experimental design, and fixed in a microtubule stabilizing buffer containing 2% formaldehyde (F79–500; Fisher Scientific, Fairlawn, NJ), 0.5% Triton X-100 (T-8717), 1 mM taxol (T-7402), aprotinin at 10 U/ml (A-6279), and 50% deuterium oxide (D-4501) at 37°C for at least 30 min [20]. Fixed oocytes were washed in washing buffer (PBS containing 3 mM NaN3 [S-8032], 0.01% Triton X-100, 0.2% nonfat dry milk, 2% normal goat serum, 0.1 M glycine [G-7126], and 2% BSA [A-3311]) three times and subsequently maintained in the same washing buffer overnight at 4°C for blocking and permeabilization. Cloned embryos were double-stained to visualize microtubules and DNA [18]. Samples were incubated overnight at 4°C with the first antibody—mouse anti-
tubulin (T-5168, 1:200 dilution). After rinsing three times in the washing buffer, reconstructed oocytes were kept at 4°C overnight in a second antibody—fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G (F-0257, 1:200 dilution). The reconstructed oocytes were mounted in PBS containing 10 µM propidium iodide (P-4170) to stain for DNA and observed under laser-scanning confocal microscopy (Leica TCS SP2; Leica, Mannheim, Germany).
Sampling of Reconstructed Bovine Embryos and Morphologic Evaluation
The present study consisted of three experimental treatments that were defined as immediate activation (IA), also called simultaneous fusion and activation; delayed activation (DA); and IA with aged oocytes (IAA). Enucleation (removal of the MII plate) began at 21 h postoocyte maturation. In the IA and DA groups, cell fusion was performed at 25 hpm. The time of completion of cell fusion (25 hpm was designated 0 h and marked as the onset for sample collection). In group IA, immediately after cell fusion (activation at 25 hpm), restructured oocytes were allocated to the activation regime of a 5-h incubation in combined CD/CHX and CHX medium, as described above. With this activation procedure, MPF activity in the oocyte decreases to a basal level within 1 h postactivation, and the oocyte is dramatically driven away from the MII phase into, presumably, the S phase [21]. In group DA, fused oocytes were incubated in M199-FBS for 4 h after cell fusion prior to parthenogenetic activation (activation at 29 hpm); subsequently, they were allocated to the same activation regime as group IA. This incubation period allowed an extended exposure of the introduced G0/G1 nucleus in the oocyte's cytoplasm that contained a high level of MPF to induce PCC. In group IAA, oocytes were enucleated at 21 hpm. Enucleated cytoplasts were subsequently cultured for 4 h before being subjected to donor cell insertion, cell fusion, and simultaneous activation. Therefore, groups DA and IAA were treated with a regime that eliminates the age difference between two groups at the time of activation (groups DA and IAA, 29 hpm). In groups IA and IAA, the activation treatments were the same, except for oocyte age difference at activation (group IA, 25 hpm vs. group IAA, 29 hpm). In all three groups, reconstructed oocytes were collected and fixed at 0, 1, 2, 4, 6, 12, 18, or 24 h postfusion (hpf). The samples were also collected from three groups at 44 hpf to determine the events of mitosis of NT embryos. The progressive changes over time of the donor nucleus in the oocyte were categorized as follows: enlarged nucleus or swelling, PCC, and the arrangement of microtubules/asters. These parameters were examined by confocal microscopy. To determine nuclear change, the nuclear area of each reconstructed embryo was analyzed by the Leica confocal software program (TCS SP2). The degree of nuclear swelling was determined by comparison to the average area value of cumulus nuclei from donor cells prior to NT. The evaluation of nuclear swelling was subjectively defined. Those nuclei with areas below (<100%) or similar to (100%–120%) the average value determined for cumulus nuclei were categorized as unswollen, while those with areas that were 120% or greater than the average value were designated swollen.
In Vivo Developmental Potentials of NT Embryos Derived from IA and DA Protocols
The cloned embryos, derived from either the IA or DA group, were transferred into recipient cows to test their in vivo viability. The NT embryos were either freshly transferred or cryopreserved by liquid nitrogen surface vitrification [19] prior to ET. Recipient cattle were synchronized by a regime of two injections (25 mg/injection i.m.) of prostaglandin F2 (Lutalyse; Upjohn Co., Kalamazoo, MI) at an interval of 11 days. The onset of standing heat estrus of recipients was monitored closely and recorded as Estrus Day 0. On Day 7 postestrus, recipients were selected by palpation per rectum to verify the presence and size of the corpus luteum (CL). Either fresh or thawed blastocysts (one or two per straw) were loaded into 0.25-ml French straws containing ViGro Holding Plus (AB Technology, Pullman, WA). One or two embryos were deposited nonsurgically into the uterine horn ipsilateral to the ovary with the CL. Pregnancy was determined by palpation per rectum or ultrasound monitoring on Day 70 after transfer. All pregnancies were allowed to carry on to term.
The data on nuclear swelling (nuclear area) were subjected to an arcsine transformation. The transformed data were then analyzed by ANOVA (General Linear Model, 11.0; SPSS, Inc., Chicago, IL) [22]. For the analysis of the in vitro and in vivo development of cloned embryos and the proportions of embryos that reached cleavage, developed to the eight-cell stage, and to the blastocyst stage, as well as the conception rates on Day 70 and calving, data were transformed by an arcsine transformation and analyzed by a Student t-test. A P value of less than 0.05 was considered statistically significant.
Nuclear Progression and PCC in Reconstructed Bovine Embryos
Cultured cumulus cells, presumably at G0/G1 [18], were designated for use in our NT experiments. The nuclear areas of introduced donor cells, in IA, DA, and IAA groups, regardless of their activation regime, were found to increase progressively and directly with the time interacted within a recipient oocyte (Fig. 1). As a comparative control, the average areas of cumulus nuclei, from donor cells prior to NT, were measured as 61 ± 11 µm2 (n = 29, Fig. 2A). There was no significant nuclear swelling in oocytes from groups IA, DA, and IAA at 0 h (Fig. 2B), 1–2 hpf (Figs. 1, panel B, and 2C), when the area was compared with the average cumulus nuclear value; however, a dramatic increase in the size of the nucleus (P < 0.05) was observed from 4 to 24 hpf (Figs. 1, panel B, and 2, D–F) in the three groups. The range of the nuclear area of donor nuclei in group DA varied from 61 ± 11 µm2 (unswollen) to as high as 680 ± 24 µm2 (hugely swollen) (Fig. 1, panel B). Pair-wise comparisons between group IA and IAA embryos did not show any difference in nuclear swelling (Fig. 1, panel B) at any of the collecting periods. With the same pair-wise comparison model between either group IA and DA or group IAA and DA embryos (Fig. 1, panel B), the degree of swelling remained similar, from 0 to 6 hpf; however, the oocytes in group DA showed significantly greater nuclear swelling than those in groups IA or IAA at 12–24 hpf (Fig. 1, panel B). The size of the nuclear area peaked at 18 hpf in groups IA (Fig. 2F), IAA, and DA (Fig. 3D) and then declined, in all three groups, by 24 hpf (Fig. 1, panel B).
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The onset of mitosis in the three groups of NT embryos began to be manifest at 12–18 hpf (Fig. 1, panel A). This was evidenced by the varied appearance of prophase (Fig. 2G), a typical metaphase (Fig. 2H), a telophase (Fig. 2I) at the first cell cycle, and a metaphase at the second cell cycle (Fig. 2J) during the period from 18 to 24 hpf (Fig. 1, panel A). The continuation of nuclear development in these NT embryos was evident by the four-cell (Fig. 2K) and eight-cell (Fig. 2L) stage embryos observed 44 h after cell fusion.
As indicated in Figure 1, panel A, PCC was not observed in group IA or IAA at any of the time points examined (0–24 hpf). In group DA (Fig. 1), PCC was not found at 0, 1, or 2 hpf; however, it appeared by 4 hpf in 72.5% of embryos and increased at 6 hpf to the peak of 91.2% of embryos. After that, the proportion of oocytes showing PCC subsequently decreased to 54.8% (12 hpf), 68.6% (18 hpf), and 46.5% (24 hpf) (Fig. 1). In addition, the morphology of PCC was discerned as one (Fig. 3A), two (Fig. 3B), and multiple (Fig. 3C) scattered chromosomal clusters; however, the number of chromosomes was difficult to examine in those PCC constructions. The percentage of chromosome condensation into a single cluster was observed to decrease in collected embryos from 75.8% (n = 29) at 4 hpf to 15.0% (n = 20) at 24 hpf. Instead, many of these embryos in group DA displayed various numbers of scattered and condensed chromosome formations between 4 and 24 hpf (Fig. 3, B and C).
Among those NT embryos having pronuclear appearances in group IA, all possessed one nucleus until 12 hpf. Embryos began to show double nuclear structure at 18 hpf; by 24 hpf, as many as 28.0% (n = 25) had two nuclei (Fig. 4), presumably indicating that these embryos had entered either the telophase or posttelophase stage of the first cell cycle (Fig. 2I). In group IAA, all embryos with nuclear appearances showed one pronuclear-like structure from 0 to 24 hpf. In contrast, a varied range of embryos in group DA (17.6%–40.0%) contained either two (Fig. 3E) or multiple (Fig. 3F) nuclear structures during 12–24 hpf (Fig. 4). Overall, irregular chromosomal clusters and the appearance of multiple nuclei were the predominant features of embryos from DA (group DA) (Fig. 3). The emission of the second polar body was not observed in all groups (IA, IAA, and DA), probably because of the treatment of cytochalasin during the activation.
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Microtubule Organization in Cloned Cattle Embryos
The microtubule organization in cloned embryos was examined under laser-scanning confocal microscopy. In groups IA and IAA, highly stained microtubule structures were evident in the donor cell prior to fusion (Fig. 2A). However, an aster or a microtubule organization center could not be seen in most reconstructed oocytes shortly after fusion (Fig. 2, B and C). With time and the progression of swelling of the introduced nucleus, one (Fig. 2D) or two (Fig. 2E) tufts of microtubules developed in the proximity of the donor nucleus at 1 hpf (Fig. 4), and these structures continued to develop into characteristic asters (two asters; Fig. 2, F and G) in the embryos collected, starting at 2 hpf (Fig. 4). At 24 hpf, all NT embryos with nuclei in group IA showed two asters (Fig. 4). Likewise, of NT embryos in group IAA, 84.2% (n = 36) showed two asters at 6 hpf, and a similar percentage of appearance of double asters was maintained until 24 hpf (90.0%, n = 10) (Fig. 4). In group DA, the aster was observed beginning at 1 hpf (Fig. 4), and it continued to develop in those embryos with one aster (Fig. 3D) and two asters (Fig. 3, E and F) around the swollen nucleus/nuclei (Fig. 4). The formation of asters seemed to be associated only with the presence of nucleus/nuclei (Fig. 3, D–F). When PCC clusters were induced in group DA, microtubules were found in the region of the chromosomal clusters; however, the typical aster structure was not observed in any of the oocytes displaying PCC (Fig. 3, A–C). All NT embryos in group DA that contained a pronuclear structure showed an aster after 4 h of incubation in M199 prior to activation (Fig. 4), indicating that aster formation was independent of parthenogenetic activation.
Developmental Potential of Cloned Embryos Derived from IA or DA
The results of NT (Table 1) indicated that a higher fusion rate was observed in aged oocytes (group IAA, fusion at 29 hpm) than in young oocytes (groups IA and DA, fusion at 25 hpm). A significant improvement of cleavage (two to eight cells) rates and the subsequent development to blastocysts (50.0% vs. 11.6%) were achieved by IA (group IA) when compared to DA (group DA). The efficiency of NT in group IA, as judged by the rate of blastocyst development, based on the number of oocytes fused, reached as high as 59.1%. Overall development of cloned embryos in group IAA was also significantly higher than that in group DA (40.7% vs. 14.2%), although the oocyte age of both treatments was the same at the time of parthenogenetic activation (29 hpm). Thus, IA proved much more effective than DA; the latter resulted in a blastocyst development rate of only 14.2% (P < 0.05).
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The cleavage between groups IA and IAA was similar, and the subsequent preimplantational development to blastocysts, as well as the overall blastocyst percentage (blastocyst/fused oocytes), was higher in group IA than in group IAA, but it was not significant (59.1% vs. 40.7%, P = 0.089).
On the basis of the similar nuclear remodeling and progression results and comparable in vitro developmental potential between groups IA and IAA, cloned embryos derived from either IA or DA were subjected to ET to examine their viability to term development. Table 2 indicates the pregnancy and calving data with either fresh NT embryos or vitrified NT embryos, derived from fibroblasts and cumulus cells as nuclear donors. In group DA, all NT embryos (of cumulus and fibroblast origin) were freshly transferred into recipients. In group IA, NT embryos derived from cumulus donors were vitrified prior to ET. In contrast, a total of 113 fibroblast-derived NT embryos in group IA were either freshly produced (n = 49) or vitrified (n = 64) and subsequently transferred into recipients (fresh ET, n = 44; vitrified ET, n = 32; total ET, n = 76; Table 2). On Day 70 of gestation, no statistically significant difference in established pregnancies was indicated between transfers of blastocysts from group IA (cumulus, 22.2%, n = 18; fibroblast, 9.2%, n = 76; overall, 11.7%) and group DA (cumulus, 11.1%, n = 18; fibroblast, 12.5%, n = 112; overall, 12.3%) (P > 0.05). There was no difference of pregnancy on Day 70 between fresh and vitrified embryos in group IA when fibroblasts were used as donors for NT (Table 2). There were two live calves born in each of groups IA (11.1%) and DA (11.1%) in which cumulus cells were used as donor cells. When fibroblast cells were used for NT, a high fetal loss was observed after Day 70 of ET in both groups IA (four abortions) and DA (nine abortions) (Table 2). One stillborn clone from each activation treatment was observed; two (2.6%) and four (3.6%) live clones were delivered from groups IA and DA, respectively. The overall term development to live clones was 4.3% for group IA (four live clones, n = 94) and 4.6% for group DA (six live clones, n = 130), respectively.
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Our NT study in cattle clearly demonstrated that an occurrence of PCC, presumably induced by MPF, is not essential for the effective reprogramming/remodeling of a somatic nucleus introduced into the cytoplasm of an MII oocyte. The direct exposure of a somatic genome to an MII oocyte rich in MPF was proven to be sufficient for the successful reprogramming of a differentiated somatic nucleus [3, 7, 15]. NEBD and PCC have been shown to occur within a short duration (usually 2–4 h) after a donor nucleus was transferred into an enucleated MII (nonactivated) mammalian oocyte [2, 5, 8, 12, 23]. Nevertheless, the mechanisms involved during the interaction of a donor nucleus with an oocyte's cytoplasmic environment, containing high levels of MPF, and the extent of chromosomal remodeling due to PCC remain obscure and quite controversial. Several previous studies reported that a prolonged exposure of a donor nucleus, particular a G0/G1 nucleus, to a nonactivated oocyte, in order to induce PCC, was beneficial for nuclear reprogramming [14, 17, 23]. In mice, Wakayama et al. [8] showed that a high proportion of enucleated oocytes developed to morulae/blastocysts when they were activated following a prolonged exposure of the adult somatic nucleus to the oocyte's cytoplasm. The inclusion of a prolonged interval between nuclear injection and oocyte activation was believed to be beneficial for both pre- and postimplantational development [24]. Somatic pig clones could be produced by the combined approaches of simultaneous NT fusion/activation and followed with serial nuclear exchange technology [25]; however, pigs [26, 27] shared a similarity with mice [24] in that the induction of PCC was in association with beneficial nuclear reprogramming. The proportion of reconstructed porcine oocytes developing to the blastocyst stage was lower when activation was immediate [14]. Wakayama et al. [8] believed one of the key steps for the successful cloning of mice was to induce PCC and the subsequent pronuclear-like vesicle formation in the injected nuclei. It was believed that in other species, such as rats, the failure to produce live clones was attributable to insufficient PCC induction [28].
Nevertheless, our results show that PCC is not a necessary process for nuclear reprogramming and subsequent embryo development in cattle. In both groups IA and IAA, when donor nuclei were exposed to the presumably MPF-rich cytoplasm for only a short time prior to chemical activation, PCC was not observed. We did observe, however, a rapid nuclear swelling that might be capable of inducing nuclear de-differentiation into a pronuclear-like stage in both groups. Our results are in agreement with those of Fulka et al. [29], who reported that exposure of the donor nucleus to the nonactivated oocyte, even for a very short time, had beneficial effects on nuclear remodeling. Our results indicate that the molecular remodeling of an introduced nucleus still occurs within the cytoplast of an activated oocyte, along with the progressively increasing nuclear swelling; however, inducing dramatic chromosomal structural reformation, such as PCC, can, and likely should, be avoided [3]. Our present study demonstrates that direct nuclear and cytoplasmic interactions are sufficient for reprogramming and subsequent embryo development, regardless of the presence of PCC.
It has been known that the formation of PCC may lead to dramatic chromosomal changes, possibly causing a range of DNA damage (e.g., fragmented chromatin, joined chromatin, chromosomal breakage) or loss of chromosomes [29, 30], especially when the donor cell cycle is not compatible with that of the recipient oocyte [5]. Our results showed that a prolonged exposure (up to 4 h) of a donor nucleus to the presumptively high MPF levels of the preactivation oocyte was not beneficial for the preimplantational blastocyst development of cloned embryos. The in vitro developmental potential was significantly lower in group DA (14.2%) than in groups IA (59.1%) and IAA (40.7%) (Table 1). The development of NT embryos, derived from cultured skin fibroblasts, was also higher with IA than with DA (unpublished results; data not shown). The effects of IA or DA in previous reports of bovine somatic cloning are controversial. Wells et al. [12] achieved a rate of 27.5% blastocysts when cultured adult mural granulosa cells were exposed to a cytoplast for a prolonged period of 4–6 h prior to activation; however, a direct comparison between IA and a 4- to 6-h incubation prior to activation (DA) was not performed in their study. The discrepancies in the results from various studies might also be explained by differences in selecting somatic donor cells (bovine embryonic stem-like cells vs. somatic cumulus cells) [31], age of oocyte recipients [32], experimental conditions [23], and protocols used [32–34]. Akagi et al. [32] found that the DA method improved the in vitro development potential of NT embryos. In the Akagi et al. study, relative aged oocytes (24 hpm) were subjected to IA; in our experiment, young oocytes at 21 hpm were arranged for IA treatment. In our IAA treatment, oocytes at a similar age (25 hpm) were used for NT and subsequent activation. In this case, the age difference between the IA and DA groups was eliminated at the time of activation (IAA and DA, activation at 29 hpm). As shown in our results, group IAA demonstrated significantly improved preimplantational development in comparison to that in group DA. While the pattern of nuclear progression and remodeling in group IAA was similar to what occurred in group IA with relatively young oocytes, blastocyst development was higher in group IA (P = 0.089). We believe that young oocytes had a more competent capability to reprogram an oocyte if better oocyte activation and preimplantational culture were used [15, 21]. On the other hand, recently, more reports have demonstrated that the proportion of embryos with normal chromosomal ploidy decreased as the incubation time prior to activation was prolonged. Decreased blastocyst development (0%–8.6%) was reported when the exposure period was longer than 3 h [17]. A plausible explanation for inferior in vitro development of NT embryos in cattle may be abnormal chromatin structure, anomalous pronuclear formation, or both [17], resulting in numeric chromosome errors, such as polyploidy and mixoploidy [35]. Our ET results demonstrated that vitrified NT bovine embryos derived from IA had in vivo survivability similar to embryos derived from a DA treatment. This suggests that reprogramming events, in the absence of PCC, did not have a detrimental effect on the in vivo viability of cloned cattle embryos. In fact, our IA procedure had resulted in fully developed and healthy newborns in the past [16, 19, 36]. In the present study, four or six live clones were produced from either IA or DA, respectively, indicating that the further implantational development potential was equivalent in the resultant cloned blastocysts, regardless of the manner of activation. Nevertheless, the efficiency of generating cloned blastocysts was significantly increased with IA (40.7%–59.1% vs. 14.2%).
Nuclear reprogramming is a complicated process, involving not only cellular nuclear-cytoplast interaction [5] but also epigenetic modification and molecular differentiation [13]. It is believed that the processes of nuclear swelling, remodeling of the somatic nucleus, and chromosomal modification are required for successful reprogramming [3]. NT oocytes in group DA had significantly larger nuclear sizes than those in groups IA and IAA during 12–24 hpf. We assumed that during the process of reformation of a pronuclear-like structure from the PCC chromatin phase, swelling factors existing in cytoplasm would be likely to more readily participate in nuclear reconstruction. As a result, a dramatically enlarged nucleus (or nuclei) could be formed in the DA group. Because only 91.2% of the NT oocytes were observed to show PCC in group DA, we cannot also exclude the possibility that some of the oocytes in group DA actually did not involve PCC-dependent nuclear reformation. A small proportion of oocytes in group DA might directly develop nuclear swelling similar to that which occurred in group IA; however, this proportion was believed to be relatively minimal. The molecular reprogramming factors, or at least the reprogramming initiation molecules, certainly reside in the cytoplast of the matured oocyte, and their function may stop postparthenogenetic activation [15]. MPF activity in an oocyte was reported to be at a basal level 1 h postactivation [3, 21]. MPF or mitogen-activated protein kinase activity was shown not to be a direct regulatory factor for reprogramming in cattle [3]. However, our results cannot rule out the possibility that MPF is acting as an initiator of reprogramming, because a somatic nucleus was introduced into a cytoplast containing high concentrations of MPF [21, 37]. In our groups IA and IAA, the nuclear envelope was observed to be intact, and the entire area of the nucleus had expanded during activation. We hypothesize that the reprogramming factors can be incorporated into the nucleus during nuclear swelling without disrupting the nuclear envelope membrane; thus, reprogramming of the nucleus occurs without dramatic chromosomal restructuring, such as PCC.
Cattle may represent a unique and excellent species as a model to extrapolate about humans, while they appear to be distant from mice and pigs with respect to IA and DA. At the very least, an improvement of the reprogramming in the context of somatic cell NT requires the erasure of cellular memory inherent in the donor cell and a reestablishment of patterns of gene expression and their regulation, such as epigenetic methylation and acetylation, for competent embryogenesis and differentiation [38]. One report demonstrated that DNA methylation supported intrinsic epigenetic memory in mammalian cells [39]. The authors of this report found that DNA methylation is not required for the establishment of the maintenance of silent chromatin status; however, it conferred to the chromatin structure a long-term, intrinsic epigenetic memory that prevents gene reactivation. Recently, a typical egg protein nucleoplasmin was reported to induce massive chromatin decondensation that resulted in nuclear swelling and to significantly influence epigenetic modification, such as histone phosphorylation and acetylation [40]. Although it is possible that molecules, such as nucleoplasmin, serve to catalyze the exchange of somatic and embryonic histones and de-repress gene expression, the long-term benefit has not been determined. A comprehensive study with molecular and cellular mechanistic approaches, in combination with a cattle cellular reconstruction system via somatic cell NT, will help address interesting questions related to reprogramming events. Among these questions is whether IA improves the process of demethylation and histone acetylation or whether induced PCC affects a delayed demethylation, alters exchange of DNA proteins, and suppresses the de-repression of genes [41].
According to our data, it was evident that the aster was associated with the introduced nucleus, suggesting that the centrosome, or microtubule organization center, was introduced into the oocyte with the donor cell via membrane fusion. We observed the duplication and splitting of the aster following NT in all groups—IA, IAA, and DA. The aster initially formed at the nuclear poles as a fusiform structure during the first mitotic phase. This phenomenon is similar to that described by Navara et al. [42]. Because of the experimental design, we were able to observe that the formation and distribution of asters were independent of parthenogenetic activation but were closely associated with pronuclear-like structures (Figs. 2 and 3). We found it most interesting that no aster formation was observed in the reconstructed oocytes that were induced to PCC by DA. The mechanisms inherent in PCC that inhibits or prevents aster formation are unknown and warrant further investigation.
In conclusion, our study has demonstrated that PCC is not an indispensable prerequisite for the competent reprogramming of a differentiated somatic genome in cattle. The direct exposure of the donor nucleus to the MII cytoplast, presumably containing high levels of MPF, for a relatively short time is sufficient to trigger a cascade of nuclear reprogramming and developmental events. A higher efficiency of blastocyst development was obtained by IA. Similar in vivo developmental potentials of NT embryos that were derived from substantially varied protocols (IA vs. DA), along with the birth of live clones from both treatments, suggest that cattle represent a unique species with a greater plasticity available for mechanical, biochemical, and physiological manipulation.
ACKNOWLEDGMENTS
We greatly thank Drs. Robert H. Foote and Myrna E. Watanabe for providing valuable discussion and comments in the preparation of this manuscript.
FOOTNOTES
3These authors contributed equally to this work. 
1Supported in part by the Research Fund from Evergen Biotechnologies, Inc., Storrs, Connecticut, awarded to F.D., a Yankee Ingenuity grant from Connecticut Innovation, Inc., Connecticut, awarded to X.Y., and a Livestock Research Institute, Council of Agriculture, Taiwan grant to S.N.L.
Correspondence: 2Fuliang Du, Evergen Biotechnologies, Inc., 1392 Storrs Rd., Unit 4213, Room 106, Storrs, CT 06269. FAX: 860 486 6628; e-mail: fuliangd{at}evergen.com
Received: 3 May 2006.
First decision: 26 May 2006.
Accepted: 31 October 2006.
REFERENCES
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