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Ruakura Research Centre, Reproductive Technologies, AgResearch Ltd., Hamilton, New Zealand
ABSTRACT
It has been postulated that mammalian nuclear transfer (NT) cloning efficiency is inversely correlated with donor cell differentiation status. To test this hypothesis, we compared genetically identical and increasingly differentiated donors within the myogenic lineage. Bovine male fetal muscle cells were cultured for 1–6 days in vitro. The proportion of cells displaying the following antigens was quantified by immunofluorescence microscopy: MYOD1, MYF5, PAX7, MYOG, DES, MYH, and 5-Bromo-2-deoxyuridine. Based on the antigen profile of both bulk populations and individually size-selected cells prepared for NT, donors serum-starved for 1, 4, and 5 days were classified as myogenic precursors (MPCs), myotubes (MTs), and muscle-derived fibroblasts (MFs) with purities of 92%, 85%, and 99%, respectively. Expression of the following transcripts was measured by RT-PCR in 1) cells selected for NT, 2) metaphase II oocytes, 3) NT couplets, 4) NT reconstructs, 5) NT two-cell embryos, and 6) NT blastocysts: MYOD1, MYF5, PAX7, MYOG, MYF6, ACTB, and 18S rRNA. Muscle-specific genes were silenced and remained undetectable up to the blastocyst stage, whereas housekeeping genes 18S and ACTB continued to be expressed. Differentiation status affected development to transferable embryos (118 [23%] of 520 vs. 93 [11%] of 873 vs. 66 [38%] of 174 for MPC vs. MT vs. MF, respectively, P < 0.001). However, there were no significant differences in pregnancy rate and development to weaning between the cell types (pregnancy rate: 14 [64%] of 22 vs. 8 [35%] of 23 vs. 10 [45%] of 22, and development: 4 [18%] of 22 vs. 2 [9%] of 23 vs. 3 [14%] of 22 for MPC vs. MT vs. MF, respectively).
assisted reproductive technology, developmental biology, early development, embryo
In the 10 years since the cloning of Dolly from a differentiated mammary gland cell [1], cloned offspring have been produced by somatic cell nuclear transfer (SCNT) in 15 mammalian species. Despite this ever-growing list of cloned species, SCNT remains a very inefficient procedure. Typically, cloning efficiency, quantified as the proportion of all embryos transferred into surrogate mothers that develop into viable offspring, is about 1%–5% [2]. A number of approaches have been devised to increase the frequency of success in donor cell reprogramming and improve somatic cattle cloning efficiency. These include treating donor cells with pharmacologic agents to alter their epigenetic marks [3, 4] or cell cycle stage [5], fusing transiently permeabilized cells containing artificially condensed chromatin [6], activating with sperm rather than artificially [7], or aggregating somatic NT embryos [8, 9]. Although some of these treatments have resulted in improved in vitro development or gene expression, their beneficial effect on in vivo development has not been conclusively demonstrated.
We have been focusing on the first step of the cloning procedure; namely, the choice of nuclear donor cell type and differentiation status [10]. During cell differentiation, somatic genomes acquire highly specialized epigenetic modifications of DNA and DNA-binding proteins [11, 12]. These changes include the appearance of DNA methylation, histone trimethylation, and other inactive chromatin marks correlating with differentiation stage-specific gene silencing [13] and a genomewide reduction in the total number of genes expressed in terminally differentiated cells [14]. It has been postulated that mammalian cloning efficiency is inversely correlated with the donor cell differentiation status [15–17]. This hypothesis is mainly supported by three lines of evidence from comparative mouse cloning experiments using 1) progressively more advanced blastomere donor nuclei from early cleavage stages [18–20], 2) embryonic stem cells [21–23], and 3) terminally differentiated somatic donor cells; namely, lymphocytes [24, 25] and neurons [26–30]. These comparisons have demonstrated that early blastomeres result in much higher cloning efficiency than somatic cells [19]. However, due to confounding parameters such as different NT procedures, genetic backgrounds, sex, and cell cycle stages, these studies have failed to conclusively determine whether differentiation status significantly affects cloning efficiency within somatic donor cell lineages [10].
In this study we have compared phenotypically distinct but genetically identical donors within the same somatic lineage in order to conclusively correlate mammalian cloning efficiency with differentiation status. We chose the skeletal muscle lineage as a model system. This lineage has been successfully used for SCNT before [31, 32]. Myogenic precursors and their terminally differentiated progeny, postmitotic myotubes, are morphologically and molecularly well defined and can be readily isolated from fetal muscle in sufficient numbers [33]. Conditions to culture and differentiate bovine fetal muscle cells in vitro have been described previously [34]. Mammalian myogenic in vitro differentiation proceeds through several stages that can be distinguished by a well-defined set of molecular markers [35]. First, quiescent muscle stem cells, or satellite cells, become activated and give rise to proliferating myogenic precursor cells (MPCs or myoblasts). Both satellite cells and their MPC derivatives express Pax7, indicating their commitment to the myogenic lineage [36]. MPCs will then stop dividing, fuse to form a syncytium, and differentiate into multinucleated myotubes (MTs). Fusion is not necessary for terminal differentiation, and both processes can be experimentally uncoupled in vitro, resulting in fully differentiated mononucleated myotubes [37]. Myogenesis is orchestrated by the basic helix-loop-helix (bHLH) DNA-binding transcription factors MYOD1, MYF5, MYOG, and MYF6 (also known as MRF4), collectively referred to as muscle regulatory factors, or MRFs. Embryologic and genetic studies in several species have delineated the complex interactions between different MRFs [38]. MYOD1 and MYF5 have similar and overlapping functions during myoblast determination, whereas MYOG and MYF6 play a later role during muscle differentiation and maintenance. Determined but undifferentiated MPCs can still respond to extracellular signals that control proliferation and migration, whereas terminally differentiated postmitotic MTs cannot. Concomitant with the transition from determination to differentiation, structural marker genes are expressed, including muscle-specific myosin heavy chain (MHY) isoforms [39] and the intermediate filament protein desmin (DES). In bovine, in contrast to rat, mouse, or chicken, DES is not detected in replicating muscle cells; only in postmitotic myotubes [40]. In addition, MPCs and MTs display large-scale differences in pericentric heterochromatin organization that are associated with epigenetic maintenance of differentiation [41]. These various markers allow specifically selecting cells of divergent differentiation status, making the muscle lineage a good candidate to test the hypothesis that cell differentiation, plasticity, and cloning efficiency may be causally related.
Chemicals were supplied by Sigma-Aldrich (Auckland, New Zealand), and all embryo manipulations were carried out at 38.5°C unless indicated otherwise. All NT experiments were direct contemporaneous comparisons (i.e., within each NT run all parameters other than nuclear donor cell type—such as pool of oocytes, activation method, and culture medium—were kept the same). Investigations were conducted in accordance with the regulations of the New Zealand Animal Welfare Act 1999.
Isolation of Nuclear Donor Cells
Donor cells were isolated from the hindquarter muscle of a male Day 150 (D 150) bovine fetus that was collected from the local abattoir. The muscle was trimmed of skin and fat, diced into 1- to 2-mm3 blocks, cryopreserved in 10% dimethyl sulphoxide, 45% fetal calf serum (FCS), and 45% culture medium (Dulbecco modified Eagle medium [DMEM]/F12 [Invitrogen, Auckland, New Zealand]), and stored in liquid nitrogen. Approximately 0.25 g tissue was cryopreserved per 1 ml cryovial (Nalge Nunc International). For MPC isolation, a vial of tissue was thawed at 38°C 48 h prior to the NT experiment and washed three times in PBS before being digested in 20 ml of 0.25% porcine trypsin 1:250 (Invitrogen) in PBS for 50 min at 38°C on a slow rocker (150 rpm). Trypsin was blocked in 30 ml warm PBS/10% FCS and the digest triturated for 3 min using a 10-ml disposable plastic pipette (Nalge Nunc International) before being filtered through 70-µm and 40-µm nylon cell strainers (BD Falcon) to remove undigested material. The suspension was centrifuged at 2500 rpm for 10 min, and the supernatant was discarded. The pellet was resuspended in 10 ml DMEM/F12 medium containing 10% FCS, 1 mM sodium pyruvate, 100 U/ml penicillin, and 0.25 µg/ml Fungizone Antimycotic (Invitrogen). Cells were preplated into a 10-cm tissue culture dish for 1 h at 38.5°C at 5% CO2 in air atmosphere to allow for attachment of contaminating fibroblasts. The cell suspension was transferred into a 10-cm tissue culture dish coated with Matrigel (Becton Dickinson Ltd., Auckland, New Zealand) and incubated overnight at 38.5°C in a humidified incubator (5% CO2 in air) to allow for attachment of MPCs. After 17–20 h, cells were washed three times with PBS and serum starved for another day in DMEM/F12 with 1.0% FCS. For myotube (MT) isolation, cells after preplating were seeded onto Matrigel-coated glass coverslips at approximately one third of the MPC culture density (to inhibit large multinucleated fibers from forming) and incubated overnight at 38.5°C in a humidified incubator (5% CO2 in air). After 17–20 h, cells were washed three times with PBS and serum starved for 4 days in DMEM/F12 with 1.0% FCS. As a control group, muscle fibroblasts (MFs) that had attached within 1 h during preplating were collected from the noncoated preplate culture. The cells were cultured for 7 days to allow for contaminating MPCs to differentiate into MTs, which were removed by brief trypsin treatment. The remaining fibroblasts were passaged and cultured for another 4 days before cryopreservation at passage 2. Prior to NT, cells were thawed, passaged, and seeded at 5.0 x 104/cm2. After 17–20 h, they were washed three times with PBS and serum starved for 5 days in DMEM/F12 with 0.5% FCS [42].
The following antigens were quantified by immunofluorescence microscopy: myogenic differentiation factor MYOD1 (mouse-monoclonal; catalog no. 554130; BD Pharmingen), myogenic factor MYF5 (rabbit-polyclonal; sc-302; Santa Cruz Biotechnology), paired box transcription factor PAX7 (mouse-monoclonal, developed by A. Kawakami, Developmental Studies Hybridoma Bank, University of Iowa), myogenin or MYOG (mouse-monoclonal F5D, developed by W. E. Wright, Developmental Studies Hybridoma Bank, University of Iowa), DES (mouse-monoclonal; Sigma D1033), and sarcomeric myosin heavy-chain MYH (mouse-monoclonal MF20, developed by D. A. Fischman, Developmental Studies Hybridoma Bank, University of Iowa). For bulk population analysis, a minimum of 150 cells for each antigen were counted per replicate. Cells were cultured in either Matrigel-coated eight-well LabTec II chamber slides (Invitrogen) or on Matrigel-coated glass coverslips in DMEM/F12 containing either 1% or 10% FCS for 1 to 6 days. Every 24 h, cells were fixed, and the proportion of Hoechst 33342-positive cells expressing the antigen was counted. Cells were fixed in 4% (w/v) paraformaldehyde/4% (w/v) sucrose in PBS for 15 min at room temperature or in 1% paraformaldehyde (w/v)/1% (w/v) sucrose in PBS overnight at 4°C. Cells were washed three times in PBS, quenched in 50 mM NH4Cl in PBS for 10 min, permeabilized in 0.1% (v/v) Triton X-100 in PBS for 10 min at room temperature, and blocked in 5% fatty acid-free BSA, 5% FCS, in PBS for at least 30 min. Primary antibodies were applied overnight at 4°C in a wet chamber (MYOD1 1:100, MYF5 1:200, PAX7 1:50, MYOG 1:100, and DES 1:100), washed three times in PBS and incubated with the following secondary antibodies for 30 min at 38.5°C: Alexa Fluor 546 goat anti-mouse IgG (A-11030; 1:300; Molecular Probes Inc.) and Cy2-conjugated AffiniPure donkey anti-rabbit IgG (711-225-152; 1:300; Jackson ImmunoResearch Laboratories Inc.) All antibodies were diluted in blocking buffer (5% BSA, 5% FCS, in PBS). During secondary antibody incubation, cells were routinely counterstained with 5 µg/ml Hoechst 33342 (B-2261; 1 mg/ml in H2O; Sigma). Preparations were washed three times in PBS and once in H2O before mounting (DAKO mounting medium; S3023; Med-Bio Ltd.). Muscle fibroblasts (passage 2) serum starved for 5 days were fixed and labeled with the muscle-specific antigens to determine the proportion of myogenic cells in the population. Quantification was done using an epifluorescence microscope (AX-70; Olympus) equipped with a Spot RT-KE slider CCD camera (Diagnostics Instruments Inc.). For immunocytochemistry on size-selected donor cells, trypsinized donors were mouth pipetted onto Matrigel-coated eight-well Lab-Tek II chamber slides (Biolab Ltd.) in approximately 20 µl H199/0.5% FCS, left to settle in the incubator for 2–4 h, cytospun (Shandon Cytospin III Cytocentrifuge; Global Medical Instrumentation Inc.) for 2 min at 200 rpm, and processed for antibody staining as above.
DNA replication was assessed using 5-Bromo-2-deoxyuridine (BrdU) incorporation. Cells were cultured on glass coverslips and labeled in culture medium containing 10 µM BrdU for 22–24 h. Cells were washed one time in PBS, fixed in 2 ml ice-cold 50 mM ethanol-glycine buffer pH 2.0 (1 M glycine solution in H2O diluted in absolute ethanol) for at least 20 min at –20°C, and washed three times in PBS, and the DNA was hydrolysed in 2 N HCl for 20 min at 38.5°C. After three PBS washes, cells were blocked, stained (using anti-BrdU IgG; 1:2000, B-2531; Sigma), mounted, and quantified as described above.
In Vitro Maturation of Oocytes (IVM)
In vitro-matured nonactivated metaphase II (MII)-arrested oocytes were derived as described previously [43]. Briefly, slaughterhouse ovaries were collected from mature cows, placed into saline (30°C), and transported into the laboratory within 2–4 h. Cumulus-oocyte complexes (COCs) were collected in Hepes-buffered medium 199 (H199; catalog no. 31100–035; Life Technologies) containing 15 mM Hepes, 5 mM NaHCO3, and 0.086 mM kanamycin monosulphate, with 925 IU/ml heparin (Artex Ltd.) and 20 µl/ml 20% (w/v) albumin concentrate (Immuno-Chemical Products [ICPbio], Auckland, New Zealand) by aspirating 3- to 12-mm follicles into a 15-ml Falcon tube using an 18-gauge needle and negative pressure (40–50 mm Hg). Only COCs with a compact, nonatretic cumulus oophorus-corona radiata and a homogenous ooplasm were selected for IVM. COCs were washed twice in H199 with 10% (v/v) FCS (H199–10) and once in bicarbonate-buffered medium M199 with 25 mM NaHCO3, 0.2 mM pyruvate, 0.086 mM kanamycin monosulphate, and 10% (v/v) FCS (B199–10). Ten COCs in 10 µl B199–10 were transferred into a 40 µl drop of IVM medium B199–10 with 10 µg/ml ovine FSH (Ovagen; ICPbio), 1 µg/ml ovine LH (ICPbio), 1 µg/ml 17-ß-estradiol, and 0.1 mM cysteamine in 6-cm dishes (Falcon 35–1007; Becton Dickinson Labware, Lincoln Park, NJ) overlaid with paraffin oil (Squibb, Princeton, NJ). Dishes were cultured in humidified 5% CO2 in air atmosphere. After IVM for 18–20 h, the cumulus-corona was dispersed by vortexing up to 180 oocytes in 500 µl of 1 mg/ml bovine testicular hyaluronidase in Hepes-buffered H199, followed by three washes in H199 containing 0.1 mg/ml cold soluble polyvinyl acetate (PVA; 10–30 kDa; H199-PVA). For zona-free NT, oocytes with a first polar body were washed three times in H199-PVA before removal of the zonae pellucidae by pronase (5 mg/ml in H199).
Bovine zona-free SCNT was performed using our previously described standard operating procedure [43]. To select mononucleated myotubes, MT cultures were briefly trypsinized to remove larger multinucleated fibers. Trypsin was blocked by two washes in H199/0.5% FCS, and the remaining cells were washed three times in PBS before transferring the coverslips into the lid of a Falcon 10-cm Petri dish. Each coverslip was covered in 20 µl H199/PVA and overlaid with mineral oil. Mononucleated MTs were selected individually under phase contrast using 400x magnification. A small volume of trypsin (0.25% trypsin; 1 mM EDTA; Invitrogen) was added around the cell using a mouth pipette and was observed until the cell began to detach. Each mononucleated MT was then transferred into 40 µl droplets of H199/0.5% FCS prior to attachment onto the cytoplasts. Individual donor cells were attached to cytoplasts in drops of 10 µg/ml phytohemagglutinin in H199-PVA. After 23–25 h after the start of maturation, couplets were automatically aligned and electrically fused at 2.0 kV/cm in hypo-osmolar fusion buffer (165 mM mannitol, 50 µM CaCl2, 100 µM MgCl2, 500 µM Hepes, 0.05% bovine albumin [ABIVP; ICPbio], pH 7.3) using a custom-made parallel-plate fusion chamber connected to an ECM 200 (BTX, San Diego, CA) at room temperature [44]. For the single-NT vs. double-NT experiments, cytoplasts were fused with either one or two muscle fibroblasts, with both groups receiving the same number of fusion pulses. Reconstructed SCNT embryos were artificially activated 3–4 h after fusion using a combination of ionomycin and 6-dimethylaminopurine. SCNT reconstructs were cultured singularly in drops of 5 µl. After 4 h in 6-dimethylaminopurine, reconstructs were washed three times in Hepes-buffered synthetic oviduct fluid (107.7 mM NaCl, 7.15 mM KCl, 0.3 mM KH2PO4, 5 mM NaHCO3, 3.32 mM sodium lactate, 0.069 mM kanamycin monosulphate, 20 mM Hepes, 0.33 mM pyruvate, 1.71 mM CaCl2.2H2O, 3 mg/ml fatty acid-free ABIVP) and transferred into AgResearch-synthetic oviduct fluid (AgR-SOF) culture medium droplets.
Reconstructed embryos were cultured singularly in vitro for 7 days (D0 = fusion) in 5 µl biphasic AgR-SOF [5]. On D4, embryos were changed into fresh AgR-SOF drops containing 10 µM 2, 4-dinitrophenol [45] to act as an uncoupler of oxidative phosphorylation. All cultures were overlaid with mineral oil and kept in a humidified modular incubation chamber (ICN Biomedicals Inc., Aurora, OH) gassed with 5% CO2, 7% O2, and 88% N2.
Karyotyping of Donor Cells and NT Embryos
Muscle fibroblasts (passage 6) at about 70% confluency were treated with KaryoMAXColcemid solution (0.1 µg/ml; Gibco Invitrogen) for 1 h, trypsinized, and centrifuged at 1000 rpm for 5 min. The pellet was carefully resuspended in 5 ml of 0.56% KCl solution and incubated at 38°C for 15 min to allow for swelling of the cells. Another 5 ml freshly made cold (–20°C) fixative (3:1 mix of methanol:acetic acid) was added, the cell suspension centrifuged for 3 min at 1000 rpm, the supernatant discarded, 5 ml of fresh fix added without disrupting the cell pellet, and incubated at 4°C for 30 min. The washing/incubation steps were repeated two more times with 5 ml fresh fixative before resuspending the pellet in 500 µl ice-cold fixative. A Pasteur pipette was flamed and pulled to produce a fine glass column, and the cell suspension was dropped from about 20 cm onto chilled, precleaned microscope slides (Biolab). Slides were left to dry for at least 1 h before staining with 10% KaryoMAXGiemsa stain in Gurr buffer, pH 6.8 (catalog no. 331932D; BDH) for 10–15 min, followed by washing under a gentle stream of tap water. Metaphase spreads were photographed using a 100x oil immersion objective and a Spot RT-KE slider CCD camera. The karyotype of D7 NT blastocysts derived after fusion with one or two MF donors was also analyzed. Embryos were incubated in KaryoMAXColcemid solution (0.1 µg/ml) in AgR-SOF for 2–3 h before being placed in 0.9% sodium citrate for at least 20 min. Using a fine-pulled Pasteur pipette, embryos were placed in a 3-cm dish with ice-cold fixative (3:2:1 mix of absolute ethanol:acetic acid:H2O). After 1–2 min, each embryo was picked up with a mouth pipette, dispensed onto a precleaned microscope slide, stained, and quantified as described above.
For each donor cell type, transcript expression was measured by RT-PCR in 1) adherent donor cells, 2) trypsinized donor cells selected for NT, 3) MII oocytes, 4) NT couplets, 5) NT reconstructs, 6) NT two-cell embryos, 7) NT blastocysts, and 8) in vitro-fertilized (IVF) control blastocysts: MYOD1, MYF5, PAX7, MYOG, MYF6, beta-actin (ACTB), and 18S rRNA. The following pooled samples were lysed in 50 µl Trizol (Invitrogen): 80–90 trypsinized MPCs and 30–33 trypsinized MT cells selected for NT, 30 MII oocytes, 10 NT couplets, 10 NT reconstructs, 10 NT two-cell embryos, and 5 NT and 5 IVF blastocysts. For the expression time course, 5 x 104 cells were trypsinized and either immediately lysed in 500 µl Trizol or replated onto Matrigel-coated tissue culture dishes and allowed to attach for 0.5, 2, 4, or 24 h before lysis. Prior to chloroform extraction, 200 ng MS2 carrier RNA and 5 pg rabbit 
-globin mRNA were added to each embryo sample as an exogenous standard. RNA was precipitated with isopropanol in the presence of 10 µg linear acrylamide, pelleted by centrifugation at 13 000 x g for 20 min, washed once with 70% ethanol, and dissolved in 8 µl DEPC-treated water. Genomic DNA was removed through digestion with 2 Kunitz units of amplification grade DNase 1 (Invitrogen) for 1 h at 37°C, followed by heat inactivation of the enzyme for 10 min at 65°C. RNA was precipitated with ethanol, pelleted by centrifugation at 13 000 x g for 20 min, washed once with 70% ethanol, and dissolved in 11 µl DEPC-treated water.
First-strand cDNA was synthesized using the SuperScript III First-Strand Synthesis System for RT-PCR kit (Invitrogen) according to the manufacturer's instructions. All RNA of each sample was reverse transcribed only once, resulting in a single batch of cDNA per sample. Briefly, 1 µl random hexamers (50 ng/µl) and 1 µl of 10 mM dNTP mix were added to 11 µl total RNA sample and incubated for 5 min at 65°C, followed by immediate quenching on ice for 1 min. Reverse transcription was performed by the addition of 10 µl master mix containing 2 µl of 10x RT buffer, 4 µl of 25 mM MgCl2, 2 µl of 0.1 M DTT, 1 µl RNaseOUT (40 U/µl), and 1 µl SuperScript III reverse transcriptase (200 U/µl). The reaction mixture was first incubated for 10 min at 25°C, then for 50 min at 50°C, and was terminated by heating to 85°C for 5 min. Finally, 1 µl Escherichia coli RNase H (20 U/µl) was added and incubated for 20 min at 37°C before storage at –80°C and use for PCR. To determine the presence of contaminating genomic DNA, reverse transcriptase was omitted in one embryo or cell sample each time a batch was processed for cDNA synthesis (designated –RT control).
An MG Thermal cycler (Bio-Rad, Auckland, New Zealand) was used for PCR amplification using the primers shown in Table 1. The PCR Mastermix consisted of sterile DEPC-treated H2O, 10x PCR buffer, 25 mM MgCl2, 10 mM dNTPs, 10 µM primers, and 5 U/µl Taq DNA polymerase (Roche). For PCR, 1 µl of cDNA (200 ng/µl in DEPC-treated H2O as determined using an ND-1000 Spectrophotometer; NanoDrop Technologies, Rockland, DE) was added to each 25 µl reaction. The PCR was performed using the following conditions: one cycle denaturation at 95°C for 5 min, followed by 35 cycles of 15 sec at 95°C, 45 sec at 52°C–60°C (see Table 1 for primer-specific annealing temperatures), 45 sec at 72°C; 4 min of extension at 72°C, and cooling to 4°C. Product identity was confirmed by the presence of a single band of the predicted amplicon size after 1.5% agarose gel electrophoresis-ethidium bromide staining.
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Embryo Transfer, Pregnancy Monitoring, and Controlled Calving
Total embryo development to compacted morula and blastocyst stages was assessed on D7, and grade 1–2 (B1–2) blastocysts [46] were selected for embryo transfer (ET). Recipient cows were synchronized as described [43]. On D7 following estrus (estrus = D0 = day of NT), a single morphologic grade 1–2 [46] cloned blastocyst (B1–2) in Emcare embryo holding solution (ICPbio) was loaded per 0.25-ml straw (Cryo-Vet, Quebriac, France) and transferred nonsurgically into the uterine lumen ipsilateral to the corpus luteum. Using ultrasonography (Aloka SSD-500 scanner with a 5-MHz linear rectal probe; Aloka Co. Ltd., Tokyo, Japan), the pregnancy status of recipient cows was determined on D35 of gestation. Development throughout gestation was monitored approximately every 30 days, from D35 to D90 by ultrasonography and thereafter by palpation per rectum. Following a regime to control parturition as described [43], cows were allowed to calve naturally if at all possible or with manual assistance to varying degrees as necessary. On rare occasions, calves were delivered by cesarean delivery. Calves were weaned when they weighed more than 100 kg (about 3 mo of age).
All values are presented as mean ± SEM unless indicated otherwise. Statistical significance was accepted at P < 0.05 and determined using the two-tailed t-test with equal variance and the two-tailed Fisher exact test for independence in 2 x 2 tables. Immunocytochemistry data were analysed using GLMM (generalized linear mixed model) with binomial distribution and logit link, with fixed effects for treatment and time and with random run effects. Raw means were plotted, as they were nearly identical to the GLMM means.
Characterization of Nuclear Donor Cells
We first investigated the effect of serum starvation on muscle donor cell populations. Serum-starved and nonstarved cells were quantified after 2, 3, 4, and 5 days in vitro (corresponding to 1, 2, 3, and 4 days of serum starvation) by immunofluorescence against the following antigens: MYOD1, MYF5, PAX7, MYOG, and DES. DNA synthesis as an indicator of proliferative capacity was measured by BrdU incorporation during a 24-h labeling period after 2, 3, and 5 days in vitro. There were no obvious differences in morphology between starved and nonstarved cells. MPCs were asymmetrically bipolar with at least one long, thin process less than half the width of the cell nucleus, and one wider process ending in an irregular growth cone-like tip, whereas myotubes were symmetrically bipolar cells with processes about the same width as their cell nucleus (Fig. 1). Fibroblasts were less elongated, with a polygonal morphology (Supplemental Fig. 1, available online at www.biolreprod.org). Serum-starved cells showed significantly different antigen presence at 4 of 20 time points measured across five antigens (Fig. 2 and Table 2). Under starvation conditions, disappearance of PAX7, a marker for undifferentiated precursors, and appearance of the differentiation markers MYOG and DES were accelerated at 5 and 2–3 days in vitro, respectively. Other MPC markers, MYOD1 and MYF5, were not affected. After 2 days in vitro, including 1 day of serum starvation, presumptive MPCs were trypsinized for NT. There were no signs of morphological cell differentiation, such as appearance of multinucleated myotubes, after 2 days in vitro. At this time point, 80% of cells within the population expressed MYOD1, and almost 50% expressed PAX7, consistent with the down-regulation of PAX7 in differentiating MYOD1-positive myoblasts [47]. Neither of these genes was expressed by contaminating muscle fibroblasts (Supplemental Fig. 1). After 5 days in vitro, the time point at which we manually selected putative mononucleated myotubes, PAX7 had almost completely disappeared from starved cultures, and MYOD1 was reduced to about 50% of cells within the population. Concomitantly with this, the proportion of morphologically differentiated myotubes was increased, making it easier to pick these cells under the microscope. The reduction in MYF5 expression at this time was an unreliable differentiation marker, as muscle-derived fibroblasts also showed staining for this antigen (Supplemental Fig. 1). The proportions of MYOG- and DES-expressing cells, mostly multinucleated myotubes, increased to 53% and 60%, respectively. Most cells present at D2 or D5 in vitro had stopped synthesizing DNA and presumably entered quiescence after serum starvation (Fig. 2 and Table 2). In summary, serum-starved muscle cell populations down-regulated precursor-specific antigens (MYOD1, PAX7), upregulated differentiation markers (MYOG, DES), and reduced BrdU incorporation with increasing time in culture. Muscle fibroblasts did not express any muscle-specific antigens.
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Following bulk characterization of donor populations, we characterized trypsinized cells actually selected for NT. Cells were selected according to their significant size differences. MTs were three times and MFs about two times larger in volume than MPCs (Table 3). A subset of size-selected donors were replated for 2–4 h, cytospun and analyzed for expression of MYOD1, PAX7, MYOG, and MYH (Fig. 3 and Table 4). After size selection, the proportion of MYOD1- and PAX7-positive MPCs increased from 80% to 100% and from 47% to 92%, respectively, indicating MPC enrichment through the selection procedure. The percentage of MYOG-positive cells decreased from 29% to 8% in size-selected MPCs. Likewise, the late-differentiation marker MYH was absent from the size-selected MPC population. Size-selected MTs were also enriched for the presence of MYOD1-positive cells (53% to 86%; Tables 2 and 4). Based on the weaker fluorescent signal after immunocytochemistry, however, the abundance of this antigen within each positive nucleus had decreased, consistent with the reported MYOD1 down-regulation during in vitro bovine myogenesis [48]. Selected MFs did not express any muscle-specific antigens. From this, we estimated the purity of donor cells for NT to be at least 92%, 85%, and 99% for MPCs, MTs, and MFs, respectively.
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We then sought to corroborate protein expression patterns in donor cell populations by determining gene expression in selected donor cells. After trypsinizing cells for NT, MYOD1 was detectable in groups of 80–90 selected MPCs, whereas MYOG was present in groups of 30–33 selected MT donors, indicating that donor cell categorization was accurate (Fig. 4A). Based on morphology, proliferation, antigen, and gene expression profile, cells serum starved for 1, 4, and 5 days were classified as MPCs, MTs, and MFs, respectively.
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Reprogramming of Donor Cell Gene Expression after NT
Despite their expression in donor cells, MYOD1 and MYOG mRNAs were undetectable by RT-PCR in pools of: NT couplets, fused reconstructs, two-cells, and blastocysts derived from MPC and MT donors, respectively (Fig. 4A). MYF5 was weakly detectable in MPCs but strongly expressed in couplets, reconstructs, two-cells (Fig. 4A), and recipient metaphase II oocytes (data not shown). None of the other muscle-specific genes were found in MII oocytes. Moreover, none of the muscle-specific genes were found in IVF blastocysts. PAX7 and MYF6 mRNAs were undetectable in trypsinized MPCs and MTs used for NT (Fig. 4B), respectively, and remained undetectable in couplets, reconstructs, two-cells, and blastocysts (data not shown). MYF5, PAX7, MYOG, and MYF6 were strongly down-regulated within 30 min after trypsinization of donor cells, but with the exception of MYF6, all re-expressed within 2 h of replating (Fig. 4B). In contrast, MYOD1 and housekeeping control genes 18S (data not shown) and ACTB were unaffected by trypsinization (Fig. 4B). Groups of 5 x 104 adherent muscle-derived fibroblasts did not contain any detectable levels of MYOD1, PAX7, and MYOG.
Effect of Donor Cell Type on In Vitro Embryo Development
In vitro development clearly depended on donor cell type (Table 5). It was highest with muscle fibroblasts, followed by MPCs, and lowest with fully differentiated MTs (57% vs. 36% vs. 20% for total development into grade 1–3 and 38% vs. 23% vs. 11% for development into transferable grade 1–2 embryos, respectively). Since up to 20% of manually selected MTs that appeared mononucleated in phase contrast contained, in fact, two or more nuclei as revealed by Hoechst 33342 staining (109 [80%] of 136 vs. 23 [17%] of 136 vs. 4 [3%] of 136 with 1, 2, or >2 nuclei, respectively; n = 3 replicates), additional experiments were conducted to control for the effect of tetraploidy. Cytoplasts were fused with either one ("single-NT") or two ("double-NT") mononucleated muscle fibroblasts and cultured in vitro. Development was significantly reduced in the double-NT group (18% vs. 39% for total development into grade 1–3 and 5% vs. 23% for development into transferable grade 1–2 embryos, respectively; Supplemental Table 1, available online at www.biolreprod.org). Karyotyping based on metaphase spreads revealed that most of the double-NT blastocysts contained tetraploid cells (17 [89%] of 19), whereas all single-NT embryos were diploid (31 [100%] of 31; Supplemental Table 2, available online at www.biolreprod.org).
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Effect of Donor Cell Type on In Vivo Embryo Development
Following transfer of morphologic grade 1 and 2 cloned embryos into recipient cows, survival did not significantly depend on donor cell differentiation (Fig. 5 and Supplemental Table 3, available online at www.biolreprod.org). Pregnancy establishment at D35 and development to weaning, expressed per embryo transferred, was not different between the three cell types (64% vs. 35% vs. 45% and 18% vs. 9% vs. 14%, for MPCs vs. MTs vs. MFs, respectively). A total of nine viable cloned calves (born June/July 2005) were obtained across all three cell types (four from MPCs, two from MTs, and three from MFs). All calves appeared healthy before being culled shortly after weaning for unrelated reasons. In a control trial for the effect of tetraploidy, blastocysts derived from single- and double-NT with MFs were also transferred. Up to midgestation (range: D142–D163), there was no significant difference between the two groups (8 [38%] of 21 vs. 2 [14%] of 14 for single- vs. double-NT, respectively; Supplemental Fig. 2, available online at www.biolreprod.org).
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We used increasingly differentiated skeletal muscle cells for SCNT and found that despite significant differences in in vitro development, muscle progenitor cells, differentiated myotubes, and muscle-derived fibroblasts had no significant effect on in vivo survival of cloned blastocysts.
Comparative NT studies require strict standardization of all donor cell selection and preparation steps in order to obtain well-defined homogenous cell populations [50]. In this study, only serum-starved somatic donor cells were used. Serum starvation served two purposes: first, it more than doubles cattle cloning efficiency for different bovine cell types (Wells and Oback, unpublished data), and second, it has been shown, together with methionine deprivation, to reproducibly synchronize myoblasts in a quiescent (G0) stage without inducing entry into myogenesis [51]. Donor cells selected for NT were characterized by immunocytochemistry and gene expression analysis. Antibodies used to determine the muscle cell antigen profile have previously proven to be specific for bovine ([52, 53] and SIGMA product information for DES) and showed the expected subcellular localization (nuclear for the transcription factors and cytoskeletal for DES). By choosing the smallest cells in the trypsinized MPC population and the largest cells in the trypsinized MT population for NT, we specifically selected against the intermediate size fibroblasts. Rather than only relying on antigen profiling of bulk populations, we determined the purity of individually selected cells actually prepared for NT. After size selection, single MPCs were essentially all MYOD1 positive and PAX7 positive as well as MYOG negative and MYH negative. Any contaminating cells were likely to be early-differentiating myogenic cells rather than fibroblasts. Size-selected MTs were mostly MYOD1 positive and MYOG positive; however, the abundance of MYOD1 within each positive nucleus had decreased, consistent with its reported down-regulation during in vitro bovine myogenesis [48]. Potentially contaminating cells within the MT group were likely to be fibroblasts rather than MYOD1- and PAX7-positive MPCs. The presence of the intermediate filament DES could not be reliably quantified in selected cells, because the replating and cytospin procedure dramatically altered cell shape. Many cytospun cells were still rounded up and did not exhibit the characteristic DES staining of adherent cells. We therefore assessed the late differentiation marker MYH instead and found it to be already expressed in 56% of selected MTs. Last, MFs were least likely to contain contaminating myogenic cell types, since they did not express any muscle-specific antigens. In summary, antigen profile analysis of size-selected trypsinized cells revealed a high degree of homogeneity within in each group of muscle-derived donor cell types.
In contrast to immunocytochemistry, donor-specific gene expression analysis could be carried out on pools of nonadherent cells. Trypsinized cells after 30 min in suspension, which is the time that it usually takes to attach cells to cytoplasts and electrically fuse them, strongly down-regulated MYF5, PAX7, MYOG, and MYF6. For all genes except MYF6, this rapid down-regulation was transient and reversible within 2 h of replating and cell attachment to the culture dish. In contrast, MYOD1 and housekeeping control genes 18S and ACTB were unaffected by trypsin. Most animal cells are anchorage dependant, and losing contact with the substrate profoundly alters their homoeostasis, leading to changes in cell adhesion molecules, cytoskeletal organization, signaling pathways, and gene expression [54], especially for genes whose mRNA has a short half-life. These data highlight that early changes in donor gene expression may simply reflect manipulation before or during NT rather than true epigenetic reprogramming, emphasizing the importance of analyzing cells in the state they are in when actually used for NT. However, even if the initial silencing of some muscle cell-specific transcription factor genes was an artefact of trypsinization, their mRNAs remained undetectable, and thus presumably repressed, until the blastocyst stage. This is similar to the specific repression of three hematopoietic stem cell genes during mouse NT [55]. For a small subset of donor genes, an "epigenetic donor cell memory" has been described (e.g., in endoderm and neuroectoderm donors during Xenopus cloning [56] and in myoblasts during mouse NT [31]). The latter study showed that myoblast-derived cloned mouse embryos continue expressing GLUT4 glucose transporters and exhibit precocious enrichment of GLUT1 at the cell surface, resulting in enhanced rates of glucose uptake. The cloned embryos grew better in somatic cell culture media favored by the donor myoblasts. We found that muscle cell-derived bovine NT embryos did not grow beyond the eight-cell stage in donor cell-specific medium (0 [0%] of 92 vs. 44 [46%] of 95 development into B1–3, n = 2, for donor cell vs. standard embryo medium, respectively) indicating that limitations on reprogramming may be less associated with cell culture requirements in bovine than they are in mouse.
In vitro development was higher with MPCs than with fully differentiated MTs. Such differences within a given somatic lineage have been described before. Natural killer T lymphocytes, for example, supported morula/blastocyst development much better than peripheral T cells or hematopoietic stem cells (71% vs. 12% vs. 6%, respectively) from the same lymphocyte lineage [25, 55]. Likewise, freshly isolated undifferentiated neural progenitors from the cerebral cortex of mouse fetuses have resulted in significantly higher morula/blastocyst development than more differentiated immature cortical neurons from the same lineage [30]. In the case of muscle cells, a confounding factor was the presence of more than one nucleus in up to 20% of manually selected MTs, with tetraploidy accounting for most of the polyploid cells. To control for the effect of tetraploidy, cytoplasts were fused with either one ("single-NT") or two ("double-NT") mononucleated diploid muscle fibroblasts and cultured in vitro. Total development was reduced more than 2-fold and transferable grade development more than 4-fold in the double-NT group (Supplemental Table 1). However, correcting the developmental data presented in Table 5 for the presence of 20% of cells with a 2- and 4-fold, respectively, reduced chance of forming an embryo had little effect on the developmental differences between cell types. The corrected developmental potential of the 80% diploid MT embryos would increase to 22.5% and 12.6% to M/B1–3 and M/B1–2, respectively, which is still significantly lower than in the MF and MPC groups. Based on one to eight metaphase spreads per embryo, most of the resulting double-NT blastocysts contained tetraploid cells (Supplemental Table 2). In order to determine whether the embryos were truly tetraploid or, in fact, mixoploid, more sensitive cytogenetic detection methods, such as karyotyping based on interphase fluorescent in situ hybridization would be needed [57]. Tetraploid embryos produced by electrofusion at the two-cell stage [58] or by parthenogenetic activation in the presence of protein kinase inhibitors [59] develop into blastocysts at the same frequency as diploid controls. This indicates that polyploidy is particularly problematic for SCNT embryos, perhaps because it requires successful epigenetic reprogramming of additional copies of somatic genomes.
Currently, it is not possible to accurately predict clone survival to adulthood from the rate of blastocyst formation. For example, apparently normal blastocyst formation can be achieved after NT with imprint-free primordial germ cells [60, 61] or cancer cell nuclei harboring nonreprogrammable mutations [62, 63], both of which are conditions that will prevent development to term. Likewise, low rates of blastocyst formation can be due to reprogramming-independent events, such as cell cycle incompatibility between donor cells in S phase and recipient oocytes [64] or simply damage of donor cells during culture or NT. In the absence of other conclusive correlations, survival into adulthood is currently the most informative and meaningful measure of extensive donor cell reprogramming. Following embryo transfer, viability of cloned embryos was not dependant on donor cell type or differentiation status. Despite all efforts, it has so far not been conclusively determined whether differentiation status affects cloning efficiency within somatic lineages [10]. Comparative data from hematopoietic [25], neuronal [30], and skin [65] cells have not provided conclusive evidence that more differentiated but genetically identical donors are harder to reprogram than less differentiated cells from the same lineage. In those lineages, the data sets were based on small numbers of surviving clones and thus lacked robust statistical significance when postblastocyst development was compared [10]. Hematopoietic stem cells even showed extremely low cloning efficiency, indicating their low reprogrammability in vivo [55].
Differences in ploidy between MPC and MF vs. MT donors could have skewed the in vivo results if the up to 20% mixoploid or tetraploid embryos derived from MT had a chance of survival different from that of diploid embryos. As a control, diploid blastocysts from single-NT and mixoploid or tetraploid blastocysts from double-NT with MFs were transferred, and their survival in utero was monitored. There was a trend, albeit insignificant, for diploid embryos to survive better until midgestation, when the pregnancies were terminated. Thus, the inclusion of mixoploid or tetraploid MT donors could have led us to underestimate postblastocyst development of MT-derived embryos. Despite the transferred double-NT blastocysts being mixoploid or tetraploid, all muscle and lung fibroblast cell lines derived from D163 double-NT fetuses were diploid (data not shown), and it is unknown when the correction in ploidy might have occurred. At least in the case of mixoploid blastocysts, it is possible that tetraploid cells were preferentially allocated to the extraembryonic membranes, as has been shown in mice [66]. In human and mouse, tetraploid embryos can develop beyond the blastocyst stage and begin implantation; however, they exhibit a high degree of abnormalities [67, 68], none of which were observed in the double-NT or myotube-derived pregnancies. Taken together, we consider it extremely unlikely that differences in ploidy had a significant effect on the cloning efficiency.
In summary, the hypothesis that NT-induced reprogramming of a cell's epigenome is inversely proportional to its differentiation status could not be confirmed within the skeletal muscle lineage. It remains to be seen whether the limited number of NT experiments reported here was simply not sufficient to detect a hierarchical relation between cell differentiation and cloning efficiency or whether, contrary to previous studies [69, 70], such a relation is not universally true. In any case, varying just one parameter alone—namely, somatic donor cell differentiation status—is unlikely to lift cloning success to the efficiency level of other assisted reproductive technologies, such as IVF or artificial insemination.
ACKNOWLEDGMENTS
We would like to thank past and present members of our cloning team (K. Leslie, P. Misica-Turner, F. Oback, J. Oliver, A. Schurmann, N. Standley, and H. Troskie) and farm staff (J. Forsyth, M. Berg, and V. Prendergast) for excellent technical assistance. We thank Drs. C. Smith and S. McCroskery for 18S rRNA, -globin, ACTB, and PAX7 primers, respectively, and N. Cox for assistance with the statistical analysis.
FOOTNOTES
1Supported by the New Zealand Foundation for Research, Science, and Technology and AgResearch. 
Correspondence: 2FAX: 64 7 838 5536; e-mail: bjorn.oback{at}agresearch.co.nz
Received: 1 November 2006.
First decision: 27 November 2006.
Accepted: 15 May 2007.
REFERENCES
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