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research-article |
AgResearch, Ruakura Research Centre, Reproductive Technologies, Private Bag 3123, Hamilton, New Zealand
ABSTRACT
Our objectives were to compare the cellular and molecular effects of aggregating bovine embryonic vs. somatic cell nuclear transfer (ECNT vs. SCNT) embryos and to determine whether aggregation can improve cattle cloning efficiency. We reconstructed cloned embryos from: 1) morula-derived blastomeres, 2) six adult male ear skin fibroblast lines, 3) one fetal female lung fibroblast line (BFF), and 4) two transgenic clonal strains derived from BFF. Embryos were cultured either singularly (1X) or as aggregates of three (3X). In vitro-fertilized (IVF) 1X and 3X embryos served as controls. After aggregation, the in vitro development of ECNT but not that of SCNT or IVF embryos was strongly compromised. The inner cell mass (ICM), total cell (TC) numbers, and ICM:TC ratios significantly increased for all the aggregates. The relative concentration of the key embryonic transcript POU5F1 (or OCT4) did not correlate with these increases, remaining unchanged in the ECNT and IVF aggregates and decreasing significantly in the SCNT aggregates. Overall, the IVF and 3X ECNT but not the 1X ECNT embryos had significantly higher relative POU5F1 levels than the SCNT embryos. High POU5F1 levels correlated with high in vivo survival, while no such correlation was noted for the ICM:TC ratios. Development to weaning was more than doubled in the ECNT aggregates (10/51 or 20% vs. 7/85 or 8% for 3X vs. 1X, respectively; P < 0.05). In contrast, the SCNT and IVF controls showed no improvement in survival. These data reveal striking biological differences between embryonic and somatic clones in response to aggregation.
assisted reproductive technology, early development, embryo, in vitro fertilization
Since the cloning of Dolly the sheep from a differentiated mammary gland cell [1], somatic cell nuclear transfer (SCNT) has virtually replaced embryonic cell nuclear transfer (ECNT) in mammalian cloning experiments. This recent ascendancy of somatic cloning can easily obscure how NT experiments for over four decades were dominated by the idea that cloning success depends on the donor cell being of embryonic origin. Historically, all initial successes in mammalian cloning were achieved with nuclei from early cleavage-stage embryos of sheep and cattle [2, 3]. In the mouse, cloning efficiencies to term were significantly higher with 1-, 2- and 4-cell donors than with somatic nuclei [4–6]. These findings are consistent with the notion that murine blastomeres up to at least the 4-cell stage remain totipotent and are able to on their own give rise to all embryonic and extra-embryonic tissues [7]. In cattle, extensive datasets on the survival of ECNT embryos derived from 8-, 8–16- or 9–15-, 16-, 16–32-, 32- and 32–64-cell donor embryos have shown that 8-cell derived NT embryos survive significantly better than all the other groups [2, 8]. As in the mouse, this is consistent with the fact that full-term development can be obtained from blastomeres isolated at the 8-cell stage in cattle [9]. Bovine morula cells are also much more efficient donors than skin fibroblasts and greatly reduce the incidence and severity of late-gestation abnormalities, such as placentome malformation, hydroallantois, and parturition difficulties [10]. The reason for this high efficiency of ECNT relative to that of SCNT is not clear.
It is often assumed that blastomeres require less or are more amenable to nuclear reprogramming than somatic cells, resulting in fewer epigenetic defects. A number of these defects have been described in somatic clones, including aberrant methylation patterns of DNA [11–14] and histones [15] and dysregulation of a large number of imprinted and non-imprinted genes [16–18]. Similar comprehensive analyses for embryonic clones have not been reported. However, a recent molecular comparison of ECNT and SCNT preimplantation embryos has identified defects in the composition of somatic spindle chromosome complexes, specifically after SCNT [19]. This provides the first molecular marker that may correlate with the different abilities of embryonic and somatic nuclei to support development after NT. We have extended these comparisons by analyzing ECNT and SCNT embryos at the cellular and subcellular levels, including allocations of inner cell mass (ICM) and trophectoderm (TE) cells and the expression of the transcription factor POU5F1 (also known as OCT4). POU5F1 is essential for the regulation of embryonic cell pluripotency [20, 21] and has been used to monitor reprogramming success after murine SCNT [22–24]. In the mouse, almost 90% of cumulus-cloned blastocysts reactivate POU5F1 transcription although only 36% of these show the correct spatio-temporal expression [22]. Candidate gene expression profiling of Pou5f1 and 10 related genes in individual cumulus-cloned blastocysts has shown that only 62% of these embryos express correctly all of these genes [23]. Failure to reactivate Pou5f1 transcription correlates with the lack of selective Pou5f1 promoter DNA demethylation [25]. In bovine preimplantation embryos, the distribution of POU5F1 transcripts is very similar to that in the mouse [26], although expression appears to be normal in bovine SCNT embryos, as assessed by qualitative RT-PCR [27].
Cattle survival rates to term of around 30% for morula-derived and around 5% for somatic donors [28] are still significantly lower than for controls after artificial insemination or transfer of in vitro fertilization (IVF)-derived embryos, which ranges from 50–70% [10, 29]. Therefore, approaches have been devised to increase the frequency of cloning success [30–34]. While some of these treatments have improved in vitro development, their beneficial effects on gene expression and in vivo development have not been demonstrated conclusively. Furthermore, their applicability to ECNT has not been reported. One of the more promising methods is embryo aggregation. In the mouse, aggregation of two or three SCNT embryos leads to increased cell numbers, normalized Pou5f1 gene expression, and eightfold higher in vivo development of the resulting single blastocysts [35]. Similarly, parthenogenetic cell development can be rescued through aggregation with normal in vivo-derived blastomeres [36, 37]. In cattle, the aggregation of parthenogenetic blastomeres with each other [38] or with IVF-derived blastomeres partially increases survival after embryo transfer [39]. Our objective was to compare the cellular and molecular effects of aggregating bovine ECNT and SCNT reconstructs, respectively, and to determine whether aggregation improves the efficiency of cattle cloning.
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 the in vitro culture (IVC) conditions (singles vs. triplets) were kept the same (e.g., the pool of donor embryos and oocytes, activation method, and culture medium). Investigations were conducted in accordance with the regulations of the New Zealand Animal Welfare Act, 1999.
In Vitro Maturation of Oocytes (IVM)
In vitro-matured, non-activated metaphase II (MII)-arrested oocytes were derived as described previously [28]. Briefly, slaughterhouse ovaries were collected from mature cows, placed in saline (30°C) and transported to the laboratory within 2–4 h. Cumulus-oocyte complexes (COCs) were collected in Hepes-buffered Medium 199 (H199; Life Technologies) that contained 15 mM Hepes, 5 mM NaHCO3, 0.086 mM kanamycin monosulfate, and 925 IU/ml heparin (Artex, Waipukurau, New Zealand) and 20 µl/ml of 20% (w/v) albumin concentrate (ICPbio, Auckland, New Zealand) by aspirating 3–12-mm follicles into a 15-ml Falcon tube using an 18G needle and negative pressure (40–50 mmHg). Only COCs with compact, non-atretic cumulus oophorus-corona radiata and homogenous ooplasm were selected for IVM. COCs were washed twice in H199 that contained 10% (v/v) fetal calf serum (FCS) (H199–10) and once in bicarbonate-buffered M199 medium that contained 25 mM NaHCO3, 0.2 mM pyruvate, 0.086 mM kanamycin monosulfate, and 10% (v/v) FCS (B199–10). Ten COCs in 10 µl of 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 17ß-estradiol, and 0.1 mM cysteamine in 6-cm dishes (Falcon 35-1007; Becton Dickinson Labware, Lincoln Park, NJ) and overlaid with paraffin oil (Squibb, Princeton, NJ). The dishes were incubated in a 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 synthetic oviduct fluid (HSOF), which consisted of 107.7 mM NaCl, 7.15 mM KCl, 0.3 mM KH2PO4, 5 mM NaHCO3, 3.32 mM sodium lactate, 0.069 mM kanamycin monosulfate, 20 mM Hepes, 0.33 mM pyruvate, 1.71 mM CaCl2.2H2O, and 3 mg/ml fatty acid-free bovine albumin (ABIVP; ICPbio), followed by three washes in H199 that contained 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 treatment with pronase (5 mg/ml in H199).
For ECNT donors and IVF controls, IVM oocytes were fertilized with frozen-thawed spermatozoa in 50-µl drops under oil for 22–24 h, as described previously [40]. During the course of the experiments, sperm from two different bulls (designated Bull 1 and Bull 2, respectively) were used for IVF. All IVF control embryos in this study were derived from Bull 2. After five days of IVC in AgResearch (AgR)-SOF (see below), precompacting morulae were dissociated into individual blastomeres. For dissociation, the zona pellucida was removed by pronase (5 mg/ml in HSOF) and the embryos were washed three times in H199–10. After washing in Ca2+- and Mg2+-free PBS that contained 1mg/ml cold soluble PVA (10–30 kDa), 0.02% EDTA (from 20% stock solution in H2O), and cytochalasin B (5 µg/ml, from 5 mg/ml stock solution in DMSO), the embryos were suspended in 30-µl droplets of dissociation buffer for 5 min before gentle mechanical dissociation with a fine-mouth pipette. Individual blastomeres of the same embryo were placed into 30-µl droplets of H199-PVA until lectin attachment (see below). Blastomeres of each donor embryo were kept separate and used to generate roughly equal proportions of cloned 1X vs. 3X blastocysts for embryo transfer (ET). For gene expression analysis, blastomeres from all the donors were pooled before NT. For SCNT, the following independent primary cell lines were used for NT: adult ear skin fibroblasts (AESF; passage 2–5) from six different bulls (AESF 1–6); lung fibroblasts (passage 3–8) from a D 60 bovine female fetus (BFF); and two lines of genetically modified BFF cells (passage 11–15) that carry copies of the human myelin basic protein gene (clonal cell strains designated MBP1 and MBP2). Adult and fetal fibroblasts were isolated and cultured as described previously [28]. Prior to NT, cells were seeded at 2.5 x 104 per cm2. After 17–20 h, the cells were washed three times with PBS and cultured for 3–7 days in medium that contained 0.5% FCS [41].
Initially, we developed a zona-free ECNT technique that increased throughput and ease of operation. The method is based on our cattle SCNT standard operating procedure [28] and adapted to a modified zona-intact ECNT protocol [42]. Modifications that increased blastocyst production included: 1) cytoplasts aged for 9 h in Emcare complete medium (ICPbio) that was supplemented with essential and non-essential amino acids (Emcare Complete Plus, ECP); 2) increased temperature during aging to eliminate cytoplast lysis during NT; and 3) IVC in biphasic AgR-SOF. Highly efficient simultaneous fusion and alignment of up to 20 couplets increased the cloned ECNT embryo production rate at least threefold compared to our previous zona-intact protocol. Briefly, oocytes were enucleated at 22–23.5 h post-IVM (hpm), and 20 cytoplasts were placed in 50-µl droplets of B199–10% FCS. At 35 hpm, aged cytoplasts were washed in ECP. About 40 cytoplasts were transferred to 40-µl droplets of ECP under oil and incubated at 15°C (as measured in the cytoplast-containing medium) in a cooled water bath overnight. At 44 hpm, the cytoplasts were washed into warm H199-PVA and attached to individual blastomeres in drops of 10 µg/ml phytohemagglutinin (PHA-P) in H199-PVA. Couplets were automatically aligned and electrically fused at 1.5 kV/cm at room temperature in hypo-osmolar fusion buffer (165 mM mannitol, 50 µM CaCl2, 100 µM MgCl2, 500 µM Hepes [pH 7.3], 0.05% ABIVP) using a custom-made parallel-plate fusion chamber connected to an ECM 200 (BTX, San Diego, CA) [43]. At about 48 hpm, reconstructed embryos were washed three times in HSOF and transferred into AgR-SOF culture medium droplets. Bovine zona-free SCNT was performed as described [28]. Reconstructed SCNT embryos were artificially activated 3–4 h postfusion, using a combination of ionomycin and 6-dimethylaminopurine (6-DMAP). SCNT reconstructs for 3X-IVC were maintained in groups of three per 10-µl drop of 6-DMAP, while 1X-IVC controls were maintained singly in drops of 5 µl. After 4 h in 6-DMAP, reconstructs were washed three times in HSOF and transferred to droplets of AgR-SOF culture medium.
Reconstructed embryos were cultured in vitro for 7 days (Day 0 was the day of fusion) in biphasic AgR-SOF [34], either singularly or as aggregates of three. For ECNT embryos, aggregation was carried out at the 1-cell stage (from the beginning of IVC) as indicated or at the 2-cell stage (cleavage scored after 24 h in IVC) for all other experiments. For SCNT and IVF controls, aggregation was carried out at the 1-cell stage. On Day 4, embryos were transferred to fresh AgR-SOF drops that contained 10 µM 2,4-dinitrophenol [40], which acts as an uncoupler of oxidative phosphorylation. For single in vitro culture (1X-IVC or 1X), one reconstruct was cultured in 5 µl of medium. For aggregate cultures (3X-IVC or 3X), three reconstructs were cultured in a 10-µl droplet of medium in a triangular arrangement, in which all reconstructs were kept in contact with each other. Zona-free IVF controls were cultured under the same conditions and aggregated at the 1-cell stage (Day 0 was the day of IVF). All cultures were overlaid with mineral oil and kept in a humidified modular incubation chamber (ICN Biomedicals Inc., Aurora, OH) that was gassed with 5% CO2, 7% O2, and 88% N2. Blastocysts were classified as 3X aggregates when the culture droplet contained only a single, relatively large, blastocyst without early cleavage-stage blastomeres. A small proportion of the 3X blastocysts appeared to be smaller overall but still contained one or two large extruded blastomeres; these blastocysts were classified as being derived from just two NT reconstructs (2X-IVC or 2X) and were also transferred.
Embryo Transfer, Pregnancy Monitoring, and Parturition
Total embryo development to compacted morula and blastocyst stages was assessed on Day 7, and morphological grade 1 to 3 (B1–3) ECNT or grade 1 to 2 (B1–2) SCNT blastocysts [44] were selected for embryo transfer (ET), even though it has not yet been established that these grading criteria are at all meaningful for cloned embryos or for zona-free embryos in particular. Recipient cows were synchronized as described previously [28]. On Day 7 following estrus (estrus defined at Day 0, the day of NT), a single B1–3 in Emcare Embryo-holding Solution (ICPbio) was loaded per 0.25-ml straw (Cryo-Vet, Quebriac, France) and transferred non-surgically 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 Day 35 of gestation. Development throughout gestation was monitored by ultrasonography approximately every 30 days, from Day 35 to Day 90, and thereafter by rectal palpation. Following the regime to control parturition described previously [28], the cows were allowed to calve naturally if possible or with manual assistance when necessary. On rare occasions, calves were delivered by Cesarean section. Calves were weaned when they weighed more than 100 kg (at about 3 mo of age).
Zona-free B1–2 blastocysts were exposed to a 1:5 dilution in PBS of rabbit anti-bovine whole serum for 1 h, rinsed in H199/PVA, and placed for 1 h in a 1:5 dilution in PBS of Guinea pig complement that contained 40 µg/ml propidium iodide and 40 µg/ml Hoechst 33342. After briefly rinsing in H199/PVA, the embryos were mounted (DAKO mounting medium; Med-Bio Ltd., New Zealand) on glass slides and examined using an epifluorescence microscope (AX-70, Olympus, Japan). ICM and TE cells were identified by blue and pink coloration, respectively [45], and cell numbers were quantified the same day using Scion Image 4.02.
Individual blastocysts were lysed in 50 µl Trizol reagent (Invitrogen). Prior to chloroform extraction, 200 ng of MS2 carrier RNA and 5 pg of rabbit 
-globin mRNA were added to each embryo sample as an exogenous standard. The 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 by 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. The total RNA of each embryo was reverse-transcribed only once, resulting in a single batch of cDNA per embryo. Briefly, 1 µl of random hexamers (50 ng/µl) and 1 µl of 10 mM dNTP mixture were added to 11 µl of 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 that contained 2 µl of 10x RT buffer, 4 µl of 25 mM MgCl2, 2 µl of 0.1 M DTT, 1 µl of RNaseOUT (40 U/µl), and 1 µl SuperScript III reverse transcriptase (200 U/µl). The reaction mixture was incubated sequentially for 10 min at 25°C, 50 min at 50°C, and 85°C for 5 min. Finally, 1 µl of Escherichia coli RNase H (20 U/µl) was added and the mixture was incubated for 20 min at 37°C before storage at –80°C and subsequent use in the PCR. To determine the presence of contaminating genomic DNA, reverse transcriptase was omitted in one embryo sample each time a batch of embryos was processed for cDNA synthesis (designated as the -RT control).
The LightCycler system (Roche Diagnostics) was used for PCR amplification and data analysis. All reactions were performed with the LightCycler FastStart DNA Master Plus SYBR Green I Kit. The following primers (forward and reverse, respectively) were used: for 18S ribosomal RNA, 5'-GACTCATGGCCCTGTAATTGGAATGAGGC-3' and 5'-GCTGCTGGCAACAGACTTG-3' (GenBank AF176811); for -globin, 5'-GCAGCCACGGTGGCGAGTAT-3' and 5'-GTGGGACAGGAGCTTGAAAT-3' (GenBank V00875); and for POU5F1, 5'-CGTTCTCTTTGGAAAGGTGTTC-3' and 5'-TGGCGCCGGTTACAGAACCA-3' (GenBank AF022987). The primers were either designed using the Vector NTI 9.0 primer design software or were published sequences [46]. For each primer combination, the MgCl2 concentration and annealing temperature were optimized experimentally using the MJ thermal cycler MPTC-200. The ready-to-use Hot Start LightCycler reaction mix consisted of 0.4 µl of each primer (10 µM), 2.0 µl LightCycler SYBR Green I master mix, 5.2 µl DEPC-treated water, and 2.0 µl cDNA template (equivalent to 9% of the RNA content of a single blastocyst, and representing about 10 or 20 cells of the 1X or 3X treatment, respectively), to give a total volume of 10 µl loaded per chilled capillary. The following four-step program was used: 1) denaturation for 10 min at 95°C; 2) amplification and quantification for 10 sec at 95°C, 10 sec at 56°C, and 10 sec (18S, -globin) or 20 sec (POU5F1) at 72°C with a single fluorescent measurement repeated 45 times; 3) a melting curve (95°C, then cooled to 65°C for 20 sec, heated at 0.2°C/sec to 95°C, while continuously measuring fluorescence); and 4) cooling to 40°C. Product identity was confirmed by amplicon size using 1.5% agarose gel electrophoresis after real-time RT-PCR (18S, POU5F1), sequence analysis of the excised RT-PCR product (POU5F1), and melting curve analysis (melting point peak at 82°C for 18S, 89°C for POU5F1, 92°C for -globin, and about 81°C for primer-dimer complexes).
Quantification of Gene Expression
External standard curves were generated from serial dilutions for each gene (100-10–4 in 1:10 steps, with each point in duplicate) and one curve of high quality (reaction efficiency = 1.939 for -globin, 1.936 for 18S, and 1.913 for POU5F1) was saved for each target gene and imported for relative quantification for all experiments with the same parameters and conditions. Standard curve data were exported into the Excel program, graphed as a scatter plot, and fitted with a logarithmic regression. The slope and intercept from the regression equation were used to transform crossing point (Cp) values into concentrations according to the equation: [concentration] = e(-(Cp-intercept)/slope). Absolute expression values were obtained by first averaging the Cp values from different replicates for each gene and embryo, converting them into an absolute concentration (in relative units) from the standard curve, and averaging those concentration values. Relative expression was calculated by dividing the absolute values for [POU5F1] by [18S] for each embryo and averaging all individual [POU5F1]/[18S] log-concentration ratios. By averaging the ratios in this manner, differences in absolute values due to cDNA batch-to-batch variation were eliminated. All templates were amplified at least twice before averaging the Cp values. Only those amplifications that fulfilled the following criteria were included in the analysis: 1) a single band of the expected size and/or a single melting peak at the expected temperature; 2) controls with H2O or sample RNA that was not reverse-transcribed (-RT) as templates had much higher Cp values and different melting peaks, which indicated the absence of contamination, non-specific product or primer-dimer complexes.
All values are presented as mean ± SD, unless indicated otherwise. Statistical significance was accepted at P < 0.05 and determined using the two-tailed t-test with equal variance (Fig. 3, Tables 2 and 3, and Supplemental Table 1 [available online at www.biolreprod.org]) or the one-tailed Fisher exact test for independence in 2 x 2 tables (Figs. 1 and 4, and Tables 1 and 4). Log ratios of the real-time RT-PCR data were analyzed using the residual maximum likelihood (REML) method [47] in GenStat (8th edition), with the treatments as fixed effects and RNA extraction and NT or IVF runs as random effects.
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In Vitro Development of 1X vs. 3X embryos
Initially, we adopted our zona-free SCNT standard operating procedure [28] for ECNT. Up to 20 zona-free blastomere-oocyte couplets were automatically aligned (665/670 = 99%) and fused (2052/2217 = 93%) simultaneously. Donor embryos were obtained after IVF with two different bulls (Bull 1 and Bull 2) that showed very similar rates of cleavage, B1–3 and transferable B1–2 development (annual averages of 4668/5940 = 79%, 1920/5940=32%, and 809/5940 = 14% for Bull 1, and 4528/5505 = 82%, 1806/5505 = 33%, and 910/5505 = 17% for Bull 2). The distribution and average number of blastomeres per Day 5 donor embryo derived from IVF with Bull 1 or 2 were not different (30 vs. 32, respectively) (Supplemental Fig. 1A [available online at www.biolreprod.org]). Likewise, development and cell counts of ECNT 1X and 3X embryos, obtained after NT with IVF donors from Bull 1 or 2, were very similar (Supplemental Fig. 1B [available online at www.biolreprod.org] and Table 1). For analyses of in vitro and in vivo development, the data from both bulls were pooled. Aggregating three embryos at the 1-cell stage did not improve the rates of cleavage to the 2-cell stage compared to non-aggregated embryos (86/123 = 70% vs. 310/448 = 69%, respectively). For all subsequent experiments, ECNT embryos were aggregated at the 2-cell stage, which significantly increased the proportion of aggregation blastocysts compared to those aggregated at the 1-cell stage (63/432 = 15% vs. 8/123 = 7%, P < 0.05) (Table 1). Since aggregated embryos typically formed a single blastocyst, the output of compacted late morulae and blastocysts of morphological grade 1 to 3 (M/B1–3) or 1 to 2 (M/B1–2) on Day 7, as measured by the proportion of the total number of embryos placed into IVC that developed to this stage, was consistently higher for 1X-IVC than for 3X-IVC (Fig. 1 and Table 1). Irrespective of the sperm source or culture conditions, NT embryos derived from different individual donor embryos differed significantly in their development to blastocysts (Supplemental Fig. 2 [available online at www.biolreprod.org] and data not shown). Blastocyst development did not correlate with the developmental stage of the donor embryo, as measured by its number of blastomeres (Supplemental Fig. 2 [available online at www.biolreprod.org)]. Donor embryos that had completed their 4th, 5th, and 6th cell division cycle produced ECNT blastocyst rates that were not significantly different (21% vs. 17% vs. 28% for 1X and 0% vs. 8% vs. 10% for 3X, respectively). SCNT reconstructs showed >90% cleavage and were aggregated at the 1-cell stage during 6-DMAP incubation. Development to M/B1–3 was better for SCNT than for ECNT or IVF embryos (48% vs. 24% vs. 39% for 1X and 29% vs. 10% vs. 24% for 3X, respectively, P < 0.001) and depended significantly on the fibroblast donor cell lines (Fig. 1 and Table 1). Development to transferable M/B1–2 was also higher for the SCNT embryos (34% vs. 13% vs. 19% for 1X and 25% vs. 9% vs. 17% for 3X, respectively, P < 0.001) (Fig. 1 and Table 1). The proportion of M/B1–2 embryos was consistently higher for 3X than for 1X ECNT, SCNT, and IVF embryos (79% vs. 58%, 86% vs. 71%, and 71% vs. 50%, respectively, P < 0.05) (Table 1). For all groups, aggregation efficiency, as measured by the proportion of cleaved embryos per droplet that were incorporated into one relatively large embryo on Days 4 and 7, was >90%. Given this high efficiency, we estimated the contribution of each NT reconstruct to the resulting aggregation blastocyst. Each ECNT 2-cell had a 0.24/0.70 = 34% probability to form a blastocyst on its own (Table 1). If aggregation does not influence the developmental competence of each reconstruct to form a blastocyst, aggregation blastocyst development will follow a binomial distribution. Thus the probability (P) that the number (n) of individual NT reconstructs is represented in the composite blastocyst from the aggregation of N reconstructs is:
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Allocation of ICM and TE Cells in 1X and 3X Embryos
We investigated in more detail the morphological differences between 1X and 3X blastocysts. SCNT (derived from MBP1), ECNT and IVF (both derived from Bull 2) blastocysts, produced by 1X-IVC vs. 3X-IVC, were differentially stained (Fig. 2), and the number of putative ICM, TE, and total cell (TC) nuclei were quantified (Table 2). Comparing the 3X and 1X treatments, ICM cell numbers were more than tripled for IVF and more than doubled for the ECNT and SCNT aggregates (136 vs. 40, 81 vs. 34, and 127 vs. 51, respectively, P < 0.001). The increase in TE cells was proportionally lower but still highly significant for IVF, ECNT, and SCNT embryos (107 vs. 78, 104 vs. 67, and 102 vs. 65, respectively, P < 0.001) from 3X-IVC vs. 1X-IVC. Consequently, the total cell number was on average about twofold higher in 3X vs. 1X for IVF, ECNT, and SCNT embryos (243 vs. 119, 185 vs. 101, and 229 vs. 116, respectively, P < 0.001). The ICM:TC ratio was also increased in IVF, ECNT, and SCNT 3X vs. 1X embryos (0.56 vs. 0.33, 0.44 vs. 0.34, and 0.55 vs. 0.44, respectively, P < 0.001). Comparing IVF, ECNT, and SCNT embryos from the same IVC conditions, the ICM and total nuclei numbers were increased in 3X IVF and SCNT compared to ECNT embryos (136 and 127 vs. 81 and 243, and 229 vs. 185, P < 0.05), although the TE numbers were very similar (107 and 102 vs. 104). As a result, the ICM:TC ratio for 3X embryos was also increased in IVF and SCNT compared to ECNT embryos (0.56 and 0.55 vs. 0.44, P < 0.05). ECNT and IVF embryos from 1X-IVC had similar cell numbers and ratios, while the ICM:TC ratio for 1X SCNT embryos was increased compared to IVF and ECNT embryos (0.44 vs. 0.33 and 0.34, P < 0.05, Table 2).
POU5F1 Expression in 1X and 3X Embryos
In order to correlate the changes in cell numbers and ratios with changes in gene expression, we measured the levels of the ICM-specific transcript POU5F1 and the housekeeping gene 18S in 1X vs. 3X blastocysts using real-time RT-PCR. Corresponding to the differences in Cp values (Fig. 3A), the absolute POU5F1 concentration (in relative units) per blastocyst increased significantly in both ECNT and IVF 3X vs. 1X embryos (15.1 vs. 10.3 and 42.8 vs. 27.8, respectively, with P < 0.05) (Table 3) but increased only marginally in SCNT aggregates (5.7 vs. 7.3). Consequently, the relative POU5F1 transcript concentration per 18S-containing blastocyst cell did not change in 1X vs. 3X ECNT and IVF embryos (37.4 vs. 48.0 and 65.9 vs. 66.5, respectively) but decreased significantly in 3X SCNT embryos (28.3 vs. 18.7, P < 0.05) (Fig. 3B and Table 3). Blastocyst cells from 1X ECNT and from 1X and 3X SCNT embryos had significantly lower relative POU5F1 transcript concentrations than 1X or 3X IVF embryos (P < 0.01) (Fig. 3B and Table 3). There was no such difference between the ECNT aggregates and IVF embryos (P = 0.13) (Fig. 3B and Table 3). The levels of POU5F1 and 18S transcripts were measured in five different pools of 100 ± 5 dissociated morula donor cells collected during three of the NT/IVF runs. The absolute POU5F1 levels were lower than in the IVF and 3X ECNT but not different from the SCNT and 1X ECNT blastocysts (P < 0.05) (Table 3). The levels of 18S were lower than in IVF and 3X SCNT but not different from 1X SCNT and ECNT blastocysts (P < 0.05) (Table 3). In pools of 100 or 5000 somatic MBP1 donor cells, the POU5F1 transcript was undetectable (Fig. 3B and Table 3).
In Vivo Development of 1X and 3X Embryos
In order to correlate the results for in vitro development, blastocyst morphology, and gene expression with in vivo developmental potential, we transferred embryos from 1X-IVC vs. 3X-IVC into recipient cows. A low number of 2X embryos (see Materials and Methods) was also transferred. Across both donor embryo sources (Bull 1 and Bull 2), pregnancy establishment at Day 35 (22/85 = 26% vs. 12/44 = 27%, respectively) was not significantly different for 1X vs. 3X ECNT embryos (Table 4). Pregnancy establishment for the 2X ECNT group was more than threefold higher (86% vs. 26% and 27%, respectively, P < 0.01) (Table 4). The majority of post-implantation losses occurred between Day 35 and Day 90, with very little loss between Day 90 and Day 120 (Fig. 4A). ECNT fetuses showed none of the typical bovine somatic clone-associated late gestation losses (e.g., due to hydroallantois) after Day 120 (Fig. 4A). Development to term (11/44 = 25% vs. 9/85 = 11%, P < 0.05) or weaning (8/44 = 18% vs. 7/85 = 8%, P=0.09) was more than doubled for the 3X vs. 1X ECNT embryos (Table 4). Likewise, the low number of 2X embryos transferred resulted in more than threefold higher cloning efficiencies to term and weaning compared to 1X embryos (3/7 = 43% vs. 9/85 = 11% and 2/7 = 29% vs. 7/85 = 8%, respectively), although these results were not statistically significant (Table 4). Taken together, all the aggregated ECNT embryos (2X and 3X combined) were significantly better at developing into calves at weaning and beyond than non-aggregated embryos (10/51 = 20% vs. 7/85 = 8%, P < 0.05). We did not observe significant differences in terms of in vivo survival between ECNT embryos derived from different individual donors; instead, survival to weaning was mostly correlated with the number of embryos transferred (Supplemental Fig. 3 [available online at www.biolreprod.org]). The average birth weights of 3X calves cloned from IVF donor embryos from Bull 1 and Bull 2 were very similar (42.2 ± 7.6 vs. 45.8 ± 6.7 kg, respectively) (Supplemental Table 1 [available online at www.biolreprod.org]). There were also no differences in average birth weights between the 1X vs. 3X ECNT calves (42.1 ± 7.3 vs. 43.7 ± 7.1 kg, respectively) (Supplemental Table 1 [available online at www.biolreprod.org]). However, the few 2X calves born were significantly heavier than those derived by 1X-IVC and 3X-IVC (51.3 ± 7.5 kg, P < 0.05) (Supplemental Table 1 [available online at www.biolreprod.org]). Male 1X calves were significantly heavier than females (47.6 ± 6.1 vs. 37.7 ± 4.9 kg, respectively, P < 0.05), while there was no difference between male and female 3X calves (43.6 ± 7.8 vs. 44.3 ± 6.6 kg, respectively). Survival beyond weaning was not different for any of the groups and all cattle, born between September 2002 and August 2005, appeared healthy.
With the SCNT and IVF embryos, we did not observe any beneficial effect of aggregation. For the MBP1 donor cell line, pregnancy establishment at Day 35 (6/21 = 29% vs. 2/5 = 40% vs. 9/20 = 45%), development to term (3/21 = 14% vs. 0/5 = 0% vs. 3/20 = 15%), and weaning (2/21 = 10% vs. 0/5 = 0% vs. 3/20 = 15%) were not different for the 1X- vs. 2X- vs. 3X- SCNT embryos, respectively (Fig. 4B and Table 4). Across all nine somatic cell lines, pregnancy establishment at D35 (52/119 = 44% vs. 16/26 = 62% vs. 51/88 = 58%), development to term (17/119 = 14% vs. 4/26 = 15% vs 16/88 = 18%), and weaning (10/119 = 8% vs. 1/26 = 4% vs. 8/88 = 9%) were not different for SCNT embryos from 1X- vs. 2X- vs. 3X-IVC, respectively (Fig. 4C and Table 4). In the IVF group there was also no difference between pregnancy establishment at Day 35 (16/27 = 59% vs. 15/27 = 56%) or development to Day 100 (14/27 = 52% vs. 12/27 = 44%) for 1X- vs. 3X embryos, respectively (Table 4). The average birth weights of all 1X vs. 2X vs. 3X SCNT calves were not significantly different (43.1 ± 7.5 vs. 48.4 ± 8.1 vs. 43.7 ± 10.6 kg, respectively). Survival beyond weaning was not different either and all cattle, born between July 2002 and August 2003, appeared healthy.
We have evaluated the effects of aggregating bovine ECNT and SCNT compared to IVF control embryos. Aggregation affected ECNT and SCNT embryos differently during development. In ECNT aggregates, in vitro development was impaired but the few developing blastocysts appeared molecularly normal and survived better to term. In SCNT aggregates, in vitro development was normal but the resulting blastocysts showed reduced POU5F1 expression and compromised in vivo survival. IVF aggregates, despite forming molecularly normal blastocysts, were also developmentally compromised in vivo. As discussed below, these differences in timing of developmental failure may be due to genetic vs. epigenetic errors in ECNT and IVF vs. SCNT embryos, respectively.
The impaired in vitro development of ECNT aggregates indicates that individual ECNT embryos harbor abnormalities that are severe enough to compromise the development of the whole aggregate. Given that these abnormalities could not be compensated for, they are likely to be genetic rather than epigenetic, and they may be caused by chromosomal alterations present in the morula donors. About 46% of Day 5 IVP embryos that contain more than 16 cells are mixoploid, with triploidy being the most frequent abnormality [48]. Such aneuploid embryos are unlikely to develop into blastocysts and may even compromise their aggregation partners. Cell cycle incompatibility between aged cytoplasts and S-phase donor cells can also contribute to chromosome abnormalities. Some cytoplasts may have sufficiently high residual levels of M-Cdk activity to induce premature condensation and rereplication of donor chromosomes, resulting in developmental failure [49, 50]. Somatic donors, which are routinely screened for karyotypic abnormalities and synchronized by serum-starvation, are unlikely to introduce genetic abnormalities that could compromise in vitro development.
Aggregation blastocysts had increased ICM:TC ratios, and this could be due to the mechanism of lineage allocation at the morula stage. We observed that morula diameter increased with aggregation. This increase in diameter would lead to an increase in surface area and volume by the power of two and three, respectively, resulting in large morulae having a smaller surface to volume ratio. According to the inside/outside-hypothesis [51], cells on the morula surface will become TE cells, while inner cells, which mainly constitute the morula volume, become ICM cells. Thus, TE cell numbers would increase disproportionately less in aggregates. Given the significant increase in total cell number of IVF and SCNT over ECNT embryos, this could also explain the relatively larger increase in ICM:TC ratios in IVF and SCNT compared to ECNT aggregates (Table 2). We did not determine whether daughter cells of the original three NT reconstructs freely intermingled and contributed equally to ICM or TE lineages in the chimeric blastocyst. Depending on reprogramming status, the daughter cells may have been preferentially allocated to one lineage. This type of developmental bias can strongly compromise the viability of the aggregation blastocyst and may have occurred in SCNT but not ECNT aggregates. However, this does not explain why the ICM:TC ratios were also significantly higher in non-aggregated SCNT vs. ECNT and IVF embryos with similar cell numbers and presumably similar embryo sizes. While our data confirm earlier findings that non-aggregated SCNT embryos have more ICM cells than IVF embryos [52], the reasons for this preferential localization of cells to the inside of the SCNT blastocyst are unclear. At least some of the outside cells became ICM instead of TE cells, which suggests that it is more difficult to reprogram somatic genomes into the TE lineage. In contrast to some reports [35, 53] but in agreement with other studies [52, 54], we observed that SCNT blastocysts contained similar numbers of nuclei as IVF controls, while the ECNT counts were lowest. This may be species-dependent and/or because our IVC system supports SCNT preimplantation development equally well, if not better, than IVF or ECNT development.
It has been speculated that fewer TE cells in SCNT embryos lead to abnormal placenta formation, and thus, the ICM:TC ratios could be used to predict postblastocyst survival [52]. Our results do not support this hypothesis. First, IVF and SCNT embryos showed increased ratios after aggregation but no reduction in survival to Day 100 or term, respectively. In our experience, Day 100 survival accurately predicts survival to term for IVF embryos. Second, despite significant ratio increases after aggregation, 3X ECNT embryos showed 2.5-fold increased development to term. Third, there was no correlation between the absolute ICM:TC ratios and survival. IVF embryos with both low (0.33) and high (0.56) ratios survived much better than ECNT embryos with low or medium (0.34, 0.44) and SCNT embryos with medium or high (0.44, 0.55) ratios. Likewise, ECNT 1X embryos with low ratios had no greater chance of survival than SCNT 1X with medium ratios or SCNT 3X with high ratios.
Despite the increased ICM:TC ratios after aggregation, the relative POU5F1 expression level did not increase in any of the groups, and even showed a significant 34% decrease in SCNT aggregates, which indicates that either each cell only had two thirds the average amount of POU5F1 mRNA or only two thirds of the cells in the aggregates expressed POU5F1 at the 1X level. This lack of correlation between POU5F1 expression and ICM size in SCNT blastocysts has been observed previously [55]. It could be interpreted as POU5F1 being not strictly ICM-specific in the Day 7 bovine blastocyst, and indeed, we have detected POU5F1 in Day 7 but not D8 trophectoderm biopsies, albeit at lower levels than in the ICM (unpublished data). To determine which cells had reduced POU5F1 expression in SCNT 3X embryos, quantitative in situ detection methods with single-cell resolution would be needed. Reprogramming differences between ECNT and SCNT embryos may explain the differences in POU5F1 expression. An ECNT blastocyst that comprised about 100 cells contained the same absolute amount (in relative units) of POU5F1 mRNA as about 100 ECNT donor cells (Tables 2 and 3). This is less than expected, which indicates that POU5F1 expression is initially downregulated and not completely reactivated at the right time or that the rates of POU5F1 synthesis vs. POU5F1 decay simply persist without any reprogramming at all. If the presence of POU5F1 transcripts is simply a carry-over from the donor cell, then it could obviously not serve as a reprogramming marker to measure epigenetic complementation or interference in ECNT blastocysts. On the other hand, POU5F1 expression in SCNT blastocysts must be due to reprogramming of the somatic donor genome because: 1) the MBP1 clonal strain of transgenic fibroblast donors had no detectable levels of POU5F1 transcript (Fig. 3B); and 2) at the blastocyst stage, maternal POU5F1 mRNA is no longer detectable in IVF [26] and SCNT bovine embryos (unpublished data). Therefore, the reduced POU5F1 expression in SCNT clones is due to insufficient reprogramming, with no indication of the cells complementing each other. Non-cell-autonomous rescue of Pou5f1 expression in somatic cells after injection into in vivo-derived early cleavage-stage mouse embryos, which is possibly cAMP-mediated via gap junctions, has been described previously [56]. Induction of Pou5f1 has been demonstrated by immunofluorescence and presumably involves upstream transactivating pathways, such as the Spalt family member Sall4 [57] and the Pou5f1/Sox2 complex [58]. However, exact quantification of the proportion of injected cells that activated Pou5f1 and its expression levels was not reported, making it difficult to compare these data to our results.
Irrespective of aggregation, the amount of POU5F1 transcript differed significantly between the three groups; expression was highest in IVF, intermediate in 3X ECNT, and lowest in 1X ECNT and SCNT embryos (Table 3). Thus, even the 36% of SCNT embryos previously identified to have normal Pou5f1 localization [22] may have had abnormal levels of Pou5f1 expression. Low average POU5F1 expression in SCNT embryos was not a consequence of outliers in either of the groups, since the variation in POU5F1 expression was actually smaller for SCNT than for ECNT and IVF embryos (Fig. 3B). In fact, even the highest relative POU5F1 levels in SCNT embryos were only just above the average expression level in IVF embryos, and the coefficient of variation was not significantly different for relative POU5F1 expression in NT and IVF embryos (data not shown). Variation between different NT/IVF experiments and cDNA batches was consistent between NT/IVF groups processed at the same time and interassay reproducibility was high (Table 3). The level of exogenous standard rabbit -globin mRNA, which is a control for similar efficiency of RNA recovery between samples and for varying amounts of RNA starting material that may affect cDNA synthesis [59], did not differ significantly between the groups (Fig. 3A). Therefore, our data lend support to the idea of the POU5F1 transcript being a molecular marker to predict blastocyst survival after embryo transfer [22], at least for the clonal donor cell strain analyzed here. In vivo survival rates were highest for IVF, intermediate for 3X ECNT, and lowest for 1X ECNT and SCNT embryos, correlating with the absolute and relative POU5F1 expression levels. Therefore, it may be possible to predict improved in vivo survival from observations of high POU5F1 expression levels. By selecting only the top 10% of POU5F1 expressers within an NT blastocyst population or using ectopically POU5F1-expressing donor cells, this predictive value could be tested directly.
Aggregation blastocysts (2X/3X) would theoretically have a two- to threefold higher chance of containing some fully reprogrammed cells. If there were no positive or negative interactions within the composite blastocyst, one would expect an n-fold increase in in vivo survival using n numbers of aggregation partners. The ECNT reconstructs were in agreement with this prediction, showing on average 2.5-fold higher cloning efficiency. In other words, there was no evidence for complementary interactions resulting in increased survival beyond what would be expected as a direct numerical consequence of aggregation. A similar increase in the in term survival of 3X ECNT embryos aggregated after 2 days in culture has been described previously, although the numbers were too low to draw definitive conclusions [60]. In contrast, none of the SCNT blastocysts showed increased cloning efficiency, indicating that they were in fact compromised compared to the ECNT aggregates. While reduced POU5F1 expression was only measured in the MBP1-derived embryos, the effect of in vivo survival was determined across nine independent fibroblast lines. Our results contrast with the data obtained for mouse SCNT clones, in which in vivo development after aggregation was eightfold greater, i.e., increased beyond what would be expected as a numerical consequence of aggregation [35]. The datasets differ in two main aspects: 1) species (cattle vs. mouse); and 2) aggregation timing (1-cell vs. 4-cell). The species difference alone is unlikely to have a large impact, given the high degree of conservation in basic cellular and developmental mechanisms in mammals. The timing of aggregation may be more critical. We aggregated as early as possible based on two assumptions: 1) mixing of blastomeres derived from the three individual NT reconstructs would be maximized at the blastocyst stage, as shown in the mouse, in which aggregating 2-cells results in chimeric blastocysts in which the descendants of the original aggregation partners intermingle and participate in both ICM and TE formation [61], while aggregating at the 8-cell stage revealed little, if any, cell mingling [62]; 2) cellular interactions resulting in beneficial complementation between individual embryos would occur very early. This was based on studies to overcome the 2-cell block in mouse, for which blocking embryos could be rescued to develop into blastocysts by aggregation with nonblocking embryos at the 2-cell stage, indicating that complementation starts immediately upon aggregation [61, 63]. However, mouse clones that have already developed to the 4-cell stage usually have a higher probability of developing into blastocysts than bovine SCNT reconstructs at the 1-cell stage. Furthermore, in our case, aggregation occurred before the onset of major embryonic genome activation (8–16-cell in cattle) rather than after this event (late 1-cell stage in the mouse). If the transcripts that arise during genome activation mediate beneficial effects, bovine aggregates would be exposed to them at least three cell cycles later. In the absence of these factors, insufficiently reprogrammed SCNT embryos may be more likely to compromise each other. It remains to be determined whether early aggregation has any negative effects on the expression of early transcripts during embryonic genome activation or on the degradation of maternal mRNA. Aggregating bovine embryos at later stages should clarify this issue.
Finally, the IVF aggregates had similar survival rates in utero compared to the 1X embryos, which indicates that they were also developmentally compromised. The IVF aggregates were true chimeras that comprised genetically rather than epigenetically (as in the case of ECNT and SCNT aggregates) distinct cell populations. For unknown reasons, the rate of livestock chimera development to term is relatively low. Even the in vitro development of chimeric embryos, aggregated from different IVF embryos, is significantly lower than after aggregation from the same IVF embryo [64]. This effect was not due to combining embryos of different sexes. Our data support a model in which negative interactions between genetically different aggregation partners also compromise postblastocyst development. Further research into the nature of these interactions, e.g., the requirement for cell-cell contact or secreted molecules, could benefit the efficiency of chimera production from the IVF embryos of livestock.
Despite its potential for agricultural applications [65], embryonic cloning faces two major obstacles, namely, the variability in developmental competence among donor embryos (Supplemental Fig. 2 [available online at www.biolreprod.org]) and the limited availability of donor nuclei or embryonic cell lines. Neither of these problems is resolved in the present study, as the cloning efficiency per donor embryo was not increased through aggregation. Each donor contained an average of 31 blastomeres, resulting in 29 reconstructs, 6.9 (2.9 for 3X) blastocysts, and 0.55 (0.58 for 3X) calves at weaning. These efficiencies are too low for widespread commercialization, which requires at least one calf, but preferentially 3–6 calves, per donor embryo. Alternatives, such as using donor embryos at later developmental stages (which contain more cells for NT), result in reduced in vitro and in vivo cloning efficiencies [66]. Similarly, during the recloning of selected embryos, the efficiency decreases with each round of NT [67]. In the future, the use of embryonic stem cell-like cells may overcome the problem of limited donor cell supply [68]. The NT embryo aggregation strategy described in the present study currently offers no commercial advantages and actually reduces the throughput in SCNT embryo and offspring production. However, from a basic research perspective, our results highlight differences between ECNT and SCNT embryos that warrant further investigation into the biological mechanisms underlying the compromising interactions following aggregation.
ACKNOWLEDGMENTS
We thank the past and present members of our cloning team (A. Green, K. Leslie, J. Oliver, A. Schurmann, and H. Troskie) and our farm staff (J. Forsyth, M. Berg, K. Cockrem, and V. Prendergast) for excellent technical assistance. We thank Dr. G. Laible for supplying the MBP donor cell line and Dr. N. Cox for assistance with the statistical analysis. We also thank Dr. W. Vivanco for initiating the bovine embryo cloning at AgResearch, and M. Olifant for technical assistance in the early stages of this project.
FOOTNOTES
3These authors contributed equally to this work. 
4Current Address: BASF Plant Science GmbH, 67 117 Limburgerhof, Germany.
3These authors contributed equally to the work.
1Supported by the New Zealand Foundation for Research, Science and Technology, Meat NZ, and AgResearch.
Correspondence: 2FAX: +64 7 838 5536; e-mail: bjorn.oback{at}agresearch.co.nz
Received: 18 February 2006.
First decision: 16 March 2006.
Accepted: 17 October 2006.
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