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Embryo |
a Section of Reproductive Biology,
b Department of Animal Breeding and Genetics, and Section for Metabolism, Growth and Lactation, Department of Animal Nutrition and Physiology, Danish Institute of Agricultural Sciences, DK-8830 Tjele, Denmark
c Monash Institute of Reproduction and Development, Monash University, Clayton, Victoria 3168, Australia
d Genetics Australia Co-operative Ltd., Bacchus Marsh, Victoria 3340, Australia
e Department of Anatomy and Physiology
f Section of Reproduction, Royal Veterinary and Agricultural University, DK-1870 Frederiksberg C, Denmark
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, embryo
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A less demanding and more productive and reliable manipulation technology for somatic cell nuclear transfer would allow more efficient production of embryos for transfer. It would also provide a more efficient method of producing material to study the basic scientific aspects of the critical limiting steps in nuclear transfer. The first published attempt to expand the conventional frontiers and exclude micromanipulators from the procedure was the bovine zona-free embryonic cell nuclear transfer method of Peura et al. [5]. Very recently, in a short preliminary communication, a technique based on the same principles but with significant technical modifications was developed for somatic cell nuclear transfer [6]. Subsequently, two additional articles were published demonstrating the application of zona-free nuclear transfer procedures, but these procedures still required micromanipulation [7, 8].
The present article is the first full publication describing somatic cell nuclear transfer done by hand, i.e., without micromanipulation. The purpose of the work was to establish a viable alternative to the traditional methods of somatic cell nuclear transfer. Efforts were focused on increasing the efficiencies of both blastocyst production and labor requirements. Separate experiments were performed to optimize the chemical environment for oocyte bisection, parameters and timing of fusion, and culture conditions regarding the protein source. To characterize possible differences between cattle serum and commercially available fetal calf serum (FCS), the mitogenic activity of these sera was tested in a bioassay based on bovine mammary epithelial cells in culture. Embryos produced were characterized by inner cell mass (ICM)-trophectodermal cell differential staining and electron microscopic investigation. The ability to establish pregnancy was investigated with transfers of single fresh or vitrified blastocysts into synchronized recipient heifers.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In Vitro Embryo Production
Methods used for oocyte maturation, fertilization, and embryo culture are described in detail elsewhere [9, 10]. Oocytes were aspirated from abattoir-derived ovaries 4–6 h after slaughter. For maturation, 25 oocytes were placed into each well of four-well dishes (Nunc, Roskilde, Denmark) for 24 h in 400 µl of bicarbonate buffered TCM-199 medium (Gibco BRL, Paisley, U.K.) supplemented with 15% cattle serum (CS; Danish Veterinary Institute, Copenhagen, Denmark), 10 IU/ml eCG, and 5 IU/ml hCG (Suigonan Vet; Intervet, Skovlunde, Denmark) under mineral oil at 39°C in 5% CO2 in humidified air. Fertilization was performed in modified Tyrode medium [11]. After 21–22 h of incubation, zygotes were vortexed at maximum speed to remove the cumulus cell layer and attached spermatozoa and then placed in groups of 50 into wells of four-well dishes each containing 400 µl culture medium consisting of SOFaaci medium [10] supplemented with 5% CS. Cultures were left undisturbed from Day 1 to Day 7 (Day 1: day of fertilization) at 38.7°C in 5% CO2, 5% O2, and 90% N2 atmosphere with maximum humidity.
Nuclear Transfer
Methods used for somatic cell nuclear transfer were based on the embryonic cell cloning procedure of Peura et al. [5] that was adapted for somatic cell cloning [6] and further modified to increase efficiency. At 21–22 h after the start of maturation, 100–150 oocyte-cumulus cell complexes were placed into a 2-ml Eppendorf tube containing 0.5 mg/ml hyaluronidase dissolved in 500 µl T0 (T indicates Hepes-buffered TCM 199 medium [9], and the number indicates percentage of CS supplementation). After a 2-min incubation at 39°C, cumulus cells were removed first with careful pipetting using a 1-ml automatic pipette for 1 min and then with vortexing at maximum speed for 3 min. From this point (except where otherwise indicated), all manipulations were performed on a heated stage adjusted to 39°C. Oocytes were separated from the dispersed cumulus cells using two washes in 35-mm Petri dishes (Nunc), each containing 4 ml T2. Oocytes with visible degenerative changes or physical damage were discarded, and the remaining oocytes were used for cytoplast preparation without any further selection.
All incubations (except where otherwise indicated) were performed in wells of four-well dishes in Hepes-buffered medium without oil overlay. Oocytes and embryos were moved using a mouth pipette with finely drawn, fire-polished glass capillaries with an inner diameter of approximately 250 µm. Oocytes were transferred first into 1.5 mg/ml pronase (Sigma protease) dissolved in 600 µl T10, and the dish was rotated using a horizontal shaker at 150 rpm and 39°C for 10–15 min, then at 80 rpm for 1 min. As a result of the low-speed rotation, zona-free oocytes were collected in the middle of the well and could thus be easily transferred into another well containing one of various concentrations of cytochalasin B dissolved in 800 µl T20, where they were incubated for at least 3 min.
For manual bisection, 40–50 zona-free oocytes were lined up in a 35-mm Petri dish with T20 and the appropriate concentration of cytochalasin B. Bisection was performed manually under stereomicroscopic control with Ultra Sharp Splitting Blades (AB Technology, Pullman, WA). Bisected oocytes were collected in the middle of the dish by swirling the dish and were then transferred into 800 µl T2 for storage. The procedure was repeated with the remaining oocytes.
After completion of the bisection, all demioocytes were stained with 10 µg/ml fluorochrome Hoechst 33342 dissolved in T2 for 5 min. Stained demioocytes were briefly washed in T2, placed into 3-µl drops of T2 on the bottom of a 60-mm Falcon Petri dish, and covered with oil (three demioocytes per drop). Using an inverted microscope and ultraviolet light, positions of half-oocytes without chromatin staining (cytoplasts) were registered using a tape recorder. Cytoplasts were then collected under a stereomicroscope and transferred into 800 µl T20 for temporary storage.
Primary granulosa cell monolayers were prepared from attached cells of cumulus-oocyte complexes in four-well dishes used 7–10 days earlier for maturation. No changes were made in the original maturation medium in these dishes during the culture period. Bovine fetal fibroblast cell line was prepared according to Korfiatis et al. [12]. After several passages in tissue culture flasks, fibroblasts were cultured in wells of four-well dishes in 400 µl Dulbecco modified Eagle medium (no. 42430-025; Gibco) supplemented with 10% FCS (no. 10106-169; Gibco) and covered with 400 µl oil. One- to 2-wk-old cultures were used for nuclear transfer donors, without previous serum starvation or medium change during the culture period. For nuclear transfer, both medium and oil were removed from one well, and cells were repeatedly washed with Ca++- and Mg++-free PBS and incubated at 39°C for 5 min in 100 µl of 0.05% trypsin dissolved in PBS. The well was then filled with 800 µl of T20, and cells were separated by vigorous pipetting and stored in 2-ml Eppendorf tubes at room temperature until fusion.
Fusions were performed 23–24 h after the start of maturation. For the first fusion, half of the total quantity of prepared cytoplasts was transferred into the first well of a four-well dish containing 800 µl T20. Five microliters of the somatic cell suspension was sedimented to the bottom of the middle compartment of a four-well dish filled with 4 ml T2. Cytoplasts were then individually transferred to the second well containing 500 µg/ml phytohemagglutinin (L 8754; Sigma) dissolved in 400 µl T2 for 3 sec and then quickly dropped over a single somatic cell settled to the bottom of the dish. Following attachment, the cytoplast-somatic cell pair was again picked up and transferred to a fusion chamber (BTX microslide 0.5-mm fusion chamber, model 450, no. 01-000209-01; VWR International ApS, Albertslund, Denmark) with 0.5-mm separation of wires. Before application, the glue was removed from the central 2-cm area of the wires with a surgical blade. Wires were covered with 2 ml of fusion medium (0.3 M mannitol, 0.1 mM MgSO4, 0.05 mM CaCl2) at 26–27°C. After incubation for 2–3 min in the fusion medium, the pair was attached to one of the wires using 15 V AC and 700 KHz (Electrofusion Machine; Genaus, Bacchus Marsh, Australia). Unless otherwise indicated, the somatic cell of each pair was positioned furthest from the wire. Fusion was performed with a double DC pulse of 65 V, each pulse for 20 µsec and 0.1 sec apart. The pair was then carefully removed and transferred to the third or fourth well of the four-well dish, both containing 800 µl T20. Unless otherwise indicated, pairs were incubated here for 15–30 min to determine whether fusion had occurred. After having fused 15–20 pairs, the fusion medium was changed to prevent damage caused by evaporation. New somatic cells were also added to the middle section of the four-well dish to avoid difficulties in pairing caused by attachment of somatic cells to the bottom of the dish.
For the second fusion, all remaining cytoplasts and fused pairs were transferred to fusion medium covering the fusion chamber. To avoid mixing, cytoplasts were placed "north" of the wires (i.e., furthest from the operator), and pairs were placed "south" of the wires. After incubation for approximately 2 min, 10 cytoplasts were aligned to one electrode using the same AC as for the first fusion. One fused pair was then attached to each cytoplast. A double fusion pulse with the same parameters but at 45 V DC was applied, then the double cytoplast-granulosa cell triplets were incubated in T20 for 20 min. These fused reconstructed embryos were then transferred into a well of a four-well dish containing 400 µl culture medium, covered with oil, and incubated in 5% CO2 in air at 39°C.
Activation was initiated 28 h after the start of maturation (approximately 4 h after the fusion). Reconstructed embryos were first incubated in 1 ml T20 containing 2 µM Ca ionophore A23187 for 5 min at room temperature. After two subsequent washings in T20, reconstructed embryos were incubated individually (to keep them from adhering to each other) in 5-µl droplets of culture medium containing 2 mM 6-dimethylaminopurine (6-DMAP), and covered with oil, and incubated in an atmosphere of 5% CO2 in air at 39°C for 6 h. Embryos were then washed twice in culture medium and cultured individually in well of the wells (WOWs) [13] in 400 µl culture medium covered with 400 µl oil. Embryo culture was performed at 39°C in 5% CO2, 5% O2, and 90% N2.
Seven days after reconstruction, the number of blastocysts per reconstructed embryo was determined using a stereomicroscope.
Experiment 1: Composition of Bisection Medium
After pronase digestion, oocytes were incubated for 3–10 min in T20 or in the same medium with the following additives commonly used during micromanipulations: 0.25 and 0.125 M sucrose or 7.5, 5.0, and 2.5 µg/ml cytochalasin B. Oocytes were then bisected manually and incubated in T20 in air for an additional 30 min, when the proportion of the lysed demioocytes was determined.
Experiment 2: Position of Cells at Cytoplast Somatic Cell Fusion
Cytoplasts were prepared and paired with granulosa cells as described above. For fusion, pairs were placed between the wires of the fusion chamber but were attaching to one wire either in a random position (group 1) or with somatic cells furthest from the wire (group 2). Fusion was performed using parameters described above, and then pairs were incubated in T20 in air as described above for 30 min, when fusion rates were determined.
Experiment 3: Time Between the First and Second Fusion
Immediately after the first fusion, pairs were randomly distributed into three groups, and a second fusion (pair + cytoplast) was performed at 5, 15, or 60 min (groups 1, 2, and 3, respectively) after the first fusion. Triplets were then incubated in T20 for 30 min, when fusion rates were determined.
In experiments 1, 2, and 3, each group consisted of 50–70 oocytes or pairs distributed to four replicates. Results were analyzed by ANOVA or chi-square test, with P < 0.05 regarded as significant.
Experiment 4: Effect of Protein Sources on Embryo Development
Two separate series of in vitro embryo production replicates were performed to test the ability of BSA or FCS versus CS to support embryo development. In vitro maturation and fertilization was performed as described above. For embryo culture, SOFaaci was supplemented with either 25 mg/ml BSA (reagent grade, ABRZ-010; Immuno Chemical Products Ltd., Auckland, New Zealand) or 5% FCS (no. 14-701E; Bio Whittaker, Vallensbæk Strand, Denmark). In control groups for both series, 5% CS supplementation was applied. Each group consisted of 25–55 embryos, with four replicates for each series. Data were analyzed with ANOVA.
Experiment 5: Bioassay with Bovine Mammary Epithelial Cells in Culture
Mammary epithelial cells isolated from prepubertal Holstein-Friesian heifers were cultured in three-dimensional collagen gels as described previously [14]. Cells were cultured for 24 h in basal serum-free medium 199 containing BSA (2.6 g/L; Sigma-Aldrich, Vallensbæk Strand, Denmark), transferrin (5 mg/L), reduced glutathione (1 mg/L), soybean trypsin inhibitor (1 mg/L), bovine insulin (10 µg/L), selenium (1 µg/L), and antibiotic solution (0.2%), followed by 4 days in treatment medium containing CS or FCS at concentrations of 0.5%, 1%, 2%, 4%, 6%, 8%, and 10%. Insulin-like growth factor I (IGF-I; Austral Biologicals, San Ramon, CA) in different concentrations (1.56, 3.12, 6.25, 12.5, 25, 50, and 100 ng/ml) was also included in the cell culture assays. This growth factor stimulates mammary epithelial cell growth in a dose-dependent manner and therefore represents a factor to evaluate the growth response in each cell culture assay. Culture medium was changed every 2 days, and 1 µCi [methyl-3H]thymidine was added for the last 24 h of the culture period. Proliferation of epithelial cells was determined using incorporation of thymidine (dpm/well) as a measure of DNA synthesis. The experimental design included three replicates per treatment. Because variation was observed in the mitogenic response to basal medium among the two independent assays, results were adjusted to a similar scale by dividing values for [3H]thymidine incorporation per well by the average basal value for incorporation in each assay. Statistical analyses were performed using the general linear models procedure (PROC GLM) of the SAS Institute [15]. The effect of CS and FCS on cell proliferation was tested in a model including the systemic effects of source of serum (n = 2), concentrations of serum (eight levels), and their interactions. The effect of assay (n = 2) was included as a blocking factor in the model. The residual mean error was used as the error term for the F-test.
Quantitative Assessment of Embryo Development
In 16 consecutive somatic cell nuclear transfer experiments, the parameters described above were used. Donor cells in the first eight and last eight experiments were primary granulosa cell monolayers and fetal fibroblasts, respectively. Seven days after reconstruction, blastocyst per reconstructed embryo rates and time of manual work required for production of one blastocyst were calculated.
Qualitative Characterization of Blastocysts Produced with the Handmade Nuclear Transfer Method
ICM-trophectodermal cell differential staining Twenty randomly selected Day 7 blastocysts produced with somatic cell nuclear transfer were subjected to differential staining according to the method of Thouas et al. [16] with modifications related to the requirements of bovine zona-free blastocysts. The procedure was performed at room temperature and in a semidark room. Embryos were first immersed for 5–7 sec in PBS containing 0.2% (v/v) Triton X-100 and 0.3 mg/ml propidium iodide and then immersed in glycerol containing 20 µg/ml Hoechst 33342. After 2–3 h of incubation, embryos were transferred in 2 µl of solution onto a microscopic slide and covered with a glass coverslip. Digital photos were taken using a Diaphot 200 inverted microscope with epifluorescent attachment and a UV-2A filter (Nikon, Tokyo, Japan), and cell numbers were determined using Image-Pro Plus version 4.0 software (Media Cybernetics, Silver Spring, MD).
Transmission electron microscopy Three Day 7 somatic cell cloned blastocysts were fixed for utrastructural examination. Embryos were placed in 3% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.2–7.4, at 4°C for 60 min, washed twice for 5 min, and stored in sodium phosphate buffer at 4°C until processed further. Blastocysts were then embedded in 4% agar individually or in groups and postfixed in 1% OsO4 in 0.1 M cacodylate buffer for 1 h at 4°C. The samples were then dehydrated by passage through an ethanol series, stained en block with uranyl, embedded in Epon, and serially sectioned into semithin sections (2 µm), which were stained with toluidine blue for bright-field light microscopy. Selected semithin sections were subsequently reembedded [17], and ultrathin sections (70 nm) were prepared, collected on copper grids, stained with uranyl acetate and lead citrate, and examined on a transmission electron microscope (JEM-1200 EX; Jeol, Tokyo, Japan).
Embryo transfer To increase the developmental ability of cloned embryos, aggregates were formed according to the method of Peura et al. [5], with modifications. At Day 4, compacted morulae produced by handmade somatic cell nuclear transfer were removed from the original WOWs and placed back in pairs, i.e., two morulae per well, and cultured together until Day 7. Single blastocysts developing from the aggregates were nonsurgically transferred to synchronized recipient heifers (one embryo/animal). Transfer was performed either with fresh embryos produced from fetal fibroblasts (seven recipients) or with open-pulled straw (OPS) vitrified, in-straw diluted [18, 19] embryos produced using primary granulosa cells (four recipients). When fresh embryos were transferred, they were transported to the site of the embryo transfer in the original culture dishes using the submarine incubation system [20] and loaded into straws <5 min before transfer. Presence of pregnancy was determined with ultrasonography on Day 28, followed by weekly scannings.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The proportion of morphologically intact demioocytes after incubation in various media and bisection was as follows (different superior letters after percentages indicate significant differences): T20, 57/65 (88%)a; 0.25 M sucrose, 29/70 (41%)b; 0.125 M sucrose, 49/67 (73%)c; 7.5 µg/ml cytochalasin B, 65/66 (98%)d; 5.0 µg/ml cytochalasin B, 64/64 (100%)d; and 2.5 µg/ml cytochalasin B, 69/70 (99%)d. According to this result and to minimize the toxic effect, T20 medium with 2.5 µg/ml cytochalasin B was used as bisection medium for subsequent experiments.
Experiment 2
The cytoplast-somatic cell fusion rates were 37/67 (55%) and 66/70 (94%) for fusions performed in random and oriented position, respectively. The difference was significant. Because of this result, all fusions in the subsequent experiments were performed in oriented position.
Experiment 3
The second fusion was equally successful (>95%) in all three groups regardless of the time between the first and second fusion. However, the completion of the first fusion was disturbed by the early second fusion (different superior letters after percentages indicate significant differences): 19/50 (38%a, 49/52 (94%)b, and 50/50 (100%)b somatic cell-cytoplast fusions were detected when second fusion was performed 5, 15, or 60 min after the first one, respectively. Consequently, in all subsequent experiments second fusions were performed 15–60 min after the first fusions.
Experiment 4
BSA supplementation of culture medium for in vitro-fertilized embryos resulted in an average 39% (32%–44%) blastocyst/oocyte rate compared with the 59% (48%–64%) achieved with CS (P < 0.01). The average blastocyst/oocyte rate achieved with FCS supplementation was 28% (9%–41%), compared with 48% (42%–52%) achieved in that series with CS supplementation (P < 0.01).
Experiment 5
Addition of 0.5%–10% serum to basal medium increased the mitogenic activity in a dose-dependent manner (Fig. 1). Maximal stimulation occurred at 10% concentration for both sera tested. No differences (P > 0.05) in mitogenic activity of CS and FCS was seen at concentrations of <4% in basal medium. However, the mitogenic activity of CS was higher (P < 0.05) than that of FCS at concentrations of 4%–10% serum in basal medium. At maximum concentration, the mitogenic activity of CS and FCS was higher than that of the basal medium (7.4-fold and 4.2-fold increase, respectively; P < 0.001).
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Quantitative Assessment of Embryo Development
To determine the number of blastocysts, embryos were removed from the WOWs and examined with a stereomicroscope. Criteria for blastocysts included a clearly visible blastocoele and a defined ICM. This evaluation was easier than with zona-included embryos because the cell debris (often misinterpreted in the case of zona-included embryos as ICM) was detached from the surface. Moreover, on Day 7 most blastocysts were at the expanded stage and possessed well-defined and clearly outlined ICM (Fig. 2). In 16 consecutive experiments, a total of 398 blastocysts developed from 790 reconstructed embryos. The blastocyst/reconstructed embryo rate varied between 43% and 64% (the average was 51%). There was no significant difference in developmental rates achieved between granulosa cell and fetal fibroblast cell donors (49% versus 52%, respectively). The average number of blastocysts produced in one experiment was 25, but this value cannot be regarded as representative because the purpose of this series of experiments was to achieve high embryo development rates and not high numbers of blastocysts. Production of one blastocyst required, on average, 6 min of manual work, including cytoplast preparation, somatic cell preparation, and fusion; therefore, 10 blastocysts person-1 hour-1 were produced.
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Qualitative Characterization of the Produced Blastocysts
ICM-trophectodermal cell differential staining The average cell number (±SD) was 216 (±52). An average of 52 (±10) cells belonged to the ICM. The minimum total cell and ICM numbers were 142 and 33, respectively. The average and minimum ICM:trophectodermal cell ratio was 0.35% and 0.17%, respectively. However, this ratio was <0.2% only in embryos where the total cell number was >300.
Transmission electron microscopy All three embryos presented an intact trophectoderm lining and well-defined and very prominent ICMs. The trophectoderm was sealed by tight junctions and desmosomes. The ICMs presented some signs of apoptosis, including condensation of chromatin into large blocks and phagocytosis by neighboring cells. In one embryo, the first differentiation of a hypoblast from the ICM was observed (Fig. 3).
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Embryo transfer Transfer of fresh embryos resulted in pregnancy in six of the seven recipients. One of four recipients was pregnant after transfer of OPS-vitrified, in-straw diluted embryos. Heartbeats were registered in all seven fetuses on Day 34. Three fetuses from fresh embryo transfer had no heartbeat on Day 42, Day 55, and Day 62, respectively, and thus had died.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The difficulties and the ways to overcome them have been discussed in detail in these previous publications. Zona pellucida removal with pronase digestion in the presence of serum is an efficient and harmless method, even when large quantities of oocytes (up to 150) are digested together. For pairing of cytoplasts and somatic cells, short preincubation of cytoplasts in phytohemagglutinin was efficient and reliable. Fusion rates (either with separate first and second fusions or with both included in one step) were superior to those generally obtained with zona-enclosed pairs. Activation (as proved by Booth et al. [7]) required lower ionophore concentration than used in traditional nuclear transfer. Individual culture systems including the WOW and glass oviduct (GO) system [6, 21] have be suitable for achieving acceptable blastocysts/reconstructed embryo rates (18–36%).
The primary aim of our present work was to improve the handmade somatic cell nuclear transfer technology by optimizing certain steps of the procedure. According to our preliminary results (unpublished), selection and exclusive use of oocytes with polar bodies did not improve blastocyst rates; therefore, all oocytes without visible degenerative changes or physical damages were processed. This approach was in accordance with the strategy of Booth et al. [7]. Mass bisection of oocytes in a large volume of medium is a unique feature of our method. Because the procedure is performed rapidly, reliability is a key question; failures can only be detected retrospectively and might lead to mass lysis of cytoplasts. There are two common constituents of the medium used for embryo manipulation: sucrose, an osmotic buffer, and cytochalasin B, a cytoskeleton relaxant [5, 7, 22–27]. According to experiment 1, sucrose incubation decreased the percentage of demioocytes surviving bisection with an intact cell membrane. Cytochalasin B, however, increased the survival. The required minimal efficient concentration was, however, lower than generally applied (2.5 µg/ml versus 7.5 or 5 µg/ml).
Although the appropriate orientation of pairs in the fusion chamber may require some skills and several additional seconds of work with each pair, as demonstrated in experiment 2, it seems to be indispensable for achieving high fusion rates, even in the zona-free situation. In experiment 3, the effect of different time intervals between the first (cytoplast-somatic cell) and second (pair-cytoplast) fusions was investigated. If the second fusion pulse occurs before completion of the first fusion, the first fusion could be disturbed, even if the second fusion were completed successfully.
Additional changes resulting in improved blastocyst/oocyte rates included prolonged incubation between reconstruction, application of serum-containing medium for Ca-ionophore incubation, and individual incubation of reconstructed zona-free embryos in DMAP. According to our unpublished observations, these changes decreased lysis rates and prevented unintended attachment of embryos to each other. Because zona-free embryos were more sensitive to chemical activation [7], the concentration of Ca-ionophore was decreased to 2 µM. The incubation period between reconstruction and activation and during DMAP exposition was prolonged to from 3 to 4 h and from 4 to 6 h, respectively, in agreement with the observations of Alberio et al. [28], Kasinathan et al. [29], and Beyhan et al. [30].
In spite of efforts to use defined components [31, 32] and some controversial effects of serum [33], protein supplementation of embryo culture medium still seems to be advisable to obtain high and consistent blastocyst rates [25, 26, 28, 33–36]. According to our observations, there is also a marked difference in the effects achieved when different protein sources are used. For BSA, a relatively high concentration was used according to the method of Peura et al. [5, 37] to promote development of zona-free embryos in culture. Our comparative embryo culture experiment demonstrated that the CS (selected previously by us from four different batches and routinely used in our laboratory) was superior to BSA and FCS for the support of embryo development.
To explain the mechanism of this effect, the mitogenic activity of the CS and a commercially available FCS was investigated in a bioassay with primary mammary epithelial cells in collagen gels. The bioassay, which is stable and reproducible, is based on incorporation of tritiated thymidine into cell DNA as a measure of cell proliferation. This bioassay is sensitive to a number of different growth factors and hormones, including members of the insulin-like, epidermal, transforming, and fibroblast growth factor families [14, 38, 39] and other factors such as vitamin A and its metabolites [39]. Furthermore, the bioassay has been used to investigate the effects of growth stimulatory and inhibitory components in biological fluids such as serum, milk, or tissue extracts [38, 40] and as a screening system for biological and toxicological factors in milk and serum from cows fed genetically modified versus conventional fodder beets [41].
Compared to the mitogenic activity of IGF-I, which at maximum was 3.2-fold higher than the mitogenic activity obtained in basal medium (results not shown), the CS was far more mitogenic in the present bioassay. This considerably higher mitogenic activity could explain why this CS was superior for producing high in vitro embryo developmental rates. Further studies are needed to determine the factors present in this specific CS. However, according to our previous experiments, embryo culture in SOFaaci medium with 5% CS did not result in increased birthweight or any other alterations related to large offspring syndrome (unpublished).
Culture of zona-free embryos requires an individual system to prevent aggregation [42]. In our preliminary study [6], the GO system was more efficient than the WOW system for the culture of zona-free somatic cell cloned embryos [13]. However, Booth et al. [7] reported high blastocyst developmental rates of zona-free somatic cell cloned embryos using the WOW system. We found increased blastocyst development with the same serum, so the difference may be attributed to the different physical, chemical, or biological characteristics of this serum.
All the factors investigated and optimized in experiments 1–5 and our developing skills in handling oocytes and embryos during manipulations contributed to the increase in efficiency achieved since the preliminary publication of the method. These skills also contributed to the increased consistency observed during the experimental period. Productivity was high, as measured by the blastocyst/reconstructed embryo and blastocyst/working hour rates, although data regarding the latter parameter are rarely published. As a result of the improved steps of the procedure, even though two cytoplasts are required for one reconstructed embryo 25–30 blastocysts can be routinely produced from 150 oocytes collected for maturation. This high productivity in addition to the low costs of equipment (no micromanipulators or related tools such as grinder, microforge, and capillary puller are required) makes this technology very economical and affordable, even for laboratories with a limited budget.
The high efficiency and low production cost is not related to compromised quality, at least according to the available information. The total cell number of blastocysts 7 days after the reconstruction is very high. All investigated blastocysts possessed a well-defined ICM with acceptable cell number, and the ICM:trophectodermal cell ratio was only low in blastocysts where the total cell number was >300, a quantity rarely observed in embryos at the same age produced in vitro or in vivo. Electron microscopic examination of blastocysts did not reveal any abnormalities that had not previously been recorded in blastocysts developed in vivo or produced in vitro.
Peura et al. [5] reported that the aggregation of zona-free embryonic cell-cloned morulae at Day 3 improved pregnancy and calving rates following embryo transfer. Consequently, aggregation of two reconstructed nuclear transfer embryos was used in an attempt to improve outcomes following the transfer of somatic cell-cloned embryos produced with our experiments. Aggregation of zona-free embryos is a simple and efficient method for chimera production [43, 44], although from a strict nomenclature perspective our genetically identical aggregates cannot be regarded as chimaeras. According to our unpublished observations, aggregation of D4 compacted morulae allows selection of embryos with a high probability of developing to the blastocyst stage. Moreover, embryo handling after compaction is easier, and in the WOW system aggregation occurs with high efficiency and without significant blastomere losses. The transfer of the blastocysts developed from these aggregates resulted in high initial pregnancy rates with both fresh and vitrified embryos. Although the number of transfers is still too low to make comparisons or conclusions, the initial in vivo results are promising. To avoid complications during pregnancy and calving, one embryo was transferred per recipient in all cases. Factors other than the cloning technique, primarily the origin, handling, and strain of the donor cells [3, 4], may profoundly influence experienced and expected losses during pregnancy and birth. Because the effect of these factors is unpredictable, in vivo development of embryos produced with handmade versus traditional cloning techniques using the same donor cells would be required to determine the role of technique in these complications.
Our data suggest that the improved handmade somatic cell nuclear transfer procedure may be a viable alternative to the traditional methods of somatic cell nuclear transfer. High efficiency, less demanding work, and low costs may be important features for a broader application of nuclear transfer for both research and commercial utilizations. The qualitative characteristics of the embryos obtained are equal to those produced with micromanipulation and culture in the zona pellucida. However, results of the ongoing pregnancies and additional embryo transfers are required to confirm (or otherwise) the initial promises of this method.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Gábor Vajta, Department of Animal Breeding and Genetics, Danish Institute of Agricultural Sciences, Research Centre Foulum, DK-8830 Tjele, Denmark. FAX: 45 89 99 13 00; gabor.vajta{at}agrsci.dk
Received: 1 July 2002.
First decision: 30 July 2002.
Accepted: 3 September 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 I. Lagutina, G. Lazzari, R. Duchi, S. Colleoni, N. Ponderato, P. Turini, G. Crotti, and C. Galli Somatic cell nuclear transfer in horses: effect of oocyte morphology, embryo reconstruction method and donor cell type Reproduction, October 1, 2005; 130(4): 559 - 567. [Abstract] [Full Text] [PDF]
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 A.M. Powell, N.C. Talbot, K.D. Wells, D.E. Kerr, V.G. Pursel, and R.J. Wall Cell Donor Influences Success of Producing Cattle by Somatic Cell Nuclear Transfer Biol Reprod, July 1, 2004; 71(1): 210 - 216. [Abstract] [Full Text] [PDF]
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131 550 dpm/well). P values for concentrations are indicated
