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Reproductive Technology |
The Fels Institute for Cancer Research and Molecular Biology3
Department of Biochemistry,4 Temple University School of Medicine, Philadelphia, Pennsylvania 19140
Division of Reproductive Endocrinology and Infertility,5 Washington University School of Medicine, St. Louis, Missouri 63130
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, early development, embryo, gene regulation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Successful cloning requires silencing of the donor cell gene expression program and the initiation of the embryonic gene expression program (nuclear reprogramming). Failure to silence completely the donor cell program could lead to expression in the early cloned embryo of gene products more typical of the donor cell. Combined with an embryo-specific pattern of protein synthesis directed by maternal mRNAs, this would create a hybrid gene expression pattern intermediate between that of normal embryos and somatic cells. This hybrid gene expression pattern would cause the cloned embryo to have an altered phenotype manifested in its physiology, metabolism, and culture medium requirements [18, 19], leading to reduced development of cloned embryos in culture media optimized for normal fertilized embryos. Our earlier studies revealed that the donor cell nucleus can affect cloned embryo phenotype, as reflected in culture medium preference, even as early as the one-cell stage [18].
This effect of the original donor cell gene expression program on cloned embryo phenotype could lead to different outcomes in cloning using different donor cell types. Earlier studies led to the suggestion that cells in the G0 compartment of the cell cycle may be better nuclear donors than actively proliferating cells, even those in G1 [6]. Clearly, nonproliferating cells differ dramatically from actively dividing cells on many levels, including gene transcription and metabolic state. Other studies have concluded that the differentiated state of the donor cell from which the nucleus is obtained may affect the efficiency of nuclear reprogramming [7, 20], and cloning with embryonic stem (ES) cells has been comparatively successful [21, 22]. Although the effect of differentiated cell state is often discussed in terms of chromatin structure, the metabolic and physiological state of the differentiated cell, if imposed on the cloned embryo, could also have an effect. Stem cells and their differentiated derivatives often occupy spatially separate domains within tissues or distinct locales within the embryo. During differentiation, basic cell parameters such as membrane potential and metabolic parameters can change dramatically. Additionally, different differentiated cell types may reside within specialized environments and be uniquely specialized to function in those environments. This specialization of donor cells of different developmental states for different environments or different functions could affect the outcome of cloning by directing uniquely altered repertoires of gene expression in the early cloned embryo. Thus, apparent difference in cloning efficiency between stem cells and differentiated cells, between proliferating and arrested cells, or between donor cells of different tissue origins could reflect, in part, differences in donor cell metabolic or physiological characteristics that persist in the cloned embryo.
Exploring the effects of the donor cell genome on cloning efficiency will require studies to be undertaken on an array of cell types. Muscle tissue presents an interesting opportunity for such cloning studies because it is an abundant tissue that is somewhat accessible and because stem cells are readily identifiable and can be isolated, cultured, and enriched in vitro [23, 24]. As undifferentiated, proliferating stem cells, myoblasts could provide a source of nuclei that would exhibit a significant ability to support clonal development. To test the hypothesis that different types of donor cells can produce cloned embryos with different characteristics, we compared cloned embryos made with myoblast nuclei to clones made with cumulus cell nuclei. We find that cloned embryos made with myoblast donor cell nuclei differ profoundly from fertilized control embryos, exhibiting a clear intolerance for embryo-optimized culture conditions and a clear preference for the same somatic cell-like culture conditions preferred by the myoblast donor cells themselves. The change in culture requirements was thus much more severe with myoblast cloned embryos than that observed for cumulus cell cloned embryos [18]. Parthenogenetic control embryos resembled fertilized embryos, as expected, and tetraploid embryos, prepared in the same manner as cloned embryos but with the oocyte genome left in place, did not show characteristics as dramatically altered, indicating that the effects of the donor cell genome can be ameliorated by the presence of the oocyte-derived genome. Along with altered culture requirements in myoblast cloned embryos, we observe an altered pattern of glucose transporter expression and localization indicative of persistent execution of the donor cell program. The significance of these observations to understanding nuclear reprogramming and cloned embryo biology is discussed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Two kinds of myoblasts were employed in these studies. C2C12 myoblasts were obtained from the American Type Culture Collection (ATCC, Manassas, VA). This is an established myoblast cell line with comparatively relaxed growth requirements (DMEM supplemented with 10% fetal bovine serum [FBS]). Myoblasts were also isolated from C57BL/6 x Mus. m. castaneus females as described [24]. Muscle tissue (adult hind limb) was thoroughly minced in PBS and then digested with 10 ml of 0.1% hyaluronidase (Sigma, St. Louis, MO) and 0.1% collagenase type V (Sigma) in PBS for 20 min at 37°C. Before the incubation, and periodically during the incubation, the tube was vigorously vortexed. The tube was then centrifuged briefly at 
350 x g for 1–2 min and the supernatant discarded. The pelleted tissue was rinsed 2x in 10 ml of PBS and digested with 0.05% trypsin/EDTA in Ca2+- and Mg2+-free Hank's balanced saline (HBSS, GIBCO/InVitrogen, Carlsbad, CA) for 20 min at 37°C with trituration using a 25-ml pipette after 10 min of incubation to facilitate digestion. After sedimenting the tissue pieces, the supernatant was collected, and 1 ml of FBS was added. The pellet was redigested in the same manner and resedimented, and the two supernatants were combined. The supernatants were then filtered through 0.5-mm mesh Nitex nylon membrane, and the cells were pelleted by centrifugation for 10 min and resuspended in myoblast enrichment medium [Ham's F10 (GIBCO), 20% FBS, 5 ng/ml basic fibroblast growth factor (bFGF, Promega, Madison, WI), 100 U/ml penicillin (GIBCO), 100 µg/ml streptomycin (GIBCO) sulfate, and 0.25 µg/ml fungizone (GIBCO)] and plated on 100- x 20-mm collagen coated dishes.
Extensive selection procedures were applied to remove contaminating cell types as described [23]. Briefly, cells were grown at 37°C in 5% CO2 in myoblast enrichment medium, which favors myoblast growth over fibroblast growth. After 2 days, the cells were fed with fresh medium. After 5 days, culture medium was replaced with PBS, and the myoblasts were selectively released by vigorously tapping the plate. The cells were pelleted, then exposed to a collagen coated dish for 10–20 min in enrichment medium, and then the medium and unattached cells were transferred to a new dish. This selective release of myoblasts was repeated eight more times as cells reached 60% confluency. Analysis of myoblast clonal colonies revealed that all (35/35) contained differentiated multinucleated myofibers (>97% myogenic). This indicates that the enrichment procedures were very effective. After enrichment for myoblasts, the cells were maintained and passaged in myoblast growth medium (1:1 mixture of Ham's F10 and DMEM, supplemented with 20% FBS, 5ng/ml bFGF, penicillin, streptomycin, and fungizone as described previously). These myoblasts are referred to hereafter as F1 myoblasts.
Nuclear Transfer and Embryo Culture
Metaphase II-arrested oocytes were obtained from superovulated (B6D2)F1 adult females (8–12 wk of age). Oocyte-cumulus cell complexes were isolated at 13–14 h post-hCG and cumulus cells removed using hyaluronidase as described [18]. Oocytes were cultured in Chatot, Ziomek, Bavister (CZB) medium [25] supplemented with 5.5. mM glucose (CZBG). Spindle-chromosome complexes (SCCs) were removed using a narrow bore pipette attached to a piezo pipette driver as described [5]. Removal of the SCCs was performed in HEPES buffered CZB medium [26] supplemented with 5.5 mM glucose as described and 2.5 µg/ml cytochalasin B [18]. Polar bodies were also removed during this operation. After removal of the SCCs, the oocytes were washed and intact myoblasts inserted under the zona pellucida. Myoblasts of the smallest size (predominantly G1) were selected manually for injection. Electrofusion of the myoblasts to the oocytes was accomplished using electrical pulses of 90 V DC in fusion medium (275 mM mannitol, 5 mM CaCl2, 10 mM MgSO4, 0.3% bovine serum albumin [BSA]). If needed, an additional one or two pulses were given at 30–45-min intervals. Typically, 90% fusion was achieved with only two pulses. At 30 min after fusion, oocytes were activated by 5 h culture in Ca2+-free CZB medium supplemented with 10 mM Sr2+ as described and 5 µg/ml cytochalasin B [5]. Diploid parthenogenetically activated embryos were obtained using the same activation protocol without removal of the SCC. The parthenotes were obtained from the same pools of oocytes used to make cloned embryos and were activated at the same time. Parthenogenetic embryos resemble normal fertilized embryos with respect to culture requirements and many aspects of gene expression (with the obvious exception of maternally imprinted genes) and have the added advantage that they develop in close temporal synchrony with the activated cloned embryos. Tetraploid embryos were prepared by introducing somatic cell nuclei into oocytes without removal of the SCC. These embryos thus undergo all the same procedures as the diploid clones, with the exception of SCC removal. They provide a means for evaluating whether the presence of an oocyte-derived genome affects embryo characteristics [18]. Additional media employed in the study were Whitten's medium (WM) [27] and potassium simplex optimized medium with (KSOMaa) or without (KSOM) amino acid supplementation [28] and the 1:1 Ham's F10:DMEM mixture used previously for myoblast culture without serum or antibiotics but supplemented with 1mM glutamine, 0.27 mM sodium pyruvate, and 0.4% bovine serum albumin. Cloned embryos prepared with cumulus cell donor nuclei were also made for comparative purposes as described [18]. It should be noted that the investigators who participated in making the cloned constructs (YGC and SG) have successfully produced live cloned offspring using cumulus cell and/or ES cell nuclei. All studies adhered to procedures consistent with the National Research Council Guide for the Care and Use of Laboratory Animals.
Blastocyst Analysis
To determine total cell number, embryos were fixed in buffered formalin, stained with DAPI, and examined under fluorescent illumination to view nuclei. Immunosurgery [29] on live blastocysts was used to isolate inner cell masses from cloned blastocysts in order to facilitate the determination of the number of cells in the inner cell mass. After immunosurgery, the ICMs were fixed, stained with DAPI, and mounted on glass slides, and the cells were counted under fluorescent illumination.
Analysis of Glucose Uptake
For glucose uptake studies, embryos were cultured overnight in CZB medium for analysis at either the late one-cell stage or the two-cell stage. Glucose uptake was assayed by culturing embryos in CZB medium (glucose free) supplemented with 0.5 mCi/ml 2-[1,2 3H]-deoxyglucose. After incubation, embryos were washed extensively in CZBG and lysed in groups of 5–10 embryos each. The incorporated label was quantified by scintillation counting for 1 h per sample. Glucose uptake was expressed as cpm per embryo per hour.
Analysis of Glucose Transporter Expression
Preimplantation embryos were freed of the zona pellucida using acidified (pH 2.5) Tyrode's medium and fixed for 10 to 15 min at room temperature in 3.7% formaldehyde in PBS. All solutions for immunofluorescence were prepared in PBS, and procedures were performed at room temperature unless specified otherwise. The fixed cells were blocked for at least 1 h in blocking buffer (3% BSA, 0.1% Triton X-100).
For the glucose transporter immunohistochemistry, the fixed embryos were placed on slides, permeabilized with 0.1% Tween, and washed with phosphate buffered saline containing 2% bovine serum albumin (PBS/BSA). Embryos were then blocked by incubating for 60 min in 20% donkey serum in PBS/BSA, followed by 1 h incubation in affinity-purified primary antibody to mouse GLUT1 or GLUT4 (both rabbit polyclonal antibodies obtained from Dr. Mike Mueckler, Washington University School of Medicine) at a dilution of 15 µg/ml. These antibodies have been used extensively in previous studies, and their specificity is well documented [30, 31]. The embryos were then washed three times for 10 min each PBS-BSA and incubated with the secondary antibody, fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG (Chemicon, Temecula, CA), at a concentration of 1:80, followed by TO-PRO-3 iodide (MolecularProbes, Eugene, OR) at a concentration of 4µM for 20 min to visualize DNA. Finally, the embryos were washed three times for 10 min each in PBS-BSA and mounted in drops of Vectosheild (Vecto Labs, Burlingame, CA) under a supported coverslip. Fluorescence was detected with a Bio-Rad MRC-600 laser-scanning confocal microscope. Confocal images were taken at 63x magnification. Total fluorescence per embryo was expressed as a number ranging from 3 to 0 as judged by two observers blinded to the identity of the embryo. The fluorescence values for each group of embryos were averaged. At least 10 embryos existed in each group, and each experiment was repeated at least twice.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We previously reported that cloned embryos prepared with cumulus cell donor nuclei, without DMSO exposure during the procedure, exhibit altered culture medium preferences relative to normal and other control mouse embryos [18]. Specifically, cumulus cell cloned embryos developed poorly in KSOMaa, CZB, or CZBG but developed better when cultured in WM until the eight-cell/morula stage and then switched to KSOMaa until blastocyst formation. Cloned embryos also exhibited an unexpected preference for glucose containing media. We therefore tested whether myoblast cloned embryos can likewise develop in the WM/KSOMaa combination or have different culture medium requirements. Cloned embryos constructed with nuclei from C2C12 myoblasts developed to blastocysts in this medium at a reduced rate as compared with cumulus cell cloned embryos (Table 1). Also unlike cumulus cell cloned embryos, about half the C2C12 myoblast cloned embryos failed to progress to the two-cell stage. Treatment with DMSO during oocyte activation, which previously improved cumulus cell cloned embryo development in CZB medium [18], failed to overcome this early block.
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Different results were obtained using F1 myoblasts as nuclear donors. Clones made with these nuclei developed to the two-cell stage efficiently in WM but then exhibited a pronounced arrest in development to the four-cell stage (Table 2). Similarly poor results were obtained with cloned embryos made with another established transgenic mouse myoblast line (data not shown). To test whether this two-cell arrest was possibly related in any way to the two-cell arrest exhibited by some strains of fertilized embryos in some media, we tried different embryo culture medium formulations. The two-cell arrest was also seen in both CZBG and KSOM. Interestingly, cumulus cell cloned embryos cultured initially in KSOM and then switched to KSOMaa formed blastocysts more efficiently under these conditions than when cultured continuously in KSOMaa ([18] and Table 2), similar to a previous study [19]. Parthenogenetic control embryos developed efficiently to the blastocyst stage in all these media ([18] and Table 2). Tetraploid embryos also develop well in these media [18]. Thus, the failure of the F1 myoblast cloned embryos to develop efficiently beyond the two-cell stage was a unique feature of these cloned embryos rather than a nonspecific effect of the culture system. These results also distinguish the established and easily cultivated C2C12 cell line from the F1 myoblasts with respect to effects of these donor nuclei on cloned embryo phenotype.
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Our previous studies with cumulus cell cloned embryos prompted the hypothesis that the somatic cell genome may be expressed in the early cloned embryo and thereby alter its phenotype [18]. Such an effect of the somatic cell genome could contribute to the overall poor success observed when culturing the cloned embryos made with F1 myoblasts in standard embryo culture media by shifting their culture medium preference toward a more somatic cell-like formulation. We therefore tested whether the somatic cell culture medium used to prepare growth medium for the F1 myoblasts could support improved cloned embryo development. This medium consisted of a 1:1 mixture of Ham's F10 and DMEM media, without serum, and was supplemented with 0.4% BSA, 1 mM glutamine, and 0.27 mM pyruvate. The results were striking. The two-cell block was virtually eliminated, and over 40% of the cloned embryos developed to the blastocyst stage (Table 2). Interestingly, DMSO treatment of oocytes during activation did not improve the outcome. However, we found that prolonging slightly the interval between hCG injection to induce superovulation and the oocyte activation step negatively affected the outcome, and DMSO treatment did have a positive effect in this situation. Not surprisingly, parthenogenetic embryos were unable to form blastocysts efficiently under these conditions. Tetraploid embryos likewise did not form blastocysts efficiently in this medium but developed well in CZBG, indicating an effect of the oocyte-derived chromosomes on embryo phenotype.
The F1 myoblast cloned blastocysts obtained by culture in the F10:DMEM medium exhibited very robust, well-expanded morphologies (Fig. 1A) with a greater average number of cells per blastocyst (59.5 ± 12.9 SD, 120 h post-hCG) than cumulus cell cloned embryos grown under the optimum WM/KSOMaa combination [18] (Fig. 1, B and C). In fact, this cell number approached that typically seen for fertilized embryos (range 68–75 cells/embryo) [18]. Out of 22 blastocysts subjected to immunosurgery, 14 yielded visible inner cell masses. Eleven of these ICMs were successfully recovered for cell counts and yielded an average of 18 cells/ICM (SD 7.8). Thus, inner cell masses were, on average, a significant fraction of the total cells contained in a majority of the blastocysts.
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We next tested whether switching the F1 myoblast cloned embryos from the F10:DMEM mixture to a standard embryo culture medium at the eight-cell/morula stage might further enhance development to the blastocyst stage (Table 2). Switching embryos to either KSOMaa or WM significantly reduced blastocyst formation in comparison to continuous culture in F10:DMEM (43% versus 18% and 25%, respectively). Switching parthenogenetic control embryos to WM but not KSOMaa had a slightly beneficial effect.
Glucose Uptake in Cloned Embryos
We previously found that cumulus cell cloned embryos favored culture media that contained a high concentration of glucose even as early as the one-cell stage [18]. The preference of F1 myoblast cloned embryos for the F10:DMEM culture medium suggested that these embryos may also manifest an alteration in glucose requirement. To test whether these two types of cloned embryos differed from control embryos with regard to glucose uptake, we exposed embryos of each type to tritiated 2-deoxyglucose and then after extensive washing monitored uptake by scintillation counting. Cloned embryos of either type exhibited a significant increase in glucose uptake at the late one-cell stage relative to control parthenogenetic embryos (Fig. 2). The cumulus cell cloned embryos also exhibited enhanced uptake of glucose relative to tetraploid control embryos and fertilized control embryos, and this pattern persisted in embryos immediately after cleavage. The tetraploid one-cell stage embryos prepared with myoblast nuclei exhibited the same enhanced rate of glucose uptake as the diploid cloned embryos at the one-cell stage. Glucose uptake was significantly elevated in myoblast cloned embryos relative to parthenogenetic embryos and tetraploid embryos at the two-cell stage, when embryo gene transcription has begun (Fig. 2D). In fact, two-cell stage myoblast cloned embryos displayed a rate of uptake that was nearly 6-fold greater than that of parthenogenetic embryos and nearly 3-fold greater than that of tetraploid embryos. Thus, while the rate of glucose uptake in diploid cloned embryos remained elevated, the enhanced rate of glucose uptake seen in tetraploid embryos at the one-cell stage was reduced. The rate of glucose uptake was similar between cloned embryos and in vivo fertilized embryos (38 h post-hCG) (Fig. 2D). It should be noted that parthenogenetic and tetraploid embryos are activated simultaneously and cultured in parallel with the cloned embryos, whereas the fertilized embryos may be slightly more advanced developmentally. These data indicate that during the two-cell stage, the rate of glucose uptake increases to a level similar to that in fertilized embryos more rapidly in myoblast cloned embryos than in parthenogenetic or tetraploid controls.
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Expression of Glucose Transporters
Given the increased rate of glucose uptake in cloned embryos relative to control embryos, we wished to ascertain whether cloned embryos display altered glucose transporter expression, indicative of continued expression of donor cell type genes. Preimplantation stage mouse embryos express GLUT1 at all stages [32], and this transporter is also expressed in many somatic cell types. We therefore examined the expression of GLUT1 in late one-cell stage cumulus and myoblast cloned embryos by immunofluorescence staining. Cumulus cell cloned embryos exhibited a modest increase in GLUT1 expression relative to parthenogenetic control embryos (Fig. 3A). Tetraploid embryos made with cumulus cell donor nuclei exhibited an intermediate level of staining. Myoblast cloned embryos did not exhibit such an increase in GLUT 1 expression at the late one-cell stage or any later stage (Fig. 3B) but at the two-cell stage exhibited enhanced plasma membrane localization of GLUT1, which normally does not occur until the eight-cell stage and which was not seen in either parthenogenetic or control embryos (Fig. 4A). Cloned embryos treated with 
-amanitin to inhibit transcription showed much reduced expression of GLUT1, indicating that the expression observed in untreated cloned embryos was transcription dependent (Fig. 3B). At the blastocyst stage, the localization of GLUT1 to the plasma membrane appeared to be reduced in myoblast cloned embryos in comparison to parthenogenetic and fertilized embryos (Fig. 4C).
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Because glucose transporter type 4 (GLUT4) is expressed in muscle but not in preimplantation stage embryos [32], we examined the expression of this transporter in myoblast cloned embryos and controls. The myoblast cloned embryos at the late one-cell stage expressed significant quantities of GLUT4, well above the low level of background fluorescence observed in the parthenogenetic and fertilized control embryos (Figs. 3C and 4B). Diploid myoblast cloned embryos treated with -amanitin showed greatly reduced signals for GLUT4 at the two-cell stage, indicating that the GLUT4 expression seen in untreated diploid cloned embryos was transcription dependent and not merely a result of protein or mRNA carryover from the donor cell. Tetraploid embryos expressed slightly elevated quantities of GLUT4 at the one-cell stage and two-cell stage, but this was less than that seen in the diploid cloned embryos (Figs. 3C and 4B). Expression declined progressively in tetraploid embryos with development to the blastocyst stage. Continued increased staining was seen in diploid myoblast cloned embryos at later stages for GLUT4, relative to parthenogenetic, tetraploid, and flushed fertilized embryos (Figs. 3C and 4D). Thus, diploid myoblast cloned embryos showed aberrant, high expression of GLUT4 throughout preimplantation development, distinguishing them from all three types of control embryos. Although enrichment of GLUT4 at the plasma membrane was not seen, such enhanced localization is often insulin dependent, and cycling of a fraction of the GLUT4 protein between cytoplasm and plasma membrane can nevertheless occur and contribute to glucose uptake.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Based on our experience with myoblasts as donor cells, it appears that those extracellular conditions that are favored by the donor cell may serve as a guide for identifying favorable conditions for the cloned embryo. The degree to which this rule applies is likely to be influenced by variables such as the degree of potency (mono-, multi-, or pluripotential) and the degree of disparity between donor cell environment and the normal oocyte/embryo environment. The degree of physiological or metabolic specializations of the donor cell may also be important. For example, cumulus cell nuclei promote a preference for glucose containing media, and cumulus cells themselves are specialized for enhanced glucose metabolism [18]. By analogy, other cellular specialization may have other effects. For example, electrically excitable cells such as differentiated neurons, cardiomyocytes, or multinucleated skeletal muscle fibers may manifest intracellular ionic properties distinct from those of nonexcitable cells, which, through continuation of the somatic cell gene expression program, could then shift the intracellular ionic properties of the cloned embryo. Such a change could be accompanied by a shift in extracellular culture requirements. It will indeed be interesting to ascertain whether cloned embryos might share other features with their corresponding nuclear donors, such as growth factor or cytokine responsiveness, and whether such characteristics can synergize with or interfere with the functions of proteins normally expressed in developing embryos.
The significant reduction in blastocyst formation observed when myoblast cloned embryos are returned to standard embryo culture media at the morula stage and the continued expression of GLUT4 through the blastocyst stage indicate that, despite an otherwise robust morphology, the cellular phenotypes in these embryos remain skewed toward that of the donor cells. This indicates that reprogramming likely requires a prolonged period extending even beyond the preimplantation period. Previously observed mosaicism in the expression of other genes such as Oct4 [33] may thus be explicable on this basis, as very slow reprogramming over the course of six or more cell cycles could lead to such heterogeneity between blastomeres. Formation of the inner cell mass and the definitive embryonic cell lineage may thus be a highly selective process that recruits only those cells that have undergone the greatest degree of nuclear reprogramming. Physiological and metabolic properties may constitute important parameters on which this selection operates. Recruitment of cells to this lineage may thus be inhibited not only by delayed or inadequate expression of regulatory proteins like Oct4 [33], which must be reactivated during clonal development, but also by persistent expression of other somatic cell-type proteins in some cells, which would make those cells unable to flourish within the specialized compartment eventually occupied by the ICM and its derivatives.
The apparently leisurely pace of nuclear reprogramming likely has important consequences for determining when cloned embryos should be returned to a reproductive tract environment and how they will fare when placed in that environment. Because the oviduct provides an environment that is highly optimized for normal embryos, this same environment could constitute a poor environment for myoblast cloned embryos during the first several cell cycles. Because the myoblast cloned embryos were only partially more tolerant of standard embryo culture media at the morula stage, this negative effect of the oviduct environment may prevail even beyond the morula stage. Thus, the overall pace of reprogramming combined with the initial degree of disparity between the preferred culture environment and the reproductive environment in situ will likely affect when cloned embryos made with various donor cells can be transferred to foster mothers. Because the period during which embryos may be transferred is limited, donor cell types for which the pace of reprogramming is unusually slow or for which the disparity between environmental requirements is largest may simply be unable to yield viable progeny at a significant rate, unless the pace of reprogramming can be improved by altering the culture environment.
Our results also reveal that procedural factors can affect the viability of cloned embryos and that these effects may be cell type specific. For example, the presence of DMSO during the activation step was able to overcome culture medium preferences in cloned embryos made with cumulus cell nuclei [18] but was unable to overcome the detrimental effects of standard embryo culture media on myoblast cloned embryos. The length of time between ovulation (related to the timing of hCG injection) and oocyte activation significantly affects blastocyst formation rates among myoblast cloned embryos, but such an effect was not readily apparent with cumulus cell cloned embryos ([5] and Y.G. Chung, unpublished results). The effect of using piezo-mediated nuclear injection versus electrofusion is significant with myoblast nuclei, possibly because of the greater size of the myoblast nucleus as compared with, for example, ES cell or cumulus cell nuclei.
Our analysis of glucose transporter gene expression in cloned embryos indicates that the altered culture medium preference is associated with continued expression of the somatic cell program. The cumulus cell cloned embryos exhibited an early increase in GLUT1 expression. The myoblast cloned embryos exhibit precocious GLUT1 localization to the plasma membrane and continue to express GLUT4 at all stages of preimplantation development examined. GLUT4 is typically expressed in muscle cells but not in preimplantation stage mouse embryos [30, 32]. This augmentation of glucose transporter expression is reminiscent of the enhanced preference for glucose containing media exhibited by cumulus cell cloned embryos [18]. We also observed increased uptake of 2'-deoxy glucose by both myoblast and cumulus cell cloned embryos, indicating that the increased transporter expression is functionally significant.
The precocious enrichment of GLUT1 at the cell surface in two-cell stage cloned embryos and the reduced plasma membrane localization of GLUT1 in cloned blastocysts reveal alterations in the normal temporal pattern of protein localization. Thus, the donor cell nucleus clearly alters a variety of basic cellular functions in the cloned embryo, including gene expression, protein localization, and glucose uptake. Such alterations are likely to be extensive and responsible for the unique phenotype observed for the cloned embryos.
It is clear that an authentic embryonic genome can exert a potent regulative effect that is noticeably absent or greatly reduced in the cloned embryo. Others have reported effects of maternal oocyte-derived chromosomes on DNA methylation [13]. We observed effects of oocyte-derived chromosomes on the expression of GLUT1 and GLUT4. Tetraploid embryos do not exhibit precocious localization of GLUT1. GLUT4 expression is lower in tetraploids than in diploid cloned embryos. Tetraploids also failed to form blastocysts efficiently in the F10:DMEM medium, preferring instead standard embryo culture conditions. Using cumulus cell donor nuclei, the rate of glucose uptake and expression of GLUT1 are lower with tetraploid embryos than with diploid cloned embryos. The silencing of donor cell genes thus may be a complex process, involving a combination of functions provided by maternally inherited factors in the oocyte and embryonically expressed factors. These observations suggest two interesting possibilities that need not be mutually exclusive. One is that the transplanted donor cell nuclei fail to express key transcriptional regulators, which may be uniquely expressed by the maternal chromosomes, or perhaps more simply by an authentic embryonic genome. Alternatively, the maternally derived chromosomes may have a unique ability to express factors involved in nuclear reprogramming. With respect to normal embryos, the oocyte-derived chromosomes may thus direct a critical part of the transcriptional programming of the embryonic genome. With respect to cloned embryos, this transcriptional programming function may be seriously compromised by removal of the maternal chromosomes. This creates the interesting possibility that retransfer of a somatic cell genome after it has been exposed to ooplasm together with a maternal set of chromosomes, or retransfer of the cloned embryo genome to enucleated blastomere cytoplasm, might result in an improved efficiency of the cloning process.
Overall, our data indicate that nuclear reprogramming during mammalian cloning by somatic cell nuclear transfer most likely occurs over a protracted period of time. As a result, many genes expressed in the somatic cell donor nucleus are likely transcribed in the cloned embryos as early as the one-cell stage. Additionally, posttranscriptional gene regulation appears to be disrupted in cloned embryos. These effects confer altered phenotypes and culture requirements on the cloned embryos that are donor cell type specific. Given the inefficient silencing of the donor cell genome and the effect of this on cloned embryo phenotype, improving the efficiency of donor cell genome silencing may constitute a critical threshold that must be overcome before the overall efficiency of cloning can be improved. Providing a culture environment that is optimized for each donor cell type will likely be critical for maintaining cloned embryo homeostasis and supporting the reprogramming process. Obviously, identifying culture conditions that support early cleavage stage development in cloned embryos is essential to the process. Additional studies seeking to refine further those conditions that support early development may help to improve long-term developmental potential. The development of multistep culture systems that allow the culture environment to change in concert with the progress of reprogramming may provide the greatest improvements to cloned embryo development. Further studies to characterize in detail the specific alterations in gene expression, metabolism, and physiology of cloned embryos made with a variety of donor cell types will be needed in order to approach the development of optimized culture systems through rationale design. Such studies should also further our understanding of the basic biology of normal fertilized embryos.
Until the effect of continued expression of the donor cell gene expression program on cloned embryo phenotype is thoroughly understood and accounted for experimentally, conclusions related to the relative developmental capacities of different types of donor cell nuclei or their ability to be reprogrammed must be viewed with caution. We show here, for example, that nuclei from differentiated cumulus cell nuclei support a greater degree of development under standard embryo culture conditions than nuclei from undifferentiated myoblasts, and yet the latter can manifest a much greater degree of developmental competence to the blastocyst stage when a different culture system is employed. Thus, it appears that reprogramming may be equally limited with either differentiated or undifferentiated cell nuclei and that the predominant effect of donor cell type on developmental capacity is the degree to which the persistent donor cell gene expression program shifts the cloned embryo phenotype away from that which is typical of normal embryos. It will be challenging to dissect inherent limitations on reprogramming that may exist within the donor cell nuclei, limitations related to donor cell type-specific effects on culture requirements, and their associated secondary effects on the pace of reprogramming. The use of developmental milestones will be inadequate for this purpose, making detailed assays that evaluate reprogramming at the level of specific gene expression while minimizing the impact of culture essential.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 215-707-1454; klatham{at}unix.temple.edu
Received: 19 December 2002.
First decision: 13 January 2003.
Accepted: 3 February 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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F. Wang, Z. Kou, Y. Zhang, and S. Gao Dynamic Reprogramming of Histone Acetylation and Methylation in the First Cell Cycle of Cloned Mouse Embryos Biol Reprod, December 1, 2007; 77(6): 1007 - 1016. [Abstract] [Full Text] [PDF]
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A.L Green, D.N Wells, and B Oback Cattle Cloned from Increasingly Differentiated Muscle Cells Biol Reprod, September 1, 2007; 77(3): 395 - 406. [Abstract] [Full Text] [PDF]
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D. K Berg, C. Li, G. Asher, D. N Wells, and B. Oback Red Deer Cloned from Antler Stem Cells and Their Differentiated Progeny Biol Reprod, September 1, 2007; 77(3): 384 - 394. [Abstract] [Full Text] [PDF]
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 B. Heindryckx, P. De Sutter, J. Gerris, M. Dhont, and J. Van der Elst Embryo development after successful somatic cell nuclear transfer to in vitro matured human germinal vesicle oocytes Hum. Reprod., July 1, 2007; 22(7): 1982 - 1990. [Abstract] [Full Text] [PDF]
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C. Smith, D. Berg, S. Beaumont, N. T Standley, D. N Wells, and P. L Pfeffer Simultaneous gene quantitation of multiple genes in individual bovine nuclear transfer blastocysts Reproduction, January 1, 2007; 133(1): 231 - 242. [Abstract] [Full Text] [PDF]
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J. Somers, C. Smith, M. Donnison, D. N Wells, H. Henderson, L. McLeay, and P L Pfeffer Gene expression profiling of individual bovine nuclear transfer blastocysts. Reproduction, June 1, 2006; 131(6): 1073 - 1084. [Abstract] [Full Text] [PDF]
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 K. Inoue, N. Ogonuki, H. Miki, M. Hirose, S. Noda, J.-M. Kim, F. Aoki, H. Miyoshi, and A. Ogura Inefficient reprogramming of the hematopoietic stem cell genome following nuclear transfer J. Cell Sci., May 15, 2006; 119(10): 1985 - 1991. [Abstract] [Full Text] [PDF]
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 L. Armstrong, M. Lako, W. Dean, and M. Stojkovic Epigenetic Modification Is Central to Genome Reprogramming in Somatic Cell Nuclear Transfer Stem Cells, April 1, 2006; 24(4): 805 - 814. [Abstract] [Full Text] [PDF]
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 T. Brambrink, K. Hochedlinger, G. Bell, and R. Jaenisch ES cells derived from cloned and fertilized blastocysts are transcriptionally and functionally indistinguishable PNAS, January 24, 2006; 103(4): 933 - 938. [Abstract] [Full Text] [PDF]
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Y. Yu, J. Yong, X. Li, T. Qing, H. Qin, X. Xiong, J. You, M. Ding, and H. Deng The proteasomal inhibitor MG132 increases the efficiency of mouse embryo production after cloning by electrofusion Reproduction, October 1, 2005; 130(4): 553 - 558. [Abstract] [Full Text] [PDF]
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K. Takeda, M. Tasai, M. Iwamoto, A. Onishi, T. Tagami, K. Nirasawa, H. Hanada, and C. A. Pinkert Microinjection of Cytoplasm or Mitochondria Derived from Somatic Cells Affects Parthenogenetic Development of Murine Oocytes Biol Reprod, June 1, 2005; 72(6): 1397 - 1404. [Abstract] [Full Text] [PDF]
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S. Eckardt, N A. Leu, S. Kurosaka, and K J. McLaughlin Differential reprogramming of somatic cell nuclei after transfer into mouse cleavage stage blastomeres Reproduction, May 1, 2005; 129(5): 547 - 556. [Abstract] [Full Text] [PDF]
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 H. D. Morgan, F. Santos, K. Green, W. Dean, and W. Reik Epigenetic reprogramming in mammals Hum. Mol. Genet., April 15, 2005; 14(suppl_1): R47 - R58. [Abstract] [Full Text] [PDF]
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B.P. Enright, L.-Y. Sung, C.-C. Chang, X. Yang, and X.C. Tian Methylation and Acetylation Characteristics of Cloned Bovine Embryos from Donor Cells Treated with 5-aza-2'-Deoxycytidine Biol Reprod, April 1, 2005; 72(4): 944 - 948. [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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S. Gao, E. Czirr, Y. G. Chung, Z. Han, and K. E. Latham Genetic Variation in Oocyte Phenotype Revealed Through Parthenogenesis and Cloning: Correlation with Differences in Pronuclear Epigenetic Modification Biol Reprod, April 1, 2004; 70(4): 1162 - 1170. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, Parthenotes; 
, Tetraploids; 
, Diploid Clones; 
, Flushed fertilized embryos; 
, 