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Reproductive Technology |
The Fels Institute for Cancer Research and Molecular Biology and Department of Biochemistry,3 Temple University School of Medicine, Philadelphia, Pennsylvania 19140
Department of Human Genetics,4 University of Pittsburgh, Pittsburgh, Pennsylvania 15261
Department of Pediatrics,5 The Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania 15213
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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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The early preimplantation mammalian embryo undergoes a process of nuclear reprogramming before it develops into a complete and normal fetus. Along with this reprogramming, essential early events related to the timely utilization of maternally inherited proteins and mRNAs must also occur. These processes require complex interactions between the transplanted donor nucleus and the ooplasm. Although nuclear reprogramming during cloning and nuclear-ooplasmic interactions in the early embryo are poorly understood, they likely involve significant epigenetic changes in the genome, with accompanying changes in gene expression and heritable patterns of DNA methylation. The abnormalities in Oct4 expression and DNA methylation in cloned embryos support the notion that reprogramming is abnormal or incomplete in cloned embryos [6–14]. The precise cause of this is unknown but might involve a diversity of processes, including inefficient cytoplasmic-nuclear protein trafficking, inefficient changes in chromatin structure, or defects in the establishment or maintenance of appropriate embryonic DNA methylation patterns.
In normal embryos, maintenance of a correct DNA methylation pattern requires both the expression and the correct, stage-specific posttranslational regulation of Dnmt1 protein [15–18]. An oocyte-derived form of Dnmt1, denoted Dnmt1o, is the only form of this protein expressed in the embryo before implantation. The Dnmt1o protein is present throughout all cleavage stages of preimplantation development but remains cytoplasmic during all stages except the eight-cell stage. During the eight-cell stage, Dnmt1o enters the nucleus just for that one cell cycle, when it is essential for maintaining imprinted gene methylation patterns [16]. The mRNA encoding the somatic form of the protein, Dnmt1s, is expressed during the preimplantation period, but Dnmt1s does not appear in normal embryos until after implantation [15, 19, 20]. Thus, either the Dnmt1s mRNA is not translated or this protein cannot accumulate in the early embryo. One important role of Dnmt1s is to maintain inherited methylation patterns, including genomic imprints, and methylation patterns established de novo in the early stages of postimplantation development [17, 18]. These results reveal that correct expression and regulation of Dnmt1 proteins in the normal embryo require correct posttranscriptional and posttranslational regulation at the levels of cytoplasmic-nuclear protein trafficking and mRNA translation or protein accumulation. Whether aberrant expression or localization of Dnmt1 proteins could contribute to methylation defects, such as those reported previously for cloned embryos, and possibly to developmental arrest at subsequent stages of development has not been examined. We therefore determined whether cloned embryos exhibit a normal pattern of posttranscriptional regulation and cytoplasmic-nuclear trafficking of Dnmt1 proteins. The results reported herein indicate that there is a highly consistent, early disruption in the regulation of Dnmt1s and Dnmt1o expression in cloned embryos. The apparent uniformity of these defects among cloned embryos indicates that these defects are likely to affect all cloned embryos and thus are likely to be a general feature of early clonal development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preimplantation embryos were freed of the zona pellucida using acidified (pH 2.5) Tyrode medium and fixed for 10 to 15 min at room temperature in 3.7% formaldehyde in PBS [15]. All solutions for the immunofluorescence of Dnmt1 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) and then incubated in either 1:100 UPT82, which specifically recognizes epitopes present in Dnmt1s but not in Dnmt1o [15], or in 1:100 UPTC21 or 1:500 PATH52, which recognize epitopes in both Dnmt1s and Dnmt1o. All antibodies were diluted in the same blocking buffer and incubated overnight at 4°C in a humidified chamber. The cells were washed three times for 5 min each in blocking buffer, then incubated in their corresponding secondary antibodies. The secondary antibody used in conjunction with UPT82 was an anti-rabbit IgG monoclonal antibody coupled with Texas Red (1:250) and the secondary antibody used in conjunction with UPTC21 was an anti-chicken IgG monoclonal antibody coupled with fluorescein (1:4000). For double staining, simultaneous staining with both primary antibodies followed by simultaneous staining with both their corresponding secondary antibodies was performed. To mount the cells for viewing, a drop of Vectashield mounting medium (Vector Laboratories, Burlingame, CA) supplemented with 0.4 µg/ml of the DNA-binding dye DAPI (Roche Diagnostics, Indianapolis, IN) was placed on a glass microscope slide, and the cells were transferred into the drop with a pipette. A coverslip was gently placed on the drop to spread the mounting medium and flatten the cells and was subsequently sealed with nail polish. Immunofluorescence was visualized using a Zeiss Axiophot, Zeiss LSM410 laser scanning confocal microscope (Carl Zeiss Microimaging, Thornwood, NY). All images were recorded under identical laser settings. Statistical differences in staining between embryo classes were evaluated using a 
2 test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine the expression patterns of Dnmt1o and Dnmt1s proteins in cloned embryos, antibodies that distinguish Dnmt1o and Dnmt1s were used. Through the combined use of these two polyclonal antibodies, the Dnmt1o and Dnmt1s proteins can be distinguished in intact oocytes and preimplantation-stage embryos or in extracts derived from these developmental stages [15]. Cloned embryos were fixed at the one-cell through blastocyst stages, permeabilized, and processed for immunofluorescence [15]. Staining with the UPT82 antibody, which specifically recognizes epitopes present in Dnmt1s but not in Dnmt1o [15] revealed aberrant Dnmt1s protein expression in blastomeres of eight-cell-stage cloned embryos (Fig. 1A). Dnmt1s was present in the cytoplasm of all blastomeres but was detected in only some blastomere nuclei. Of 35 cloned embryos examined in seven experiments, all displayed staining for the somatic form of the protein. No nuclear staining for Dnmt1s was observed for cloned embryos at either the one-cell or two-cell stages, indicating that the staining seen at later stages was not likely the result of carryover of protein from the donor nucleus. Some cytoplasmic staining persisted at the morula and blastocyst stages, but no nuclear staining was seen (Fig. 1A). In contrast, no nuclear staining for Dnmt1s was ever observed in any of the 21 control embryos examined, all of which were derived from normally fertilized eggs ([15] and Fig. 1B), thus indicating a significant difference between cloned and fertilized embryos (P < 1 x 10-6).
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Tetraploid embryos were constructed using methods like those used to make diploid cloned embryos: by transferring cumulus cell nuclei into unfertilized eggs in which the maternal chromosome-spindle complex was left in place followed by cytochalasin B treatment to maintain a diploid complement of maternal chromosomes and of the donor genome. These embryos provide an informative means for evaluating possible regulatory deficiencies in cloned embryos; they possess the somatic donor cell as do diploid clones but possess in addition oocyte-derived chromosomes, which may provide regulatory functions not supported by a somatic cell genome. None of the eight eight-cell-stage tetraploid embryos examined displayed nuclear staining for Dnmt1s (Fig. 1C), which differed significantly from diploid cloned embryos (P < 1 x 10-6). These findings indicate that endogenous oocyte-derived chromosomes (which comprise part of an authentic embryonic genome) prevented the aberrant nuclear expression of the Dnmt1s protein that was observed in diploid cloned embryos. As expected, none of the seven control diploid parthenogenetic embryos examined showed Dnmt1s nuclear staining (Fig. 1D). The absence of Dnmt1s protein in the nuclei of tetraploid embryos also indicated that the nuclear expression of Dnmt1s seen in cloned embryos was neither a result of protein carryover from the donor cell nor an artifact of the injection procedure or the culture system, since the tetraploid embryos experienced the identical series of treatments as cloned embryos except for removal of the oocyte spindle-chromosome complex. The aspect of the maternal chromosomes that confers the embryonic type of Dnmt1s regulation in the tetraploid cloned embryos is not known. Nonetheless, we speculate that the maternal chromosomes express regulatory factors that are lacking in cloned embryos containing only somatic nuclei from differentiated cells.
Dnmt1o Expression Pattern in Cloned Embryos
Nuclear Dnmt1s protein is seen in cloned embryos at the same developmental stage (eight-cell) at which nuclear Dnmt1o protein is seen in normal embryos [16]. This observation raised the possibility that abnormalities in Dnmt1o expression or localization may underlie the aberrant Dnmt1s expression. Using the PATH52 antibody, which detects both forms of the Dnmt1 protein [19], abundant cytoplasmic protein (predicted likely to be mostly Dnmt1o) was seen in all preimplantation stages of cloned embryos (Fig. 2A). However, only a few nuclei of eight-cell-stage clones were stained with PATH52 (e.g., six of eight nuclei in the embryo shown in Fig. 2D). This pattern of staining is similar to the mosaic pattern of Dnmt1s expression seen with the UPT82 antibody (Fig. 1A) and contrasts with the presence of Dnmt1o in all eight-cell-stage nuclei of normal diploid embryos ([16] and Fig. 2E). Thus, at the preimplantation stage during which Dnmt1o probably functions to maintain methylation patterns on imprinted genes, cloned embryos have nuclei that lack either form of the Dnmt1 methyltransferase. The expected outcome of this deficiency would be the inability of some eight-cell-stage blastomeres to maintain sex-specific methylation patterns on imprinted genes.
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There are different explanations for the mosaic patterns of UPT82 (Fig. 1A) and PATH52 (Fig. 2, A and D) staining in eight-cell clones. PATH52-stained nuclei may contain only Dnmt1s, only Dnmt1o, or a mixture of Dnmt1o and Dnmt1s. To distinguish among these possibilities, single eight-cell cloned embryos were stained with both the UPT82 rabbit anti-Dnmt1s antibody and the UPTC21 chicken antibody, which detects both Dnmt1s and Dnmt1o [15]. Because no antibody that is specific for Dnmt1o can be generated, dual immunofluorescent labeling combined with confocal microscopic image analysis constitutes the best method for determining which Dnmt1 proteins reside in the blastomere nuclei. Staining with the UPTC21 antibody revealed that the amount of nuclear staining seen in cloned embryos was far below that seen for control fertilized embryos (Fig. 3A). Consequently, it was necessary to examine more intensely exposed images to ascertain the likely composition of Dnmt1 proteins in the nuclei. As shown in Figure 3B, there are areas of cytoplasm that stain with UPTC21 but do not stain with UPT82, indicating that both Dnmt1o and Dnmt1s are in the cytoplasm. All of the nuclei that stain with UPTC21 also stain with UPT82, since all nuclei displayed yellow coloration in the merged images (Fig. 3B). This observation is consistent with the presence of only Dnmt1s or a mixture of Dnmt1o and Dnmt1s in stained nuclei of cloned eight-cell embryos. It is not consistent with the presence of only Dnmt1o in any of the stained nuclei or with a large excess of Dnmt1o over Dnmt1s if nuclei contain a mixture of the two proteins. Given the overall low intensity of staining of nuclei in cloned embryos with these antibodies, it appears that if any Dnmt1o enters the nuclei of cloned embryos, the amount is greatly diminished compared with normal embryos. We conclude from this analysis that none of the blastomeres examined in cloned embryos exhibited the appropriate embryonic pattern of nuclear Dnmt1 staining.
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Because trafficking of the Dnmt1o protein from the cytoplasm to the nucleus in eight-cell-stage blastomeres is greatly reduced, Dnmt1s expression in these nuclei may be a consequence of the reduced nuclear abundance of Dnmt1o. To examine this possibility, we measured Dnmt1s expression in fertilized embryos that lack the Dnmt1o protein because of their derivation from homozygous mutant Dnmt1
1o oocytes [16]. Intense UPT82 staining for Dnmt1s was seen in all nuclei of 4-cell, 8-cell, and 16-cell mutant embryos (Fig. 3, C and D). This is consistent with the hypothesis that the absence of Dnmt1o in the nucleus leads to aberrant expression of Dnmt1s. Despite the expression of the Dnmt1s protein in these embryos, they remain nonviable and show a loss of methylation on imprinted genes, indicating a lack of functional compensation for the missing Dnmt1o protein [16]. Based on this observation, the Dnmt1s protein in nuclei of eight-cell cloned embryos may also be unable to maintain methylation patterns on alleles of imprinted genes.
Mosaic Pattern of Nuclear Staining for Dnmt1o and Dnmt1s in Cloned Embryos
The mosaic pattern of nuclear UPT82 staining in the eight-cell cloned embryos was analyzed further. All 35 of the cloned embryos examined exhibited mosaic nuclear staining for Dnmt1s at the eight-cell stage. There was a wide range of staining intensities among nuclei of individual clones (Fig. 4, A and C). The average number of nuclei per cloned embryo showing moderate-to-intense levels of UPT82 staining was 4.4 ± 1.03 (mode, 4; median, 4.0). Similar degrees of mosaic protein expression were seen in cloned embryos stained with PATH52 (Fig. 2, A and D), with 5.4 ± 0.70 nuclei stained per embryo (mode, 6; median, 5.5) (Fig. 4D). We also made clones using an immediate oocyte activation protocol, which avoids the possibility of chromosome loss [22]. Embryos from this immediate-activation protocol also showed a mosaic pattern of Dnmt1s expression with a wide range of nuclear staining intensities (Fig. 4, B and C). Mosaicism appeared to be reduced slightly, with 5.3 ± 0.82 nuclei showing moderate-intense staining (mode, 6; median, 5.5), but this reduction was not statistically significant. Thus, the mosaic Dnmt1s nuclear staining cannot be explained by the loss of chromosomes, consistent with the expression of Dnmt1s protein in the cytoplasm of all blastomeres of cloned embryos. Mosaic Dnmt1s nuclear staining in cloned embryos contrasts significantly (P < 1 x 10-6) with the Dnmt1s staining of embryos from Dnmt1o-deficient mothers (n = 5 examined), in which all of the nuclei were within approximately 80% of the intensity of the most heavily stained nucleus (Fig. 3, C and D, and Fig. 4C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Dnmt1s protein is expressed in clones at the eight-cell stage, whereas this protein is normally not expressed until much later in development. One possible explanation for this is that cloned embryos lack the appropriate posttranscriptional gene regulatory mechanisms that normally prevent Dnmt1s protein expression in preimplantation embryos [15]. Such a defect in regulating Dnmt1s mRNA translation or protein accumulation could reflect a broader defect in cloned embryos, affecting multiple maternal mRNAs and their proteins. If this is the case, then this would seriously disrupt the essential stage-specific utilization of maternal mRNAs. Because the correct utilization of maternal mRNAs is important for such basic processes as embryonic genome activation [23], such a defect would severely compromise cloned embryo viability.
An alternative explanation is that the somatic cell nucleus in cloned embryos directs a higher rate of Dnmt1 gene transcription or accumulation of the somatic form of Dnmt1 mRNA, exceeding the capacity of the cell to repress its translation. The same donor genome transcription, however, would be expected to occur in tetraploid constructs, which do not show Dnmt1s protein expression. Thus, it appears that even if there is a transcriptional component to the aberrant regulation of Dnmt1 gene expression in cloned embryos, other levels of regulation related to Dnmt1s mRNA accumulation or translation must also be defective. These other levels of regulation can be provided in tetraploid constructs by the presence of an authentic oocyte-derived set of chromosomes. The presence of an oocyte nucleus in tetraploid embryos also overcomes the inefficient demethylation of satellite regions in cloned blastocysts [11]. Thus, it appears that cloned constructs may simply lack key regulatory functions that can only be supplied by an authentic embryonic genome.
Because there is abundant cytoplasmic Dnmt1o in both normal and cloned eight-cell embryos but little or no nuclear uptake of Dnmt1o in cloned embryos, there appears to be a significant defect in the cytoplasmic-to-nuclear trafficking of Dnmt1o protein in clones. Indeed, the mosaic pattern of Dnmt1s nuclear localization may indicate defects in the trafficking of both Dnmt1o and Dnmt1s into nuclei of eight-cell clones. This defect in cytoplasmic-nuclear protein trafficking could result from a failure of the somatic nucleus to express essential nuclear import proteins uniquely required in the early embryo or an incompatibility between somatic cell–derived chromatin or residual nuclear membrane components and embryonically expressed proteins. Although nuclear envelope breakdown and remodeling of some nuclear envelope proteins occur [24–26], some somatic components could remain. The defect in cytoplasmic-nuclear protein trafficking could affect nuclear reprogramming and contribute to the mosaic patterns of gene expression reported herein and elsewhere [6, 27].
The failure of intracellular trafficking of Dnmt1o and the expression of Dnmt1s in the cloned embryos could contribute to defects in DNA methylation. No evidence exists currently that Dnmt1s and Dnmt1o display different specificities in DNA methylating activities. The Dnmt1s form is, however, intrinsically less stable than the Dnmt1o form [28]. The relative instability of Dnmt1s, combined with the near absence of Dnmt1o from the nucleus, could lead to an overall reduction in Dnmt1 activity within cloned eight-cell blastomere nuclei and hence a loss of methylation at numerous sites, as occurs on alleles of imprinted genes in Dnmt1o-deficient embryos [16]. Moreover, mosaic expression of Dnmt1s at the eight-cell stage could lead to heterogeneous DNA methylation patterns among blastomeres and eventually to heterogeneity in methylation patterns among the cells and tissues of an individual cloned animal or among cloned offspring.
In summary, our results reveal defects in the regulation of Dnmt1 gene expression and cytoplasmic-nuclear trafficking that precede any defects thus far reported for cloned embryos of any species. These early defects clearly lead to additional defects, such as aberrant expression of Dnmt1 proteins and possibly aberrant Oct4 expression, which collectively must prevent normal development in most clones. Because embryos from homozygous Dnmt11o mothers form blastocysts readily, it is unlikely that disrupted expression of Dnmt1 proteins by itself can account for the reduced formation of blastocysts among cloned embryos. The defects that lead to altered Dnmt1 protein expression and the defects affecting cytoplasm-nuclear Dnmt1o protein transport may, however, be indicative of other, more severe homeostatic or regulative defects underlying an early developmental arrest. The observed defects may have later effects as well. The mosaic expression of Oct4, Dnmt1, and transgenes may be mechanistically related and would provide the foundation for the observed variability in phenotypes of the rare surviving cloned embryos. Overall, none of the cells examined in cloned embryos in this study appeared to express the correct pattern of nuclear Dnmt1 staining, and thus none of the cells appeared to have been reprogrammed correctly by this criterion. If defects in posttranscriptional gene regulation, cytoplasmic-nuclear protein trafficking, and expression of DNA methyltransferases also exist in clones of other species and are associated with similar blocks in reprogramming, then this would raise obvious concerns about the use of current cloning technologies for broader purposes such as therapeutic cloning.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Keith E. Latham, The Fels Institute for Cancer Research and Molecular Biology, Department of Biochemistry, 3307 North Broad St., Room 302, Philadelphia, PA 19140. FAX: 215 707 1454; klatham{at}unix.temple.edu
Received: 6 December 2002.
First decision: 3 January 2003.
Accepted: 5 February 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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