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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
* State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences; and
Graduate School, Chinese Academy of Sciences, Beijing, China
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: histone acetylation • rabbit fertilized embryos • somatic cell nuclear transfer embryos • Trichostatin A
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Kang et al., 2002).
Because Trichostatin A (TSA) keeps the chromatin in an accessible state (histone hyperacetylation), it can inhibit histone deacetylases (HDAC) from making the gene active (Lee et al., 1993). Therefore, it may improve the nuclear reprogramming and increase the cloned blastocyst rate. Previous studies showed that treatment of donor cells with TSA or sodium butyrate (NaBu), another HDAC inhibitor, resulted in a great increase in the cloned blastocyst rate compared with that of untreated cells (Enright et al., 2003b; Shi et al., 2003a). However, it has been reported that the distribution pattern of histone acetylation in somatic cell NT (SCNT) embryos derived from NaBu- or TSA-treated donor cells does not match that of normal embryos (Wee et al., 2006; Yang et al., 2007a). In addition, there have been reports demonstrating that TSA treatment of cloned embryos after activation could greatly improve both the quantity and quality of cloned blastocysts and full-term development (Kishigami et al., 2006, 2007; Rybouchkin et al., 2006). It is of interest that after TSA treatment following activation, the patterns of histone acetylation in cloned mouse embryos were similar to those in normal embryos in the first cell cycle (Wang et al., 2007). To our knowledge, the effects of TSA on the distribution pattern of histone acetylation in cloned embryos after activation have not been thoroughly examined. The current study was designed to investigate the effects of TSA on the distribution patterns of acetylation in SCNT rabbit embryos after TSA treatment during activation.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Oocyte Collection
Mature female Japanese big-eared white rabbits (4 to 6 mo old) were superovulated by administering pregnant mare’s serum gonadotropin and human chorionic gonadotropin (hCG; Institute of Zoology, Chinese Academy of Sciences). Each rabbit was injected with 100 IU of hCG at 96 h after injection of 80 IU of pregnant mare’s serum gonadotropin. Rabbits were killed 14 h after hCG treatment. Mature metaphase II stage oocytes were collected by flushing the oviducts with warm M199 medium (Gibco BRL, Grand Island, NY). After exposure to 300 IU/mL of hyaluronidase (Sigma, St. Louis, MO) for 2 to 3 min, cumulus cells were stripped from the oocyte by repeated gentle pipetting.
Collection of In Vivo Fertilized Embryos
In vivo fertilized zygotes were collected 18 h post-hCG treatment from the oviductal ampullae of super-ovulated females that had been mated with the same strain of males just after hCG treatment. After removing the cumulus cells with 300 IU/mL of hyaluronidase in M199 medium, zygotes were cultured in M199 containing 10% fetal bovine serum (FBS; Gibco; culture medium). Embryos at different developmental stages were then fixed for further experimentation.
Preparation of Donor Cells
Fibroblast cells were collected from an earskin biopsy of a mature female rabbit. Primary cell culture and preparation of donor cells were performed by using the same method as described previously (Han et al., 2001). Fibroblasts at passage 4 to 10 were used as donor cells.
Oocyte Enucleation, NT, Fusion, and Activation
Oocyte enucleation, NT, fusion, and activation were carried out as described previously (Yan et al., 2006). Briefly, the zona pellucida of the cumulus-free oocyte was dissected by introducing a slit near the first polar body, and the cytoplasm containing the metaphase II spindle was squeezed out.
Next, a donor cell was transferred into the perivitelline space of the enucleated oocyte. The couplets were then transferred to the fusion chamber containing fusion medium (0.25 M sorbitol, 0.5 mM HEPES, 0.1 mM Ca(CH3COO)2, 0.5 mM Mg(CH3COO)2, and 1 mg/mL of BSA), and fusion was induced by 2 direct-current pulses (1.4 kV/cm, 80 µs each, 1 s apart) emitted from an ECM 2001 Electrocell Manipulator (BTX Inc., San Diego, CA). The fusion results were examined 30 min later, and fused couplets were activated by double DC pulses of 1.2 kV/cm for 20 µs at 3 h after fusion. The activated embryos were then washed 3 times with M199 supplemented with 10% FBS and were used for further experimentation.
TSA Treatment
Trichostatin A solution prepared in dimethylsulfoxide (DMSO) was diluted in culture medium to yield a final concentration of 100 nM. When the embryos were treated with TSA, correspondingly, 0.1% DMSO was included as a control. The reconstructed embryos were cultured in the M199 supplemented with 10% FBS with or without 100 nM TSA for 6 h after activation. The TSA-treated cloned embryos were then washed 3 times with the culture medium and cultured in a humidified atmosphere of 5% CO2 in air at 38° C. Five embryos were collected for immunochemistry at 6 h after activation, and the embryos at different developmental stages were fixed for further experimentation.
Immunofluorescent Confocal Microscopy
After removing the zona pellucida in acidic M2 medium (Sigma; pH 2.5), the embryos were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 1 h, permeabilized with 0.5% Triton X-100 at 4° C overnight, and then blocked in 1% BSA-supplemented PBS for 1 h at room temperature. Next, the embryos were incubated with rabbit polyclonal primary antibodies (1:300; Upstate Biotechnology, Lake Placid, NY) at 4° C overnight. Goat-anti-rabbit fluorescein isothiocyanate-conjugated secondary antibody (1:100; Jackson ImmunoResearch Laboratories Inc., West Grove, PA) was then applied for 1 h at room temperature. Finally, the samples were mounted between a coverslip and a glass slide supported by 4 columns of a mixture of petroleum jelly and paraffin (9:1, vol/vol). Each experiment was repeated at least 3 times, and at least 5 randomly selected, reconstructed embryos were examined each time. Slides were scanned by using a confocal laser-scanning microscope (Zeiss LSM 510 META, Carl Zeiss, Jena, Germany) with an argon-krypton laser at 488 and 563 nm and 2-channel scanning for detection of fluorescein isothiocyanate and propidium iodide, respectively.
Quantitative Analysis
The nuclear intensities of integrated fluorescence were measured by manually outlining all nuclei, 20 nuclei per morula and 30 nuclei per blastocyst (Suteevun et al., 2006). The total fluorescence intensity emitted by each individual nucleus was measured on antibody-stained images, after background subtraction, by ImageJ software (image processing and analysis in Java, http://rsb.info.nih.gov/ij/) and averaged per embryo (Carmona et al., 2007; Yang et al., 2007b). With SPSS software (SPSS Inc., Chicago, IL), a paired t-test was used to compare the values of different embryo stages, and P < 0.05 was considered significant.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Distribution Patterns of AcH3/K14 in Fertilized, Cloned, and TSA-Treated Cloned Embryos
The AcH3/K14 signal was detected in both male and female pronuclei in fertilized embryos (Figure 1, panel I, A and A' ). In SCNT embryos, the signal was observed at 6 h after activation (Figure 1, panel I, G and G' ), and TSA-treated SCNT embryos displayed a relatively strong staining (Figure 1, panel I, M and M' , and panel III). In normal embryos, the staining was completely undetectable at the 2-cell stage and reappeared after the 4-cell stage (Figure 1, panel I, B to E and B' to E' , and panel II), whereas in cloned embryos, the signals were maintained at an intense level in all of the subsequent development stages (Figure 1, panel I, H to K and H' to K' , and panel II). At the 2-cell stage, the intensity of the AcH3/K14 signal in TSA-treated cloned embryos was much weaker than that of embryos at 6 h of TSA-treatment (Figure 1, panel I, N and N' , and panel III, P < 0.05), with a slight increase from the 4-cell to the morula stage (Figure 1, panel I, O to Q and O' to Q' , and panel II). Strikingly, strong signals were observed in metaphase and anaphase stage blastomeres (Figure 1, panel I, C, H, and K, arrow). At the blastocyst stage, the signals in all fertilized embryos, cloned embryos, and TSA-treated cloned embryos were focused in the ICM (Figure 1, panel I, F and F' , L and L' , and R and R' ). The distribution patterns of histone acetylation in DMSO-control embryos were indistinguishable from those in untreated cloned embryos.
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At the pronuclear stage, AcH4/K12 staining was associated with the chromatin at equal levels in both male and female pronuclei in fertilized embryos (Figure 2, panel I, A and A' ). At 6 h after activation in cloned embryos, an intense signal was detected (Figure 2, panel I, G and G' ) and a much stronger staining was observed at 6 h in TSA-treated cloned embryos (Figure 2, panel I, M and M' , and panel III, P < 0.05). Both normal and TSA-treated cloned embryos displayed a high level of AcH4/K12 at the 2-cell stage (Figure 2, panel I, B and B' , N and N' , metaphase stage blastomere, arrow); however, the staining decreased slightly in cloned embryos (Figure 2, panel I, H and H' , and panel III). During the 4-cell stage, the staining disappeared completely in fertilized embryos (Figure 2, panel I, C and C' , and panel II) and that in TSA-treated embryos was greatly reduced (Figure 2, panel I, O and O' , and panel II, P < 0.05), whereas the staining in cloned embryos increased and was maintained until the morula stage (Figure 2, panel I, I to K and I' to K' , and panel II), including the strong staining of the metaphase stage blastomere (Figure 2, panel I, J, arrow). With preimplantation embryo development, the signal reappeared or was enhanced in 8-cell fertilized embryos and TSA-treated cloned embryos, and further increased until the morula stage (Figure 2, panel I, D to E, D' to E' , P to Q, and P' to Q' , and panel II). At the blastocyst stage, strong signals were found in the blastomeres of the ICM in fertilized embryos (Figure 2, panel I, F and F' ), whereas they were detected in both the trophectodermal blastomeres and the ICM in cloned embryos (Figure 2, panel I, L and L' ). More important, TSA-treated cloned embryos showed the same staining pattern as fertilized embryos and the staining was limited to the ICM (Figure 2, panel I, R and R' ).
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Staining of the AcH4/K5 modification exhibited yet another distinct pattern. In normal embryos, the AcH4/K5 staining was almost uniform in parental pronuclei (Figure 3, panel I, A and A' ). In cloned embryos, the staining was observed at 6 h after activation (Figure 3, panel I, G and G' ), when an intense signal was detected in TSA-treated cloned embryos (Figure 3, panel I, M and M' ). With the development of embryos, the complete deacetylation of histone took place at the 2-cell stage in all groups (Figure 3, panel I, B and B' , H and H' , N and N' , and panel III). Reacetylation occurred from the 4- to 8-cell stage (Figure 3, panel I, C to D, C' to D' , O to P, and O' to P' , and panel II), and the staining was barely visible at the morula and blastocyst stages (Figure 3, panel I, E to F, E' to F' , Q to R, and Q' to R' , and panel II) in both normal embryos and TSA-treated cloned embryos. Interestingly, the AcH4/K5 modification did appear to be associated with the fertilized metaphase chromatin (Figure 1
, panel I, F, arrow), suggesting that HDAC activity might be deficient during the M phase of the blastomeres at the blastocyst stage. In cloned embryos, the signal was absent from 4- to 8-cell embryos (Figure 3, panel I, I to J and I' to J' ) and reacetylation was observed at the morula stages (Figure 3, panel I, K and K' , and panel II). At the blastocyst stage in particular, the staining in TSA-treated cloned blastocysts was more similar to that in fertilized embryos than in cloned embryos (Figure 3, panel I, F and F' , L and L' , R and R' , and panel III). Together, TSA-treated cloned embryos improved the histone acetylation in a manner similar to that in normal embryos.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Epigenetic reprogramming, which occurs in normal preimplantational embryos, is apparently species-specific (Dean et al., 2001; Beaujean et al., 2004a,b; Shi et al., 2004) and lysine-specific (Kim et al., 2003; Akiyama et al., 2006). Consistent with a previous study in rabbit fertilized embryos (Yang et al., 2007a), with preimplantation embryonic development, the lysine residue on histone H3 was characteristically reduced and de novo acetylation occurred in normal embryos. In their study, AcH3K9/14 became hypoacetylated at the 2- and 8-cell stages and hyperacetylated at the 4-cell stage and after the 16-cell stage. The different distribution patterns of histone acetylation between Yang et al. (2007a) and the current experiment might be due to the different antibodies used (AcH3K9/14 vs. AcH4/K12, AcH3/K14, and AcH4/K5). However, in the current study the deactylation-reacetylation change occurred in both experiments as well as in other species (Suteevun et al., 2006; Wee et al., 2006). It seemed that all species shared the "erase-and-rebuild" pattern in normal embryo development (Sun et al., 2007). In this theory, the deacetylation process generates a relatively nai 've chromatin and the reacetylation process rebuilds the embryonic transcription program de novo, making the embryo ready for subsequent full-term development. Thus, in the process of genome reprogramming, acetylated lysine should be deacetylated to erase any information (i.e., cell memory) and to create the undifferentiated or totipotent zygotes for the next generation.
It was of additional interest to investigate the histone acetylation distribution pattern at the blastocyst stage, when lineage allocation first occurred and differentiation was beginning to take place. The ICM gives rise to all adult tissues and the trophectoderm brings about most placental tissues. Lineage-specific hyperacetylation of the ICM was observed at both AcH3/K14 and AcH4/K12, but not AcH4/K5, in fertilized embryos, indicating the epigenetic asymmetry between embryonic and extra-embryonic lineages.
Proper distribution patterns of histone acetylation, which characterize specific chromatin modifications, play a key role in nuclear reprogramming after SCNT and can predict the efficiency of SCNT. Unlike fertilized embryos, with embryonic development, there was no obvious deacetylation-reacetylation process of AcH3/K14 and AcH4/K12 in cloned rabbit embryos. This observation was in agreement with the reports by Suteevun et al. (2006) and Enright et al. (2003a), who reported that AcH3/K18 was observed in the nuclei of SCNT Swamp Buffalo and bovine embryos throughout preimplantation development. Nevertheless, AcH3K9/K14 rabbit cloned embryos were hyperacetylated at all stages except for being hypoacetylated at the 4- and 8-cell stages (Yang et al., 2007a). These results indicate that some intrinsic species-specific and lysine-specific differences on histone acetylation patterns may exist in cloned embryos. Additionally, AcH4/K5 underwent a longer deacetylation process compared with the normal embryos. Earlier studies have shown that the acetylation of histone H4 occurs initially at lysine 16 (AcH4/K16), and then at K8 or K12, and ultimately at K5 (Turner and Fellows, 1989). Therefore, the acetylated AcH4/K5 reflects the hyperacetylated state in histone H4 and is strongly correlated with the activation of genes (Grunstein, 1997; Rundlett et al., 1998). Thus, it is reasonable to infer that some genes associated with the embryonic development are not activated in untreated cloned rabbit embryos, which eventually results in the low rate of successful cloning (Solter, 2000).
The AcH4/K12 failed to establish epigenetic asymmetry at the blastocyst stage with the trophectoderm being as highly acetylated as the ICM, suggesting a widespread gene dysregulation in extra-embryonic tissue in cloned rabbit embryos, thereby resulting in placental dysfunction as in other species of cloned animals (Hill et al., 1999; De Sousa et al., 2001; Inoue et al., 2002; Ohgane et al., 2004). Notably, the distribution patterns of histone acetylation in SCNT rabbit embryos in the TSA treatment were more similar to those of in vivo fertilized embryos than to those of untreated cloned embryos.
Considering the fact that during activation, TSA-treated cloned mouse embryos showed the greatest pre- or postimplantation developmental potential (Kishigami et al., 2006; Rybouchkin et al., 2006; Kishigami et al., 2007), it was reasonable to expect that the histone acetylation patterns of these embryos would be more similar to those of in vivo fertilized embryos than to those of untreated cloned embryos. To address this view, we detected changes in the histone acetylation patterns of rabbit TSA-treated cloned embryos during activation. As expected, TSA-treated rabbit cloned embryos during activation remodeled the somatic cell nucleus at the epigenetic level to mimic normal embryo development. Our results provide further information about the distribution patterns of histone acetylation in SCNT rabbit embryos after TSA treatment during activation. In agreement with our results, Wang et al. (2007) also reported that similar acetylation patterns occurred in cloned mouse embryos after TSA treatment following activation in the first cell cycle. The distribution patterns of acetylation in TSA-treated cloned embryos were more similar to those in fertilized embryos than to those in SCNT embryos, suggesting that hyperacetylated chromatin occurring at the time of oocyte activation might be beneficial to the nuclear reprogramming of SCNT embryos. Hyper-acetylation loosened chromatin packaging and correlated with transcriptional activity to facilitate embryonic development (Shi et al., 2003b). Therefore, a fraction of the total genome was affected by TSA, which is critical to the proper development of embryos (Kim et al., 2003) and gives rise to correction of abnormal acetylation patterns in NT rabbit embryos.
Our results did not confirm the results of Yang et al. (2007a), in which the distribution patterns of histone acetylation in rabbit cloned embryos did not resemble those in normal embryos following pretreatment of donor cells with NaBu. Similarly, Wee et al. (2006) detected that histone acetylation patterns in cloned bovine embryos derived from TSA-treated donor fibroblast cells did not match those of in vitro-fertilized embryos. In fact, the histone acetylation patterns in rabbit cloned embryos after TSA treatment during activation were more similar to normal embryos than to those in cloned embryos derived from the donor cells with TSA pretreatment, suggesting that hyperacetylation during activation contributes greatly to the nuclear reprogramming of cloned embryos. However, details of the mechanism of nuclear reprogramming of TSA-treated cloned embryos are still unclear and need further investigation.
On the basis of our findings, we propose that abnormal acetylation events during the preimplantation development of clones may significantly affect the epigenetic reprogramming, resulting in cloning inefficiency. In addition, we found that TSA could improve rabbit somatic cell nuclear reprogramming and enhance SCNT rabbit embryo development.
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Footnotes |
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2 Corresponding author: chendy{at}ioz.ac.cn
Received for publication November 9, 2007. Accepted for publication January 17, 2008.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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