Embryonic development and gene expression of porcine SCNT embryos treated with sodium butyrate

Incomplete epigenetic modification is one of important reasons of inefficient reprogramming of the donor cell nuclei in ooplasm after somatic cell nuclear transfer (SCNT). It may also underlie the observed reduced viability of cloned embryos. Sodium butyrate (NaBu) is a natural histone deacetylase inhibitor that is produced in the intestine. In the current study, we evaluated the effects of NaBu on preimplantation development, histone acetylation, and gene expression in porcine SCNT embryos. Our results showed that the blastocyst rate (24.88 ± 2.09) of cloned embryos treated with 1.0 mM NaBu for 12 hr after activation was significantly higher (P < 0.05) than that of untreated cloned embryos (13.15 ± 3.07). In addition, treated embryos displayed a global acetylated histone H3 at lysine 14 profile similar to that of in vitro fertilized (IVF) embryos during preimplantation development. Lower levels of Oct4 and Bcl-2, but higher levels of Hdac1, in SCNT embryos at the two-cell and blastocyst stages were observed, compared with those in the IVF counterparts. The four-cell embryos showed no differences in the levels of these genes among IVF embryos or SCNT embryos treated with or without NaBu; however, the levels of Dnmt3b were significantly different. NaBu-treated SCNT embryos showed similar levels of Oct4, Bcl-2, and Dnmt3b as in IVF blastocysts. These results indicated that NaBu treatment in SCNT embryos alters their histone acetylation pattern to provide beneficial effects on in vitro developmental competence and gene expression.     

Oocytes selected using BCB staining enhance nuclear reprogramming and the in vivo development of SCNT embryos in cattle

The selection of good quality oocytes is crucial for in vitro fertilization and somatic cloning. Brilliant cresyl blue (BCB) staining has been used for selection of oocytes from several mammalian species. However, the effects of differential oocyte selection by BCB staining on nuclear reprogramming and in vivo development of SCNT embryos are not well understood. Immature compact cumulus-oocyte complexes (COCs) were divided into control (not exposed to BCB), BCB+ (blue cytoplasm) and BCB- (colorless cytoplasm) groups. We found that BCB+ oocytes yielded a significantly higher somatic cell nuclear transfer (SCNT) blastocyst rate and full term development rate of bovine SCNT embryos than the BCB- and control oocytes. BCB+ embryos (embryos developed from BCB+ oocytes) showed increased acetylation levels of histone H3 at K9 and K18 (AcH3K9, AcH3K18), and methylation levels of histone H3 at K4 (H3K4me2) than BCB- embryos (embryos developed from BCB- oocytes) at the two-cell stage. Furthermore, BCB+ embryos generated more total cells, trophectoderm (TE) cells, and inner cell mass (ICM) cells, and fewer apoptotic cells than BCB- embryos. The expression of SOX2, CDX2, and anti-apoptotic microRNA-21 were up-regulated in the BCB+ blastocysts compared with BCB- blastocysts, whereas the expression of pro-apoptotic gene Bax was down-regulated in BCB+ blastocysts. These results strongly suggest that BCB+ oocytes have a higher nuclear reprogramming capacity, and that BCB staining can be used to select developmentally competent oocytes for nuclear transfer.     

Treatment of donor cells with recombinant KDM4D protein improves preimplantation development of cloned ovine embryos

Incomplete epigenetic reprogramming is one of the major factors affecting the development of embryos cloned by somatic cell nuclear transfer (SCNT). Histone 3 lysine 9 (H3K9) trimethylation has been identified as a key barrier to efficient reprogramming by SCNT. The aim of this study was to explore a method of downregulating H3K9me3 levels in donor cells by using histone lysine demethylase (KDM) protein. When sheep fetal fibroblast cells were treated with recombinant human KDM4D protein (rhKDM4D), the levels of H3K9 trimethylation and dimethylation were both significantly decreased. After SCNT, rhKDM4D-treated donor cells supported significantly higher percentage of cloned embryos developing into blastocysts as compared to non-treated control cells. Moreover, the blastocyst quality was also improved by rhKDM4D treatment of donor cells, as assessed by the total cell number in blastocysts and the expression of developmental genes including SOX2, NANOG and CDX2. These results indicate that treatment of donor cells with recombinant KDM4D protein can downregulate the levels of H3K9 trimethylation and dimethylation and improve the developmental competence of SCNT embryos. This strategy may be convenient to be used in KDM4-assisted SCNT procedure for improving the efficiency of cloning.     

Epigenetic states of donor cells significantly affect the development of somatic cell nuclear transfer (SCNT) embryos in pigs

The type and pattern of epigenetic modification in donor cells can significantly affect the developmental competency of somatic cell nuclear transfer (SCNT) embryos. Here, we investigated the developmental capacity, gene expression, and epigenetic modifications of SCNT embryos derived from porcine bone marrow‐derived mesenchymal stem cells (BMSCs) and fetal fibroblasts (FFs) donor cells compared to embryos obtained from in vitro fertilization (IVF). Compared to FFs, the donor BMSCs had more active epigenetic markers (Histone H3 modifications: H3K9Ac, H3K4me3, and H3K4me2) and fewer repressive epigenetic markers (H3K9me3, H3K9me2, and DNA methyltransferase 1). Embryos derived from BMSC nuclear‐transfer (BMSC‐NT embryos) and IVF embryos had significantly higher cleavage and blastocyst rates (BMSC‐NT: 71.3 ± 3.4%, 29.1 ± 2.3%; IVF: 69.2 ± 2.2%, 30.2 ± 3.3%; respectively) than FF‐NT embryos (58.1 ± 3.4%, 15.1 ± 1.5%, respectively). Bisulfite sequencing revealed that DNA methylation at the promoter regions of NANOG and POU5F1 was lower in BMSC‐NT embryos (30.0%, 9.8%, respectively) than those in FF‐NT embryos (34.2%, 28.0%, respectively). We also found that BMSC‐NT embryos had more H3K9Ac and less H3K9me3 and 5‐methylcytosine than FF‐NT embryos. In conclusion, our finding comparing BMSCs versus FFs as donors for nuclear transfer revealed that differences in the initial epigenetic state of donor cells have a remarkable effect on overall nuclear reprogramming of SCNT embryos, wherein donor cells possessing a more open chromatin state are more conducive to nuclear reprogramming.     

Trichostatin A improves histone acetylation in bovine somatic cell nuclear transfer early embryos

Epigenetic aberrancies likely preclude correct and complete nuclear reprogramming following somatic cell nuclear transfer (SCNT), and may underlie the observed reduced viability of cloned embryos. In the present study, we tested the effects of the histone deacetylase inhibitor (HDACi), trichostatin A (TSA), on development and histone acetylation of cloned bovine preimplantation embryos. Our results indicated that treating activated reconstructed SCNT embryos with 50 nM TSA for 13 h produced eight-cell embryos with levels of acetylation of histone H4 at lysine 5 (AcH4K5) similar to fertilized counterparts and significantly greater than in control NT embryos (p < 0.005). Further, TSA treatment resulted in SCNT embryos with preimplantation developmental potential similar to fertilized counterparts, as no difference was observed in cleavage and blastocyst rates or in blastocyst total cell number (p > 0.05). Measurement of eight selected developmentally important genes in single blastocysts showed a similar expression profile among the three treatment groups, with the exception of Nanog, Cdx2, and DNMT3b, whose expression levels were higher in TSA-treated NT than in in vitro fertilized (IVF) embryos. Data presented herein demonstrate that TSA can improve at least one epigenetic mark in early cloned bovine embryos. However, evaluation of development to full-term is necessary to ascertain whether this effect reflects a true increase in developmental potential.     

Epigenetic marks in cloned rhesus monkey embryos: comparison with counterparts produced in vitro

Until now, no primate animals have been successfully cloned to birth with somatic cell nuclear transfer (SCNT) procedures, and little is known about the molecular events that occurred in the reconstructed embryos during preimplantation development. In many SCNT cases, epigenetic reprogramming of the donor nuclei after transfer into enucleated oocytes was hypothesized to be crucial to the reestablishment of embryonic totipotency. In the present study, we focused on two major epigenetic marks, DNA methylation and histone H3 lysine 9 (H3K9) acetylation, which we examined by indirect immunofluorescence and confocal laser scanning microscopy. During preimplantation development, 67% of two-cell- and 50% of eight-cell-cloned embryos showed higher DNA methylation levels than their in vitro fertilization (IVF) counterparts, which undergo gradual demethylation until the early morula stage. Moreover, whereas an asymmetric distribution of DNA methylation was established in an IVF blastocysts with a lower methylation level in the inner cell mass (ICM) than in the trophectoderm, in most cloned blastocysts, ICM cells maintained a high degree of methylation. Finally, two donor cell lines (S11 and S1-04) that showed a higher level of H3K9 acetylation supported more blastocyst formation after nuclear transfer than the other cell line (S1-03), with a relatively low level of acetylation staining. In conclusion, we propose that abnormal DNA methylation patterns contribute to the poor quality of cloned preimplantation embryos and may be one of the obstacles to successful cloning in primates.     

Dynamic reprogramming of histone acetylation and methylation in the first cell cycle of cloned mouse embryos

Epigenetic reprogramming is thought to play an important role in the development of cloned embryos reconstructed by somatic cell nuclear transfer (SCNT). In the present study, dynamic reprogramming of histone acetylation and methylation modifications was investigated in the first cell cycle of cloned embryos. Our results demonstrated that part of somatic inherited lysine acetylation on core histones (H3K9, H3K14, H4K16) could be quickly deacetylated following SCNT, and reacetylation occurred following activation treatment. However, acetylation marks of the other lysine residues on core histones (H4K8, H4K12) persisted in the genome of cloned embryos with only mild deacetylation occurring in the process of SCNT and activation treatment. The somatic cloned embryos established histone acetylation modifications resembling those in normal embryos produced by intracytoplasmic sperm injection through these two different programs. Moreover, treatment of cloned embryos with a histone deacetylase inhibitor, Trichostatin A (TSA), improved the histone acetylation in a manner similar to that in normal embryos, and the improved histone acetylation in cloned embryos treated with TSA might contribute to improved development of TSA-treated clones. In contrast to the asymmetric histone H3K9 tri- and dimethylation present in the parental genomes of fertilized embryos, the tri- and dimethylations of H3K9 were gradually demethylated in the cloned embryos, and this histone H3K9 demethylation may be crucial for gene activation of cloned embryos. Together, our results indicate that dynamic reprogramming of histone acetylation and methylation modifications in cloned embryos is developmentally regulated.     

Scriptaid Treatment Decreases DNA Methyltransferase 1 Expression by Induction of MicroRNA-152 Expression in Porcine Somatic Cell Nuclear Transfer Embryos

Abnormal epigenetic reprogramming of donor nuclei after somatic cell nuclear transfer (SCNT) is thought to be the main cause of low cloning efficiencies. A growing body of evidence has demonstrated a positive role of Scriptaid, a histone deacetylase inhibitor (HDACi) that belongs to an existing class of hydroxamic acid-containing HDACis, on the development competence of cloned embryos in many species. The present study investigated the effects of Scriptaid on the development of porcine SCNT embryos in vitro and its mechanism. Treatment with 300 or 500 nM Scriptaid for 20 h after activation significantly increased the percentage of SCNT embryos that developed to the blastocyst stage and the total number of cells per blastocyst and significantly decreased the percentage of apoptotic cells in blastocysts. Scriptaid treatment significantly increased the level of histone H3 acetylated at K9 and the conversion of 5-methylcytosine into 5-hydroxymethylcytosine and significantly decreased the level of histone H3 trimethylated at K9 at the pronuclear stage. As a potential mechanism for the DNA methylation changes, our results showed that the expression of DNA methyltransferase 1 was frequently down-regulated in Scriptaid-treated embryos in comparison with untreated embryos and was inversely correlated to endogenous microRNA-152 (miR-152). Taken together, these findings illustrated a crucial functional crosstalk between miR-152 and DNMT1. Meanwhile, mRNA and protein levels of POU5F1 and CDX2 were increased in Scriptaid-treated embryos. mRNA levels of Caspase3, and Bax were significantly decreased and that of Bcl-xL was significantly increased in Scriptaid-treated embryos. In conclusion, these observations would contribute to uncover the nuclear reprogramming mechanisms underlying the effects of Scriptaid on the improvement of porcine SCNT embryos.     

The histone deacetylase inhibitor Scriptaid improves in vitro developmental competence of ovine somatic cell nuclear transferred embryos

Although the success rate of sheep cloning remains extremely low, using a histone deacetylase (HDAC) inhibitor to increase histone acetylation in SCNT embryos has significantly enhanced developmental competence in several species. The objective was to determine whether HDAC inhibitors trichostatin A (TSA) and the novel inhibitor Scriptaid enhance cloning efficiency in sheep cumulus cell (passage 2) reconstructed embryos. In this study, 0.2 μmol/L Scriptaid yielded a high blastocyst development rate, almost twice that of the untreated group (25/103 [24.3%] vs. 12/101 [11.9%]; P < 0.05). Furthermore, 0.2 μmol/L Scriptaid was more effective than 0.05 μmol/L TSA in terms of the blastocyst percentage for cloned ovine embryos in vitro (17/66 [25.7%] vs. 11/65 [16.8%]; P < 0.05). Furthermore, treatment with Scriptaid increased acetylation (compared with the Control, P < 0.05) at lysine residue 12 of histone H4 (acH4K12) and lysine residue 9 of histone H3 (acH3K9) in one-, two-, four-, and eight-cell stages, as well as blastocyst stages, in cloned embryos. In conclusion, Scriptaid was more effective than TSA to enhance in vitro developmental competence in ovine SCNT embryos; furthermore, Scriptaid improved epigenetic status.     

Oxamflatin significantly improves nuclear reprogramming, blastocyst quality, and in vitro development of bovine SCNT embryos

Aberrant epigenetic nuclear reprogramming results in low somatic cloning efficiency. Altering epigenetic status by applying histone deacetylase inhibitors (HDACi) enhances developmental potential of somatic cell nuclear transfer (SCNT) embryos. The present study was carried out to examine the effects of Oxamflatin, a novel HDACi, on the nuclear reprogramming and development of bovine SCNT embryos in vitro. We found that Oxamflatin modified the acetylation status on H3K9 and H3K18, increased total and inner cell mass (ICM) cell numbers and the ratio of ICM∶trophectoderm (TE) cells, reduced the rate of apoptosis in SCNT blastocysts, and significantly enhanced the development of bovine SCNT embryos in vitro. Furthermore, Oxamflatin treatment suppressed expression of the pro-apoptotic gene Bax and stimulated expression of the anti-apoptotic gene Bcl-XL and the pluripotency-related genes OCT4 and SOX2 in SCNT blastocysts. Additionally, the treatment also reduced the DNA methylation level of satellite I in SCNT blastocysts. In conclusion, Oxamflatin modifies epigenetic status and gene expression, increases blastocyst quality, and subsequently enhances the nuclear reprogramming and developmental potential of SCNT embryos.     

Epigenetic reprogramming,gene expression and in vitro development of porcine SCNT embryos are significantly improved by a histone deacetylase inhibitor—m-carboxycinnamic acid bishydroxamide (CBHA)

Insufficient epigenetic reprogramming of donor nuclei is believed to be one of the most important causes of low development efficiency of mammalian somatic cell nuclear transfer (SCNT). Previous studies have shown that both the in vitro and in vivo development of mouse SCNT embryos could be increased significantly by treatment with various histone deacetylase inhibitors (HDACi), including Trichostatin A, Scriptaid, and m-carboxycinnamic acid bishydroxamide (CBHA), in which only the effect of CBHA has not yet been tested in other species. In this paperweexamine the effect ofCBHAtreatment on the development of porcine SCNT embryos. We have discovered the optimum dosage and time for CBHA treatment: incubating SCNT embryos with 2 μmol/L CBHA for 24 h after activation could increase the blastocyst rate from 12.7% to 26.5%. Immunofluorescence results showed that the level of acetylation at histone 3 lysine 9 (AcH3K9), acetylation at histone 3 lysine 18 (AcH3K18), and acetylation at histone 4 lysine 16 (AcH4K16) was raised after CBHAtreatment. Meanwhile,CBHAtreatment improved the expression of development relating genes such as pou5f1, cdx2, and the imprinted genes like igf2. Despite these promising in vitro results and histone reprogramming, the full term development was not significantly increased after treatment. In conclusion, CBHA improves the in vitro development of pig SCNT embryos, increases the global histone acetylation and corrects the expression of some developmentally important genes at early stages. As in mouse SCNT, we have shown that nuclear epigenetic reprogramming in pig early SCNTembryos can be modified by CBHA treatment.     

Pluripotent-related gene expression analyses in single porcine recloned embryo

The developmental ability among embryos produced by three different techniques were examined: there were no significant differences in the developmental rate in porcine embryos produced by in vitro fertilization (IVF) and first generation of somatic cell nucleus transfer (SCNT), but the developmental rate dropped sharply at the 2- to four-cell stage in recloned (second generation of SCNT) embryos. In most recloned embryos, Oct4 and Klf4 were under-expressed at all stages, whereas Sox2 and Nanog were over-expressed at the two-cell stage. In contrast, Nanog was absent in IVF and SCNT embryos at the two-cell stage. The recloned embryos were treated with valproic acid to enhance developmental capacity and this led to an increase in the rate of blastocyst formation and total cell number compared with the findings for untreated recloned embryos (29.8 vs. 12.4 %, 39 vs. 25, respectively, p < 0.05).     

PCI-24781 can improve in vitro and in vivo developmental capacity of pig somatic cell nuclear transfer embryos

Objective

To examine the effect of PCI-24781 (abexinostat) on the blastocyst formation rate in pig somatic cell nuclear transferred (SCNT) embryos and acetylation levels of the histone H3 lysine 9 and histone H4 lysine 12.

Results

Treatment with 0.5 nM PCI-24781 for 6 h significantly improved the development of cloned embryos, in comparison to the control group (25.3 vs. 10.5 %, P < 0.05). Furthermore, PCI-24781 treatment led to elevated acetylation of H3K9 and H4K12. TUNEL assay and Hoechst 33342 staining revealed that the percentage of apoptotic cells in blastocysts was significantly lower in PCI-24781-treated SCNT embryos than in untreated embryos. Also, PCI-24781-treated embryos were transferred into three surrogate sows, one of whom became pregnant and two fetuses developed.

Conclusion

PCI-24781 improves nuclear reprogramming and the developmental potential of pig SCNT embryos.

     

Developmental rate and ploidy of embryos produced by nuclear transfer with different activation treatments in cattle

Bovine oocyte activation is one of the essential elements that determine the success of nuclear transfer and the subsequent development of cloned embryos. Three methods for oocyte activation, including 5 microM ionomycin (5 min, Group 1) alone, ionomycin+1.9 mM 6-dimethylaminopurine (DMAP, 3h, Group 2), and ionomycin+10 microg/ml cycloheximide (CHX, 3h, Group 3) were compared for the development of embryos produced by somatic nuclear transfer (SCNT) to parthenotes and IVF counterparts. At 19-h post-activation/insemination (hpa/hpi), 27.5% of oocytes in Group 2 cleaved and this rate was greater (P<0.05) than other groups (Group 1, 2.1%; Group 3, 3.0%). None of the oocytes in the IVF control group cleaved at 19-22 hpi. At 24 hpa, the rates of cleavage of oocytes in Group 2 (52.1%) were greater (P<0.05) than those in Groups 1 and 3 (7 and 38.3%, respectively). Only six oocytes (3.3%) in the IVF control group cleaved at 24 hpi. The overall cleavage rates of oocytes in Group 2 (85.5%) at 48 hpa were greater (P<0.05) than other treatments, but it did not show any difference when compared with the IVF control group (75.0%). The development rate to two-cell stage embryos of Group 2 was consistently greater at all observation points followed by Groups 3 and 1. Similar results were obtained in SCNT embryos, but the rates of cleavage at 48 hpi and blastocyst development in Group 2 (68.4 and 16.3%, respectively) did not differ from Group 3 (63.0 and 13.1%, respectively). The chromosomal composition in the parthenotes and SCNT embryos differed (P<0.05) among treatments. In Groups 1 and 3, greater percentages of haploid parthenotes (86 and 71%, respectively) were observed. In contrast, 84% of parthenotes in Group 2 had abnormal ploidy (44% polyploid and 40% mixoploid). In the case of SCNT embryos, Groups 1 and 3 had greater percentages of diploid chromosomal sets (77 and 70%, respectively), whereas 54% in Group 2 were polyploid or mixoploid. These results indicate that DMAP treatment after ionomycin greatly increases the developmental rates of parthenotes, but did not differ in blastocyst development compare with CHX treatment. However, DMAP treatment increased the time-dependent cleavage rate to two-cell stage embryos. Further, it greatly enhanced the incidence of chromosomal abnormalities in parthenotes and SCNT embryos. Hence, it is concluded that CHX combined with ionomycin is more desirable than DMAP for oocyte activation during nuclear transfer in cattle.