Hong-Thuy Bui

The histone deacetylase inhibitor scriptaid enhances nascent mRNA production and rescues full-term development in cloned inbred mice

Since the birth of Cumulina, the first mouse clone produced by somatic cell nuclear transfer (SCNT), the success rate of cloning in mice has been extremely low compared with other species and most of the inbred mouse strains have never been cloned. Recently, our laboratory has found that treatment of SCNT mouse embryos with trichostatin A, a histone deacetylase inhibitor (HDACi), improved the full-term development of B6D2F1 mouse clones significantly. However, this was not effective for the inbred strains. Here, we show for the first time that by treating SCNT embryos with another HDACi, scriptaid, all the important inbred mouse strains can be cloned, such as C57BL/6, C3H/He, DBA/2, and 129/Sv. Moreover, the success of somatic nuclear reprogramming and cloning efficiency via nuclear transfer technique is clearly linked to the competent de novo synthesis of nascent mRNA in cloned mouse embryos.

Production of cloned mice by somatic cellnuclear transfer

Although it has now been 10 years since the first cloned mammals were generated from somatic cells using nuclear transfer (NT), the success rate for producing live offspring by cloning remains <5%. Nevertheless, the techniques have potential as important tools for future research in basic biology. We have been able to develop a stable NT method in the mouse, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Although manipulation of the piezo unit is complex, once mastered it is of great help not only in NT experiments but also in almost all other forms of micromanipulation. In addition to this technique, embryonic stem (ES) cell lines established from somatic cell nuclei by NT can be generated relatively easily from a variety of mouse genotypes and cell types. Such NT-ES cells can be used not only for experimental models of human therapeutic cloning but also as a backup of the donor cells genome. Our most recent protocols for mouse cloning, as described here, will allow the production of cloned mice in ≥3 months.

Efficient establishment of mouse embryonic stem cell lines from single blastomeres and polar bodies.

Recently, ES cell lines were established from single blastomeres taken from eight‐cell embryos in mice and humans with success rates of 4% and 2%, respectively, which suggests that the method could be used in regenerative medicine to reduce ethical concerns over harm to embryos. However, those studies used other ES cells as supporting cells. Here, we report a simple and highly efficient method of establishing mouse ES cell lines from single blastomeres, in which single blastomeres are simply plated onto a feeder layer of mouse embryonic fibroblasts with modified ES cell medium. A total of 112 ES cell lines were established from two‐cell (establishment rate, 50%–69%), early four‐cell (28%–40%), late four‐cell (22%), and eight‐cell (14%–16%) stage embryos. We also successfully established 18 parthenogenetic ES cell lines from first (36%–40%) and second polar bodies (33%), the nuclei of which were reconstructed to embryos by nuclear transfer. Most cell lines examined maintained normal karyotypes and expressed markers of pluripotency, including germline transmission in chimeric mice. Our results suggest that the single cells of all early‐stage embryos or polar bodies have the potential to be converted into ES cells without any special treatment.

Effect of Trichostatin A on Chromatin Remodeling, Histone Modifications, DNA Replication, and Transcriptional Activity in Cloned Mouse Embryos

Our group and others have found that the treatment of embryos with trichostatin A (TSA) after cloning by somatic cell nuclear transfer (SCNT) results in a significant improvement in efficiency. We believe that TSA treatment improves nuclear remodeling via histone modifications, which are important in the epigenetic regulation of gene silencing and expression. Some studies found that treatment of SCNT-generated embryos with TSA improved lysine acetylation of core histones in a manner similar to that seen in normally fertilized embryos. However, how histone methylation is modified in TSA-treated cloned embryos is not completely understood. In the present study, we found that TSA treatment caused an increase in chromosome decondensation and nuclear volume in SCNT-generated embryos similar to that in embryos produced by intracytoplasmic sperm injection. Histone acetylation increased in parallel with chromosome decondensation. This was associated with a more effective formation of DNA replication complexes in treated embryos. We also found a differential effect of TSA on the methylation of histone H3 at positions K4 and K9 in SCNT-generated embryos that could contribute to genomic reprogramming of the somatic cell nuclei. In addition, using 5-bromouridine 5′-triphosphate-labeled RNA, we showed that TSA enhanced the levels of newly synthesized RNA in 2-cell embryos. Interestingly, the amount of SCNT-generated embryos showing asymmetric expression of nascent RNA was reduced significantly in the TSA-treated group compared with the nontreated group at the 2-cell stage. We conclude that the incomplete and inaccurate genomic reprogramming of SCNT-generated embryos was improved by TSA treatment. This could enhance the reprogramming of somatic nuclei in terms of chromatin remodeling, histone modifications, DNA replication, and transcriptional activity.

Regulation of chromatin and chromosome morphology by histone H3 modifications in pig oocytes

Oocyte growth, maturation, and activation are complex processes that include transcription, heterochromatin formation, chromosome condensation and decondensation, two consecutive chromosome separations, and genomic imprinting. The objective of this study was to investigate changes in histone H3 modifications in relation to chromatin/chromosome morphology in pig oocytes during their growth, maturation, and activation. During the growth phase, histone H3 was acetylated at lysines 9, 14, and 18 (K9, K14, and K18), and became methylated at K9 when the follicles developed to the antral stage (oocyte diameter, 90 mum). When the fully grown oocytes (diameter, 120 mum) started their maturation, histone H3 became phosphorylated at serine 28 (S28) and then at S10, and deacetylated at K9, K14, and K18 as the chromosomes condensed. After the electroactivation of mature oocytes, histone H3 was reacetylated and dephosphorylated concomitant with the decondensation of the chromosomes. Histone H3 kinase activity increased over a similar time course to that of the phosphorylation of histone H3-S28 during oocyte maturation, and this activity decreased as histone H3-S10 and H3-S28 began to be dephosphorylated after the activation of the mature oocytes. These results suggest that the chromatin morphology of pig oocytes is regulated by the acetylation/deacetylation and the phosphorylation/dephosphorylation of histone H3, and the phosphorylation of histone H3 is the key event in meiotic chromosome condensation in oocytes. The inhibition of histone deacetylase with trichostatin A (TSA) inhibited the deacetylation and phosphorylation of histone H3, and chromosome condensation. Therefore, the deacetylation of histone H3 is thought to be required for its phosphorylation in meiosis. Although histone H3 acetylation and phosphorylation were reversible, the histone methylation that was established during the oocyte growth phase was stable throughout the course of oocyte maturation and activation.

Normal specification of the extraembryonic lineage after somatic nuclear transfer

To examine the establishment and maintenance of trophectoderm (TE) lineage in somatic cloned blastocysts, the expression of Cdx2, a key molecule for specification of TE fate, was immunohistochemically examined simultaneously with Oct4 expression. Cloned mouse embryos were made by nuclear transfer using cumulus cells, tail‐tip fibroblasts, and embryonic stem cells. After 96 h of culture, the rates of Oct4‐expressing blastocysts were as low as 50% and 60% for cumulus and fibroblast clones, respectively. However, regardless of Oct4 expression, the majority of those cloned blastocysts (>90%) normally expressed Cdx2. Thus, even though somatic cloned embryos have reduced potential to produce the inner cell mass lineage, the TE lineage can be established and maintained.

The cytoplasm of mouse germinal vesicle stage oocytes can enhance somatic cell nuclear reprogramming

In mammalian cloning, evidence suggests that genomic reprogramming factors are located in the nucleus rather than the cytoplasm of oocytes or zygotes. However, little is known about the mechanisms of reprogramming, and new methods using nuclear factors have not succeeded in producing cloned mice from differentiated somatic cell nuclei. We aimed to determine whether there are functional reprogramming factors present in the cytoplasm of germinal vesicle stage (GV) oocytes. We found that the GV oocyte cytoplasm could remodel somatic cell nuclei, completely demethylate histone H3 at lysine 9 and partially deacetylate histone H3 at lysines 9 and 14. Moreover, cytoplasmic lysates of GV oocytes promoted somatic cell reprogramming and cloned embryo development, when assessed by measuring histone H3-K9 hypomethylation, Oct4 and Cdx2 expression in blastocysts, and the production of cloned offspring. Thus, genomic reprogramming factors are present in the cytoplasm of the GV oocyte and could facilitate cloning technology. This finding is also useful for research on the mechanisms involved in histone deacetylation and demethylation, even though histone methylation is thought to be epigenetically stable.

Involvement of Histone H3 (Ser10) Phosphorylation in Chromosome Condensation Without Cdc2 Kinase and Mitogen-Activated Protein Kinase Activation in Pig Oocytes

Abstract When oocytes resume meiosis, chromosomes start to condense and Cdc2 kinase becomes activated. However, recent findings show that the chromosome condensation does not always correlate with the Cdc2 kinase activity in pig oocytes. The objectives of this study were to examine 1) the correlation between chromosome condensation and histone H3 phosphorylation at serine 10 (Ser10) during the meiotic maturation of pig oocytes and 2) the effects of protein phosphatase 1/2A (PP1/ PP2A) inhibitors on the chromosome condensation and the involvement of Cdc2 kinase, MAP kinase, and histone H3 kinase in this process. The phosphorylation of histone H3 (Ser10) was first detected in the clump of condensed chromosomes at the diakinesis stage and was maintained until metaphase II. The kinase assay showed that histone H3 kinase activity was low in oocytes at the germinal vesicle stage (GV) and increased at the diakinesis stage and that high activity was maintained until metaphase II. Treatment of GV-oocytes with okadaic acid (OA) or calyculin-A (CL-A), the PP1/PP2A inhibitors, induced rapid chromosome condensation with histone H3 (Ser10) phosphorylation after 2 h. Both histone H3 kinase and MAP kinase were activated in the treated oocytes, although Cdc2 kinase was not activated. In the oocytes treated with CL-A and the MEK inhibitor U0126, neither Cdc2 kinase nor MAP kinase were activated and no oocytes underwent germinal vesicle breakdown (GVBD), although histone H3 kinase was still activated and the chromosomes condensed with histone H3 (Ser10) phosphorylation. These results suggest that the phosphorylation of histone H3 (Ser10) occurs in condensed chromosomes during maturation in pig oocytes. Furthermore, the chromosome condensation is correlated with histone H3 kinase activity but not with Cdc2 kinase and MAP kinase activities.

Histone Deacetylase Inhibition Improves Activation of Ribosomal RNA Genes and Embryonic Nucleolar Reprogramming in Cloned Mouse Embryos

Our group found that the treatment of embryos with histone deacetylase inhibitors (HDACi), including trichostatin A, Scriptaid, suberoylanilide hydroxamic acid, and oxamflatin, after cloning by somatic cell nuclear transfer (SCNT) resulted in significantly improved efficiency. Although many researchers have investigated the use of HDACi treatment to improve the quality of cloned mouse embryos, the mechanism underlying this treatment has not been completely understood. We believe that the effect of HDACi on embryonic gene activation (EGA) is important for normal development of cloned embryos. In the present study, using highly sensitive fluorescence in situ hybridization (FISH) with probes complementary to mouse rDNA, the effect of Scriptaid on the onset of rRNA synthesis was examined in cloned embryos. In addition, to determine how Scriptaid affects pre-rRNA processing machinery in SCNT embryos with activated rDNA transcription, functional nucleolar formation was analyzed in detail by combined assessment of rRNA synthesis and nucleolar protein allocation in preimplantation embryos. In this experiment, at least part of the rRNA localization by FISH was substituted by 5-bromouridine 5′-triphosphate staining after alpha-amanitin treatment. The results show that in the late 2-cell stage, a number of SCNT embryos initiated transcriptional activation while having one blastomere showing inactivated rRNA transcription and another blastomere showing activated rRNA transcription and despite both nuclei being in interphase. In addition, in some SCNT embryos, the same nuclei contained a mixture of inactively and actively transcribed rRNA, which was rarely observed in intracytoplasmic sperm injection embryos. This asynchronous transcription induced a delay of one cell cycle in SCNT embryo activation of functional nucleoli. Scriptaid can overcome this failure in the timely onset of embryonic gene transcription by activation of rRNA genes and promotion of nucleolar protein allocation during the early phase of EGA.

Epigenetic reprogramming in somatic cells induced by extract from germinal vesicle stage pig oocytes

Genomic reprogramming factors in the cytoplasm of germinal vesicle (GV) stage oocytes have been shown to improve the efficiency of producing cloned mouse offspring through the exposure of nuclei to a GV cytoplasmic extract prior to somatic cell nuclear transfer (SCNT) to enucleated oocytes. Here, we developed an extract of GV stage pig oocytes (GVcyto-extract) to investigate epigenetic reprogramming events in treated somatic cell nuclei. This extract induced differentiation-associated changes in fibroblasts, resulting in cells that exhibit pluripotent stem cell-like characteristics and that redifferentiate into three primary germ cell layers both in vivo and in vitro. The GVcyto-extract treatment induced large numbers of high-quality SCNT-generated blastocysts, with methylation and acetylation of H3-K9 and expression of Oct4 and Nanog at levels similar to in vitro fertilized embryos. Thus, GVcyto-extract could elicit differentiation plasticity in treated fibroblasts, and SCNT-mediated reprogramming reset the epigenetic state in treated cells more efficiently than in untreated cells. In summary, we provide evidence for the generation of stem-like cells from differentiated somatic cells by treatment with porcine GVcyto-extract.