Comparison of electro-fusion and intracytoplasmic nuclear injection methods in pig cloning

This paper methodologically compares the electro-fusion (EF) and intracytoplasmic injection (ICI) methods, as well as simultaneous fusion/activation (SA) and delayed activation (DA), in somatic nuclear transfer in pigs using fetal fibroblast cells. Comparison of the remodeling pattern of donor nuclei after nuclear transfer by ICI or EF showed that a high rate (80-100%) of premature chromosome condensation occurred in both cases whether or not Ca2+ was present in the fusion medium. Formation of pseudo-pronuclei tended to be lower for nuclear transfer performed by the ICI method (65% vs. 85-97%, p < 0.05). In vitro developmental potential of nuclear transfer embryos reconstructed with IVM oocytes using the EF method was higher than that of those produced by the ICI method (blastocyst formation: 19 vs. 5%, p < 0.05), and it was not improved using in vivo-matured oocytes as recipient cytoplasts. Embryos produced using SA protocol developed to blastocysts with the same degree of efficiency as those produced under the DA protocol (11 vs. 12%). Use of the EF method in conjunction with SA was shown to be an efficient method for producing cloned pigs based on producing a cloned normal pig fetus. However, subtle differences in nuclear remodeling patterns between the SA and DA protocols may imply variations in their nuclear reprogramming efficiency.     

Cloned endangered species takin (Budorcas taxicolor) by inter-species nuclear transfer and comparison of the blastocyst development with yak (Bos grunniens) and bovine

Interspecies cloning might be used as an effective method to conserve endangered species and to support the study of nuclear-cytoplasm interaction. In this study, we describe the development of takin-bovine embryos in vitro produced by fusing takin ear fibroblasts with enucleated bovine oocytes and examine the fate of mitochondrial DNA in these embryos. We also compare the blastocyst development of takin-bovine embryos with yak-bovine and bovine-bovine embryos and compare the cell numbers of the blastocyst. Our results indicate that: (1) takin-bovine cloned embryos can develop to the blastocyst stage in vitro (5%), (2) blastocyst mitochondria DNA are derived primarily from bovine oocytes in spite of a little takin donor cell mitochondrial DNA, (3) using the same cloned protocol, development efficiency is significantly different between bovine-bovine cloning, yak-bovine, and takin-bovine cloning (48 vs. 28% vs. 5%, P < 0.01), and (4) cell numbers in the blastocysts of the three species of embryos were not different. These results suggest that the bovine oocytes can reprogram the takin, yak, and bovine fibroblast nuclei. However, the development efficiency of intra-species cloning tends to be higher than inter-species cloning; the more close the species of the donor cell is to the recipient oocyte (yak versus takin), the greater the blastocyst development in vitro.     

细胞核重编程对克隆的影响

细胞核重编程是哺乳动物正常受精胚胎和克隆胚胎发育过程中的一个重要特性,主要是对表观遗传学特征进行重新编写,包括染色质重塑、组蛋白修饰、DNA甲基化、印记基因表达、X染色体失活等表观遗传修饰的改变。通过细胞核重编程,首先,受精卵和克隆胚胎的供体核停止其特有的基因表达程序,恢复为全能状态的基因表达程序;然后,受精胚胎和克隆胚胎的细胞再从全能状态重新进入分化状态,最终形成各种组织和器官。近年来,不少研究表明,克隆胚胎的细胞核重编程存在不同程度的表观遗传修饰异常,可能对克隆及其农业和医学应用有着重要影响。本文就正常和克隆胚胎细胞核重编程的研究进展以及克隆胚胎的细胞核重编程异常对克隆的影响作一综述,并对目前有关治疗性克隆前景的不同看法进行了讨论。     

核移植重构胚染色体重塑的分析研究进展

重构胚核染色体重塑对于核移植的成功是必要的。供体核在卵胞质内将会发生一系列的形态学变化,包括核膜破裂、成熟前的染色体凝集,然后重新形成新的核结构,指导着整个胚胎的发育过程。本综述对发生在核移植重构胚染色体中的生物学事件进行了简要阐述。     

Increased pre-implantation development of cloned bovine embryos treated with 5-aza-2'-deoxycytidine and trichostatin A

Limited success of somatic cell nuclear transfer is attributed to incomplete reprogramming of transferred nuclei. The objective was to determine if 5-aza-2'-deoxycytidine (5-aza-dC) and trichostatin A (TSA) promoted reprogramming and improved development. Relative to untreated controls, treatment of donor cells, cloned embryos, and continuous treatment of both donor cells and cloned embryos with a combination of 0.01microM 5-aza-dC and 0.05microM TSA significantly increased the blastocyst rate (11.9% vs 31.7%, 12.4% vs 25.6%, and 13.3% vs 38.4%, respectively) and total cell number (73.2 vs 91.1, 75.2 vs 93.7, and 74.6 vs 96.7). Moreover, blastocyst rate and inner cell mass (ICM) cell number of embryos continuously exposed to both reagents were significantly higher than that of a TSA-treated group (38.4% vs 23.9% and 27.4 vs 18.2). The DNA methylation level of 2-cell embryos was decreased significantly, whereas the histone acetylation level increased dramatically after donor cell treatment and continuous treatment with both reagents. However, these epigenetic features of cloned blastocysts were not significantly different than the untreated control group. Following embryo treatment, DNA methylation and histone acetylation levels of cloned blastocysts were unchanged, except for the group given 0.5microM TSA (acetylation level was significantly increased, but development potential was reduced). In conclusion, development of cloned bovine embryos was enhanced by 5-aza-dC and TSA; furthermore, the combination was more effective than either one alone.     

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.     

Cloned calves from chromatin remodeled in vitro

We have developed a novel system for remodeling mammalian somatic nuclei in vitro prior to cloning by nuclear transplantation. The system involves permeabilization of the donor cell and chromatin condensation in a mitotic cell extract to promote removal of nuclear factors solubilized during chromosome condensation. The condensed chromosomes are transferred into enucleated oocytes prior to activation. Unlike nuclei of nuclear transplant embryos, nuclei of chromatin transplant embryos exhibit a pattern of markers closely resembling that of normal embryos. Healthy calves were produced by chromatin transfer. Compared with nuclear transfer, chromatin transfer shows a trend toward greater survival of cloned calves up to at least 1 mo after birth. This is the first successful demonstration of a method for directly manipulating the somatic donor chromatin prior to transplantation. This procedure should be useful for investigating mechanisms of nuclear reprogramming and for making improvements in the efficiency of mammalian cloning.     

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.     

The effects of delayed activation and MG132 treatment on nuclear remodeling and preimplantation development of embryos cloned by electrofusion are correlated with the age of recipient cytoplasts

The electrofusion method, used extensively in livestock cloning, cannot be used in mice, because it is believed that the mouse oocytes are more susceptible to detrimental effects of electrical stimulus than oocytes from other species. Reports on whether a delayed activation after electrofusion and a premature chromosome condensation (PCC) is essential for efficient cloning are inconclusive. To address these issues, effects of pulsing on activation and MPF activity of nonenucleated oocytes and effects of delayed activation and MG132 treatment on donor nuclear PCC and preimplantation development of embryos cloned by electrofusion or nuclear injection were compared between different cytoplast ages in mice and goats. The results indicated that the use of oocytes collected early after donor stimulation would make it possible to conduct somatic cell nuclear transfer in mice by electrofusion. Whether a delayed activation is essential was dependent upon the age, or rather, the level, of MPF activity of the cytoplasts at the time of electrofusion, as was the requirement for MG132 treatment. The competence for blastocyst formation of cloned embryos was highly correlated with the level of donor nuclear PCC in recipient cytoplasts. The nuclear injection technique was more adaptable to older cytoplast ages, and hence less dependent on drugs for inhibition of MPF inactivation, compared to electrofusion.     

Gene expression and in vitro development of inter-species nuclear transfer embryos

This study examined the chromatin morphology, in vitro development, and expression of selected genes in cloned embryos produced by transfer of mouse embryonic fibroblasts (MEF) into the bovine ooplasm. After 6 hr of activation, inter-species nuclear transfer (NT) embryos (MEF-NT) had one (70%) or two pronuclei (20%), respectively. After 72 hr of culture in vitro, 62.6% of the MEF-NTs were arrested at the 8-cell stage, 31.2% reached the 2- to 4-cell stage, and only 6.2% had more than eight blastomeres, but none of these developed to the blastocyst stage. Whereas, 20% of NT embryos derived from bovine embryonic fibroblast fused with bovine ooplasm (BEF-NT) reached the blastocyst stage. Donor MEF nuclei expressing an Enhanced Green Fluorescent Protein (EGFP) transgene resulted in 1- to 8-cell stage MEF-NT that expressed EGFP. The expression of selected genes was examined in 8-cell MEF-NTs, 8-cell mouse embryos, enucleated bovine oocytes, and MEFs using RT-PCR. The mRNA for heat shock protein 70.1 (Hsp 70.1) gene was detected in MEF-NTs and MEF, but not in mouse embryos. The hydroxy-phosphoribosyl transferase (HPRT) mRNA was found in normal mouse embryos and MEF but not in MEF-NTs. Expression of Oct-4 and embryonic alkaline phospatase (eAP) genes was only detected in normal mouse embryos and not in the inter-species NT embryos. Abnormal gene expression profiles were associated with an arrest in the development at the 8-cell stage, but MEF-NT embryos appeared to have progressed through gross chromatin remodeling, typical of intra-species NT embryos. Therefore, molecular reprogramming rather than chromatin remodeling may be a better indicator of nuclear reprogramming in inter-species NT embryos.     

鱼类核移植与重编程

鱼类核移植是动物克隆研究的一个重要领域, 我国学者在上世纪60年代首创了鱼类的核移植研究。以斑马鱼为模式动物, 进行核移植与再程序化研究具有独特的优势。文章总结了鱼类细胞核移植研究的历史、斑马鱼核移植研究概况、以及影响核移植胚胎发育的因素, 特别是核移植胚胎基因组的表观遗传修饰, 如基因组DNA甲基化及组蛋白乙酰化和甲基化等的研究, 将有助于完善克隆技术并提高克隆的成功率, 推动克隆技术的广泛开展和应用。     

Production of cloned pigs by nuclear transfer of preadipocytes established from adult mature adipocytes

The aim of the present study was to determine whether porcine preadipocytes can be efficient donor cells for somatic cell nuclear transfer (SCNT) in pigs. Primary culture of porcine preadipocytes was established by de-differentiating mature fat cells taken from an adult pig. The cell cycle of the preadipocytes could be synchronized by serum starvation for 1 day, with a higher efficiency than control fetal fibroblasts. Incidence of premature chromosome condensation following nuclear transfer (NT) of preadipocytes was as high as that observed after NT with fetal fibroblasts. In vitro developmental rate of the NT embryos reconstructed with preadipocyte was equivalent to that of the fetal fibroblast derived embryos. Transfer of 732 NT embryos with preadipocytes to five recipients gave rise to five cloned piglets. These data demonstrate that preadipocyites collected from an adult pig are promising nuclear donor cells for pig cloning.     

Epigenetic characteristics and development of embryos cloned from donor cells treated by trichostatin A or 5-aza-2'-deoxycytidine

Development to blastocyst following nuclear transfer is dependent on the donor cell's ability to reprogram its genome to that of a zygote. This reprogramming step is inefficient and may be dependent on a number of factors, including chromatin organization. Trichostatin A (TSA; 0-5 microM), a histone deacetylase inhibitor, was used to increase histone acetylation and 5-aza-2'-deoxycytidine (5-aza-dC; 0-5 microM), a DNA methyl-transferase inhibitor, was used to decrease methylation of chromatin in donor cells in an attempt to improve their reprogrammability. Adult fibroblast cells treated with 1.25 or 5 microM TSA had elevated histone H3 acetylation compared to untreated controls. Cells treated with 0.3 microM 5-aza-dC had decreased methylation compared to untreated controls. Both drugs at 0.08 microM caused morphological changes of the donor cells. Development to blastocysts by embryos cloned from donor cells after 0.08 or 0.3 microM 5-aza-dC treatments was lower than in embryos cloned from untreated control cells (9.7% and 4.2%, respectively, vs. 25.1%), whereas 0.08 microM TSA treatment of donor cells increased blastocyst development compared to controls (35.1% vs. 25.1%). These results indicate that partial erasure of preexisting epigenetic marks of donor cells improves subsequent in vitro development of cloned embryos.     

Nuclear and cytoskeletal dynamics during oocyte maturation and development of somatic cell cloned pig embryos injected with membrane disintegrated donor cells

The objectives of this study were to characterize the nuclear and cytoskeletal changes of pig oocytes during in vitro maturation (IVM) and the development of the reconstructed embryos after injection with membrane intact or disintegrated donor cells. Cumulus-oocyte complexes (COCs) were collected from abattoir ovaries by follicle (2-8mm) aspiration. In Experiment 1, COCs were cultured in NCSU-23 medium for 0, 11, 22, 33, and 44 h. Oocytes were fixed at different time points for nuclear and cytoskeletal labeling. Forty-three percent and 75% oocytes progressed to MII stage at 33 and 44 h after IVM culture, respectively. Dynamic shift of spindle and cytoplasmic microtubules was evident. In Experiment 2, matured oocytes were injected with either the whole cumulus cell with or without intact cell membranes after enucleation. The reconstructed oocytes were fixed at 0, 2, or 4 h after cell injection for nuclear and cytoskeletal evaluation. When an intact cumulus cell was injected, the injected cell remained intact within 4h after injection. When a cell with disintegrated membrane was injected, 59-63% (n=146) of the injected cell underwent premature chromosome condensation (PCC). In Experiment 3, the reconstructed pig oocytes received membrane-disintegrated cumulus cells or fetal fibroblasts were cultured in PZM medium. The blastocyst rate of the fibroblast-injected embryos was 10%, which was lower than the non-cloned parthenotes (33%, P<0.05) but higher than the cumulus cell-injected embryos (2.7%). These results suggest that pig oocytes are subjected to nuclear and cytoskeletal reorganization during maturation. Pig oocytes injected with membrane-disintegrated fibroblast cells support better blastocyst development of the cloned embryos.     

Comparison of potency between histone deacetylase inhibitors trichostatin A and valproic acid on enhancing in vitro development of porcine somatic cell nuclear transfer embryos

Epigenetic modification influences reprogramming and subsequent development of somatic cell nuclear transfer (SCNT) embryos. Such modification includes an increase in histone acetylation. Histone deacetylase inhibitors (HDACi), such as trichostatin A (TSA) and valproic acid (VPA), have been known to maintain a high cellular level of histone acetylation. Hence, treatment of nuclear transfer embryos with HDACi may increase the efficiency of cloning. The present study attempted direct comparison of TSA and VPA with regard to the potency of enhancement of in vitro development in porcine SCNT embryos. Reconstructed oocytes using fetal fibroblasts were cultured in PZM-3 containing no HDACi (control), 5 mM VPA, or 50 nM TSA for 24 h, and another 5 d thereafter without HDACi. The frequency of blastocyst formation was significantly higher (P<0.05) in embryos treated with VPA than the frequencies with TSA and without HDACi (125/306, 40.8% vs. 94/313, 30.2% vs. 80/329, 23.4%). In addition, VPA treatment significantly increased (P<0.05) the number of inner cell mass (ICM) cells compared with the control (15.6 ± 1.7 vs. 10.8 ± 2.6), whereas no differences were observed between the TSA treatment and control groups (12.9 ± 3.0 vs. 10.8 ± 2.6). The present study demonstrates that VPA enhances in vitro development of porcine SCNT embryos, particularly by an increase in blastocyst formation and in the number of ICM cells, suggesting that VPA may be more potent than TSA in supporting developmental competence of cloned embryos.     

in vitro development of porcine enucleated oocytes reconstructed by the transfer of porcine fetal fibroblasts and cumulus cells

In the pig little information is available on cytoplasmic events during the reprogramming of oocytes reconstructed with somatic nuclei. The present study was conducted to determine the developmental potential of porcine cumulus cells (CC) and fetal fibroblasts (FF) after they were transferred into enucleated oocytes. Non-quiescent FF were fused to the enucleated oocytes using electrical pulse, whereas CC were directly injected into the oocytes. Transferred nuclei from both CC and FF underwent premature chromosome condensation (PCC), nuclear swelling and pronucleus formation. The remodeled oocytes developed to the mitotic and 2-cell stage at 18 to 24 h after nuclear transfer. The pattern of nuclear remodeling was similar regardless of the sources of karyoplasts or nuclear transfer methods. However, using FF, 24% of nuclear transferred embryos developed to the morula or blastocyst stage, whereas only 8% of those using CC developed to the morula or blastocyst stage. These results suggest that porcine oocyte cytoplasm can successfully reprogram somatic cell nuclei and support the development of nuclear transferred embryos to the blastocyst stage.     

Relationship between nuclear remodeling and subsequent development of mouse embryonic nuclei transferred to enucleated oocytes

The present study was conducted to examine the relationship between nuclear remodeling and subsequent embryonic development in nuclear transplant mouse embryos. Metaphase II oocytes were enucleated without staining and fused with transferred donor nuclei from two-, four-, or eight-cell embryos. Fusion and oocyte activation were performed by means of electric fields. High rates of enucleation (89.1%), fusion (88.0–91.6%), and activation (95.2–96.9%) were obtained using this system. Nuclear remodeling was characterized by premature chromosome condensation (PCC), followed by various pronuclear-like formations upon oocyte activation. Development to blastocysts was obtained from both PCC (17.9%) and non-PCC (NPCC; 52.9%) embryos fused with the two-cell nuclei. However, development to term was obtained only in PCC embryos with a single pronucleus-like structure and a polar body (12.5%). In vitro development of nuclear transplant embryos with four- and eight-cell nuclei was limited. All the NPCC embryos examined had tetraploid chromosome constitutions, but chromosome constitutions of PCC embryos varied. Only 37.5% of the PCC embryos had diploid chromosome constitutions. The results indicated that the development of nuclear transplant embryos is affected by the types of nuclear remodeling and that oocyte activation in relation to their chromosome constitutions. The results also indicated that the PCC of the donor nucleus in nonactivated cytoplasm is important for the development of the nuclear transplant embryos. © 1994 Wiley-Liss, Inc.     

Epigenetic marking correlates with developmental potential in cloned bovine preimplantation embryos

During differentiation, somatic nuclei acquire highly specialized DNA and chromatin modifications, which are thought to result in cellular memory of the differentiated state. Upon somatic nuclear transfer into oocytes, the donor nucleus may have to undergo reprogramming of these epigenetic marks in order to achieve totipotency. This may involve changes in epigenetic features similar to those that occur in normal embryos during early development. However, there is accumulating evidence that epigenetic reprogramming is severely deficient in cloned embryos. Several reports reveal inefficient demethylation and inappropriate reestablishment of DNA methylation in quantitative and qualitative patterns on somatic nuclear transfer. Here we examine histone H3 lysine 9 (H3-K9) methylation and acetylation in normal embryos and in those created by somatic nuclear transfer. We find that H3-K9 methylation is reprogrammed in parallel with DNA methylation in normal embryos. However, the majority of cloned embryos exhibit H3-K9 hypermethylation associated with DNA hypermethylation, suggesting a genome-wide failure of reprogramming. Strikingly, the precise epigenotype in cloned embryos depends on the donor cell type, and the proportion of embryos with normal epigenotypes correlates closely with the proportion developing to the blastocyst stage. These results suggest a mechanistic link between DNA and histone methylation in the mammalian embryo and reveal an association between epigenetic marks and developmental potential of cloned embryos.     

Methylation and acetylation characteristics of cloned bovine embryos from donor cells treated with 5-aza-2'-deoxycytidine

Differentiated somatic cells and embryos cloned from somatic cells by nuclear transfer (NT) have higher levels of DNA methylation than gametes and early embryos produced in vivo. Reducing DNA methylation in donor cells before NT by treating them with chemicals such as the DNA methyl-transferase inhibitor (5-aza-2'-deoxycytidine; 5-aza-dC) may improve cloning efficiency of NT embryos by providing donor cells with similar epigenetic characteristics as in vivo embryos. Previously, high levels of this reagent were used to treat donor cells, and decreased development of cloned embryos was observed. In this study, we tested a lower range (0.005 to 0.08 microM) of this drug and used cell cycle distribution changes as an indicator of changes in the characteristics of donor cells. We found that at 0.01 microM 5-aza-dC induced changes in the cycle stage distribution of donor cells, increased the fusion rate of NT embryos, and had no deleterious effect on the percentage of blastocyst development. Levels of 5-aza-dC greater than 0.01 microM significantly decreased embryo development. Embryos cloned from donor cells treated with a low dose of 5-aza-dC had higher levels of DNA methylation than embryos produced by in vitro fertilization, but they also had higher levels of histone acetylation. Although 5-aza-dC at 0.04 microM or higher reduced DNA methylation and histone acetylation levels to those of in vitro-fertilized embryos, development to blastocyst was reduced, suggesting that this concentration of the drug was detrimental. In summary, 5-aza-dC at 0.01 microM altered donor cell characteristics while showing no deleterious effects on embryos cloned from treated cells.