Biomedical subjects

Da Yuan Chen

Publications and source records attributed to Da Yuan Chen.

6 recordsLinked to original sources

PubMed2003

Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes.

To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and 60 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of the donor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PCR and immunocytochemistry with probes that distinguish between the various species. The ntES cells maintain the capability of sustained growth in an undifferentiated state, and form embryoid bodies, which, on further induction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that express markers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NT to rabbit eggs retain phenotypes similar to those of conventional human ES cells, including the ability to undergo multilineage cellular differentiation.

Adult↗

PubMed2003

Effects of MEK inhibitor U0126 on meiotic progression in mouse oocytes: microtuble organization, asymmetric division and metaphase II arrest.

In this study we used U0126, a potent and specific inhibitor of MEK, to study the roles of MEK/ERK/p90rsk signaling pathway in the meiotic cell cycle of mouse oocytes. The phosphorylation of MAP kinase and p90rsk in the oocytes treated with 1.5 microM U0126 was the same as that in oocytes cultured in drug-free medium. With 1.5 microM U0126 treatment, the spindles appeared normal as they formed in oocytes, but failed to maintain its structure. Instead, the spindle lost one pole or elongated extraordinarily. After further culture, some oocytes extruded gigantic polar bodies (>30 microm) that later divided into two small ones. Some oocytes underwent symmetric division and produced two equal-size daughter cells in which normal spindles formed. In oocytes with different division patterns, MAP kinase was normally phosphorylated. When the concentration of U0126 was increased to 15 mM, the phosphorylation of both MAPK and p90rsk were inhibited, while symmetric division was decreased. When incubating in medium containing 15 microM U0126 for 14 h, oocytes were activated, but part of them failed to emit polar bodies. MII oocytes were also activated by 15 microM U0126, at the same time the dephosphorylation of MAP kinase and p90rsk was observed. Our results indicate that 1) MEK plays important but not indispensable roles in microtubule organization; 2) MEK keeps normal meiotic spindle morphology, targets peripheral spindle positioning and regulates asymmetric division by activating some unknown substrates other than MAP kinase /p90rsk; and 3) activation of MEK/ERK/p90rsk cascade maintains MII arrest in mouse oocytes.

Animals↗

PubMed2003

[Roles of protein kinase C in mouse oocyte maturation and fertilization in vitro].

Protein kinase C is an important family of serine/threorine protein kinase. In this experiment the possible roles of PKC in meiotic maturation, fertilization and egg activation were studied in mouse oocytes. Two isoforms of PKC, PKC alpha and beta 1, were localized in mouse oocytes. PKC activator PMA inhibited the resumption of meiosis in oocytes at GV stage, whose effect could be overcomed by PKC inhibitor calphostin C, neither the phospholipase C inhibitor U73122 nor the PKC delta-specific inhibitor rottlerin. The result of Western bolt indicates that both PKC alpha and beta 1 express stably during oocyte development. The translocation of PKCs and cortical granule exocytosis were observed by confocal microscopy. The results show that PKC alpha translocate from the cytoplasm to the membrane after fertilization or drug-stimulation, accompanied by CG exocytosis. This fact suggested that PKC alpha might be involved in the regulation of cortical reaction in mouse oocytes. Roles of PLC gamma in mouse oocyte during fertilization and localization of PKCs in mouse ovary were also investigated in this experiment. The result suggested that there did not exist a feedback regulation of PLC gamma on PKC during fertilization.

Animals↗

PubMed2003

[Effects of 6-dimethylaminopurine and passages of embryonic stem cells on the development in vitro of interspecific reconstructed oocytes].

The development of reconstructed oocytes and the survival rate of cloned animal were affected by many factors during nuclear transfer. The genetic constitution and the genetic state of donor nucleus were proposed to be primary factors, which affected the survival rate of cloned animal. In addition, the survival rate of cloned animal might be influenced by nuclear transfer technique itself and passages of donor cells as well as the activation methods of oocytes. We reconstructed oocytes with outbreeding Kunming albino mouse ES cells and enucleated rabbit oocytes, and analyzed the effects of the passages of ES cells and 6-DMAP on the development of interspecific reconstructed oocytes. The interspecific reconstructed ES-rabbit oocytes were activated either by combined two set electric pulses and 6-DMAP or by two set electric pulses alone. The rate of cleavage was significantly higher for the group (86.2%) treated with 6-DMAP than the group (64.2%, P < 0.05) treated with electric pulses only, and the rate of blastocysts was 17.0% and 13.4% respectively, which were not significantly different between two groups. When ES cells that had been passed for 24 and 14 generations were used as donors, the cleavage rates of the reconstructed oocytes were 88.5% and 82.1%, respectively (P > 0.05), and the rates of blastolation were 16.7% and 15.4%, respectively (P > 0.05). The results show that 6-DMAP increases the cleavage rate of reconstructed oocytes derived from ES cells, and affects slightly the developmental rate of blastocysts. There are no differences when high passage and low passage ES cells are used as nuclear donors.

Adenine↗

PubMed2002

[Interspecies nuclear transfer using nonquiescent somatic cell].

It is the point at issue in intraspecies nuclear transfer whether quiescence is necessary for development of nuclear transfer reconstructed embryos. In the interspecies nuclear transfer, some reports have proved that quiescent cell is able to support preimplantation development of the interspecies reconstructed embryos. Are non-quiescent cells able to support preimplantation development of the interspecies reconstructed embryos? We used non-quiescent somatic cells from C57BL/6 mice and giant pandas as donors to transfer into enucleated rabbit oocytes. After electrofusion (the electrofusion rates were 62.2% and 71.6%, respectively) and electrical activation, 5.1% of those mouse-rabbit reconstructed embryos developed to blastocyst in vitro, and 4.2% of panda-rabbit reconstructed embryos developed to blastocyst after transferring into ligated rabbit oviduct. These results indicate that non-quiescent cell from C57BL/6 mouse and giant panda could be dedifferentiated in enucleated rabbit oocytes and support early embryo development.

Animals↗

PubMed2002

[Effects of different treatments on oocytes activation and parthenogenesis in mouse].

In order to study effects of electro-fusion and strontium chloride (SrCl2) activation in nuclear transfer experiment on activation and development of mouse oocytes, concentration and treatment duration of SrCl2, electro-pulse and electro-pulse combining SrCl2 were used to activate mouse oocytes which were obtained after hCG 17h. Activated oocytes were cultured in vitro in CZB medium. The results were as follows: 82.4% activation percentage was obtained when the oocytes were treated with 10mmol/L SrCl2 for 6h, it was significantly (P>0.05) higher than those obtained from that treated with the 5mmol/L or 10mmol/L SrCl2 for 4h. The activation percentage was not significantly different between 5mmol/L and 10mmol/L SrCl2 for 6h, but the percentage of morula and blastocyst in 10mmol/L SrCl2 6h group was significantly (P > 0.05) higher than those in 5mmol/L SrCl2 6h group. In the groups of treatment with electro-pulse, the best activation percentage (70.9%) was obtained when the oocytes were treated with 1.0kv/cm, 320micros, 3 pulses, but M + B percentage (7.9%) was low. In the groups of treatment with electro-pulse combining with SrCl2, the best result was acquired (activation and M + B percentage were 75.0% and 57.3% separately) when the oocytes were treated in 10mmol/L SrCl2 for 6h interval 1h after treated with 1.8kv/cm, 10s, 1pulse. These results show that the treatment with electro-pulse combining SrCl2 is a better way to mouse parthenogenesis.

Animals↗