Biomedical subjects

Shu-Zhen Liu

Publications and source records attributed to Shu-Zhen Liu.

11 recordsLinked to original sources

PubMed2006

Different fates of donor mitochondrial DNA in bovine-rabbit and cloned bovine-rabbit reconstructed embryos during preimplantation development.

The functions of mitochondria depend on precise interaction between nuclear and cytoplasmic genomes. Non-balance of mtDNA has been reported in most nuclear transfer embryos and offspring. The reason of the degradation of donor mtDNA is still not clear. To further investigate the mechanism, in this study, we designed an experiment as follows. Two fibroblast cell lines sharing same nuclear genome but different mitochondria genome backgrounds, namely cells from ear tissues of cloned bovine and its donor, were choose as donor cells and introduced into enucleated rabbit oocytes. Similar developmental potential was observed in cloned bovine-rabbit (clone group) and bovine-rabbit (non-clone group) embryos. Real-time PCR assay showed that, in non-clone group, bovine mtDNA decreased during the development of reconstructed embryo, and that a sharp decrease was detected at the blastocyst stage. In clone group, bovine mtDNA decreased slightly, and the abrupt reduction of donor mtDNA did not occur during preimplantation development. In addition, an obvious increase in rabbit mtDNA was observed in both groups at the blastocyst stage. Our results demonstrate that: 1) the fates of donor mtDNAs in bovine-rabbit and cloned bovine-rabbit reconstructed embryos were different; and 2) recipient mtDNAs replicate at blastocyst regardless of the difference of donor cells.

Active Transport, Cell Nucleus↗

PubMed2006

Dynamic changes in NuMA and microtubules in monkey-rabbit nuclear transfer embryos.

Previous reports have indicated that failure in cloning monkey is attributed to the removal of nuclear mitotic apparatus (NuMA) during enucleation and subsequent abnormal organization of mitotic apparatus. This study investigated the transformation and assembly of tubulin and NuMA protein during the first cell cycle of cloned monkey embryos reconstructed by using enucleated rabbit oocytes as recipients. After the oocyte fused with a fibroblast, extensive microtubule organization was observed around the introduced nucleus in most reconstructed embryos, suggesting the introduction of a somatic cell centrosome. A high proportion of fibroblast nuclei transferred into non-activated oocytes underwent premature chromosome condensation (PCC), transient spindle organization and chromosomes separation, followed by the formation of two pronucleus-like structures. In contrast, fibroblast nuclei in pre-activated ooplasm rarely underwent PCC, but formed a swollen pronucleus-like structure. Normal spindles were observed in about one third of the cloned embryos reconstructed by both methods. After transferring monkey fibroblasts into NuMA-removed enucleated rabbit oocytes, NuMA was localized in pseudo-pronuclei and gradually moved to mitotic spindle poles at the first mitotic spindle poles. NuMA antibody microinjection resulted in spindle disorganization and chromosome misalignment, but did not significantly affect early cleavage. Our findings indicate that: 1. NuMA in donor monkey fibroblast may contribute to form a normal spindle in enucleated rabbit oocyte; 2. when non-activated cytoplasts and pre-activated cytoplasts are used as recipients, the donor nuclei undergo different morphological changes, but yield similar early embryo development; 3. although abnormal spindle organization and chromosome alignment may cause low efficiency of animal cloning, these abnormalities do not significantly affect early cleavage.

Animals↗

PubMed2005

Parthenogenetic and nuclear transfer rabbit embryo development and apoptosis after activation treatments.

Previous studies mainly evaluated the effect of culture conditions on preimplantation embryo apoptosis. In order to inhibit apoptosis of nuclear transfer (NT) embryos, putative apoptosis inhibitors were used to treat donor cells. However, little is known about the effect of activation treatments on embryo apoptosis. We firstly investigated the effect of various parthenogenetic activation (PA) treatments on embryo development, blastocyst cell number, and apoptosis, and then one of these activation treatments proved to be most efficient was selected for activation rabbit NT embryos. The activation by electrical pulses and 30 min later, electroporation with 25 muM D-myoinositol 1,4,5-trisphosphate (IP3) in Ca(2+)- and Mg(2+)-free PBS, then exposure to 2.0 mM 6-dimethylaminopurine (6-DMAP) for 3 hr effectively activated rabbit oocytes, and resulted in significantly a higher blastocyst development rate (72.7%) and total cell number (175 +/- 14.1), and markedly lower apoptosis level of blastocyst (4.3 +/- 0.5) than all the other groups. When the same activation protocol was applied in NT embryo activation, we found that exposure of the embryos to 6-DMAP for 3 hr could decrease the apoptosis level of blastocyst and increase blastocyst rate and cell number. The results demonstrate that oocyte activation affects not only embryo development and quality but also embryo apoptosis.

Adenine↗

PubMed2005

Active demethylation of individual genes in intracytoplasmic sperm injection rabbit embryos.

Intracytoplasmic sperm injection (ICSI), as an assisted reproduction technique, has been widely used in animal and human. However, its possible effect on epigenetic changes has not been well studied. To investigate whether ICSI can induce aberrant DNA methylation changes in rabbit preimplantation embryos, we examined the methylation status of the SP-A promoter region and the satellite sequence Rsat IIE by bisulfite-sequencing technology. The SP-A promoter region was extensively demethylated before the first round of DNA replication commences, and the unmethylated status was maintained until morula when dynamic remethylation occurred. A similar but more moderate demethylation process was observed in satellite sequence Rsat IIE. These results are in contrast with the previous reports of no active demethylation in normal rabbit embryos, suggesting that the active demethylation we observed may be induced by ICSI.

Animals↗

PubMed2005

Effects of oocyte collection time and injection position on pronucleus formation and blastocyst development in round spermatid injection in mouse.

The injection of spermatozoa into mouse, human and rabbit oocytes at specific times and positions can result in different rates of viable embryo development. However, it is not clear how the timing and position of round spermatid injection (ROSI) affect pronucleus (PN) formation and blastocyst development of mice. First, we determined the changes in relative position of the first polar body and the spindle, carried out ROSI from 11.5 to 13 h post-hCG administration, then activated by Sr2+, and finally compared the development of ROSI zygotes, including the formation of pronuclei and development of blastocyst. Between 11.5 and 13 h post-hCG administration, the rate of 2PN formation by ROSI at 3 o'clock was the highest among all treated oocytes. Moreover, the blastocyst rate of zygotes with two pronuclei (2PN) was up to 27.41%. These results suggest that the time and position of ROSI can significantly influence the formation of 2PN, that the rates of 2PN formation are closely correlated with blastocyst formation and that the formation of 2PN is necessary for later embryo development.

Animals↗

PubMed2005

Apoptosis in rabbit embryos produced by fertilization or nuclear transfer with fibroblasts and cumulus cells.

In this study, we investigated the development, the cell number of the blastocyst, and apoptosis in rabbit nuclear transfer (NT) embryos derived from adult fibroblasts and cumulus cells as compared with embryos derived from in vivo fertilization and in vitro culture. The developmental rate and the total cell number of the blastocyst were significantly lower in NT embryos than in fertilized embryos (FEs). The type of donor cells did not affect the embryonic developmental rate and the total cell number of blastocysts in NT groups. The present study investigated the onset and the frequency of apoptosis in NT embryos and FEs by using a terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling (TUNEL) assay. The earliest positive TUNEL signals were detected at the eight-cell stage in NT embryos and at the morula stage in FEs. The apoptotic index of the total blastocysts, the inner cell mass and the trophoderm was greatly higher in the NT embryos than in FEs. Moreover, the apoptotic index of the blastocyst from fibroblasts was significantly higher than that of the blastocyst from cumulus cells.

Animals↗

PubMed2004

The DNA methylation events in normal and cloned rabbit embryos.

To study the DNA methylation events in normal and cloned rabbit embryos, we investigated the methylation status of a satellite sequence and the promoter region of a single-copy gene using bisulfite-sequencing technology. During normal rabbit embryo development, both sequences maintained hypermethylation status until the 8- to 16-cell stage when progressive demethylation took place. In cloned embryos, the single-copy gene promoter sequence was rapidly demethylated and precociously de novo methylated, while the satellite sequence maintained the donor-type methylation status in all examined stages. Our results indicate that unique sequences as well as satellite sequences may have aberrant methylation patterns in cloned embryos.

Animals↗

PubMed2004

Blastocysts produced by nuclear transfer between chicken blastodermal cells and rabbit oocytes.

Interspecies nuclear transfer (INT) has been used as an invaluable tool for studying nucleus-cytoplasm interactions; and it may also be a method for rescuing endangered species whose oocytes are difficult to obtain. In the present study, we investigated interaction of the chicken genome with the rabbit oocyte cytoplasm. When chicken blastodermal cells were transferred into the perivitelline space of rabbit oocytes, 79.3% of the couplets were fused and 9.7% of the fused embryos developed to the blastocyst stage. Both M199 and SOF medium were used for culturing chicken-rabbit cloned embryos; embryo development was arrested at the 8-cell stage obtained in SOF medium, while the rates of morulae and blastocysts were 12.1 and 9.7%, respectively, in M199 medium. Polymerase chain reaction (PCR) amplification of nuclear DNA and karyotype analyses confirmed that genetic material of morulae and blastocysts was derived from the chicken donor cells. Analysis mitochondrial constitution of the chicken-rabbit cloned embryos found that mitochondria, from both donor cells and enucleated oocytes, co-existed. Our results suggest that: (1) chicken genome can coordinate with rabbit oocyte cytoplasm in early embryo development; (2) there may be an 8- to 16-cell stage block for the chicken-rabbit cloned embryos when cultured in vitro; (3) mitochondrial DNA from the chicken donor cells was not eliminated until the blastocyst stage in the chicken-rabbit cloned embryos; (4) factors existing in ooplasm for somatic nucleus reprogramming may be highly conservative.

Animals↗

PubMed2004

Somatic nucleus remodelling in immature and mature Rassir oocyte cytoplasm.

Successful production of cloned animals derived from somatic cells has been achieved in sheep, cattle, goats, mice, pigs, rabbits, etc. But the efficiency of nuclear transfer is very low in all species. The present study was conducted to examine somatic nucleus remodelling and developmental ability in vitro of rabbit embryos by transferring somatic cells into enucleated germinal vesicle (GV), metaphase I (MI) or metaphase II (MII) oocytes. Microtubules were organized around condensed chromosomes after the nucleus had been transferred into any of the three types of cytoplasm. A bipolar spindle was formed in enucleated MII cytoplasm. Most of the nuclei failed to form a normal spindle within GV and MI cytoplasm. Some chromosomes scattered throughout the cytoplasm and some formed a monopolar spindle. Pseudopronucleus formation was observed in all three types of cytoplasm. Reconstructed embryos with MI and MII cytoplasm could develop to blastcysts. Nuclei in GV cytoplasm could develop only to the 4-cell stage. These results suggest that (1) GV material is important for nucleus remodelling after nuclear transfer, and (2) oocyte cytoplasm has the capacity to dedifferentiate somatic cells during oocyte maturation.

Animals↗

PubMed2004

The effects of chemical enucleation combined with whole cell intracytoplasmic injection on panda-rabbit interspecies nuclear transfer.

Conventional methods of somatic cell nuclear transfer either by electrofusion or direct nucleus injection have very low efficiency in animal cloning, especially interspecies cloning. To increase the efficiency of interspecies somatic cell nuclear transfer, in the present study we introduced a method of whole cell intracytoplasmic injection (WCICI) combined with chemical enucleation into panda-rabbit nuclear transfer and assessed the effects of this method on the enucleation rate of rabbit oocytes and the in vitro development and spindle structures of giant panda-rabbit reconstructed embryos. Our results demonstrated that chemical enucleation can be used in rabbit oocytes and the optimal enucleation result can be obtained. When we compared the rates of cleavage and blastocyst formation of subzonal injection (SUZI) and WCICI using chemically enucleated rabbit oocytes as cytoplasm recipients, the rates in the WCICI group were higher than those in the SUZI group, but there was no statistically siginificant difference (p > 0.05) between the two methods. The microtubule structures of rabbit oocytes enucleated by chemicals and giant panda-rabbit embryos reconstructed by WCICI combined with chemical enucleation were normal. Therefore the present study suggests that WCICI combined with chemical enucleation can provide an efficient and less labor-intensive protocol of interspecies somatic cell nuclear transfer for producing giant panda cloned embryos.

Animals↗

PubMed2004

The fate of mitochondria in Ibex-hirus reconstructed early embryos.

Inter-species nuclear transfer could be used to preserve North Goat (Capra ibex), an endangered species. We established the culture conditions for ibex-hirus reconstructed embryos and optimized the method for DNA extractions of a single cell and early cloned embryo. By using mitochondria-specific probes of ibex and hirus respectively we found that mitochondria of donor cells can co-exist with recipients in 1-cell and 2-cell stages of the reconstructed embryos but not in the following developmental stages.

Animals↗