Biomedical subjects

Da-Yuan Chen

Publications and source records attributed to Da-Yuan Chen.

At least 55 records · Page 3Linked to original sources

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.

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PubMed2004

Regulation of ubiquitin-proteasome pathway on pig oocyte meiotic maturation and fertilization.

Degradation of proteins mediated by the ubiquitin-proteasome pathway (UPP) plays essential roles in the eukaryotic cell cycle. The main aim of the present study was to analyze the functional roles and regulatory mechanisms of the UPP in pig oocyte meiotic maturation, activation, and early embryo mitosis by drug treatment, Western blot analysis, and confocal microscopy. By using the hypoxanthine-maintained meiotic arrest model, we showed that the meiotic resumption of both cumulus-enclosed oocytes and denuded oocytes was stimulated in a dose- and time-dependent manner by two potent and cell-permeable proteasome inhibitors. Both the mitogen-activated protein kinase (MAPK) kinase inhibitor U0126 and the maturation-promoting factor inhibitor roscovitine overcame the stimulation of germinal vesicle breakdown induced by proteasome inhibitors. The phosphorylation of MAPK and p90rsk and the expression of cyclin B1 increased in a dose- and time-dependent manner when treated with proteasome inhibitors during oocyte in vitro-maturation culture. Both U0126 and roscovitine inhibited the phosphorylation of MAPK and p90rsk, and the synthesis of cyclin B1 stimulated by proteasome inhibitors. When matured oocytes were pretreated with proteasome inhibitors and then fertilized or artificially activated, the second polar body emission and the pronuclear formation were inhibited, and the dephosphorylation of MAPK and p90rsk as well as the degradation of cyclin B1 that should occur after oocyte activation were also inhibited. We also investigated, to our knowledge for the first time, the subcellular localization of 20S proteasome alpha subunits at different stages of oocyte and early embryo development. The 20S proteasome alpha subunits were accumulated in the germinal vesicle, around the condensed chromosomes at prometaphase, with spindle at metaphase I and II, the region between the separating chromosomes, and especially the midbody at anaphase I and telophase I, the pronucleus, and the nucleus in early embryonic cells. In conclusion, our results suggest that the UPP is important at multiple steps of pig oocyte meiosis, fertilization, and early embryonic mitosis and that it may play its roles by regulating cyclin B1 degradation and MAPK/p90rsk phosphorylation.

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PubMed2004

Mitochondrial DNA heteroplasmy in calves cloned by using adult somatic cell.

Adult somatic cell cloned calves were produced by somatic cell nuclear transfer prepared by fusion of cultured ear fibroblast from a Holstein cow into enucleated oocytes of Luxi Yellow cow. In order to determinate the source of mitochondrial DNA of cloned calves, we designed the breed-specific PCR primers by aligning the known D-loop sequences of Bos taurus and analyzed the displacement loop sequences of five live cloned calves by breed-specific primers PCR. The results demonstrated that mtDNA originated from Holstein breed and that from Luxi breed co-exist in all five live calves.

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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.

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PubMed2004

Ubiquitin-proteasome pathway modulates mouse oocyte meiotic maturation and fertilization via regulation of MAPK cascade and cyclin B1 degradation.

Degradation of proteins mediated by ubiquitin-proteasome pathway (UPP) plays important roles in the regulation of eukaryotic cell cycle. In this study, the functional roles and regulatory mechanisms of UPP in mouse oocyte meiotic maturation, fertilization, and early embryonic cleavage were studied by drug-treatment, Western blot, antibody microinjection, and confocal microscopy. The meiotic resumption of both cumulus-enclosed oocytes and denuded oocytes was stimulated by two potent, reversible, and cell-permeable proteasome inhibitors, ALLN and MG-132. The metaphase I spindle assembly was prevented, and the distribution of ubiquitin, cyclin B1, and polo-like kinase 1 (Plk1) was also distorted. When UPP was inhibited, mitogen-activated protein kinase (MAPK)/p90rsk phosphorylation was not affected, but the cyclin B1 degradation that occurs during normal metaphase-anaphase transition was not observed. During oocyte activation, the emission of second polar body (PB2) and the pronuclear formation were inhibited by ALLN or MG-132. In oocytes microinjected with ubiquitin antibodies, PB2 emission and pronuclear formation were also inhibited after in vitro fertilization. The expression of cyclin B1 and the phosphorylation of MAPK/p90rsk could still be detected in ALLN or MG-132-treated oocytes even at 8 h after parthenogenetic activation or insemination, which may account for the inhibition of PB2 emission and pronuclear formation. We also for the first time investigated the subcellular localization of ubiquitin protein at different stages of oocyte and early embryo development. Ubiquitin protein was accumulated in the germinal vesicle (GV), the region between the separating homologous chromosomes, the midbody, the pronuclei, and the region between the separating sister chromatids. In conclusion, our results suggest that the UPP plays important roles in oocyte meiosis resumption, spindle assembly, polar body emission, and pronuclear formation, probably by regulating cyclin B1 degradation and MAPK/p90rsk phosphorylation.

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PubMed2004

Effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection rabbit embryos.

This study assessed the effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection (ICSI) rabbit embryos. Oocytes were recovered from female rabbits superovulated with PMSG and hCG, and epididymal sperm were collected from a fertile male rabbit. The oocyte was positioned with the first polar body at 12 o'clock position, and a microinjection needle containing a sperm was inserted into the oocyte at 3 o'clock. Oolemma breakage was achieved by aspirating ooplasm, and the aspirated ooplasm and sperm were re-injected into the oocyte. The injected oocytes were cultured in M199 medium containing 10% fetal calf serum at 38 degrees C with 5% CO2 in air. The results showed that oocytes injected at 1 h post-collection produced a higher (p < 0.05) fertilization rate than those injected at 4 or 7 h post-collection. Blastocyst rate in the 1 h group was higher (p < 0.05) than in the 7 h group. Denuded oocytes (group A) and oocytes with cumulus cells (group B) were injected, respectively. Rates of fertilization and development of ICSI embryos were not significantly different (p > 0.05) between the two groups. Four ICSI methods were applied in this experiment. In methods 1 and 2, the needle tip was pushed across half the diameter of the oocyte, and oolemma breakage was achieved by either a single aspiration (method 1) or repeated aspiration and expulsion (method 2) of ooplasm. In methods 3 and 4, the needle tip was pushed to the oocyte periphery opposite the puncture site, and oolemma breakage was achieved by either a single aspiration (method 3) or repeated aspiration and expulsion (method 4) of ooplasm. Fertilization rate in method 2 was significantly higher (p < 0.05) than in methods 1 and 3. Blastocyst rates were not significantly different (p > 0.05) among methods 1, 3 and 4, but method 2 produced a higher (p < 0.05) blastocyst rate than method 3.

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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.

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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.

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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.

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PubMed2004

Development of goat embryos reconstituted with somatic cells: the effect of cell-cycle coordination between transferred nucleus and recipient oocytes.

The developmental ability and the nucleus and microtubule dynamics of nuclear transplanted goat embryos derived from in vitro matured oocytes were studied while controlling cell-cycle coordination of donor embryonic nuclei and recipient cytoplasts. Three groups of transfers were studied: G0/G1 (after the fibroblast cells grew to 100% confluence) and G2/M (nocodazole treated) phase fibroblasts transferred to MII cytoplasts (G0/G1-->MII and G2/M-->MII group, respectively), and G0/G1 phase fibroblasts transferred to preactivated cytoplasts, mostly at S-phase, (G0/G1-->Pre group) by electrical fusion. The results showed that fusion and developmental ability did not differ between G0/G1-->MII and G0/G1-->Pre groups. However the developmental rate of embryos in the G0/G1-->MII group was significantly higher than that of the G2/M-->MII group. Most fibroblast nuclei (G0/G1 and G2/M) transferred into MII oocytes underwent premature chromosome condensation (PCC). Normal spindle were only detected in the G0/G1-->MII group. In contract, fibroblast nuclei in pre-activated oocytes rarely underwent PCC, but formed a swollen nuclear structure. The data suggest that in vitro matured goat oocytes can support the development of somatic fibroblasts after nuclear transfer, G0/G1 -->MII and G0/G1-->S nuclear transfer might be effective ways for improving the developmental competence of the reconstituted embryos, and that G2/M-->MII nuclear transfer by electrical fusion (even in Ca2+-free fusion medium) induces abnormal chromosome ploidy.

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PubMed2004

Localization of gamma-tubulin in mouse eggs during meiotic maturation, fertilization, and early embryonic development.

Gamma-tubulin, a member of the tubulin superfamily, is a peri-centriolar component which is considered to be essential for microtubule nucleation. The dynamics of gamma-tubulin during mouse oocyte meiotic maturation, fertilization, and early cleavage as well as the co-localization of gamma-tubulin and alpha-tubulin during the formation of the meiotic I spindle were studied by confocal microscopy. We found that gamma-tubulin was evenly distributed in the germinal vesicle (GV) stage oocyte. After germinal vesicle breakdown (GVBD) gamma-tubulin dots were localized in both the cytoplasm and the vicinity of the condensed chromosomes, and aligned at both poles of the meiotic spindle at prometaphase I and metaphase I. At anaphase I and telophase I, gamma-tubulin was detected between the separating chromosomes, while it was absent in the midbody. At the MII stage, gamma-tubulin was again accumulated at the spindle poles. Alpha-tubulin had a similar distribution pattern as gamma-tubulin in the cytoplasm and radiated from gamma-tubulin foci close to the chromosomes during the meiotic spindle formation. After fertilization, gamma-tubulin was translocated from spindle poles to the area between separating chromatids and distributed around the pronuclei. It aggregated into some dots during the interphase, but was distributed on the mitotic spindle poles in early embryos. Our results suggest that gamma-tubulin is essential for microtubule nucleation and spindle formation during mouse oocyte meiosis, fertilization, and early embryo cleavage.

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PubMed2004

Quantitative analysis of mitochondrial DNAs in macaque embryos reprogrammed by rabbit oocytes.

In cloned animals where somatic cell nuclei and oocytes are from the same or closely related species, the mitochondrial DNA (mtDNA) of the oocyte is dominantly inherited. However, in nuclear transfer (NT) embryos where nuclear donor and oocyte are from two distantly related species, the distribution of the mtDNA species is not known. Here we determined the levels of macaque and rabbit mtDNAs in macaque embryos reprogrammed by rabbit oocytes. Quantification using a real-time PCR method showed that both macaque and rabbit mtDNAs coexist in NT embryos at all preimplantation stages, with maternal mtDNA being dominant. Single NT embryos at the 1-cell stage immediately after fusion contained 2.6 x 10(4) copies of macaque mtDNA and 1.3 x 10(6) copies of rabbit mtDNA. Copy numbers of both mtDNA species did not change significantly from the 1-cell to the morula stages. In the single blastocyst, however, the number of rabbit mtDNA increased dramatically while macaque mtDNA decreased. The ratio of nuclear donor mtDNA to oocyte mtDNA dropped sharply from 2% at the 1-cell stage to 0.011% at the blastocyst stage. These results suggest that maternal mtDNA replicates after the morula stage.

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PubMed2003

Aurora-A is a critical regulator of microtubule assembly and nuclear activity in mouse oocytes, fertilized eggs, and early embryos.

Aurora-A is a serine/threonine protein kinase that plays a role in cell-cycle regulation. The activity of this kinase has been shown to be required for regulating multiple stages of mitotic progression in somatic cells. In this study, the changes in aurora-;A expression were revealed in mouse oocytes using Western blotting. The subcellular localization of aurora-A during oocyte meiotic maturation, fertilization, and early cleavages as well as after antibody microinjection or microtubule assembly perturbance was studied with confocal microscopy. The quantity of aurora-A protein was high in the germinal vesicle (GV) and metaphase II (MII) oocytes and remained stable during other meiotic maturation stages. Aurora-A concentrated in the GV before meiosis resumption, in the pronuclei of fertilized eggs, and in the nuclei of early embryo blastomeres. Aurora-A was localized to the spindle poles of the meiotic spindle from the metaphase I (MI) stage to metaphase II stage. During early embryo development, aurora-A was found in association with the mitotic spindle poles. Aurora-A was not found in the spindle region when colchicine or staurosporine was used to inhibit microtubule organization, while it accumulated as several dots in the cytoplasm after taxol treatment. Aurora-A antibody microinjection decreased the rate of germinal vesicle breakdown (GVBD) and distorted MI spindle organization. Our results indicate that aurora-A is a critical regulator of cell-cycle progression and microtubule organization during mouse oocyte meiotic maturation, fertilization, and early embryo cleavage.

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PubMed2003

Protein kinase C and mitogen-activated protein kinase cascade in mouse cumulus cells: cross talk and effect on meiotic resumption of oocyte.

Protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in cumulus cells are involved in FSH-induced meiotic resumption of cumulus-enclosed oocytes (CEOs), but their regulation and cross talk are unknown. The present experiments were designed to investigate 1) the possible involvement of MAPK cascade in PKC-induced meiotic resumption; 2) the regulation of PKC on MAPK activity in FSH-induced oocyte maturation; and 3) the pattern of PKC and MAPK function in induced meiotic resumption of mouse oocytes. PKC activators, phorbol 12-myristate 13-acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol (OAG), induced the meiotic resumption of CEOs and activation of MAPK in cumulus cells, whereas this effect could be abolished by PKC inhibitors, calphostin C and chelerythrine, or MEK inhibitor U0126. These results suggest that PKC might induce the meiotic reinitiation of CEOs by activating MAPK in cumulus cells. Both PKC inhibitors and U0126 inhibited the FSH-induced germinal vesicle breakdown (GVBD) of oocytes and MAPK activation in cumulus cells, suggesting that PKC and MAPK are involved in FSH-induced GVBD of mouse CEOs. Protein synthesis inhibitor cycloheximide (CHX) inhibited FSH- or PMA-induced oocyte meiotic resumption, but not the MAPK activation in cumulus cells. FSH and PKC activators induced the GVBD in denuded oocytes cocultured with cumulus cells in hypoxanthine (HX)-supplemented medium, and this effect could be reversed by U0126. Thus, when activated by FSH and PKC, MAPK may stimulate the synthesis of specific proteins in cumulus cells followed by secretion of an unknown positive factor that is capable of inducing GVBD in oocytes.

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PubMed2003

Involvement of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes.

Calcium signal is important for the regulation of meiotic cell cycle in oocytes, but its downstream mechanism is not well known. The functional roles of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes were studied by drug treatment, Western blot analysis, kinase activity assay, indirect immunostaining, and confocal microscopy. The results indicated that meiotic resumption of both cumulus-enclosed and denuded oocytes was prevented by CaMKII inhibitor KN-93, Ant-AIP-II, or CaM antagonist W7 in a dose-dependent manner, but only germinal vesicle breakdown (GVBD) of denuded oocytes was inhibited by membrane permeable Ca2+ chelator BAPTA-AM. When the oocytes were treated with KN-93, W7, or BAPTA-AM after GVBD, the first polar body emission was inhibited. A quick elevation of CaMKII activity was detected after electrical activation of mature pig oocytes, which could be prevented by the pretreatment of CaMKII inhibitors. Treatment of oocytes with KN-93 or W7 resulted in the inhibition of pronuclear formation. The possible regulation of CaMKII on maturation promoting factor (MPF), mitogen-activated protein kinase (MAPK), and ribosome S6 protein kinase (p90rsk) during meiotic cell cycles of pig oocytes was also studied. KN-93 and W7 prevented the accumulation of cyclin B and the full phosphorylation of MAPK and p90rsk during meiotic maturation. When CaMKII activity was inhibited during parthenogenetic activation, cyclin B, the regulatory subunit of MPF, failed to be degraded, but MAPK and p90rsk were quickly dephosphorylated and degraded. Confocal microscopy revealed that CaM and CaMKII were localized to the nucleus and the periphery of the GV stage oocytes. Both proteins were concentrated to the condensed chromosomes after GVBD. In oocytes at the meiotic metaphase MI or MII stage, CaM distributed on the whole spindle, but CaMKII was localized only on the spindle poles. After transition into anaphase, both proteins were translocated to the area between separating chromosomes. All these results suggest that CaMKII is a multifunctional regulator of meiotic cell cycle and spindle assembly and that it may exert its effect via regulation of MPF and MAPK/p90rsk activity during the meiotic maturation and activation of pig oocytes.

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PubMed2003

Characterization of Polo-like kinase-1 in rat oocytes and early embryos implies its functional roles in the regulation of meiotic maturation, fertilization, and cleavage.

Polo-like kinase 1 (Plk1) is a family of serine/threonine protein kinases that play important regulatory roles during mitotic cell cycle progression. In this study, Plk1 expression, subcellular localization, and possible functions during rat oocyte meiotic maturation, fertilization, and embryonic cleavages were studied by using RT-PCR, Western blot, confocal microscopy, drug-treatments, and antibody microinjection. Both the mRNA and protein of this kinase were detected in rat maturing oocytes and developing embryos. Confocal microscopy revealed that Plk1 distributed abundantly in the nucleus at the germinal vesicle (GV) stage, was associated with spindle poles during the formation of M-phase spindle, and was translocated to the spindle mid-zone at anaphase. In fertilized eggs, Plk1 was strongly stained in the cytoplasm between the apposing male and female pronuclei, from where microtubules radiated. Throughout cytokinesis, Plk1 was localized to the division plane, both during oocyte meiosis and embryonic mitosis. The specific subcellular distribution of Plk1 was distorted after disrupting the M-phase spindle, while additional aggregation dots could be induced in the cytoplasm by taxol, suggesting its intimate association with active microtubule assembly. Plk1 antibody microinjection delayed the meiotic resumption and blocked the emission of polar bodies. In conclusion, Plk1 may be a multifunctional kinase that plays pivotal regulatory roles in microtubule assembly during rat oocyte meiotic maturation, fertilization, and early embryonic mitosis.

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PubMed2003

Asynchronous cytoplast and karyoplast transplantation reveals that the cytoplasm determines the developmental fate of the nucleus in mouse oocytes.

The relationship between nucleus and cytoplasm can be well revealed by nuclear transplantation. Here, we have investigated the behavior changes of the reconstructed oocytes after transferring the karyoplasts from mouse GV, MI, and MII oocytes into the cytoplasts at the different developmental stages. When the GV cytoplast was used as recipient and MI or MII karyoplast was used as donor (MI-GV pair and MII-GV pair), the reconstructed pairs extruded a polar body after electrofusion and culture. Both the cytoplasm and the polar body had a metaphase spindle in the MI-GV pair, while only a clutch of condensed chromatin was observed in the cytoplasm and polar body of the MII-GV pair. When the MI cytoplast was used as recipient and GV or MII karyoplast was used as donor (GV-MI pair and MII-MI pair), the reconstructed pairs also extruded a polar body. Each had one spindle and a group of metaphase chromosomes in the cytoplasm and polar body, respectively. When the MII cytoplast was used as recipient and GV or MI karyoplast was used as donor (GV-MII pair and MI-MII pair), the reconstructed pairs were activated, became parthenogenetic embryos and even developed to hatching blastocysts after electrofusion. The result from immunoblotting showed that MAP kinase activity was high in the MI and MII cytoplasts, while not detected in GV cytoplast. The results demonstrate that the cytoplasmic environment determines the behavior of asynchronous donors.

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PubMed2003

In vitro development and mitochondrial fate of macaca-rabbit cloned embryos.

Interspecies cloning may be used as an effective method to conserve highly endangered species and to support the development of non-human primate animal models for studying therapeutic cloning and nuclear-cytoplasm interaction. The use of the monkey model for biomedical research can avoid legal, ethical, and experimental limitations encountered in a clinical situation. We describe in this study the in vitro development of macaca-rabbit embryos produced by fusing macaca fibroblasts with enucleated rabbit oocytes and examine the fate of mitochondrial DNA in these embryos. We show that macaca-rabbit cloned embryos can develop to the blastocyst stage when cultured in vitro in HECM(10) +10% FBS and that mitochondrial DNA derived from donor somatic cells was detectable in cloned embryos throughout preimplantation development. These results suggest that (1) macaca fibroblast nuclei can dedifferentiate in enucleated metaphase II rabbit oocytes; (2) HECM(10) +10% FBS can break through the development block and support the development of macaca-rabbit cloned embryos to blastocysts; and (3) donor-cell-derived mitochondrial DNA is not eliminated until blastocyst stage.

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