RESUMEN
The developmental potential of hybrid embryos produced by transferring panda or cat fibroblasts into nucleated rabbit oocytes was assessed. Both the panda-rabbit and the cat-rabbit hybrid embryos were able to form blastocysts in vitro. However, the rates of attaining the two-cell, four-cell, eight-cell, morula, or blastocyst stages for panda-rabbit hybrids were significantly greater than those of cat-rabbit hybrids (P<0.05). Transferring the rabbit fibroblasts into nucleated rabbit oocytes, 31.0% of the blastocyst rate was obtained, which was significantly higher than that of both the panda-rabbit and the cat-rabbit hybrid embryos (P<0.05). Whether or not the second polar body (PB2) was extruded from the one-cell hybrid embryos (both panda-rabbit and cat-rabbit hybrids) significantly affected their developmental capacity. Embryos without an extruded PB2 showed a higher capacity to develop into blastocysts (panda-rabbit: 19.2%; cat-rabbit: 4.3%), while embryos with extruded PB2 could only develop to the morula stage. The hybrid embryos formed pronucleus-like structures (PN) in 2-4 hr after activation, and the number of PN in one-cell embryos varied from one to five. Tracking of the nucleus in the egg after fusion revealed that the somatic nucleus could approach and aggregate with the oocyte nucleus spontaneously. Chromosome analysis of the panda-rabbit blastocysts showed that the karyotype of the hybrid embryos (2n=86) consisted of chromosomes from both the panda (2n=42) and the rabbit (2n=44). The results demonstrate that (1) it is possible to produce genetic hybrid embryos by interspecies nuclear transfer; (2) the developmental potential of the hybrid embryos is highly correlated to the donor nucleus species; and (3) the hybrid genome is able to support the complete preimplantation embryonic development of the hybrids.
Asunto(s)
Gatos , Fibroblastos/fisiología , Hibridación Genética , Técnicas de Transferencia Nuclear , Oocitos/fisiología , Conejos , Ursidae , Animales , Células Cultivadas , Desarrollo Embrionario/genética , Desarrollo Embrionario/fisiología , Femenino , Fertilización In Vitro/veterinaria , CariotipificaciónRESUMEN
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.
Asunto(s)
Blastocisto/metabolismo , Blastodermo/metabolismo , Técnicas de Transferencia Nuclear , Oocitos/metabolismo , Animales , Núcleo Celular/genética , Embrión de Pollo , Citocromos b/genética , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Reacción en Cadena de la Polimerasa , ConejosRESUMEN
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.
Asunto(s)
Blastocisto/fisiología , Clonación de Organismos/métodos , ADN Mitocondrial/análisis , Embrión de Mamíferos/fisiología , Macaca mulatta , Conejos , Animales , Blastocisto/citología , Células Cultivadas , Cromosomas de los Mamíferos , Técnicas de Cultivo , Desarrollo Embrionario y Fetal , Femenino , Fibroblastos/fisiología , Macaca mulatta/embriología , Macaca mulatta/genética , Técnicas de Transferencia Nuclear , Oocitos/fisiología , Conejos/embriología , Conejos/genéticaRESUMEN
Interspecies nuclear transfer is an invaluable tool for studying nucleus-cytoplasm interactions; and at the same time, it provides a possible alternative to clone animals whose oocytes are difficult to obtain. In the present study, we investigated the possibility of cloning cat embryos using rabbit oocytes, and compared the developmental capacity; the timing of embryogenesis of the cat-rabbit cloned embryos with that of the cat-cat or the rabbit-rabbit cloned embryos. When cultured in M199, the rate of blastocyst formation of the cat-rabbit embryos was 6.9%, which was not significantly different than that of the cat-cat embryos (10.5%). However, the rate of blastocyst formation of rabbit-rabbit embryos (22.9%) was significantly greater than that of both the cat-rabbit and the cat-cat embryos (P < 0.05). The timing of the first three cleavages for the cat-rabbit embryos was similar to that of the rabbit-rabbit embryos, but significantly faster than that of the cat-cat embryos (P < 0.05), while the time to form blastocysts was similar to that of cat-cat embryos, but significantly slower than that of the rabbit-rabbit embryos (P < 0.05). Both M199 and SOF medium were evaluated for culturing cat-rabbit embryos; the rate of blastocyst formation in SOF (14.5%) was significantly greater than that in M199 (6.9%) (P < 0.05). These results demonstrate that: (1) the cat-rabbit embryos possess equal developmental capacity as cat-cat embryos; (2) the timing of the first three cleavages for the cat-rabbit embryos is recipient-specific, while the time to form blastocysts is donor nucleus-specific; and (3) SOF medium may be beneficial to overcome the morula-to-blastocyst block for cat-rabbit cloned embryos.
Asunto(s)
Clonación de Organismos , Técnicas de Transferencia Nuclear , Oocitos , Animales , Gatos , Transferencia de Embrión/veterinaria , Cariotipificación , Conejos , Factores de TiempoRESUMEN
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.
Asunto(s)
Núcleo Celular/fisiología , Citoplasma/trasplante , Oocitos/citología , Oocitos/fisiología , Huso Acromático/trasplante , Animales , Citoplasma/fisiología , Femenino , Técnica del Anticuerpo Fluorescente , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismoRESUMEN
Abnormal oocyte spindle is frequently associated with the infertility of aged women. Directly manipulating the metaphase I (MI) spindle may be a feasible method to overcome this kind of problem. Here, we report that the MI meiotic spindle can be removed from MI mouse oocytes and will autonomously divide into two daughter cells with the same size, morphology and an equal number of chromosomes after culture for 5 h in maturation medium. The division rate of the MI spindle reached 56% after 10-15 h of culture. After transferring the MI meiotic spindle into synchronous ooplasm by electrofusion, about 61% of the reconstructed oocytes continued to complete the first meiosis and extruded a normal first polar body. The matured reconstructed oocytes can also be fertilised. Approximately 50% of the 2-cell embryos developed to the morula stage after in vitro culture.
Asunto(s)
Embrión de Mamíferos/fisiología , Metafase/fisiología , Oocitos/fisiología , Huso Acromático/trasplante , Animales , Femenino , Meiosis/fisiología , Ratones , Ratones Endogámicos C57BLRESUMEN
Somatic cell nuclei of giant pandas can dedifferentiate in enucleated rabbit ooplasm, and the reconstructed eggs can develop to blastocysts. In order to observe whether these interspecies cloned embryos can implant in the uterus of an animal other than the panda, we transferred approximately 2300 panda-rabbit cloned embryos into 100 synchronized rabbit recipients, and none became pregnant. In another approach, we cotransferred both panda-rabbit and cat-rabbit interspecies cloned embryos into the oviducts of 21 cat recipients. Fourteen recipients exhibited estrus within 35 days; five recipients exhibited estrus 43-48 days after embryo transfer; and the other two recipients died of pneumonia, one of which was found to be pregnant with six early fetuses when an autopsy was performed. Microsatellite DNA analysis of these early fetuses confirmed that two were from giant panda-rabbit cloned embryos. The results demonstrated that panda-rabbit cloned embryos can implant in the uterus of a third species, the domestic cat. By using mitochondrial-specific probes of panda and rabbit, we found that mitochondria from both panda somatic cells and rabbit ooplasm coexisted in early blastocysts, but mitochondria from rabbit ooplasm decreased, and those from panda donor cells dominated in early fetuses after implantation. Our results reveal that mitochondria from donor cells may substitute those from recipient oocytes in postimplanted, interspecies cloned embryos.
Asunto(s)
Clonación de Organismos/métodos , Implantación del Embrión/fisiología , Mitocondrias/metabolismo , Ursidae/fisiología , Músculos Abdominales/citología , Músculos Abdominales/ultraestructura , Animales , Secuencia de Bases , Gatos , Núcleo Celular/ultraestructura , Células Cultivadas , ADN Mitocondrial/metabolismo , Transferencia de Embrión , Desarrollo Embrionario y Fetal/fisiología , Femenino , Repeticiones de Microsatélite , Datos de Secuencia Molecular , Embarazo , ConejosRESUMEN
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.