Activation with ionomycin followed by dehydroleucodine and cytochalasin b for the production of parthenogenetic and cloned bovine embryos

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In this work, Dehydroleucodine [DhL] was evaluated as a chemical activator of bovine oocytes and somatic cell nuclear transfer [SCNT] reconstituted embryos. Oocytes were activated with 5 uM Ionomycin [Io] and exposed for 3h to 1 or 5 uM DhL alone [Io-Dhl1 or Io-DhL5] or combined with Cytochalasin B...

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Detalles Bibliográficos
Otros Autores: Canel, Natalia Gabriela, Bevacqua, Romina Jimena, Fernández Martín, Rafael, Salamone, Daniel Felipe
Formato: Artículo
Lenguaje:Inglés
Materias:
CYTOCHALASIN B
DEHYDROLEUCODINE
LONOMYCIN A
SESQUITERPENE LACTONE
UNCLASSIFIED DRUG
ANIMAL CELL
BLASTOCYTE
BLASTOMERE
CONTROLLED STUDY
COW
CUMULUS CELL
DIPLOIDY
DRUG ACTIVITY
EMBRYO DEVELOPMENT
EMBRYO TRANSFER
KARYOTYPE
MOLECULAR CLONING
NONHUMAN
OOCYTE DEVELOPMENT
PARTHENOGENESIS
POLYPLOIDY
SOMATIC CELL
ANIMALS
BLASTOCYST
CATTLE
CELL NUCLEUS
CELLS, CULTURED
CLONING, ORGANISM
EMBRYONIC DEVELOPMENT
FEMALE
FERTILIZATION IN VITRO
IONOMYCIN
IONOPHORES
LACTONES
NUCLEAR TRANSFER TECHNIQUES
OOCYTES
SESQUITERPENES
BOVINAE
Acceso en línea:http://ri.agro.uba.ar/files/intranet/articulo/2010Canel.pdf
LINK AL EDITOR
Aporte de:Registro referencial: Solicitar el recurso aquí
Biblioteca Central (FAUBA) de Universidad de Buenos Aires
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245 1 0 |a Activation with ionomycin followed by dehydroleucodine and cytochalasin b for the production of parthenogenetic and cloned bovine embryos 
520 |a In this work, Dehydroleucodine [DhL] was evaluated as a chemical activator of bovine oocytes and somatic cell nuclear transfer [SCNT] reconstituted embryos. Oocytes were activated with 5 uM Ionomycin [Io] and exposed for 3h to 1 or 5 uM DhL alone [Io-Dhl1 or Io-DhL5] or combined with Cytochalasin B [Io-DhL1/CB; Io-DhL5/CB]. Control groups were Io [Io], Io followed by 1.9mM 6-Dimethylaminopurine [Io-6DMAP], and embryos produced by in vitro fertilization [IVF]. Pronuclear formation and development to blastocysts of activated oocytes were evaluated. Embryos obtained by the DhL concentration that induced the highest blastocyst rates [1 uM] were karyotyped. An additional treatment based in Io-DhL1 plus lengthened [6-h] exposure to CB [Io-DhL1/long CB] was included to improve the proportion of diploid blastomeres. Finally, DhL combined with CB was employed to assist cloning by intracytoplasmic injection of whole cumulus cells. Results showed that DhL induces a pronuclear formation dynamic that was more similar to IVF-produced embryos than DMAP. Development to blastocyst stage was higher after activation with 1 uM DhL than with 5 uM DhL, either for groups combined or not with CB [19.15; 21.74 vs. 6.82; 0 percent, respectively] [p less than 0.05]. Io-DhL1 and Io-DhL1/CB treatments induced blastocyst-cleaved embryo ratios not statistically different from those of Io-DMAP [35.85 percent] and IVF [33.33 percent] groups [p greater than 0.05]. Io-DhL1/long CB induced higher diploid blastomere rates than Io-Dhl1, Io-DhL1/CB and Io-DMAP [63.8 vs. 36.8; 40 and 31.6 percent, respectively] [p less than 0.05]. Moreover, all DhL treatments resulted in polyploidy rates that were lower than Io-DMAP [5.2, 12.0, 10.6, and 31.6 percent, respectively] [p less than 0.05]. Io-DhL1/CB and Io-DhL1/long CB induced cloned embryo blastocyst rates that were not significantly different from Io-DMAP [6.1, 9.4, and 18.3 percent, respectively] [p less than 0.05]. Our results indicate that Io-DhL1/long CB protocol could be useful for SCNT programs. 
653 0 |a CYTOCHALASIN B 
653 0 |a DEHYDROLEUCODINE 
653 0 |a LONOMYCIN A 
653 0 |a SESQUITERPENE LACTONE 
653 0 |a UNCLASSIFIED DRUG 
653 0 |a ANIMAL CELL 
653 0 |a BLASTOCYTE 
653 0 |a BLASTOMERE 
653 0 |a CONTROLLED STUDY 
653 0 |a COW 
653 0 |a CUMULUS CELL 
653 0 |a DIPLOIDY 
653 0 |a DRUG ACTIVITY 
653 0 |a EMBRYO DEVELOPMENT 
653 0 |a EMBRYO TRANSFER 
653 0 |a KARYOTYPE 
653 0 |a MOLECULAR CLONING 
653 0 |a NONHUMAN 
653 0 |a OOCYTE DEVELOPMENT 
653 0 |a PARTHENOGENESIS 
653 0 |a POLYPLOIDY 
653 0 |a SOMATIC CELL 
653 0 |a ANIMALS 
653 0 |a BLASTOCYST 
653 0 |a CATTLE 
653 0 |a CELL NUCLEUS 
653 0 |a CELLS, CULTURED 
653 0 |a CLONING, ORGANISM 
653 0 |a CYTOCHALASIN B 
653 0 |a EMBRYONIC DEVELOPMENT 
653 0 |a FEMALE 
653 0 |a FERTILIZATION IN VITRO 
653 0 |a IONOMYCIN 
653 0 |a IONOPHORES 
653 0 |a LACTONES 
653 0 |a NUCLEAR TRANSFER TECHNIQUES 
653 0 |a OOCYTES 
653 0 |a PARTHENOGENESIS 
653 0 |a SESQUITERPENES 
653 0 |a BOVINAE 
700 1 |9 38039  |a Canel, Natalia Gabriela 
700 1 |9 67357  |a Bevacqua, Romina Jimena 
700 1 |9 33720  |a Fernández Martín, Rafael 
700 1 |9 61021  |a Salamone, Daniel Felipe 
773 |t Cellular Reprogramming  |g Vol.12, no.4 (2010), p.491-499 
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900 |a ^tActivation with ionomycin followed by dehydroleucodine and cytochalasin b for the production of parthenogenetic and cloned bovine embryos 
900 |a ^aCanel^bN. 
900 |a ^aBevacqua^bR. 
900 |a ^aFernández-Martín^bR. 
900 |a ^aSalamone^bD.F. 
900 |a ^aCanel^bN. 
900 |a ^aBevacqua^bR. J. 
900 |a ^aFernández Martín^bR. 
900 |a ^aSalamone^bD. F. 
900 |a ^aCanel^bN.^tLaboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires-CONICET, Av. San Martín 4453, Buenos Aires, 1417, Argentina 
900 |a ^aBevacqua^bR. 
900 |a ^aFernández-Martín^bR. 
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900 |a Vol. 12, no. 4 
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900 |a CYTOCHALASIN B 
900 |a DEHYDROLEUCODINE 
900 |a LONOMYCIN A 
900 |a SESQUITERPENE LACTONE 
900 |a UNCLASSIFIED DRUG 
900 |a ANIMAL CELL 
900 |a BLASTOCYTE 
900 |a BLASTOMERE 
900 |a CONTROLLED STUDY 
900 |a COW 
900 |a CUMULUS CELL 
900 |a DIPLOIDY 
900 |a DRUG ACTIVITY 
900 |a EMBRYO DEVELOPMENT 
900 |a EMBRYO TRANSFER 
900 |a KARYOTYPE 
900 |a MOLECULAR CLONING 
900 |a NONHUMAN 
900 |a OOCYTE DEVELOPMENT 
900 |a PARTHENOGENESIS 
900 |a POLYPLOIDY 
900 |a SOMATIC CELL 
900 |a ANIMALS 
900 |a BLASTOCYST 
900 |a CATTLE 
900 |a CELL NUCLEUS 
900 |a CELLS, CULTURED 
900 |a CLONING, ORGANISM 
900 |a CYTOCHALASIN B 
900 |a EMBRYONIC DEVELOPMENT 
900 |a FEMALE 
900 |a FERTILIZATION IN VITRO 
900 |a IONOMYCIN 
900 |a IONOPHORES 
900 |a LACTONES 
900 |a NUCLEAR TRANSFER TECHNIQUES 
900 |a OOCYTES 
900 |a PARTHENOGENESIS 
900 |a SESQUITERPENES 
900 |a BOVINAE 
900 |a In this work, Dehydroleucodine [DhL] was evaluated as a chemical activator of bovine oocytes and somatic cell nuclear transfer [SCNT] reconstituted embryos. Oocytes were activated with 5 uM Ionomycin [Io] and exposed for 3h to 1 or 5 uM DhL alone [Io-Dhl1 or Io-DhL5] or combined with Cytochalasin B [Io-DhL1/CB; Io-DhL5/CB]. Control groups were Io [Io], Io followed by 1.9mM 6-Dimethylaminopurine [Io-6DMAP], and embryos produced by in vitro fertilization [IVF]. Pronuclear formation and development to blastocysts of activated oocytes were evaluated. Embryos obtained by the DhL concentration that induced the highest blastocyst rates [1 uM] were karyotyped. An additional treatment based in Io-DhL1 plus lengthened [6-h] exposure to CB [Io-DhL1/long CB] was included to improve the proportion of diploid blastomeres. Finally, DhL combined with CB was employed to assist cloning by intracytoplasmic injection of whole cumulus cells. Results showed that DhL induces a pronuclear formation dynamic that was more similar to IVF-produced embryos than DMAP. Development to blastocyst stage was higher after activation with 1 uM DhL than with 5 uM DhL, either for groups combined or not with CB [19.15; 21.74 vs. 6.82; 0 percent, respectively] [p less than 0.05]. Io-DhL1 and Io-DhL1/CB treatments induced blastocyst-cleaved embryo ratios not statistically different from those of Io-DMAP [35.85 percent] and IVF [33.33 percent] groups [p greater than 0.05]. Io-DhL1/long CB induced higher diploid blastomere rates than Io-Dhl1, Io-DhL1/CB and Io-DMAP [63.8 vs. 36.8; 40 and 31.6 percent, respectively] [p less than 0.05]. Moreover, all DhL treatments resulted in polyploidy rates that were lower than Io-DMAP [5.2, 12.0, 10.6, and 31.6 percent, respectively] [p less than 0.05]. Io-DhL1/CB and Io-DhL1/long CB induced cloned embryo blastocyst rates that were not significantly different from Io-DMAP [6.1, 9.4, and 18.3 percent, respectively] [p less than 0.05]. Our results indicate that Io-DhL1/long CB protocol could be useful for SCNT programs. 
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