title: The effect of activation of Mammalian oocytes on remodeling of donor nuclei after nuclear transfer.
abstract: Activation of bovine oocytes by experimental procedures that closely mimic normal fertilization is essential both for intracytoplasmic sperm injection and for nuclear transfer (NT). Therefore, with the goal of producing haploid activated oocytes, we evaluated whether butyrolactone I and bohemine, either alone or in combination with ionomycin, are able to activate young matured mammalian oocytes. Furthermore, the effect on the patterns of DNA synthesis after pronuclear formation as well as changes in histone H1 kinase and MAP kinase activities during the process of activation were studied. Our results with bohemine show that the specific inhibition of cyclin-dependent kinases (CDKs) in metaphase II bovine oocytes induces parthenogenetic activation in a dose dependent manner (25, 50, and 100 microM, respectively), either alone (3%, 30%, and 50%) or in combination with ionomycin (30%, 70%, and 87.5%). The effect of two activation protocols on nuclear remodeling, DNA synthesis during the first cell cycle, chromosome segregation after first mitosis, and development to blastocyst of embryos produced by somatic nuclear transfer were studied. Pronuclear formation was significantly higher when activation lasted 5 h compared to 3 h for both ethanol-cycloheximide and ionomycin-bohemine treatment. Initiation of DNA synthesis was delayed in ethanol-cycloheximide group, however, after 12-h labeling 100% of embryos synthesized DNA in both groups. Analysis of two-cell embryos with DNA probes for chromosome 6, 7, and 15 by fluorescence in situ hybridization showed that at least 50% of NT embryos were of normal ploidy, independent of the activation protocol.
authors.affiliation: Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Libechov, Czech Republic. motlik@iapg.cas.cz
authors.forename: Jan
authors.initials: J
authors.lastname: Motlik
authors.forename: Ramiro
authors.initials: R
authors.lastname: Alberio
authors.forename: Valeri
authors.initials: V
authors.lastname: Zakhartchenko
authors.forename: Miodrag
authors.initials: M
authors.lastname: Stojkovic
authors.forename: Michal
authors.initials: M
authors.lastname: Kubelka
authors.forename: Eckhard
authors.initials: E
authors.lastname: Wolf
mesh_terms: D000818:Animals; D002417:Cattle; D002467:Cell Nucleus / Q000502:physiology* / Q000648:ultrastructure*; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004261:DNA Replication / Q000502:physiology*; D005260:Female; D005306:Fertilization; D006107:Granulosa Cells / Q000166:cytology / Q000502:physiology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D004676:Myelin Basic Protein / Q000378:metabolism; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology / Q000502:physiology*; D011494:Protein Kinases / Q000378:metabolism; D020554:Sperm Injections, Intracytoplasmic
chemical_list: D004676:Myelin Basic Protein; D011494:Protein Kinases; C102333:histone H1 kinase; D018844:Cyclin-Dependent Kinases; D020928:Mitogen-Activated Protein Kinases