title: Effect of the enucleation procedure on the reprogramming potential and developmental capacity of mouse cloned embryos treated with valproic acid.
abstract: Mouse recipient cytoplasts for somatic cell nuclear transfer (SCNT) are routinely prepared by mechanical enucleation (ME), an invasive procedure that requires expensive equipment and considerable micromanipulation skills. Alternatively, oocytes can be enucleated using chemically assisted (AE) or chemically induced (IE) enucleation methods that are technically simple. In this study, we compared the reprogramming potential and developmental capacity of cloned embryos generated by ME, AE, and IE procedures and treated with the histone deacetylase inhibitor valproic acid. A rapid and almost complete deacetylation of histone H3 lysine 14 in the somatic nucleus followed by an equally rapid and complete re-acetylation after activation was observed after the injection of a cumulus cell nucleus into ME and AE cytoplasts. In contrast, histone deacetylation occurred at a much lower level in IE cytoplasts. Despite these differences, the cloned embryos generated from the three types of cytoplasts developed into blastocysts of equivalent total and inner cell mass mean cell numbers, and the rates of blastocyst formation and embryonic stem cell derivation were similar among the three groups. The cloned embryos produced from ME and AE cytoplasts showed an equivalent rate of full-term development, but no offspring could be obtained from the IE group, suggesting a lower reprogramming capacity of IE cytoplasts. Our results demonstrate the usefulness of AE in mouse SCNT procedures, as an alternative to ME. AE can facilitate oocyte enucleation and avoid the need for expensive microscope optics, or for potentially damaging Hoechst staining and u.v. irradiation, normally required in ME procedures.
authors: Departament de Biologia Cel.lular, Fisiologia i Immunologia, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.
authors: Nuno
authors: N
authors: Costa-Borges
authors: Sheyla
authors: S
authors: Gonzalez
authors: Josep
authors: J
authors: Santaló
authors: Elena
authors: E
authors: Ibáñez
mesh_terms: D000107:Acetylation; D000704:Analysis of Variance; D000818:Animals; D001724:Birth Weight; D001755:Blastocyst / Q000187:drug effects / Q000378:metabolism; D002460:Cell Line; D065150:Cellular Reprogramming / Q000187:drug effects*; D016009:Chi-Square Distribution; D042002:Chromatin Assembly and Disassembly / Q000187:drug effects; D002999:Clone Cells; D046149:Embryo Culture Techniques; D010064:Embryo Implantation; D004624:Embryo Transfer; D004622:Embryo, Mammalian / Q000187:drug effects* / Q000378:metabolism; D047108:Embryonic Development; D005260:Female; D005313:Fetal Death; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D005865:Gestational Age; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D006657:Histones / Q000378:metabolism; D050498:Live Birth; D008239:Lysine; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D053652:Nuclear Transfer Techniques; D011247:Pregnancy; D027724:Reproductive Techniques, Assisted; D014635:Valproic Acid / Q000494:pharmacology*
chemical_list: D056572:Histone Deacetylase Inhibitors; D006657:Histones; D014635:Valproic Acid; D008239:Lysine