title: Evaluation of in vitro cultured rat oocytes, from different strains, by spindle morphology and maturation-promoting-factor activity combined with nuclear-transfer experiments.
abstract: Although successful nuclear transfer (NT) has been reported in the rat 6 years ago, somatic cell nuclear transfer (SCNT) in the rat could not be repeated. Our experiments with rat SCNT reveal the difficulties related to rat cloning. We first focussed on the most appropriate rat strain that could be used as an oocyte donor. Then we describe how rat oocytes can be kept in a nonactivated state during in vitro culture, because the latter undergo spontaneous partial activation through rapid extrusion of the second polar body after isolation from the oviduct. In the SCNT experiments performed with the one-step manipulation technique it was possible to produce rat embryos, which developed in vivo up to the blastocyst stage. In addition, we identified the implantation sites of SCNT rat embryos reconstructed with Sprague-Dawley (SD) oocytes. Furthermore, different rat strains were used as oocyte donors and their oocytes were cultured under different conditions to establish a stable nonactivating oocyte culture system. The ratio of activated to nonactivated oocytes was measured by spindle-stability and maturation promoting factor (MPF) activity. These measurements indicated that a substrain of the SD rat strain, the so-called OFA-SD strain, is the one providing the most stable oocytes, when their oocytes are cultured in the presence of the proteasome inhibitor MG132. However, it was not possible to obtain any implantation sites with reconstructed oocytes derived from the OFA-SD strain transferred to foster mothers. This goal was not achieved, even when the trichostatin A (TSA) treatment was used, which is known to enhance the cloning efficiency of reconstructed mouse, porcine, bovine, and rabbit oocytes both in vitro and in vivo by enhancing the reprogramming efficiency of the recipient nucleus.
authors.affiliation: Institute of Laboratory Animal Science, University of Zurich, Zurich, Switzerland. OSterthaus@uhbs.ch
authors.forename: Oliver
authors.initials: O
authors.lastname: Sterthaus
authors.forename: Ewa
authors.initials: E
authors.lastname: Skoczylas
authors.forename: Christian
authors.initials: C
authors.lastname: De Geyter
authors.forename: Kurt
authors.initials: K
authors.lastname: Bürki
authors.forename: Birgit
authors.initials: B
authors.lastname: Ledermann
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism; D002417:Cattle; D019976:Cloning, Organism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D005260:Female; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006877:Hydroxamic Acids / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D016200:Maturation-Promoting Factor / Q000378:metabolism*; D000090204:Mesothelin; D051379:Mice; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology* / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D013045:Species Specificity; D008941:Spindle Apparatus / Q000378:metabolism*; D013552:Swine
chemical_list: D015853:Cysteine Proteinase Inhibitors; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D007976:Leupeptins; C000715528:Msln protein, mouse; D061988:Proteasome Inhibitors; C012589:trichostatin A; D016200:Maturation-Promoting Factor; D046988:Proteasome Endopeptidase Complex; D000090204:Mesothelin; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde