title: Nuclear replacement of in vitro-matured porcine oocytes by a serial centrifugation and fusion method.
abstract: The objective of the present study was to establish a method for nuclear replacement in metaphase-II (M-II) stage porcine oocytes. Karyoplasts containing M-II chromosomes (K) and cytoplasts without chromosomes (C) were produced from in vitro-matured oocytes by a serial centrifugation method. The oocytes were then reconstructed by fusion of one karyoplast with 1, 2, 3 or 4 cytoplasts (K + 1C, K + 2C, K + 3C and K + 4C, respectively). Reconstructed oocytes, karyoplasts without fusion of any cytoplast (K) and zona-free M-II oocytes (control) were used for experiments. The rates of female pronucleus formation after parthenogenetic activation in all groups of reconstructed oocytes (58.2-77.4%) were not different from those of the K and control groups (58.2% and 66.0%, respectively). In vitro fertilization was carried out to assay the fertilization ability and subsequent embryonic development of the reconstructed oocytes. The cytoplast : karyoplast ratio did not affect the fertilization status (penetration and male pronuclear formation rates) of the oocytes. A significantly high monospermy rate was found in K oocytes (p < 0.05, 61.6%) compared with the other groups (18.2-32.8%). Blastocyst formation rates increased significantly as the number of the cytoplasts fused with karyoplasts increased (p < 0.05, 0.0-15.3%). The blastocyst rate in the K + 4C group (15.3%) was comparable with that of the control (17.8%). Total cell numbers in both the K + 3C and K + 4C groups (16.0 and 15.3 cells, respectively) were comparable with that of the control (26.2 cells). Our results demonstrate that a serial centrifugation and fusion (Centri-Fusion) is an effective method for producing M-II chromosome transferred oocytes with normal fertilization ability and in vitro development. It is suggested that the number of cytoplasts fused with a karyoplast plays a critical role in embryonic development.
authors.affiliation: Division of Animal Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Maedomari
authors.forename: K
authors.initials: K
authors.lastname: Kikuchi
authors.forename: T
authors.initials: T
authors.lastname: Nagai
authors.forename: M
authors.initials: M
authors.lastname: Fahrudin
authors.forename: H
authors.initials: H
authors.lastname: Kaneko
authors.forename: J
authors.initials: J
authors.lastname: Noguchi
authors.forename: M
authors.initials: M
authors.lastname: Nakai
authors.forename: M
authors.initials: M
authors.lastname: Ozawa
authors.forename: T
authors.initials: T
authors.lastname: Somfai
authors.forename: L V
authors.initials: LV
authors.lastname: Nguyen
authors.forename: J
authors.initials: J
authors.lastname: Ito
authors.forename: N
authors.initials: N
authors.lastname: Kashiwazaki
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000502:physiology; D002467:Cell Nucleus; D046149:Embryo Culture Techniques; D005260:Female; D005307:Fertilization in Vitro; D008297:Male; D009865:Oocytes / Q000502:physiology*; D010312:Parthenogenesis / Q000502:physiology; D013552:Swine / Q000502:physiology*