Nuclear Replacement of In Vitro-Matured Porcine Oocytes by a Serial Centrifugation and Fusion Method

• Published in: Reproduction in domestic animals Vol. 45; no. 4; pp. 659 - 665
• Main Authors: Maedomari, N, Kikuchi, K, Nagai, T, Fahrudin, M, Kaneko, H, Noguchi, J, Nakai, M, Ozawa, M, Somfai, T, Nguyen, LV, Ito, J, Kashiwazaki, N
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 01.08.2010; Blackwell Publishing Ltd; Blackwell
• Subjects: Animals; Biological and medical sciences; Blastocyst - physiology; Cell Nucleus; Cells; Embryo Culture Techniques; Female; Fertilization in Vitro; Fundamental and applied biological sciences. Psychology; Fusion; Genes; Male; Mammalian reproduction. General aspects; Nuclear physics; Oocytes - physiology; Parthenogenesis - physiology; Swine - physiology; Vertebrates: reproduction; Vertebrata; Mammalia; Animal; Replacement; Artiodactyla; Germinal cell; In vitro; Ungulata; Oocyte; Pig
• ISSN: 0936-6768; 1439-0531
• DOI: 10.1111/j.1439-0531.2008.01324.x

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.