title: Effective Oocyte Vitrification and Survival Techniques for Bovine Somatic Cell Nuclear Transfer.
abstract: Bovine somatic cell nuclear transfer (SCNT) using vitrified-thawed (VT) oocytes has been studied; however, the cloning efficiency of these oocytes is not comparable with that of nonvitrified (non-V) fresh oocytes. This study sought to optimize the survival and cryopreservation of VT oocytes for SCNT. Co-culture with feeder cells that had been preincubated for 15 h significantly improved the survival of VT oocytes and their in vitro developmental potential following SCNT in comparison to co-culture with feeder cells that had been preincubated for 2, 5, or 24 h (p<0.05). Spindle assessment via the Oosight Microscopy Imaging System and microtubule staining revealed that vitrified metaphase II oocytes (VT group) were not suitable for SCNT. However, enucleating and/or activating oocytes prior to freezing enhanced their developmental potential and suitability for SCNT. The cloning efficiency of the enucleated-activated-vitrified-thawed (EAVT) group (21.6%) was better than that of the other vitrification groups [enucleated-vitrified-thawed (EVT) group, 13.7%; VT group, 15.0%; p<0.05] and was comparable with that of the non-V group (25.9%). The reactive oxygen species level was significantly lower in the EAVT group than in the other vitrification groups (p<0.05). mRNA levels of maternal genes (ZAR1, BMP15, and NLRP5) and a stress gene (HSF1) were lower in the vitrification groups than in the non-V group (p<0.05), whereas the level of phospho-p44/42 mitogen-activated protein kinase did not differ among the groups. Among the vitrification groups, blastocysts in the EAVT group had the best developmental potential, as judged by their high mRNA expression of developmental potential-related genes (POU5f1, Interferon-tau, and SLC2A5) and their low expression of proapoptotic (CASP3) and stress (Hsp70) genes. This study demonstrates that SCNT using bovine frozen-thawed oocytes can be successfully achieved using optimized vitrification and co-culture techniques.
authors.affiliation: 1 Mirae Cell Bio Inc., Seoul 143-854, Korea.|2 Jeju National University Stem Cell Research Center , Seoul 143-854, Korea.|3 Faculty of Biotechnology, College of Applied Life Sciences, Jeju National University , Jeju 690-756, Korea.|5 These authors contributed equally to this work.
authors.forename: Min Jee
authors.initials: MJ
authors.lastname: Park
authors.affiliation: 2 Jeju National University Stem Cell Research Center , Seoul 143-854, Korea.|3 Faculty of Biotechnology, College of Applied Life Sciences, Jeju National University , Jeju 690-756, Korea.|5 These authors contributed equally to this work.
authors.forename: Seung Eun
authors.initials: SE
authors.lastname: Lee
authors.affiliation: 1 Mirae Cell Bio Inc., Seoul 143-854, Korea.|2 Jeju National University Stem Cell Research Center , Seoul 143-854, Korea.|3 Faculty of Biotechnology, College of Applied Life Sciences, Jeju National University , Jeju 690-756, Korea.
authors.forename: Eun Young
authors.initials: EY
authors.lastname: Kim
authors.affiliation: 4 Shin Woman's Hospital , Uijeongbu 480-848, Korea.
authors.forename: Jun Beom
authors.initials: JB
authors.lastname: Lee
authors.affiliation: 4 Shin Woman's Hospital , Uijeongbu 480-848, Korea.
authors.forename: Chang Jin
authors.initials: CJ
authors.lastname: Jeong
authors.affiliation: 1 Mirae Cell Bio Inc., Seoul 143-854, Korea.|2 Jeju National University Stem Cell Research Center , Seoul 143-854, Korea.|3 Faculty of Biotechnology, College of Applied Life Sciences, Jeju National University , Jeju 690-756, Korea.
authors.forename: Se Pill
authors.initials: SP
authors.lastname: Park
mesh_terms: D000818:Animals; D002417:Cattle; D002470:Cell Survival; D019976:Cloning, Organism / Q000379:methods*; D018920:Coculture Techniques; D005260:Female; D018507:Gene Expression Regulation, Developmental; D059471:In Vitro Oocyte Maturation Techniques; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000378:metabolism / Q000502:physiology*; D058989:Vitrification