Expression of ganglioside GT1b in mouse embryos at different developmental stages after cryopreservation |
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Authors: | Bo-Hyun Kim Ji-Ung Jung Kisung Ko Won-Sin Kim Sun-Mi Kim Jae-Sung Ryu Jung-Woo Jin Hyo-Jung Yang Ji-Su Kim Hyuck-Chan Kwon Sang-Yoon Nam Dong-Hoon Kwak Yong-II Park Deog-Bon Koo Young-Kug Choo |
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Institution: | (1) Obstetric and gynecology clinic, Mirae-Heemang Hospital, Seoul 135-888, Korea;(2) College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungbuk National University, Cheongju, 361-763, Korea;(3) Department of Biotechnology, Catholic University, Bucheon, 420-749, Korea;(4) Center for Development and Differentiation, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 305-806, Korea;(5) Department of Biological Science, College of Natural Sciences, Wonkwang University, 344-2 Shinyong-dong, Iksan, Jeonbuk, 570-749, Korea |
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Abstract: | Gangliosides are a family of sialic acid-containing glycosphingolipids that are abundant in neurons and have a variety of
functions in developing and mature tissues. We examined the expression of ganglioside GT1b in the embryonic preimplantation
stage after freezing and thawing processes to determine the regulatory roles of ganglioside GT1b in early embryonic development.
ICR mouse embryos at the two-cell stage obtained by flushing the oviducts were frozen by two cryopreservation procedures,
slow freezing using a programmable freezer or vitrification by direct plunging into liquid nitrogen. Slow freezing was conducted
with equilibration in 1.5 M 1,2-propanediol or 5% equilibration glycerol. Vitrification was applied with a 10–15 min equilibration
in 7.5% ethylene glycol (EG), 7.5% dimethylsulfoxide (DMSO), and 30 sec in a solution of 15% EG, 15% DMSO and 0.5 M sucrose.
Immediately after thawing, the survival rate of the embryos was assessed by their morphology and ability to develop to blastocysts
in culture. The survival rate of vitrified and thawed embryos (92%) was significantly higher than that of slow frozen and
thawed embryos (76%) (P<0.05). A tendency of higher blastocyst rate was found in the vitrified and thawed embryos compared
to that of the slow frozen and thawed embryos. Confocal immunofluorescence staining confirmed that surviving embryos expressed
ganglioside GT1b, with the strongest expression at the compacted eight-cell or later stage embryos. Ganglioside GT1b was not
observed in the TUNEL-positive, apoptotic embryos, suggesting that cryopreservation had induced DNA breaks in them. These
results suggest that ganglioside GT1b may play an important role in embryo survival or development.
These authors contributed equally to this work. |
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Keywords: | Slow freezing Vitrification Ganglioside GT1b |
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