Expression of ganglioside GT1b in mouse embryos at different developmental stages after cryopreservation.
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.
Kim
Bo-Hyun
BH
Department of Biological Science, College of Natural Sciences, Wonkwang University, Iksan 570-749, Korea.
Jung
Ji-Ung
JU
Ko
Kisung
K
Kim
Won-Sin
WS
Kim
Sun-Mi
SM
Ryu
Jae-Sung
JS
Jin
Jung-Woo
JW
Yang
Hyo-Jung
HJ
Kim
Ji-Su
JS
Kwon
Hyuck-Chan
HC
Nam
Sang-Yoon
SY
Kwak
Dong-Hoon
DH
Park
Yong-Il
YI
Koo
Deog-Bon
DB
Choo
Young-Kug
YK
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001755:Blastocyst / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D015925:Cryopreservation; D047108:Embryonic Development / Q000502:physiology*; D005260:Female; D005455:Fluorescent Antibody Technique; D005732:Gangliosides / Q000096:biosynthesis*; D020287:In Situ Nick-End Labeling; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D009028:Morula / Q000378:metabolism; D011247:Pregnancy; D015044:Zona Pellucida / Q000187:drug effects / Q000648:ultrastructure
D005732:Gangliosides; C016481:trisialoganglioside GT1