Expression of ganglioside GT1b in mouse embryos at different developmental stages after cryopreservation

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.

Effects of Gangliosides on Spermatozoa, Oocytes, and Preimplantation Embryos

“…Several reports have proposed that DNA fragmentation can be mainly assigned to cryopreservation-produced ROS [63][64][65]. We reported that ganglioside GT1b was expressed in surviving murine embryos during the development of both fresh and frozen embryos [66]. These results demonstrate that GT1b suppresses DNA damage and participates in embryo survival and growth.…”

Different chromatin and energy/redox responses of mouse morulae and blastocysts to slow freezing and vitrification

“…After equilibration in a medium containing 10% glycerol, Dulbecco phosphate bufferd saline (DPBS + 10% FCS + 10% glycerol) for 10–15 min, the embryos were sucked up into straws (5 embryo per straw) [ 34 - 37 ]. The straws containing the embryos were then transferred into a Planer freezing machine (PLANER R 205, Planer, Sunbury-on-Thames, Middlesex UK) pre-cooled to minus 7°C.…”

Ganglioside GM1 influences the proliferation rate of mouse induced pluripotent stem cells

“…These gangliosides are thought to be involved in the control of several biological processes, including apoptosis, mouse embryonic development, cell proliferation, cell surface interactions, cell differentiation, and transmembrane signaling (10,11). GM3 is mostly expressed during embryogenesis in mice (12,13), but its expression is decreased during brain development (14). GD1a is also expressed during late embryogenesis (E9-E16), and increased expression of GD1a is observed during mouse brain development (12,14).…”