Magnetic Fe۳O۴ Nanoparticles Modulate The Expression of Pluripotent Genes in Embryos Derived fromVtrified GV Oocytes

Background: Recently, nanoparticles is widely used to improvethe cryopreservation of different cells and tissues specialsex cells (sperm and oocyte). According to studies conductedin this area, the vitrification of oocyte alongside with nanoparticlesis recognized as a promising technique to cope with theproblems which encountered in the freezing process. As respectto deleterious effect of vitrification on oocyte as well as destructionon the embryo development, it can thus be consideredwhether these nanoparticles are advantageous or not?Materials and Methods: This study was conducted on thevitrification of immature oocytes with and without magneticnanoparticles (Fe۳O۴) (Vit and Vit+NP groups) as well as thecontrol group (fresh oocytes). Immature oocytes (GVs) werederived from ۶-۸ weeks-old NMRI mice and exposed to equilibrationmedium (۷.۵%EG plus ۷.۵%DMSO (v/v)) ۵ min, thentreated with vitrification solution (VS) (۱۵%DMSO, ۱۵%EG,۰.۵ mol/L sucrose) supplemented by nanoparticles (۵μgr/ml) ۱min, then they were exposed to warming solutions in three-stepsucrose dilution: W۱ (۱.۰ mol/L sucrose) ۳۷۰c, W۲ (۰.۵ mol/Lsucrose) and W۳ (۰.۲۵ mol/L sucrose) RT for ۱, ۳ and ۳ min.In the following, in vitro maturation (IVM) of germinal vesicleoocytes (GV) as well as in vitro fertilization (IVF) of obtainedmature oocytes were performed after warming in orderto achieve ۲PN embryos. In ۲PN stage we studied pluripotencygenes (Oct-۴, Nanog, Sox-۲ and Cdx۲) were amplified usingqRT-Realtime PCR. Housekeeping gene for normalization ofmRNAs was GAPDH. Expression was calculated using equation;Expression=(۲^-ΔΔCt).Results: The expression levels of pluripotent genes, Oct۴ andSox-۲ were significantly increased in ۲PN embryos derivedfrom vitrified oocytes comparing with non-vitrified embryosbut Cdx۲ was decreased analytically in Vit+NP group. Nanogdidn’t has any signal in ۲PN embryos. Based on scientific documentsthis increase does not optimum for embryo development.But after adding ۵μgr/ml magnetic Fe۳O۴ nanoparticles to vitrificationsolution, the expression levels of pluripotent genes(Oct-۴ and Sox-۲) returned to the natural level.Conclusion: Using of magnetic Fe۳O۴ nanoparticles to vitrificationsolution could modulates the expression levels ofpluripotent genes (Oct-۴ and Sox-۲) in pronuclear stage mouseembryos.Background: Recently, nanoparticles is widely used to improvethe cryopreservation of different cells and tissues specialsex cells (sperm and oocyte). According to studies conductedin this area, the vitrification of oocyte alongside with nanoparticlesis recognized as a promising technique to cope with theproblems which encountered in the freezing process. As respectto deleterious effect of vitrification on oocyte as well as destructionon the embryo development, it can thus be consideredwhether these nanoparticles are advantageous or not?Materials and Methods: This study was conducted on thevitrification of immature oocytes with and without magneticnanoparticles (Fe۳O۴) (Vit and Vit+NP groups) as well as thecontrol group (fresh oocytes). Immature oocytes (GVs) werederived from ۶-۸ weeks-old NMRI mice and exposed to equilibrationmedium (۷.۵%EG plus ۷.۵%DMSO (v/v)) ۵ min, thentreated with vitrification solution (VS) (۱۵%DMSO, ۱۵%EG,۰.۵ mol/L sucrose) supplemented by nanoparticles (۵μgr/ml) ۱min, then they were exposed to warming solutions in three-stepsucrose dilution: W۱ (۱.۰ mol/L sucrose) ۳۷۰c, W۲ (۰.۵ mol/Lsucrose) and W۳ (۰.۲۵ mol/L sucrose) RT for ۱, ۳ and ۳ min.In the following, in vitro maturation (IVM) of germinal vesicleoocytes (GV) as well as in vitro fertilization (IVF) of obtainedmature oocytes were performed after warming in orderto achieve ۲PN embryos. In ۲PN stage we studied pluripotencygenes (Oct-۴, Nanog, Sox-۲ and Cdx۲) were amplified usingqRT-Realtime PCR. Housekeeping gene for normalization ofmRNAs was GAPDH. Expression was calculated using equation;Expression=(۲^-ΔΔCt).Results: The expression levels of pluripotent genes, Oct۴ andSox-۲ were significantly increased in ۲PN embryos derivedfrom vitrified oocytes comparing with non-vitrified embryosbut Cdx۲ was decreased analytically in Vit+NP group. Nanogdidn’t has any signal in ۲PN embryos. Based on scientific documentsthis increase does not optimum for embryo development.But after adding ۵μgr/ml magnetic Fe۳O۴ nanoparticles to vitrificationsolution, the expression levels of pluripotent genes(Oct-۴ and Sox-۲) returned to the natural level.Conclusion: Using of magnetic Fe۳O۴ nanoparticles to vitrificationsolution could modulates the expression levels ofpluripotent genes (Oct-۴ and Sox-۲) in pronuclear stage mouseembryos.

کلیدواژه ها:

Oocyte Vitrification ، Fe۳O۴ Nanoparticles ، Embryo ، Pluripotency