بررسي انجماد شيشه اي برفراساختار سلولي بلاستوسيست اوليه موش

Ultrastructural Study of Mouse Early Blastocyst After Vitrification

The aim of this study was to investigate vitrification effects on ultrastructure of mice baistocysts by transmission electron microscopy. Early blastocysts were flushed from excised uteri of NMRI female mice. Embryos were divided into control and experimental groups. Embryos in experimental group were suspended in a solution of ethlene glycol, ficoll 70, and sucrose (EFS40) for two minutes and loaded into 0.25 ml straws. Held on liquid nitrogen vapour for 3-5 minutes and then immersed into liquid nitrogen. Straws were thawed for 15 seconds and were plunged into 20°C water and were equilibrated in 0.5 mole sucrose solution for 5 minutes. Both control and experimental groups were prepared for transmission electron microscopy. Embryos were fixed in a solution of 3% glutaraldehyde, 1% tannic acid followed by 1% osmium tetroxide. Thirty to 50 nm sections were stained by 25% methanolic uranyl acetate followed by 0.2% lead citrate. Sections were studied with transmission electron microscope at different magnifications. Cellular damages induced by cryopreservation included loss of microvilli, disruption of plasma membrane, disarrangement of cytoskeleton, mitochondrial changes, and swelling of rough endoplasmic reticulum and golgi apparatus. However, junctional complex regions remained intact. The untreated embryos showed normal structure. Our results indicate that vitrefication damages mouse early blastocysts particularly membrane bound structures that involved in metabolic and synthetic activities. Therefore delay in resumption of normal development of vitrified embryos is accountable. Such damages may either be due to ethylene glycol toxicity or change in osmotic pressure during freezing and thawing. The extent and nature of these damages depends on the vitrification procedure.