Characteristics of the cell membrane fluidity, actin fibers, and mitochondrial dysfunctions of frozen‐thawed two‐cell mouse embryos

Abstract

Physical and chemical alterations caused by the freezing and thawing and their effects on survivals/developments in vitro were investigated. Of a total of 452 two-cell mouse embryos, the overall survival rate of the frozen-thawed embryos was 76.1% (344/452). The blastocyst formation of the frozen-thawed embryos was 32.6% (44/136) compared to 74.5% (117/157) in the fresh embryos (P<0.05). The total number of cells in a blastocyst also decreased from 96.0 +/- 19.0 (n=26) in the fresh embryos to 42.0 +/- 11 .34 (n=30) in the frozen-thawed embryos (P<0.05). Fluorescence recovery after photobleaching (FRAP) measurement revealed about 5-fold decrease in the cell membrane fluidity with a characteristic time constant (tau) of 1.46 +/- 0.13 sec (n=5) in the frozen-thawed embryos as opposed to 0.28 +/- 0.04 sec (n=5) in the fresh embryos (P<0.05). The relative amount of H(2)O(2) in an embryo as quantified by the fluorescence intensity of 2',7'-dichlorofluorescein (DCF) showed 62.8 +/- 23.5 (n=24) and 34.2 +/- 14.5 (n=20) in the frozen-thawed embryos and in the fresh embryos, respectively (P<0.05). The distribution of actin filaments in the frozen-thawed embryos revealed an uneven distribution, particularly discontinuities at the "actin band," which contrasted to an even distribution shown in the fresh embryos. Mitochondrial staining by Rhodamine 123 showed that there was no significant difference between the two treatments in the number and in the distribution of viable mitochondria, but a marked aggregation was seen in the arrested embryos. No Annexin V binding was detected in two-cell or four-cell embryos while the binding was positive in the arrested embryos. The mitochondrial membrane potential measured by a membrane potential-sensitive fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol- carbocyanine iodide (JC-1) revealed a marked depolarization in the frozen-thawed embryos. Finally, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) was employed to quantify the DNA fragmentation. In 75.0% cells of blastocysts (n=24) in the frozen-thawed embryos, the DNA fragmentation was detected as opposed to 37.0% in the fresh embryos (n=20) (P<0.05). Taken together, it is proposed that during the cryopreservation, two-cell mouse embryos are subjected to physical and chemical alterations, including destruction of the cell membrane integrity, redistribution of actin fibers, mitochondrial depolarizations, and increased reactive oxygen species (ROS) productions, which then may trigger the apoptotic cascade leading to a decrease in the survival rate and in the developmental rate of the embryos.

Effect of vitrification versus slow freezing of human day 3 embryos on β-hCG levels

“…This would imply that cytotrophoblast and syncytiotrophoblast cells resulting from vitrified embryos might proliferate at a lower rate in contrast to those from slow-freezing embryos. Results from previous studies on mouse embryo showed that vitrification reduced mitochondrial dynamics and increased reactive oxygen species (ROS) production in embryos [25][26][27]. Oxidative stress may trigger the apoptosis cascade in the developmental rate of embryos after thawing [26], which may be an important mechanism underlying the decreased capacity of the vitrified embryo to hCG secretion.…”

Mitochondria in human oogenesis and preimplantation embryogenesis: engines of metabolism, ionic regulation and developmental competence

“…In MII mouse and human oocytes and embryos (Ahn et al 2002, 2003, Jones et al 2004, the highest intensity of J-aggregate fluorescence is reported to occur in the pericortical/subplasmalemmal cytoplasm (arrows, Fig. 1K).…”

ATP-induced apoptosis of human granulosa luteal cells cultured in vitro

“…The apoptotic nuclei were brown after TUNEL staining (arrow events would be triggered from mitochondria, including release of caspase activators (such as cytochrome c), changes in the electron transport, loss of the mitochondrial transmembrane potential, altered cellular oxidation-reduction, and activation of proapoptotic and antiapoptotic Bcl-2 family proteins. Accompanying the underlying biochemical events are many apparent morphologic changes, including cell membrane shrinkage and subsequent loss of membrane integrity (42,43), the translocation of membrane phosphatidylserine (44), and DNA fragmentation (45). Thus, the translocation of membrane phosphatidylserine, the mitochondrial membrane depolarization, and DNA fragmentation detected by the annexin V binding assay, JC-1 staining, and the TUNEL assay, respectively, are good indicators of cellular apoptosis.…”