Effect of vitrification on mitochondrial distribution and membrane potential in mouse two pronuclear (2-PN) embryos.

The present study was designed to investigate the effect of vitrification on mitochondrial distribution, membrane potential (Δψ) and microtubule distribution in mouse 2-PN embryos, as well as to document the relationship between mitochondrial distribution and developmental ability of those embryos. Mitochondrial distribution was examined by fluorescence microscopy technology. Results indicated that: (1) The rate of mitochondrial ring formation around pronuclei in vitrified 2-PN embryos was significantly lower than in fresh ones (67.3 ± 3.0% vs. 84.9 ± 3.1%) (P < 0.05). (2) Blastocyst development rate of vitrified 2-PN embryos without mitochondrial rings (61.7 ± 4.5%) was significantly lower than that of vitrified embryos with mitochondrial rings (82.1 ± 2.8%). (3) Following staining by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbo-cyanine iodide (JC-1), most red-colored mitochondria (high Δψ) were distributed peripherally around pronuclei and along cell membranes of fresh 2-PN embryos. Conversely, red-colored mitochondria were greatly diminished in vitrified embryos, with green mitochondria (low Δψ) evenly distributed throughout the cytoplasm. The proportion of fresh 2-PN embryos with obvious aggregation of high Δψ mitochondria (84.2 ± 2.2%) was significantly higher than that of vitrified embryos (26.7 ± 3.0%) (P < 0.05). (4) The proportion of fresh embryos with microtubules distributed around pronuclei (83.5 ± 3.4%) was similar to that of vitrified embryos (74.7 ± 2.5%). In conclusion, vitrification affected mitochondrial distribution and decreased the mitochondrial membrane potential in mouse 2-PN embryos, events which may affect subsequent developmental viability of such embryos. Mol. Reprod. Dev. 76: 1056–1063, 2009. © 2009 Wiley-Liss, Inc.