Rapid Movement of Water and Cryoprotectants in Pig Expanded Blastocysts via Channel Processes: Its Relevance to Their Higher Tolerance to Cryopreservation1

Abstract

Pig oocytes and embryos are highly sensitive to cryopreservation; however, tolerance to cryopreservation increases in embryos at the expanded blastocyst stage. This increased tolerance may be attributed to a decrease in cytoplasmic lipid droplets at this stage. We previously showed that an increase in the permeability of the plasma membrane in mouse oocytes to water and cryoprotectants, caused by the artificial expression of aquaporin 3, an aquaglyceroporin, enhanced tolerance to cryopreservation. In the present study, we investigated whether membrane permeability was also involved in the tolerance of pig embryos to cryopreservation. The permeability of oocytes and morulae to water and glycerol was low, whereas that of expanded blastocysts was high. Activation energy for permeability to water, glycerol, ethylene glycol, and dimethyl sulfoxide was markedly lower for expanded blastocysts than for oocytes. This suggests that water and these cryoprotectants move through expanded blastocysts predominantly by facilitated diffusion and through oocytes predominantly by simple diffusion. Aquaporin 3 mRNA was expressed in expanded blastocysts abundantly, but less so in oocytes. On the other hand, the permeability of expanded blastocysts to propylene glycol was as low as that of oocytes, and activation energy for its permeability was similar to that of oocytes, which suggests that propylene glycol moves through oocytes and embryos predominantly by simple diffusion. These results suggest that the higher tolerance of pig expanded blastocysts to cryopreservation is also related to high membrane permeability due to the expression of water/cryoprotectant channels, in addition to the decrease in cytoplasmic lipid droplets.

The movement of water and cryoprotectants across the plasma membrane of mammalian oocytes and embryos and its relevance to vitrification

“…In pigs, the L P value of oocytes at 25°C is low (1.0 μm/min/atm), and its E a value is high (19 kcal/mol), similar to that of mouse oocytes [25]. However, unlike mouse and bovine morulae, in pig morulae, the L P value remains low, [25], probably because pig embryos become compacted at the 4-cell stage.…”

“…However, unlike mouse and bovine morulae, in pig morulae, the L P value remains low, [25], probably because pig embryos become compacted at the 4-cell stage. Therefore, water moves through pig oocytes and morulae principally by simple diffusion.…”

“…Therefore, water moves through pig oocytes and morulae principally by simple diffusion. In pig blastocysts, the L P value increases significantly and increases further in expanded blastocysts (3.4 μm/min/atm) at 25°C [25]. The E a value for the permeability of expanded blastocysts (7 kcal/mol) is close to 6 kcal/mol.…”

“…Therefore, water moves through pig expanded blastocysts principally by facilitated diffusion via water channels. Since pig expanded blastocysts abundantly express aquaporin 3 mRNA, aquaporin 3 might be involved in the facilitated diffusion of water [25]. …”

The effects of over expressing aquaporins on the cryopreservation of hepatocytes

“…[12,15,23]. The osmotic gradient created during the freezing process and the limitation of water transport results in increased incidence of intracellular ice formation leading to higher incidence of cell death [13,26].…”