Pathway for the Movement of Water and Cryoprotectants in Bovine Oocytes and Embryos1

Abstract

The permeability of cells is important for cryopreservation. Previously, we showed in mice that the permeability to water and cryoprotectants of oocytes and embryos at early cleavage stages (early embryos) is low because these molecules move across the plasma membrane predominantly by simple diffusion through the lipid bilayer, whereas permeability of morulae and blastocysts is high because of a water channel, aquaporin 3 (AQP3). In this study, we examined the pathways for the movement of water and cryoprotectants in bovine oocytes/embryos and the role of AQP3 in the movement by determining permeability, first in intact bovine oocytes/embryos, then in bovine morulae with suppressed AQP3 expression, and finally in mouse oocytes expressing bovine AQP3. Results suggest that water moves through bovine oocytes and early embryos slowly by simple diffusion, as is the case in mice, although channel processes are also involved in the movement. On the other hand, water appears to move through morulae and blastocysts predominantly by facilitated diffusion via channels, as in mice. Like water, cryoprotectants appear to move through bovine oocytes/early embryos mostly by simple diffusion, but channel processes could also be involved in the movement of glycerol and ethylene glycol, unlike that in mice. In bovine morulae, although glycerol and ethylene glycol would move predominantly by facilitated diffusion, mostly through AQP3, as in mice, dimethylsulfoxide appears to move predominantly by simple diffusion, unlike in mice. These results indicate that permeability-related properties of bovine oocytes/embryos are similar to those of mouse oocytes/embryos, but species-specific differences do exist.

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

“…This suggests that cryoprotectants permeate mouse embryos through different pathways, which depend not only on the developmental stage, but also on the cryoprotectant itself. Similar results were obtained in bovine oocytes and embryos, although species specificity does exist [15]; the permeability of embryos to water, glycerol, and ethylene glycol increased markedly at the morula stage, whereas that to acetamide, DMSO, and propylene glycol did not. Two pathways have been described for the movement of water and cryoprotectants across the plasma membrane.…”

“…We previously suggested that the permeability of mouse and bovine embryos to water and cryoprotectants increased markedly at the morula stage and that this increased permeability affected the conditions suitable for cryopreservation [13][14][15]. We showed that aquaporin 3 played an important role in this increased permeability [13][14][15].…”

“…One is simple diffusion through the lipid bilayer, and the other is facilitated diffusion via channel processes. We previously showed that water and cryoprotectants moved through the oocytes and embryos of mice and cows at early cleavage stages predominantly by simple diffusion, and that water and some cryoprotectants moved through embryos at later stages (morulae and possibly blastocysts) predominantly by facilitated diffusion via channel processes [13][14][15]. We also demonstrated that aquaporin 3 was a major contributor to facilitated diffusion by water, glycerol, and ethylene glycol [14,15].…”

“…To examine whether aquaporin 3 plays an important role in the rapid movement of water, glycerol, and ethylene glycol in pig expanded blastocysts similar to that in mouse morulae and bovine morulae [13][14][15], we investigated the expression of aquaporin 3 mRNA in oocytes and parthenogenetically developed embryos. The expression was low in oocytes and morulae, but was high in expanded blastocysts (Fig.…”

“…We previously showed that water and cryoprotectants moved through the oocytes and embryos of mice and cows at early cleavage stages predominantly by simple diffusion, and that water and some cryoprotectants moved through embryos at later stages (morulae and possibly blastocysts) predominantly by facilitated diffusion via channel processes [13][14][15]. We also demonstrated that aquaporin 3 was a major contributor to facilitated diffusion by water, glycerol, and ethylene glycol [14,15]. Therefore, the increased tolerance of pig embryos to cryopreservation at the expanded blastocyst stage may depend not only on the decrease in cytoplasmic lipid droplets, but also on an increase in the permeability of the plasma membrane to water and cryoprotectants via channel processes.…”