The human ATP-binding cassette (ABC) transporter ABCB6 has been described as a mitochondrial porphyrin transporter essential for heme biosynthesis1, but is also suspected to contribute to anticancer drug resistance2–4, as do other ABC transporters located at the plasma membrane. We identified ABCB6 as the carrier of the blood group antigen Lan on red blood cells, but also at the plasma membrane of hepatocellular carcinoma (HCC) cells, and established that ABCB6 actually encodes a new blood group system (Langereis, Lan). Targeted sequencing of ABCB6 in 12 unrelated individuals of the blood type Lan− identified 10 different ABCB6 null mutations. This is the first report of deficient alleles of this human ABC transporter gene. Surprisingly, Lan− (ABCB6−/−) individuals do not suffer any clinical consequences, albeit their deficiency in ABCB6 may place them at risk when defining drug dosage.

Abstract.To assess the permeability of mouse oocytes and embryos, matured oocytes and embryos at various stages of development were placed in five cryoprotectant solutions at 25 C for 25 min. From the cross-sectional areas of the oocytes/embryos, the relative change in volume was analyzed. In oocytes, shrinkage was least extensive and recovery was quickest in the propylene glycol solution, showing that propylene glycol permeates the oocytes most rapidly. Dimethyl sulfoxide, acetamide, and ethylene glycol permeated the oocytes slightly more slowly than propylene glycol. The oocytes in glycerol shrunk extensively and then expanded marginally, indicating slow permeation. The volume changes of 1-cell and 2-cell embryos were similar to those of oocytes, showing little change in permeability. In 8-cell embryos, the volume recovered much faster than in the earlier stages especially in glycerol and acetamide. In morulae, the volume recovery was much faster in glycerol and in ethylene glycol; in ethylene glycol, the extent of shrinkage was small and the recovery was fast, indicating an extremely rapid permeation. Although the permeability of oocytes/embryos generally increased as embryo development proceeded, the degree of increase varied greatly among the cryoprotectants. Interestingly, the volume change in propylene glycol was virtually unaffected by the stage of development. Such information will be valuable for determining a suitable protocol for the cryopreservation of oocytes/embryos at different stages of development. Key words: Mouse, Embryo, Oocyte, Permeability, Cryoprotectant (J. Reprod. Dev. 51: 235-246 , 2005) he survival of mammalian oocytes and embryos after cryopreservation varies with the stage of maturation and development. In general, oocytes/embryos at earlier stages appear to be more sensitive to cryopreservation. In the mouse, the rate of survival after cryopreservation is lower for unfertilized oocytes than embryos and increases as development proceeds up to the 8-cell or morula stage, although it may decrease as the blastocoel enlarges Cryoprotectants for mammalian oocytes and embryos are virtually limited to these five agents both in slow freezing and in vitrification [8].Although the protective action of cryoprotectants is considered colligative [9], each agent has its own specific properties. Among them, the permeating p r o p e r t y i s o f g r e a t i m p o r t a n c e , b e c a u s e permeation of the cell with a cryoprotectant is critical for the successful cryopreservation of

The cryosensitivity of mammalian embryos depends on the stage of development. Because permeability to water and cryoprotectants plays an important role in cryopreservation, it is plausible that the permeability is involved in the difference in the tolerance to cryopreservation among embryos at different developmental stages. In this study, we examined the permeability to water and glycerol of mouse oocytes and embryos, and tried to deduce the pathway for the movement of water and glycerol. The water permeability (L(P), microm min(-1) atm(-1)) of oocytes and four-cell embryos at 25 degrees C was low (0.63-0.70) and its Arrhenius activation energy (E(a), kcal/mol) was high (11.6-12.3), which implies that the water permeates through the plasma membrane by simple diffusion. On the other hand, the L(p) of morulae and blastocysts was quite high (3.6-4.5) and its E(a) was quite low (5.1-6.3), which implies that the water moves through water channels. Aquaporin inhibitors, phloretin and p-(chloromercuri) benzene-sulfonate, reduced the L(p) of morulae significantly but not that of oocytes. By immunocytochemical analysis, aquaporin 3, which transports not only water but also glycerol, was detected in the morulae but not in the oocytes. Accordingly, the glycerol permeability (P(GLY), x 10(-3) cm/min) of oocytes was also low (0.01) and its E(a) was remarkably high (41.6), whereas P(GLY) of morulae was quite high (4.63) and its E(a) was low (10.0). Aquaporin inhibitors reduced the P(GLY) of morulae significantly. In conclusion, water and glycerol appear to move across the plasma membrane mainly by simple diffusion in oocytes but by facilitated diffusion through water channel(s) including aquaporin 3 in morulae.

The permeability to water and cryoprotectants of the plasma membrane is crucial to the successful cryopreservation of embryos. Previously, we have shown in mouse morulae that water and glycerol move across the plasma membrane by facilitated diffusion, and we have suggested that aquaporin 3 plays an important role in their movement. In the present study, we clarify the contribution of aquaporin 3 to the movement of water and various cryoprotectants in mouse morulae by measuring the Arrhenius activation energies for permeability to cryoprotectants and water, through artificial expression of aquaporin 3 using Aqp3 cRNA in mouse oocytes, and by suppressing the expression of aquaporin 3 in morulae by injecting double-stranded RNA of Aqp3 at the one-cell zygote stage. The results show that aquaporin 3 plays an important role in the facilitated diffusion of water, glycerol, and ethylene glycol, but not of acetamide and dimethylsulfoxide. On the other hand, in a propylene glycol solution, aquaporin 3 in morulae transported neither propylene glycol nor water by facilitated diffusion, probably because of strong water-solute interactions. These results provide important information for understanding the permeability of the plasma membrane of the mouse embryo.

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