A trial to cryopreserve immature medaka (Oryzias latipes) oocytes after enhancing their permeability by exogenous expression of aquaporin 3

Abstract

Fish oocytes have not been cryopreserved successfully, probably because it is difficult to prevent intracellular ice from forming. Previously, we have shown in medaka that immature oocytes are more suitable for cryopreservation than mature oocytes or embryos, in terms of permeability. We have also shown in immature medaka oocytes that the exogenous expression of aquaporin 3 (AQP3), a water/cryoprotectant channel, promotes the movement of water and cryoprotectants through the plasma membrane. In the present study, we attempted to cryopreserve immature medaka oocytes expressing AQP3. We first examined effects of hypertonic stress and the chemical toxicity of cryoprotectants on the survival of the AQP3-expressing oocytes. Exposure to hypertonic solutions containing sucrose decreased the survival of oocytes, but the expression of AQP3 did not affect sensitivity to hypertonic stress. Also, AQP3 expression did not markedly increase sensitivity to the toxicity of cryoprotectants. Of the four cryoprotectants tested, propylene glycol was the least toxic. Using a propylene glycol-based solution, therefore, we tried to cryopreserve immature oocytes by vitrification. During cooling with liquid nitrogen, all intact oocytes became opaque, but many AQP3-expressing oocytes remained transparent. This indicates that the expression of AQP3 is effective in preventing intracellular ice from forming during cooling. During warming, however, all the AQP3-expressing oocytes became opaque, indicating that intracellular ice formed. Therefore, the dehydration and permeation by propylene glycol were still insufficient. Further studies are necessary to realize the cryopreservation of fish oocytes.

Fig. 1.

The effect of hypertonic sucrose solutions on the viability of immature medaka oocytes. Intact (open) and aquaporin 3-expressing (shaded) oocytes were exposed to 90% TCM199 (control) and 90% TCM199 containing 0.2, 0.3 or 0.4 M sucrose for 30 min at 25 C. Their viability was assessed by the ability to mature (A), to be fertilized (B) and to develop till hatching within 14 days of culture (C) at 26 C. Data are indicated as means ± SD from triplicate determinations. For each treatment, 23–25 oocytes were used. Bars with different superscripts differ significantly within each criterion (P<0.05).

Fig. 2.

The effect of cryoprotectants on the viability of immature medaka oocytes. Intact (open) and aquaporin 3-expressing (shaded) oocytes were exposed to 90% TCM199 (control) and 90% TCM199 containing 8% (v/v) ethylene glycol (EG), 10% (v/v) propylene glycol (PG) or 9.5% (v/v) Me₂SO (Me₂SO) for 60 min at 25 C. Their viability was assessed by the ability to mature (A), to be fertilized (B) and to develop till hatching within 14 days of culture (C) at 26 C. Data are indicated as means ± SD from triplicate determinations. For each treatment, 32–33 oocytes were used. Bars with different superscripts differ significantly within each criterion (P<0.05).

Fig. 3.

The effect of a high concentration of propylene glycol on the viability of immature medaka oocytes. Intact (open) and aquaporin 3-expressing (shaded) oocytes were exposed to 90% TCM199 (control) and 90% TCM199 containing 30% (v/v) propylene glycol for 3, 5 or 10 min at 25 C. Their viability was assessed by the ability to mature (A), to be fertilized (B) and to develop till hatching within 14 days of culture (C) at 26 C. Data are indicated as means ± SD from triplicate determinations. For each treatment, 35–45 oocytes were used. Bars with different superscripts differ significantly within each criterion (P<0.05).

Fig. 4.

The effect of a propylene glycol-based vitrification solution on the viability of immature medaka oocytes. Intact (open) and aquaporin 3-expressing (shaded) oocytes were exposed to the vitrification solution, a mixture of 30% (v/v) propylene glycol and 70% (v/v) FS solution, for 3, 5 or 10 min at 25 C. FS solution was 90% TCM199 medium containing 10% (w/v) Ficoll PM70 and 0.2 M sucrose. Their viability was assessed by the ability to mature (A), to be fertilized (B) and to develop till hatching within 14 days of culture (C) at 26 C. Data are indicated as means ± SD from triplicate determinations. For each treatment, 29–34 oocytes were used. Bars with different superscripts differ significantly within each criterion (P<0.05).

Fig. 5.

The morphological change of immature medaka oocytes after vitrification with cryoloops. Aquaporin 3-expressing oocytes were exposed to the pretreatment solution (90% TCM199 medium containing 10% (v/v) propylene glycol) for 60 min at 25 C, suspended in the vitrification solution containing 30% (v/v) propylene glycol for 3 min at 25 C, and vitrified with liquid nitrogen. A, an aquaporin 3-expressing oocyte before exposure to the pretreatment solution; B, a vitrified aquaporin 3-expressing oocyte just after removal of cryoprotectants; C, a vitrified aquaporin 3-expressing oocyte after 1 h of culture at 26 C. Similar morphology was observed in vitrified intact oocytes.