Shyoso Ogawa

A simple and rapid method for cryopreservation of mouse 2-cell embryos by vitrification: Beneficial effect of sucrose and raffinose on their cryosurvival rate

Survival of mouse 2-cell embryos was evaluated after exposure to 1.38, 2.75 or 5.5 M single cryoprotectants [dimethylsulphoxide (DMSO), acetamide (Ac) and propylene glycol (PG)], components frequently utilized as a vitrification solution, for 0.5, 1, 2 and 10 minutes at room temperature prior to vitrification. More than 80 % of the treated embryos developed to normal blastocysts in culture, after exposure to 1.38-2.75 M of each reagent for 0.5 minutes, although Ac tended to provide with have a deleterious effect on their survival. When the embryos were vitrified with solutions containing DP (2.75 M DMSO and 2.75 M PG) plus 0, 0.5 and 1.0 M Ac after a 0.5-minute exposure, their in vitro survival rates to the blastocysts were 44, 41 and 37%, respectively, showing no significant difference among them (x(2)=0.1-0.6, P>0.05). This indicates that the presence of Ac is not always needed for vitrifying mouse 2-cell embryos. Embryos, that had been vitrified with DP solution supplemented with 1.0 M sucrose (DPS) after a 0.5- minute exposure, exhibited significantly higher in vitro survival rate (82%) than those vitrified with DP (44%) (x(2)=23.4, P<0.001). Similar high survival rate (81%) was obtained when they were vitrified with DP plus 0.16 M raffinose (DPR) (x(2)=28.3, P<0.001). In vivo survival of embryos vitrified with DPS or DPR after a 0.5-minute exposure was both 49%, and there was no significant difference comparing to the unvitrified control group (60%). This method is rapid, efficient and reliable, and thus may be of practical use for cryopreserving mouse 2-cell embryos.

The survival of whole and bisected rabbit morulae after cryopreservation by the vitrification method.

The survival of whole and bisected rabbit morulae cryopreserved by the vitrification method was investigated. The embryos were loaded in a column of vitrification solution (VS, a mixture of 25% glycerol and 25% 1, 2-propanediol in PBS+16% calf serum), which was located between two columns of 1 M sucrose solution in a plastic straw. The embryos were frozen by being plunged into liquid nitrogen and thawed in a water bath at 20 degrees C. Two methods of loading embryos into straws were used: the single and double column vitrification solution methods. The embryonic survival rates between these two methods were compared. Seventy-one (86.6%) out of 82 morulae vitrified in double column straws developed into the blastocyst stage in vitro. Eleven (18.3%) live fetuses were obtained after the transfer of 60 frozen-thawed morulae to four recipients. By contrast, the survival rate (36.5%, 27 74 ) of embryos vitrified in the single column straws was significantly lower (P<0.05). The vitrification solution of the single column straws became opaque, indicating ice-crystal formation, upon thawing in 5 of 11 straws, which was assumed to have damaged the embryos. More than 80% (29 36 ) of the bisected morulae frozen and thawed in the double column straws developed to the blastocyst stage in vitro when cryoprotectant was diluted stepwise with 1 M and 0.25 M sucrose solution. When the cryoprotectant was removed by one-step dilution with 1 M sucrose solution, swelling in blastomeres was observed and the development rate of the recovered embryos decreased (45.8%, 11 24 ). These results indicate that the vitrification method employed in our experiment is not only efficient for the cryopreservation of rabbit morulae, but it can also be used for the preservation of bisected rabbit morulae, which had not been successful using previous methods.

Production of transgenic mice by microinjection of DNA into vitrified pronucleate stage eggs

Vitrification is a technique for cryopreserving cells without crystallization due to elevation of the viscosity during the cooling process. We have developed a rapid and convenient mean of, cryopreserving mouse preimplantation embryos by vitrification using a solution (hereafter named DPS) consisting of 2.75m dimethylsulfoxide, 2.75m propylene glycol and 1.0m sucrose.In vitro fertilized pronucleate stage eggs were used because a large number of stage-matched eggs can be obtained at once. Only successfully fertilized eggs were collected and vitrified in DPS. After warming, two DNA constructs were injected into a total of 257 cryopreserved eggs, of which 175 (68%) survived the injection and were transferred into six recipients. All recipients became pregnant and gave birth to a total of 20 pups. When these DNA constructs were concomitantly injected into fresh eggs, 18% of eggs that were transferred developed into live pups, which was the same as the 18% figure for the cryopreserved eggs. With respect to transgenesis, 40% of the pups (8/20) developed from vitrified eggs were transgenic. In terms of the injected eggs that had been transferred, 4.5% of the 213 fresh eggs and 3.1% of the 112 vitrified eggs developed into transgenic mice. These results indicate that the efficiency of production of transgenic mice from vitrified eggs is comparable to that from fresh eggs.

Production of normal piglets from microsurgically split morulae and blastocysts

The embryo splitting technique was applied to pig embryos, and the developmental ability of the split embryos was examined by means of in vitro culture and transfer. Morulae, early blastocysts and blastocysts were collected from Landrace x Large White F(1) gilts which had been mated to Duroc boars. The embryos were bisected with a fine glass or alloy (PtIr) needle after the softening of zonae pellucidae. The halved-embryos, which had either been placed in zonae pellucidae or not, were transferred to recipient gilts immediately after the micromanipulation (Experiment 1) or after cultivation for 15 to 20 h (Experiment 2). In Experiment 1, two fetuses were obtained from one of three recipients which had received 12 half-embryos. In Experiment 2, three of five recipients became pregnant, and in one recipient, seven piglets of a litter were obtained from 12 zona-free half-embryos produced from the original seven blastocysts. The results obtained indicate that a simple method not requiring the encasing of split embryos into zonae pellucidae is satisfactory to produce viable half-embryos.

Are fluorescent bodies of Y-spermatozoa detectable in common with mammalan species?*

An attempt was made to detect the fluorescent bodies (F-body), using Quinacrine mustard (Q-M) staining in the spermatozoa from eight mammalian species (human, bull, boar, dog, rabbit, rat, mouse, and mastomys) as well as in the cock (used as negative control). Sperm suspension, prepared after rinsing by repeated centrifugation with phosphate buffered saline (PBS), was either stained with Q-M for 24 h or treated with protease and then stained with Q-M for 60 min. The final concentration of Q-M in the mixed staining sperm suspension was 0.025 mg/ml. The examination using a reflecting fluorescent microscope revealed that the F-body found in human sperm was also present in the sperm of all the mammals but not in the cock after 24 h of staining. The enzyme-treated specimens showed higher incidences of F-bodies than specimens stained for 24 h without enzymatic digestion. These findings strongly suggest that the F-body is commonly present in the spermatozoa of many mammalian species.

Personal computer-controlled microsurgery of fertilized eggs and early embryos

The microsurgery of mouse and rat eggs and early embryos was attempted using a micromanipulator driven by three pulse motors. The pulse signals that regulate the three pulse motors for the X, Y, and Z axes were controlled according to the personal computer programs produced on the basis of the displayed data. As a result, the following was found. 1) The computer-controlled operation was possible in the X and Y plane on a specimen previously suctioned and retained by a holding pipet. A microinjection pipet was inserted into the male pronucleus of a fertilized egg and the morula was bisected using a microblade; these microtools were moved horizontally. 2) A more complicated micromanipulation in two dimensions (X and Z axes), which is very difficult manually, was possible by using this system. 3) microsurgery (microinjection of a fluorescent material (FITC) into the male pronucleus, enucleation of a fertilized egg, and vertical or horizontal bisection of morulae) was carried out successfully by a student who had no practical experience in this field. These facts suggest that the system markedly facilitates microsurgery, without need for full training in the manual procedures.