MATERIALS AND METHODS

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Develop. Growth and Differ. Vol. 21 No. 5 1979 pp. 423-430. CRYOPRESERVATION OF SEA URCHIN EMBRYOS AND SPERM EIZO ASAHINA AND TSUNEO TAKAHASHI Minami 5.

1 Develop. Growth and Differ. Vol. 21 No pp CRYOPRESERVATION OF SEA URCHIN EMBRYOS AND SPERM EIZO ASAHINA AND TSUNEO TAKAHASHI Minami 5. Nishi 24 Sapporo 060 Japan and The American National Red Cross Blood Research Laboratory 9312 Old Georgetown Road Bethesda MD USA A simple method for preserving live sea urchin embryos and sperm in liquid nitrogen (LN) was developed through the.use of DMSO as a cryoprotective additive. Samples of late embryos in double test tubes were cooled to C by two-step freezing first to -76 C and then by plunging in LN. In the case of fertilized eggs samples were previously frozen to -WC at which temperature the samples were kept for 15 min; they were then transferred into LN. After preservation in LN for various lengths of time samples in the double test tubes were thawed in water at 15 C. The post-thaw survival was more than 90% for late embryos and about 10% for fertilized eggs. Difference in the length of the cryopreservation period did not affect survival. Post-thaw development in cryopreserved embryos often showed abnormalities in. structure. Sperm with 1.5 M DMSO was successfully preserved in LN by a similar method to the one used for cryopreservation of late embryos. Fertilizability in cryopreserved sperm was complete regardless of the length of the preservation period. Nearly all the eggs fertilized by cryopreserved sperm developed to normal plutei. It has been well established that a wide variety of organisms can be successfully cryopreserved at very low temperatures for use in experimental biology clinical medicine and the animal breeding industry. Developments in methods of cryopreservation permit easy use of various biological materials in laboratories at any time and place if a liquid nitrogen container is available. Since there are many developmental biologists using sea urchin embryos and sperm as experimental materials easy preservation of these materials in a frozen condition may be of use for their work. In our previous paper a remarkable freezing tolerance of sea urchin sperm and embryos from the one-cell stage to pluteus was demonstrated (7). This suggested the possibility of cryopreservation of these materials at very low temperatures. In this paper we shall describe a simple method of preserving live sea urchin embyos and sperm in liquid nitrogen. Except for the highly successful case of the sperm the present method of cryopreservation is still inadequate in maintaining the structure and activity of embryos after freeze-thawing. However the present work may we hope offer useful clues leading to a completely successful cryopreservation of sea urchin embryos. MATERIALS AND METHODS Materials Strongylocentrotus intermedius from Oshoro Hokkaido was the main material used. Hemicentrotus pulcherrimus from Misaki Kanagawa and Strongylocentrotus nudus from Oshoro Hokkaido were also used for additional experiments. These materials were kept in an aquarium at 15 C for about a week prior to experimentation. Eggs were obtained by KCI-induced spawning and washed three times with filtered sea water. Sperm was obtained as dry sperm from dissected testes. Eggs suspended in sea water were inseminated by the addition of a very diluted sperm suspension (1: 10000). The embryos thus fertilized were raised in Millipore-filtered sea water with gentle stirring at 15 C. 423

424 EIZO ASAHINA AND TSUNEO TAKAHASHI Protective additives In our previous work the following compounds were examined as potential cryoprotectants : sucrose polyvinylpyrrolidone glycerol ethylene

2 424 EIZO ASAHINA AND TSUNEO TAKAHASHI Protective additives In our previous work the following compounds were examined as potential cryoprotectants : sucrose polyvinylpyrrolidone glycerol ethylene glycol (EG) and dimethyl sulfoxide (DMSO). The last two proved to afford good protection to frozen sea urchin embryos in liquid nitrogen (7). A preliminary experiment indicated that DMSO was better than EG as a protectant for sea urchin embryos if the embryos were kept at 0 C throughout the equilibration period. No significant difference in the cryoprotective effect of DMSO between concentrations of 1 and 2 M was observed with sea urchin embryos. 1 M DMSO in sea water was therefore exclusively used as a cryoprotective medium. Embryo suspension in sea water previously cooled to OT was mixed with the same amount of 2 M DMSO in sea water at 0 C to yield the samples to be used. In this sample medium many embryos once dehydrated and contracted recovered their normal appearance within about 10 min at 0 C as DMSO penetrated the cells of the embryos. The embryos especially in early stages however became increasingly susceptible to DMSO as the time of immersion lengthened beyond 1 hr. Half-ml samples containing 200 to 500 embryos were placed in small test tubes (10 x 110 mm) and maintained in an ice water bath for an equilibration period of less than 30 min prior to freezing. General freeze-thaw procedure and determination of sample temperature Samples in single or double test tubes were cooled in air in a refrigerator or in an alcohol bath with solid carbon dioxide at various temperatures from -25 to -78 C (see Fig. 2). Cooling to C was performed by two-step freezing first to about -70 C and then by plunging in liquid nitrogen (LN) in which test tubes containing samples were kept for at least 10 min. During this time a sample temperature of C is actually attained. The temperature of each sample was measured by inserting the fine tip of a thermocouple connected to an electronic recorder into the embryo suspension. Since samples with 1 M DMSO always froze spontaneously at temperatures above - 10 C during gradual cooling seeding with ice crystals which is otherwise necessary to prevent an excessive supercooling in samples was not performed. The cooling rate of the freezing samples was measured mainly from the tangent of the freezing curve (temperature vs. time curve) at -20"C since any intracellular freezing which except in the case of extra rapid freeze-thawing (5) is fatal to the cells in the sample is most likely to occur in a temperature range between - 10 and -45 C. In the present procedure the cooling rate measured at -20 C was very similar to the average cooling rate between -10 and -40 C. Test tubes containing frozen samples with the tip of a thermocouple inserted were cooled to predetermined temperatures and then warmed in air or water at 15 C. The rate of cooling and warming of the sample was controlled by changing the bath temperature and sometimes the amount of embryo suspension. An increased cooling or warming rate was also obtained by changing the test tube in which the sample was contained from double to single. Since cryoprotected sea urchin embryos were observed to be unsusceptible to a rapid rate of warming in the temperature range between -50 and -196 C during thawing (7) the warming rate was measured at -20 C. Post-thaw procedure Immediately after thawing a dilution of the cryoprotectant in the sample medium to a concentration innocuous to the embryo at room temperatures was made by a gradual washing with filtered fresh sea water. Survival was defined as the percentage of recovered embryos that developed to plutei. In the case of post-blastula embryos and sperm active swimming was also used as an additional index for recovery. RESULTS AND DISCUSSION Tolerable rates of cooling and warming The initial cooling rate is one of the most important factors in determining the optimum procedure of cryopreservation of living organisms since cooling too rapidly is apt to cause intracellular freezing ( ). The effect of the cooling rate on post-thaw survival was examined in cryoprotected embryos at various stages during freeze-thawing to and from - 40 C. Thawing was performed by shaking double tubes containing frozen samples in water at 15"C yielding a warming rate of 5-1O0C/min. The results represented in Fig. 1 indicate that maximal survival was obtained at cooling rates between 7 and 20"C/min in the pluteus while in fertilized

$At cooling rates above 30"C/ '\ h min a marked decrease in survival was invariably \ \ observed.$

3 CRYOPRESERVATION OF THE SEA URCHIN EMBRYO 425 eggs cooling rates between 2 and 10"C/min appeared tolerable. At cooling rates above 30"C/ '\ h min a marked decrease in survival was invariably \ \ observed. In fertilized eggs frozen at such a *I0 '\ high cooling rate cytolysis with the characteristic 60 - pattern of destroyed protoplasm which results from rapid intracellular freezing (3) was clearly observed after thawing. On the other hand > a cryomicroscopic observation revealed that : 20 - cryoprotected embryos invariably underwent a remarkable contraction with irregular or flattened shapes during slow freezing to - 30 C (6). They appeared as translucent as intact unfrozen embyos C.R. 'chin throughout the freeze-thaw process suggesting Fig. 1. Effect of cooling rates (C. R.) on the that ice formation was extracellular and that no Of frozen-thawed embryos to and ice formed within the blastocoel during slow from -40 C with 1 M DMSO in sea water* (s. inte.medius). PIuteus (.-.) fertilized freezing. If the length of freezing time was not egg (0--0). Warming rate: 5-1O0C/min at too long the post-thaw survival of these slowly -20 C. frozen embryos under the cryomicroscope was * Data are averages based on 3 experiments nearly complete. with replicated samples. Since sea urchin eggs are known to be very susceptible to an excessively rapid thawing (3 12) warming rates were also examined for their effect on the survival of cryoprotected embryos which were frozen to - 40 C at 10"C/min. As a result of this examination frozen embryos were observed to be tolerant of a broad range of warming rates. The best survival was obtained at warming rates between 5 and 5O"C/min in fertilized eggs whereas in plutei the optimum warming rates were observed between 5 and 90"C/min. It appears that plutei are much more tolerant of a rapid rate of both freezing and thawing than fertilized eggs. In addition embryos in blastula and gastrula stages were observed to have nearly the same degree of tolerance as that of plutei to both freezing and thawing rates. The surface-to-volume ratio of individual cells in a sea urchin embryo is much larger in late than in early stages since the number of cells increases to more than 1000 during embryonic development to the blatula stage without any appreciable increase in total cell volume. Therefore if the membrane permeability to water is approximately the same in both stages both release and absorption of cellular water during freeze-thawing will proceed more readily in a late embryo than in an early embryo. Since the difficulties of easy permeation of water through cell membrane during freeze-thawing are causally related to the mechanisms of freeze-thaw injury ( ) the above-mentioned characters of late embryos may contribute to their high tolerance of both rapid freezing and rapid thawing. Minimum tolerable freezing temperatures Based on the above observations the minimum tolerable freezing temperatures in cryoprotected embryos were determined. Embryos in double tubes were frozen-thawed to and from various predetermined final temperatures. Immediately after reaching the final temperatures the double tubes containing samples were warmed in water at 15 C. The cooling rates were

4 426 EIZO ASAHINA AND TSUNEO TAKAHASHI about 10 and 4O"C/min at -20 and -80"C respectively and the warming rate at -20 C was about 10"C/min. The results represented in Table 1 show that the freezing tolerance in cryoprotected embryos increases in advanced developmental stages later than blastula. A previous work demonstrated that un- Table 1. Minimum tolerable temperatures in fertilized eggs were much more susceptible to cryoprotected embryos (S. intermedius)*. freezing than fertilized ones in the absence of cryoprotectant (3). Cryoprotection of unfertilized eggs was therefore tried through the Stage of embryo** use of EG since EG easily penetrates the eggs F M B G Pr PI within 10 min at 0 C and is more harmless than any other cryoprotectant used. When * Minimum examined temperature was frozen to a temperature below -20 C with C. 1.5 M EG in sea water at a cooling rate of 1 O"C/min nearly all unfertilized eggs were ** F fertilized egg; M morula (8-16 cell stage); B blastula; G gastrula; Pr prism; PI pluteus. observed intact immediately after thawing. Some of them could even form fertilization membranes following insemination although the membrane was subnormal in appearance. However no formation of aster was observed in these eggs and they gradually cytolysed usually with blister formation. Since the minimum tolerable temperature of slowly frozen (I"C/min) unfertilized eggs of S. intermedius was reported to be - 15 C (12) the presence of EG does not appear to affect the freezing tolerance of unfertilized eggs. This is very intersting in comparison with the results obtained from the cryopreservation of unfertilized mammalian eggs. In mouse eggs both DMSO and glycerol were very effective in keeping unfertilized eggs alive at low temperatures (9 13). Although fertilized mouse eggs were more permeable to cryoprotectants than unfertilized ones good permeation of cryoprotectant resulted in good protection against freeze-thaw injury even in unfertilized eggs (9). Some factors other than mere penetration of a cryoprotectant into the cell may therefore be involved in the mechanisms of freezing tolerance in unfertilized sea urchin eggs. Cryopreservation of' late embryos From the results described above the following procedure was developed as a simple method of cryopreservation of sea urchin embryos. Cryopreservation of late embryos in the prism and pluteus stages is described first since late embryos are very tolerant to both rapid rates of freeze-thawing and very low freezing temperatures in the presence of cryoprotectant. Two Dewar flasks 300 and 500 ml in capacity were prepared as cold baths. The smaller contained ethyl alcohol and solid carbon dioxide to keep the bath temperature below -70 C. The larger was filled with LN. A double test tube was prepared as a sample container during freezing and thawing to control both cooling and warming rates. This tube consisted of a 21 x 110 mm outer glass tube and a 10 x 110 mm inner glass tube wihch is supported in the former by a piece of polyurethane foam to form a uniform air mantle surrounding the inner bube (Fig. 2). Embryos were equilibrated with 1 M DMSO in sea water at 0 C for 20 min. Half-ml samples of embryo suspension were placed in the double tubes and cooled by immersing the double tubes in the - 70 C alcohol bath described above by which treatment the sample was cooled down to -70 C within about 16 min. The double tubes were subsequently immersed in the LN bath

'T Fig. 2. Freezing vessel an assembly. A alcohol; C solid carbon dioxide; D Dewar vessel; I inner glass tube; 0 outer glass tube; P polyurethane foam; S sample suspension; T thermocouple.

5 'T Fig. 2. Freezing vessel an assembly. A alcohol; C solid carbon dioxide; D Dewar vessel; I inner glass tube; 0 outer glass tube; P polyurethane foam; S sample suspension; T thermocouple. CRYOPRESERVATION OF THE SEA URCHIN EMBRYO 427 cryopreserved for 53 days in LN. to embryos in blastula and gastrula stages. Cryopreservation of fertilized eggs by holding the top of the tubes by which a final sample temperature below C was attained within about 6 min. The samples cooled to C or below were immediately transferred to 20 1 LN container by directly dropping the inner tubes into LN in which they were stored for various lengths of time. After a predetermined period of time a tube containing the frozen sample was taken from the LN container and quickly set in an outer tube to form a double tube. The samples in double tubes were thawed in water at 15 C. One of the typical freezing and thawing curves obtained from the freezethaw procedure described above is represented in Fig. 3. On this curve cooling rates of 10 and 40"C/min at -20 and -8O"C respectively and warming rates of 40 and 7"Clmin at - 80 and - 20"C respectively are indicated. In the present procedure the length of the thawing period can safely be shortened when the samples are rewarmed to about -25"C by directly immersing the inner tubes containing frozen samples in water at 15"C yielding a warming rate of about 80"C!min. In samples thawed from freezing to - 196"C embryos gradually began to swim within 2 hr at 15 C. The post-thaw survival of cryopreserved embryos in the present treatment was usually more than 90%. No difference in survival rate was observed between embryos thawed immediately after cooling to -196 C and those The present method of cryopreservation was also applicable Since fertilized sea urchin eggs could not survive one-step freezing to temperatures below -40 C (Table I) the same method of two-step freezing described above for late embryos is not applicable to fertilized eggs. Therefore a prefreezing method was employed. This method was developed based on the hypothesis that some organisms if they were extracellularly frozen at appropriate tolerable temperatures for a certain length of time would become tolerable to subsequent rapid cooling to very low temperatures because of the decrease in intracellular freezable water during the previous freezing (4). Such a prefreezing method has proved effective in both individual insects and tiny pieces of glycerolated molluscan tissues (1). Preliminary experiments revealed that freezing at - 40 C became increasingly harmful to fertilized sea urchin eggs within one hr even in the presence of cryoprotectant and that previous freezing at -40 C for a period less than 10 min was almost useless for keeping the eggs alive during subsequent freezing to LN temperature. In view of the above observations the following procedure was employed for the cryopreservation of fertilized eggs. Fertilized eggs at 5 min after insemination were exclusively used since the freezing tolerance of sea urchin eggs increases

6 428 EIZO ASAHINA AND TSUNEO TAKAHASHI remarkably within a few minutes after fertilization because of the very rapid change in surface protoplasmic membrane (8 12). The eggs were equilibrated with 1 M DMSO in sea water for 20 min at 0 C. One-ml samples of cryoprotected egg suspension were placed in double tubes. They were cooled in a - 70 C alcohol bath for 12 min during which treatment the temperature of the sample approached -40 C. Double tubes containing samples were then rapidly transferred to another alcohol-solid carbon dioxide bath in which a constant temperature of - 40 C was maintained. After being kept for 15 min at - 40"C the double tubes were immersed into a LN bath by which a final sample temperature below C was attained within about 13 min. The procedure then used for preservation and thawing was entirely the same as that described for late embryos. An example of the freezing curves obtained from the present procedure for fertilized eggs is also represented in Fig. 3. Cooling rates of 5.5"C/min and 30"C/min at -20 and - 50"C respectively are indicated on this curve m 'C rnin Fig. 3. Freezing curves of embryo suspensions with 1 M DMSO in sea water (S. intermedius). P 0.5 rnl sample of pluteus; E 1.0 ml sample of fertilized egg; LN liquid nitrogen temperature C. Six replicated samples of cryoprotected fertilized eggs were stored in LN for various periods of time from several minutes to a full day. The post-thaw survival of these eggs was observed to be about 10 % on the average and 19 % in the best sample. No difference in the length of time of cryopreservation affected the post-thaw survival. Nearly all the surviving eggs grew to plutei. The prefreezing method described above is also applicable to cryopreservation of embryos in the morula stage. Prefreezing at temperatures between - 40 and - 60 C was also effective in keeping the late embryos alive during subsequent cooling to C. However post-thaw survival of late embryos was always worse than that in the two-step freezing method

7 CRYOPRESERVATION OF THE SEA URCHIN EMBRYO 429 described before (ASAHINA unpublished). Embryos after cryopreservation Post-thaw development in cryopreserved embryos was subnormal regardless of the length of the preservation period. In early embryos the formation of gastrula was generally delayed. When they grew to post-blastula stages about one third of the embryos were nearly normal while the others contained many isolated cells in the blastocoel which came out from the body wall so that the whole embryo appeared much darker than normal. In late embryos frozen and thawed many cells in the body wall gradually came off both sides of the wall resulting in a dark appearance of the blastocoel and a shortening of arms and cap in the whole pluteus. Cryopreservation of sperm Cryopreservation of sea urchin sperm is much easier than that of embryos since sea urchin spermatozoa are more tolerant of cryoprotectants than are sea urchin embryos. A drop (about 0.02 ml) of dry sperm was mixed with 0.5 ml of 1.5 M DMSO in sea water and equilibrated for 50min at 0 C. The freezing procedure was similar to that described for late embryos. After preservation at C for various lengths of time from a few minutes to 3 days the frozen samples Fig. 4. A surviving pluteus after cryopreservation for 53 days in in tubes were rapidly liquid nitrogen (H. pulcherrimus). x 400. thawed by immersing and stirring the tubes in water at 15 C. Immediately after thawing the same amount of fresh sea water was added to thawed samples at 0 C to dilute the cryoprotectant in the medium. Active swimming of spermatozoa was observed within about 5 min after thawing. Normal unfrozen eggs in suspension were inseminated by adding a drop of the thawed and diluted sperm suspension. The fertilizability in cryopreserved sperm was observed to be entirely the same as that in unfrozen control sperm regardless of the length of the preservation period. Nearly all the eggs fertilized by cryopreserved sperm developed to normal plutei. Cryopreservation of embryos of other sea urchin species A procedure of cryopreservation similar to the one described above was applied to embryos in various stages of both H. pulcherrimus and S. nudus. The former responded with the same degree of success as S. intermedius while the latter was scarcely protected by any cryoprotectant

8 430 EIZO ASAHINA AND TSUNEO TAKAHASHI used during freeze-thawing to and from a temperature below -40 C. This is very interesting since the egg of S. nudus under both fertilized and unfertilized conditions was known to be one of the most freezing tolerant materials in the absence of cryoprotectant among the various kinds of sea urchin eggs (3 12). We thank the directors and staff of Oshoro Marine Biological Station Hokkaido University and Misaki Marine Biological Station University of Tokyo for supplying materials. REFERENCES I. ASAHINA E Nature ~ Nature ~ In Cellular Injury and Resistance in Freezing Organisms (E. Asahina ed.) Institute of Low Temperature Science Hokkaido University Sapporo ~ and K. AOKI Nature ~ K. SHIMADA and Y. HISADA Exptl. Cell Res ~ and T. TAKAHASHI Photomicrographs projected at the 1976 Annual Meeting (Hiroshima) of The Zoological Society of Japan. 7. ~ and ~ Cryobiology ~ and K. TANNO Exptl. Cell Res JACKOWSKI S. C. and S. P. LEIBO Cryobiology LEIBO S. P. J. J. MCGRATH and E. G. CRAVALHO Cryobiology MAZUR P J. gen. Physiol TAKAHASHI T Contr. Inst. Low Temp. Sci. Ser. B WHIITINGHAM D. G J. Reprod. Fert (Received: April ) (Revised version received: May )

Title. Author(s)TAKAHASHI, Tsuneo. CitationContributions from the Institute of Low Temperature. Issue Date Doc URL. Type.

Title. Author(s)TAKAHASHI, Tsuneo. CitationContributions from the Institute of Low Temperature. Issue Date Doc URL. Type. Title On the Mechanism of Freezing njury in Extracellular Author(s)TAKAHASH, Tsuneo CitationContributions from the nstitute of Low Temperature ssue Date 1978-05-22 Doc URL http://hdl.handle.net/2115/20271