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Cryopreservation of Totipotent Nuclei from Honeybee (Apis mellifera) Embryos by Rapid Freezing
35, 41–45 (1997) CY972023
CRYOBIOLOGY ARTICLE NO.
Cryopreservation of Totipotent Nuclei from Honeybee (Apis mellifera) Embryos by Rapid Freezing Yu Ronglin, Arne Hagen, and Stig W. Omholt1 Department of Animal Science, Agricultural University of Norway, P.O. Box 5025, 1432 AAS, Norway Here we report a method to cryopreserve totipotent preblastoderm nuclei from honeybee (Apis mellifera) embryos by rapid freezing without the addition of a specific cryoprotectant or other additive. By making feasible long-term storage of ooplasm biopsied from preblastoderm embryos, the method could become a key element for the development of an efficient and practical procedure for cloning and other genetic studies of honeybees. Donor ooplasm to be cryopreserved was extracted from the anterior pole of 8.0- to 9.0-h preblastoderm embryos with a transplantation pipet. The anterior end of the pipet was plugged with a small ball of cotton fiber, before it was fastened with a tape to a fine iron wire and plunged into liquid nitrogen. After being stored in liquid nitrogen for at least 24 h, the pipet was thawed rapidly in a water bath at 257C. The ooplasm was injected at the anterior pole of recipient embryos (0.5 to 3.5 h) at room temperature and at a relative humidity ú70%. Chimerism was assayed on 3-day larvae by making use of the polymorphic malate dehydrogenase locus and isoelectric focusing. Of 618 recipient embryos injected with cryopreserved nuclei, 157 (25.4%) hatched into larvae. Of the 157 larvae, 16 were chimeras carrying both donor and recipient genotypes and 2 expressed only the donor genotype and thereby were presumed to be embryo clones. The chimeric frequency (11.5%) was 85% of the frequency achieved when using noncryopreserved nuclei and the same transplantation protocol. q 1997 Academic Press
To develop an efficient embryo cloning technology of practical use for honeybees (Apis mellifera), long-term storage of ooplasm biopsied from preblastoderm embryos has to be mastered. As far as we know, such a method has not been developed for any insect. The honeybee egg is about 1.6 to 1.8 mm long, with a maximum diameter of 0.35 mm, which gives an egg volume of about 135 nl (about 10 times that of a Drosophila egg (1)). The outer and inner egg layers (chorion and vitelline membrane) are about 0.1 and 0.25 mm thick, respectively. The early development of the embryo involves 10 synchronous cleavage mitoses where the resulting nuclei migrate into the peripheral egg layer during a parasynchronous 11th mitosis (8 to 9 h). Thereafter, a peripheral cell layer, the blastoderm, is formed, as is the case for nearly all pterygote insects (5). The complete embryogenesis is amenable to direct observation, as honeybee
embryos become highly transparent when placed in paraffin oil, resolving individual cells and even nuclei (4). The developmental period is normally 72 h at 357C. Incubation of nonmanipulated eggs above a layer of 21% H2SO4 (w/w) in a closed plastic chamber normally yields a hatching rate above 90% (unpublished results). The larvae may be reared to adult workers and queens by in vitro feeding with semiartificial diets (13, 14). The usefulness of this technique is still somewhat restricted as the morphological variation of sister queens or workers reared by in vitro feeding is considerably higher than in normally reared sister groups (unpublished results). However, it works very well to feed larvae hatching from manipulated eggs for 1 to 2 days in vitro and then produce queens from these larvae by standard queen-rearing techniques (12, 18). An efficient nuclear transplantation methodology for honeybee embryos was recently described by Omholt et al. (12), and it was shown that nuclei from 8- to 9-h preblastoderm embryos are totipotent. Here we report a method to cryopreserve such totipotent pre-
Received July 22, 1996; accepted April 5, 1997. 1 To whom correspondence should be addressed. Fax: /47 64947960. E-mail: [email protected]. 41
0011-2240/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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blastoderm nuclei by rapid freezing without The rear end of the pipet was plugged with the addition of a specific cryoprotectant or a small ball of cotton fiber before it was fasother additive. tened with tape to a fine iron wire and plunged Combined with the fact that biopsied em- into liquid nitrogen. After being stored in liquid bryos develop into normal honeybee queens nitrogen for at least 24 h, the pipet was thawed (18), we are now in principle able to test the rapidly in a water bath at 257C. After removal breeding, developmental, and behavioral po- of the cotton plug and mounting the pipet to the tential of honeybee genomes stored in liquid micromanipulator, the ooplasm was injected at nitrogen. Though an efficient embryo cloning the anterior pole of recipient 0.5- to 3.5-h emtechnology for honeybees is still not within bryos (containing 1 to 4 nuclei) previously prereach, we view this result as a notable step pared for injection. This was done by mounting toward this goal. In addition, until cryopreser- 10 plastic cell cups with recipients (11) on a vation of honeybee embryos is mastered, as strip of modeling clay on the back side of a is already the case for Drosophila (10, 15), 90-mm petri dish (counted as one series) and our cryopreservation method may be used for keeping the petri dish at 357C and relative hulong-term storage of mutant honeybee strains midity ú80% until the injections started. The obtained by natural means or by genetic trans- injections were done at room temperature (20 to 307C) inside a moist plastic chamber beneath formation. the stereo microscope (relative humidity MATERIALS AND METHODS ú70%). The chamber was made such that the The experiments relied on a continuous eggs on the petri dish could be properly oriensupply of eggs throughout the year from colo- tated with the left hand before each injection, nies kept in a flight room with appropriate while the micromanipulator could be operated light conditions and where the temperature from the outside with the right hand. and the humidity are strictly controlled (12). The aspirations and transplantations of The eggs are sampled from small hives con- ooplasm were performed with an Oxford mitaining a queen and about 2500 workers. The cromanipulator (Singer Instrument Co., Roadhives are designed such that the eggs laid by water, Somerset, England), a microinjector the queen can be sampled from the outside. (PLI-100, Medical Systems Corp., Greenvale, Without this type of hive it is extremely diffi- NY, USA), and an ordinary stereo microcult to obtain eggs of a given age at appro- scope. We normally operated with an injection priate amounts (11). time of 0.6 s, an injection pressure of 8.0 kPa, Donor ooplasm to be cryopreserved was ex- and a balance pressure in the pipet of 3.0–4.0 tracted from the anterior pole of 8.0- to 9.0-h kPa. The average amount injected into each embryos with a transplantation pipet made embryo was estimated to be 2 to 4 nl or 1.5 from a borosilicate capillary tube (o.d. Å 1.0 to 3.0% of the total egg volume (4). mm, i.d. Å 0.58 mm) (Sutter Instrument Co., Embryos were incubated for 70–73 h at Novato, CA, USA). The tip was beveled to an 357C above a layer of 21% H2SO4 (w/w) in angle of 157 with a K. T. Brown Type Micropi- closed plastic chambers, until hatching. All larpet Beveler (Sutter Instrument Co.) and then vae from each series (i.e., max 10) were placed forged with a laboratory-made microforge. The in a small plastic cap (12 1 3 mm) filled with inner diameter of the pipet tip was 16 to 18 0.4 ml food mixture, ensuring worker bee demm. The amount of donor ooplasm extracted velopment (13), and incubated at 357C above from each donor embryo varied somewhat, but a layer of 7% H2SO4 (w/w) in a closed plastic we tried to keep it in the range 50 to 80 nl. chamber. The larvae were given fresh preThese volumes were based on previous calcula- warmed food mixture twice a day for 3 days, tions of pipet volumes estimated by measure- whereupon they were placed in separate Epments of the inner diameter of the pipets and pendorf tubes and stored at 0207C. This temthe length of column of ooplasm. perature seems to be sufficiently low to prevent
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damage of the protein used for assaying chimerism for several months (see below). Normally about 80% of the hatched individuals develop into 3-day larvae by following this procedure. However, the optimal time window for harvesting larvae after hatching is rather narrow (about 1h), and collecting the larvae too early or too late increases the mortality dramatically (data not shown). We allowed some remnants of the diet to stay on the larvae as previous tests have shown that this food mixture does not contain any detectable amount of the enzyme used for assaying chimerism (12). To determine chimerism, we assayed the polymorphic malate dehydrogenase (Mdh) locus, where three alleles (a, b, c) have been identified in larvae and adult worker bees (17). Isoelectric focusing (IEF) was used to determine genotypes. Each larva was homogenized in a small volume of distilled and deionized H2O (20 to 150 ml depending on the size of the larva), and after removing particulate material by centrifugation (14000g, 10 min, 47C), 20 ml of the extract was loaded onto an IEF gel (pH 3.5 to 9.5). After isoelectric focusing at 25 W constant power for 75 min at 107C on a Multiphor II electrophoresis system (Pharmacia LKB Biotechnology, Sweden), Mdh activity was assayed by incubating the gel at 377C for 3 h in 100 ml 0.1 M Tris–HCl buffer (pH 8.3), containing 12 mg nitroblue tetrazolium (NBT) (Sigma), 16 mg phenazine methosulfate (PMS) (Sigma), 24 mg nicotinamide adenine dinucleotide (NAD) (Sigma), and 24 mg malic acid (Sigma). Unrelated A. mellifera carnica queens with different Mdh genotypes were bred from screened queens from ordinary production colonies and artificially inseminated with semen of appropriate genotype. In this way we obtained four different genotypes (aa, ab, bc, and cc), which were used as donors as well as recipients. RESULTS AND DISCUSSION
Of 618 recipient embryos injected with cryopreserved nuclei, 157 (25.4%) hatched and developed into 3-day larvae. Of the 157 larvae, 16 were chimeras carrying both donor
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FIG. 1. A representational diagram of the typical IEF banding pattern of the Mdh assay. As the active enzyme is a dimer, the homozygote aa constitutes a single band on an IEF gel (lane 1), while, for example, the heterozygote bc in lane 3 expresses three bands (bb, bc, and cc). Lanes 1 to 4 show the expression patterns of the genotypes used as donors and recipients. Lanes 5 to 8 show typical chimeric expression patterns from the genotype combinations aa 1 cc, aa 1 bc, ab 1 bc, and ab 1 cc. The column to the right shows the positions of the various dimers. The positions of the dimers will be as depicted when the negative pole of the IEF field is at the top.
and recipient genotypes and 2 expressed only the donor genotypes, and were hence presumed to be embryo clones (12) (Fig. 1). This gives a chimeric frequency of 11.5%, which is 85% of the frequency obtained when using noncryopreserved nuclei (Table 1). When using fresh ooplasm our results are similar to those obtained on Drosophila with regard to hatching rate of manipulated embryos and frequency of chimeras (8). Considering the extra problems associated with using toxic cryoprotectants that have to be removed immediately after thawing (16), and stabilization of the nuclei (6), our method seems to offer some benefits by its operational simplicity. In a preliminary study using ethylene glycol as a cryoprotectant we experienced several of the problems associated with this. Fluorescent staining of these nuclei showed that many had a different shape compared to controls (39%), and some of these were of different size as well as showing loss of membrane integrity. Of 1135 recipients, only 96 larvae hatched and survived 3 days of in vitro feeding. Of these, 4 larvae expressed both donor and recipient genotypes and 1 expressed only the donor genotype. Thus, the overall chimeric frequency with reference to total number of manipulated embryos was only 15% of what was achieved by the other
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RONGLIN, HAGEN, AND OMHOLT TABLE 1 Chimeras Obtained by Nuclear Transplantation of Fresh and Cryopreserved Ooplasm
Ooplasm
Eggs injected
Larvae hatching/3day larvae
Larvae expressing recipient genotype only
Larvae expressing recipient and donor genotypes
Larvae expressing donor genotype only
Fresh Cryopreserved
830 618
214/89* 197/157
77 139
10 16
2 2
Note. The data for fresh ooplasm are taken from a previous report (12). The asterisk denotes that only a fraction of the surviving larvae was analyzed in this case.
method. However, we suspect that an appropriate buffer was not used in this experiment, and this may explain the low success ratio. For real long-term storage it may be necessary to seal the pipet containing ooplasm more properly than we did or to use other devices to store the ooplasm, but this should represent only a minor technical challenge. Although a chimeric frequency which is 85% of that achievable with fresh ooplasm is encouraging, it may be possible to improve this figure by making use of nitrogen slush instead of liquid nitrogen (15) and by increasing the temperature of the water bath to 357C for thawing. Storage of the ooplasm in a better heat-conducting material than glass may also improve the result (15). It is reasonable to believe that every change of the transplantation protocol using fresh ooplasm that increases the frequency of chimeras and embryo clones will give the same relative increase with cryopreserved ooplasm. It should be noted that we made use of no cryoprotectants. Considering prevailing opinions reflected in the cryobiological literature (2, 9, 16), it seems to be somewhat counterintuitive that this should work as well as it does. However, an apparently analogous result was obtained by Kalinina and Gromov when transplanting cryopreserved nuclei of amoeba to noncryopreserved enucleated amoeba. They found that the cytoplasm was irreparably damaged by freezing, while minor ultrastructural damages of the nuclei were repaired after transferal (7). We made no detailed studies of the cryopreserved ooplasm to determine the degree of ice
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formation or whether the ooplasm was preserved in a structurally arrested state. However, some preliminary results were obtained by studying the structure of cryopreserved and noncryopreserved nuclei stained with 0.5 mg/ ml Hoechst 33342 (Sigma) in 0.85% NaCl. After incubation under a layer of paraffin oil at 357C for 30 min the two groups were compared with respect to nuclear shape and nuclear membrane integrity by using a Leitz Aristoplan microscope (Leitz Wetzlar, Germany). There were no observable differences in these parameters (data not shown). This indicates that at least low-resolution structural effects due to osmotic stress and intranuclear ice formation must have been very moderate (2). In principle, by combining a technique for making biopsies of preblastoderm honeybee embryos (18) and by letting these embryos develop into queens that are inseminated and tested for individual or colony level traits, we now seem able to test the breeding, developmental, and behavioral potential of honeybee genomes stored in liquid nitrogen. However, before this can be realized, we will have to develop efficient and nonharmful procedures for enucleation of recipient honeybee embryos before transplantation of cryopreserved ooplasm. This is technically rather challenging, and to the best of our knowledge this has not been achieved on Drosophila embryos either, despite the fact that a nuclear transplantation methodology has been available for more than 25 years (19). Assuming that we will be able to solve the enucleation problem, the next step will be to increase the survival rate of the injected embryos. The sur-
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vival rate of embryos injected at the 0.5- to 2-h stage is considerably lower than those injected at 2 to 4.5 h of age (data not shown). We do not know the reason for this, but it may be that the physical damage caused by the penetration of the pipet at the anterior pole is greater for the very young embryo due to a more delicate and less resilient structure at this stage. This interpretation is partially supported by the fact that injection of a 0.9% salt solution into very young embryos with smaller pipets (3 to 4 mm) results in a survival rate above 85% (data not shown). This might imply that reducing the size of the transplantation pipets will increase the survival rate to an acceptable level. However, we have yet no data on how far down we can go before the nuclei are irreparably damaged during aspiration and transplantation. In a preliminary experiment we used centrifugation to concentrate the nuclei of 8- to 9-h donor embryos to a small band within the embryo, whereby we were able to aspirate ú500 nuclei into the pipet. The nuclear shape and membrane integrity were compared with nuclei taken from unmanipulated embryos of the same age by using a Leitz Aristoplan microscope (Leitz Wetzlar, Germany), and we did not observe any differences (data not shown). However, we were not able to significantly increase the chimeric frequency, despite the fact that each embryo in this case received on average many more nuclei. This indicates that the number of nuclei may not be as critical as the synchronization of the mitotic stage of recipient and donor (3). REFERENCES
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6.
7.
8.
9.
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1. Ashburner, M. ‘‘Drosophila: A Laboratory Handbook.’’ Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. 2. Bischof, J. C., and Rubinsky, B. Large ice crystals in the nucleus of rapidly frozen liver cells. Cryobiology 30, 597–603 (1993). 3. Collas, P., Balise, J. J., and Robl, J. M. Influence of cell cycle stage of the donor nucleus on development of nuclear transplant rabbit embryos. Biol. Reprod. 46, 492–599 (1992). 4. DuPraw, E. J. The honeybee embryo. In ‘‘Methods in Developmental Biology’’ (F. H. Wilt and N. K.
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18.
19.
Wessels, Eds.), pp. 183–217. Crowell Press, New York, 1967. Fleig, R., and Sander, K. Blastoderm development in honeybee embryogenesis as seen in the scanning electronmicrosope. Int. J. Invertebr. Reprod. Dev. 8, 279–286 (1985). Graham, J. M., Ford, T., and Rickwood, D. Isolation of the major subcellular organelles from mouse using Nycodenz gradients without the use of an ultracentrifuge. Anal. Biochem. 187, 318–323 (1990). Kalinina L. V., and Gromov, D. B. The nucleus of nonviable frozen–thawed amoeba has normal morphology and high functional activity (nuclear transplantation and electron microscopical studies). Cryo-Lett. 12, 87–92 (1991). Lawrence, P. A., and Johnston, P. Observation on cell lineage of internal organs of Drosophila. J. Embryol. Exp. Morphol. 27, 69–83 (1986). MacFarlane, D. R., Forsyth, M., and Barton, C. A. Vitrification and devitrification in cryopreservation. In ‘‘Advances in Low-Temperature Biology,’’ Vol. 1, pp. 221–278. JAI Press, 1992. Mazur, P., Cole, K. W., Hall, J. W., Schreuders, P. D., and Mahowald, A. P. Cryobiological preservation of Drosophila embryos. Science 258, 1932– 1935 (1992). Omholt, S. W., Hagen, A., Elmholdt, O., and Rishovd, S. A laboratory hive for frequent collection of honeybee eggs. Apidologie 26, 297–304 (1995). Omholt, S. W., Rishovd, S., Hagen, A., Elmholdt, O., Dalsgard, B., and Fromm, S. Successful production of chimeric honeybee larvae. J. Exp. Zool. 272, 410–412 (1995). Peng, Y. S. C., Mussen, A., Fong, A., Montague, M. A., and Tyler, T. Effect of chlortetracycline of honey bee worker larvae reared in vitro. J. Invertebr. Pathol. 60, 127–133 (1992). Rembold, H., and Lackner, B. Rearing of honeybee larvae in vitro: Effect of yeast extract on queen differentiation. J. Apic. Res. 20, 165–171 (1981). Steponkus, P. L., and Caldwell, S. An optimized procedure for the cryopreservation of Drosophila melanogaster embryos. Cryo-Lett. 14, 375–380 (1993). Steponkus, P. L., Langis, R., and Fujikawa, S. Cryopreservation of plant tissues by vitrification. In ‘‘Advances in Low-Temperature Biology,’’ Vol. 1, pp.1–61. JAI Press, 1992. Sylvester, H. A. Biochemical genetics. In ‘‘Bee Genetics and Breeding’’ (T. E. Rinderer, Ed.), pp.177–203. Academic Press, New York, 1986. Yu, R., Hagen, A., and Omholt, S. W. Queen production from biopsied preblastoderm honeybee (Apis mellifera) embryos. [Submitted for publication] Zalokar, M. Transplantation of nuclei in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 68, 1539–1541 (1971).
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Cryopreservation of Totipotent Nuclei from Honeybee (Apis mellifera) Embryos by Rapid Freezing Yu Ronglin, Arne Hagen, and Stig W. Omholt1 Department of Animal Science, Agricultural University of Norway, P.O. Box 5025, 1432 AAS, Norway Here we report a method to cryopreserve totipotent preblastoderm nuclei from honeybee (Apis mellifera) embryos by rapid freezing without the addition of a specific cryoprotectant or other additive. By making feasible long-term storage of ooplasm biopsied from preblastoderm embryos, the method could become a key element for the development of an efficient and practical procedure for cloning and other genetic studies of honeybees. Donor ooplasm to be cryopreserved was extracted from the anterior pole of 8.0- to 9.0-h preblastoderm embryos with a transplantation pipet. The anterior end of the pipet was plugged with a small ball of cotton fiber, before it was fastened with a tape to a fine iron wire and plunged into liquid nitrogen. After being stored in liquid nitrogen for at least 24 h, the pipet was thawed rapidly in a water bath at 257C. The ooplasm was injected at the anterior pole of recipient embryos (0.5 to 3.5 h) at room temperature and at a relative humidity ú70%. Chimerism was assayed on 3-day larvae by making use of the polymorphic malate dehydrogenase locus and isoelectric focusing. Of 618 recipient embryos injected with cryopreserved nuclei, 157 (25.4%) hatched into larvae. Of the 157 larvae, 16 were chimeras carrying both donor and recipient genotypes and 2 expressed only the donor genotype and thereby were presumed to be embryo clones. The chimeric frequency (11.5%) was 85% of the frequency achieved when using noncryopreserved nuclei and the same transplantation protocol. q 1997 Academic Press
To develop an efficient embryo cloning technology of practical use for honeybees (Apis mellifera), long-term storage of ooplasm biopsied from preblastoderm embryos has to be mastered. As far as we know, such a method has not been developed for any insect. The honeybee egg is about 1.6 to 1.8 mm long, with a maximum diameter of 0.35 mm, which gives an egg volume of about 135 nl (about 10 times that of a Drosophila egg (1)). The outer and inner egg layers (chorion and vitelline membrane) are about 0.1 and 0.25 mm thick, respectively. The early development of the embryo involves 10 synchronous cleavage mitoses where the resulting nuclei migrate into the peripheral egg layer during a parasynchronous 11th mitosis (8 to 9 h). Thereafter, a peripheral cell layer, the blastoderm, is formed, as is the case for nearly all pterygote insects (5). The complete embryogenesis is amenable to direct observation, as honeybee
embryos become highly transparent when placed in paraffin oil, resolving individual cells and even nuclei (4). The developmental period is normally 72 h at 357C. Incubation of nonmanipulated eggs above a layer of 21% H2SO4 (w/w) in a closed plastic chamber normally yields a hatching rate above 90% (unpublished results). The larvae may be reared to adult workers and queens by in vitro feeding with semiartificial diets (13, 14). The usefulness of this technique is still somewhat restricted as the morphological variation of sister queens or workers reared by in vitro feeding is considerably higher than in normally reared sister groups (unpublished results). However, it works very well to feed larvae hatching from manipulated eggs for 1 to 2 days in vitro and then produce queens from these larvae by standard queen-rearing techniques (12, 18). An efficient nuclear transplantation methodology for honeybee embryos was recently described by Omholt et al. (12), and it was shown that nuclei from 8- to 9-h preblastoderm embryos are totipotent. Here we report a method to cryopreserve such totipotent pre-
Received July 22, 1996; accepted April 5, 1997. 1 To whom correspondence should be addressed. Fax: /47 64947960. E-mail: [email protected]. 41
0011-2240/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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RONGLIN, HAGEN, AND OMHOLT
blastoderm nuclei by rapid freezing without The rear end of the pipet was plugged with the addition of a specific cryoprotectant or a small ball of cotton fiber before it was fasother additive. tened with tape to a fine iron wire and plunged Combined with the fact that biopsied em- into liquid nitrogen. After being stored in liquid bryos develop into normal honeybee queens nitrogen for at least 24 h, the pipet was thawed (18), we are now in principle able to test the rapidly in a water bath at 257C. After removal breeding, developmental, and behavioral po- of the cotton plug and mounting the pipet to the tential of honeybee genomes stored in liquid micromanipulator, the ooplasm was injected at nitrogen. Though an efficient embryo cloning the anterior pole of recipient 0.5- to 3.5-h emtechnology for honeybees is still not within bryos (containing 1 to 4 nuclei) previously prereach, we view this result as a notable step pared for injection. This was done by mounting toward this goal. In addition, until cryopreser- 10 plastic cell cups with recipients (11) on a vation of honeybee embryos is mastered, as strip of modeling clay on the back side of a is already the case for Drosophila (10, 15), 90-mm petri dish (counted as one series) and our cryopreservation method may be used for keeping the petri dish at 357C and relative hulong-term storage of mutant honeybee strains midity ú80% until the injections started. The obtained by natural means or by genetic trans- injections were done at room temperature (20 to 307C) inside a moist plastic chamber beneath formation. the stereo microscope (relative humidity MATERIALS AND METHODS ú70%). The chamber was made such that the The experiments relied on a continuous eggs on the petri dish could be properly oriensupply of eggs throughout the year from colo- tated with the left hand before each injection, nies kept in a flight room with appropriate while the micromanipulator could be operated light conditions and where the temperature from the outside with the right hand. and the humidity are strictly controlled (12). The aspirations and transplantations of The eggs are sampled from small hives con- ooplasm were performed with an Oxford mitaining a queen and about 2500 workers. The cromanipulator (Singer Instrument Co., Roadhives are designed such that the eggs laid by water, Somerset, England), a microinjector the queen can be sampled from the outside. (PLI-100, Medical Systems Corp., Greenvale, Without this type of hive it is extremely diffi- NY, USA), and an ordinary stereo microcult to obtain eggs of a given age at appro- scope. We normally operated with an injection priate amounts (11). time of 0.6 s, an injection pressure of 8.0 kPa, Donor ooplasm to be cryopreserved was ex- and a balance pressure in the pipet of 3.0–4.0 tracted from the anterior pole of 8.0- to 9.0-h kPa. The average amount injected into each embryos with a transplantation pipet made embryo was estimated to be 2 to 4 nl or 1.5 from a borosilicate capillary tube (o.d. Å 1.0 to 3.0% of the total egg volume (4). mm, i.d. Å 0.58 mm) (Sutter Instrument Co., Embryos were incubated for 70–73 h at Novato, CA, USA). The tip was beveled to an 357C above a layer of 21% H2SO4 (w/w) in angle of 157 with a K. T. Brown Type Micropi- closed plastic chambers, until hatching. All larpet Beveler (Sutter Instrument Co.) and then vae from each series (i.e., max 10) were placed forged with a laboratory-made microforge. The in a small plastic cap (12 1 3 mm) filled with inner diameter of the pipet tip was 16 to 18 0.4 ml food mixture, ensuring worker bee demm. The amount of donor ooplasm extracted velopment (13), and incubated at 357C above from each donor embryo varied somewhat, but a layer of 7% H2SO4 (w/w) in a closed plastic we tried to keep it in the range 50 to 80 nl. chamber. The larvae were given fresh preThese volumes were based on previous calcula- warmed food mixture twice a day for 3 days, tions of pipet volumes estimated by measure- whereupon they were placed in separate Epments of the inner diameter of the pipets and pendorf tubes and stored at 0207C. This temthe length of column of ooplasm. perature seems to be sufficiently low to prevent
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damage of the protein used for assaying chimerism for several months (see below). Normally about 80% of the hatched individuals develop into 3-day larvae by following this procedure. However, the optimal time window for harvesting larvae after hatching is rather narrow (about 1h), and collecting the larvae too early or too late increases the mortality dramatically (data not shown). We allowed some remnants of the diet to stay on the larvae as previous tests have shown that this food mixture does not contain any detectable amount of the enzyme used for assaying chimerism (12). To determine chimerism, we assayed the polymorphic malate dehydrogenase (Mdh) locus, where three alleles (a, b, c) have been identified in larvae and adult worker bees (17). Isoelectric focusing (IEF) was used to determine genotypes. Each larva was homogenized in a small volume of distilled and deionized H2O (20 to 150 ml depending on the size of the larva), and after removing particulate material by centrifugation (14000g, 10 min, 47C), 20 ml of the extract was loaded onto an IEF gel (pH 3.5 to 9.5). After isoelectric focusing at 25 W constant power for 75 min at 107C on a Multiphor II electrophoresis system (Pharmacia LKB Biotechnology, Sweden), Mdh activity was assayed by incubating the gel at 377C for 3 h in 100 ml 0.1 M Tris–HCl buffer (pH 8.3), containing 12 mg nitroblue tetrazolium (NBT) (Sigma), 16 mg phenazine methosulfate (PMS) (Sigma), 24 mg nicotinamide adenine dinucleotide (NAD) (Sigma), and 24 mg malic acid (Sigma). Unrelated A. mellifera carnica queens with different Mdh genotypes were bred from screened queens from ordinary production colonies and artificially inseminated with semen of appropriate genotype. In this way we obtained four different genotypes (aa, ab, bc, and cc), which were used as donors as well as recipients. RESULTS AND DISCUSSION
Of 618 recipient embryos injected with cryopreserved nuclei, 157 (25.4%) hatched and developed into 3-day larvae. Of the 157 larvae, 16 were chimeras carrying both donor
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FIG. 1. A representational diagram of the typical IEF banding pattern of the Mdh assay. As the active enzyme is a dimer, the homozygote aa constitutes a single band on an IEF gel (lane 1), while, for example, the heterozygote bc in lane 3 expresses three bands (bb, bc, and cc). Lanes 1 to 4 show the expression patterns of the genotypes used as donors and recipients. Lanes 5 to 8 show typical chimeric expression patterns from the genotype combinations aa 1 cc, aa 1 bc, ab 1 bc, and ab 1 cc. The column to the right shows the positions of the various dimers. The positions of the dimers will be as depicted when the negative pole of the IEF field is at the top.
and recipient genotypes and 2 expressed only the donor genotypes, and were hence presumed to be embryo clones (12) (Fig. 1). This gives a chimeric frequency of 11.5%, which is 85% of the frequency obtained when using noncryopreserved nuclei (Table 1). When using fresh ooplasm our results are similar to those obtained on Drosophila with regard to hatching rate of manipulated embryos and frequency of chimeras (8). Considering the extra problems associated with using toxic cryoprotectants that have to be removed immediately after thawing (16), and stabilization of the nuclei (6), our method seems to offer some benefits by its operational simplicity. In a preliminary study using ethylene glycol as a cryoprotectant we experienced several of the problems associated with this. Fluorescent staining of these nuclei showed that many had a different shape compared to controls (39%), and some of these were of different size as well as showing loss of membrane integrity. Of 1135 recipients, only 96 larvae hatched and survived 3 days of in vitro feeding. Of these, 4 larvae expressed both donor and recipient genotypes and 1 expressed only the donor genotype. Thus, the overall chimeric frequency with reference to total number of manipulated embryos was only 15% of what was achieved by the other
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RONGLIN, HAGEN, AND OMHOLT TABLE 1 Chimeras Obtained by Nuclear Transplantation of Fresh and Cryopreserved Ooplasm
Ooplasm
Eggs injected
Larvae hatching/3day larvae
Larvae expressing recipient genotype only
Larvae expressing recipient and donor genotypes
Larvae expressing donor genotype only
Fresh Cryopreserved
830 618
214/89* 197/157
77 139
10 16
2 2
Note. The data for fresh ooplasm are taken from a previous report (12). The asterisk denotes that only a fraction of the surviving larvae was analyzed in this case.
method. However, we suspect that an appropriate buffer was not used in this experiment, and this may explain the low success ratio. For real long-term storage it may be necessary to seal the pipet containing ooplasm more properly than we did or to use other devices to store the ooplasm, but this should represent only a minor technical challenge. Although a chimeric frequency which is 85% of that achievable with fresh ooplasm is encouraging, it may be possible to improve this figure by making use of nitrogen slush instead of liquid nitrogen (15) and by increasing the temperature of the water bath to 357C for thawing. Storage of the ooplasm in a better heat-conducting material than glass may also improve the result (15). It is reasonable to believe that every change of the transplantation protocol using fresh ooplasm that increases the frequency of chimeras and embryo clones will give the same relative increase with cryopreserved ooplasm. It should be noted that we made use of no cryoprotectants. Considering prevailing opinions reflected in the cryobiological literature (2, 9, 16), it seems to be somewhat counterintuitive that this should work as well as it does. However, an apparently analogous result was obtained by Kalinina and Gromov when transplanting cryopreserved nuclei of amoeba to noncryopreserved enucleated amoeba. They found that the cytoplasm was irreparably damaged by freezing, while minor ultrastructural damages of the nuclei were repaired after transferal (7). We made no detailed studies of the cryopreserved ooplasm to determine the degree of ice
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formation or whether the ooplasm was preserved in a structurally arrested state. However, some preliminary results were obtained by studying the structure of cryopreserved and noncryopreserved nuclei stained with 0.5 mg/ ml Hoechst 33342 (Sigma) in 0.85% NaCl. After incubation under a layer of paraffin oil at 357C for 30 min the two groups were compared with respect to nuclear shape and nuclear membrane integrity by using a Leitz Aristoplan microscope (Leitz Wetzlar, Germany). There were no observable differences in these parameters (data not shown). This indicates that at least low-resolution structural effects due to osmotic stress and intranuclear ice formation must have been very moderate (2). In principle, by combining a technique for making biopsies of preblastoderm honeybee embryos (18) and by letting these embryos develop into queens that are inseminated and tested for individual or colony level traits, we now seem able to test the breeding, developmental, and behavioral potential of honeybee genomes stored in liquid nitrogen. However, before this can be realized, we will have to develop efficient and nonharmful procedures for enucleation of recipient honeybee embryos before transplantation of cryopreserved ooplasm. This is technically rather challenging, and to the best of our knowledge this has not been achieved on Drosophila embryos either, despite the fact that a nuclear transplantation methodology has been available for more than 25 years (19). Assuming that we will be able to solve the enucleation problem, the next step will be to increase the survival rate of the injected embryos. The sur-
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RAPID FREEZING OF NUCLEI FROM HONEYBEE EMBRYOS
vival rate of embryos injected at the 0.5- to 2-h stage is considerably lower than those injected at 2 to 4.5 h of age (data not shown). We do not know the reason for this, but it may be that the physical damage caused by the penetration of the pipet at the anterior pole is greater for the very young embryo due to a more delicate and less resilient structure at this stage. This interpretation is partially supported by the fact that injection of a 0.9% salt solution into very young embryos with smaller pipets (3 to 4 mm) results in a survival rate above 85% (data not shown). This might imply that reducing the size of the transplantation pipets will increase the survival rate to an acceptable level. However, we have yet no data on how far down we can go before the nuclei are irreparably damaged during aspiration and transplantation. In a preliminary experiment we used centrifugation to concentrate the nuclei of 8- to 9-h donor embryos to a small band within the embryo, whereby we were able to aspirate ú500 nuclei into the pipet. The nuclear shape and membrane integrity were compared with nuclei taken from unmanipulated embryos of the same age by using a Leitz Aristoplan microscope (Leitz Wetzlar, Germany), and we did not observe any differences (data not shown). However, we were not able to significantly increase the chimeric frequency, despite the fact that each embryo in this case received on average many more nuclei. This indicates that the number of nuclei may not be as critical as the synchronization of the mitotic stage of recipient and donor (3). REFERENCES
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