Recent research activity

Surrogate propagation of fish

　The term, surrogate, is usually used as “surrogate mother” in reproductive medicine.  Surrogate mother is a mother who bears a child on behalf of another woman by the implantation in her womb of a fertilized egg from the other woman.  The word surrogate is also used as “surrogate production” in the field of animal husbandry.  For example, fertilized eggs from beef cattle that have high market value are transplanted into cows to induce milk production as well as beef cattle baby.  Surrogate production in the aquaculture can also be possible by applying similar technique to the teleost fish. 

Surrogate production in teleost fish is achieved by inducing the germ-line chimeras.  Primordial germ cells (PGCs) are the germ cells before their arrival at the genital anlage during embryonic stage.  To make germ-line chimeras, the PGCs are isolated and transplanted into the embryo of other species.  The hosts become germ-line chimeras if the transplanted PGCs migrated to genital anlage and differentiated into functional gametes.  In consequence, donor genotypes are restored into the next generation.  The productivity of fish seed is expected to increase by surrogate production using germ-line chimeras by two different species whose biological properties are different each other.  For example, life cycle of the fish could be extremely shortened if the surrogate parents that have shorter life cycle are produced. 

Germ-line chimeras in teleost fish are induced by the following methods: (1) blastomere transplantation, (2) transplantation of a blastoderm graft, and (3) transplantation of isolated PGCs.  In blastomere transplantation, blastomeres at the blastula stage are randomly sucked by glass micro-needle, and transplanted into the embryo of other species at the same stage.  In this method, donor cells do not always contain PGCs due to the random separation of cells from donor blastoderm.  In the transplantation of a blastoderm graft, lower part of the blastula blastoderm, in which PGCs are contained, is transplanted into the host embryo.  In this method, germ-line chimeras can be always induced.  In both cases, however, the donor cells do not always contribute the development of resultant chimeric embryos, because selective aggregation of donor cells frequently occurs.

More effective method is the transplantation of isolated early germ cells, such as oogonia and spermatogonia.  In salmonids, germ-line chimeras are successfully induced, when the germ cells at early stage isolated from the ovary and testis are transplanted into the abdominal cavity of the hatched embryos.  In this case, functional gametes developed from donor germ cells were obtained from the host parents.  

In this Station, surrogate production has been studied by using PGCs from many kinds of teleost fish species.

Procedures inducing germ-line chimera

　Germ-line is induced by transplantation of early stage germ cells into host embryos.  These germ cells are required to maintain motile activity to host gonad and developmental potency to gametes.  Therefore, PGCs and gonia are appropriate as donor cells to induce germ-line chimera.

Isolated PGCs and germ cells are transplanted into host embryo by using micro-glass needle under the stereomicroscope.  The size of PGCs is about 10 to 20 mm in diameter.  The position in blastoderm where PGCs are transplanted is important factor for successful migration of PGCs to host gonad. Therefore, the transplantation is important, very skillful and laborious work.  PGCs from different kinds of teleost species, such as eel, flatfish, and sturgeon, have migration activity to the gonad of zebrafish or goldfish. 

PGCs migrated successfully to host gonad do not always differentiate to germ cells, because of intervention or non-support between donor germ cell and host somatic cells.  Functional gametes are induced in interspecific donor and host combinations, while only functional sperm in interfamilial.  It is difficult to differentiation functional sperm in inter-order transplantation, because of extinction of donor PGCs during subsequent development.

Visualization and isolation of teleost PGCs　

PGCs differentiate at early stage of development in teleost fish.  As difficulty to distinguish them from other somatic cells in early embryo, visualization of PGCs is required for their isolation and implantation.  Injection of the artificial mRNA, in which GFP sequence is conjugated with zebrafish nos1 3’ UTR to the fertilized eggs can induce PGC visualization after the somitogenesis stage.  We have succeeded to visualize PGCs in 17 fish species, such as herring, loach and ice goby, belonging to 6 order, using this technique.  Visualized PGCs are able to isolate from somatic cells by using cell-sorter. 

When the visualized PGCs were isolated and transplanted into hetero-chronous host blastula, donor PGCs migrated to genital anlage in the chimeras between loach and goldfish (inter-familial transplantation).  However, more detailed studies on molecular mechanisms of PGCs differentiation and migration is required to introduce the donor PGCs into xenogeneic host genital anlage. 

Functional gametes from donor PGCs can be intra-specifically obtained through germ-line chimeras in many teleost.  Functional eggs were also obtained from inter-subspecific chimeras induced by graft transplantation of blastoderm between diploid goldfish and triploid crucian carp.  Sperm production in the inter-specific chimeric fish between donor masu salmon and host rainbow trout was also reported.

　
Cryopreservation and vitrification of PGCs

　Today genetic diversity decreases rapidly.  It is important to preserve PGCs.  As PGCs contain genetic factors in cytoplasm, diversity of mitochondrial DNAs is also preserved.  PGCs preservation means that of genetic diversity.  Genetic diversity also decreases during pedigree preservation necessarily.  Genetic properties in existence will be useful order to improve genetically future strains and to induce through germ-line chimera whenever.

Collection of genetic diversity from natural population

　If PGCs are isolated from larva, we may be able to collect large genetic diversity from natural population in spawning area.  Many marine species spawn large number of eggs around spawning area, but almost all offspring die during early stage of development.  Therefore, collection of PGCs around this stage does not damage the natural genetic resource.  Moreover, genetic diversity of this stage is larger than that of individuals at later stages.  Individuals that are not at all afraid of men, are very useful for aquaculture, but are difficult to survive in natural condition.  But it is not performed yet to isolate PGCs from larva collected offshore.  It is important to elucidate basic biological properties of PGCs, because of establishment of the new isolation technique of them.

Analysis of biological property of PGCs

　As described above, all donor PGCs do not always move to gonadal area in host embryos and differentiate in host gonad.  The migration pathway and differentiation of PGCs are not always clear in many teleost fish species yet.  Basic properties of PGCs in many kinds of teleost fish are required for establishment of surrogate propagation techniques.  Now, migration pathway has been analysis in about 20 species, including barfin flounder, loach, ice-goby, rainbow smelt, herring, eel and sturgeon.

Sterilization of host germ cells

　In the germ-line chimeras, however, it is impossible to avoid differentiation of gametes derived from host PGCs; two types of gametes, one from the donor and the other from the host are obtained.  This is not efficient for the surrogate production.  Endogenous PGCs should be removed by artificial sterilization of the host.  It has been reported that sterile fish are induced by hybrid crossing between two different species.  For example, hybrid males between goldfish and common carp are completely sterile.  Applying this technique, only sperms from the donor were obtained from the germ-line chimeras, in which goldfish PGCs were transplanted into the host blastula of the hybrid between goldfish and common carp.  Inhibition of differentiation by morpholino treatment is also effective to inhibit host PGCs differentiation in surrogate production.

Fusion technique between surrogate production and other bio-technology

　In early stage of development, PGCs are only cell to differentiate gametes thorough germ-line chimera.  Modified changes in are confirmed phenotypically in the next generation.  Therefore, artificial modification in PGCs will produce individuals with new properties.  For example, tetraploid PGCs may be induced by cell-fusion technology, resulting in diploid gametes.  These diploid gametes are useful for triploid and amphidiploid induction by normal fertilization and that followed by inhibition of second maturation division.  Now, tetraploid PGCs have not induced yet, because it is difficult to fuse a single pair of PGCs.  A new idea is required.

Conclusion

　In Japan, many teleost fish are used as food.  The diversity in fish used is wider than that in domestic animals.  In the future, several investigations are required in the following points; 1) improvement of PGC isolation technique, 2) enhancement of PGC integration efficiency into host genital anlage, 3) investigation of xenogeneic host which enabled donor PGCs to differentiate, and 4) sterilization of host endogenous PGCs.

　

Manipulation apparatus and techniques

　In this section, we introduce our techniques and apparatus for manipulation of fish embryos, using Nanae Fresh-Water Laboratory.  In many case, goldfish is used as the material.  Please check the references described in each section, too, if you want to know the detailed procedures.

Artificial fertilization

　Embryos are prepared by artificial fertilization, because of synchronized development and subsequent treatments, such as dechorionation.  It is important for cell and blastoderm transplantation to remove the chorion.  Dechorionation procedures are described below.  Quality of eggs is also important for subsequent development, but it depends on many factors, such as cultivation of maternal fish and treatments from ovulation.
　Eggs are striped onto the appropriated sheet or bowl by gentle pressing from ovulated maternal fish.  Sperms are collected into the capillary tube in case of small fish, or to the small Falcon tubes in case of large fish.  It is recommended that collected sperm is diluted with artificial seminal plasm and kept in the refrigerator or on ice before use.  Dilution prolongs the sperm motility.  In goldfish, sperm collected in the micro-capillary tube loses their activity for several hours, but it keeps the activity for one week under diluted and lower temperature conditions.  Eggs are inseminated with sperm and fertilized by adding appropriate medium for fertilization.
　In the case of goldfish, eggs are collected onto the polyethylene or other film and sperm is collected into the micro-capillary tube and diluted about 50-100 times with artificial seminal plasm (5.61 g NaCl, 5.23 g KCl, 0.33 g CaCl2・2H2O, 0.22 g MgCl2・6H2O, 0.2 g NaHCO3, diluted with 1 litter DDW).

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Dechorionation

　Chorion of fertilized eggs should be removed by appropriate treatment.  Eggs are inseminated with sperm and fertilized by adding appropriate medium for fertilization.  In the case of loach and zebrafish, eggs are fertilized in tap water, while in goldfish, inseminated eggs are fertilized in tap water containing 0.2% urea and 0.24% NaCl, because of removal of adhesive materials around surface of chorion.  Controls of pH condition or physical treatment of fertilized eggs are occasionally required before dechorionation in other teleost species.  We can remove chorion of fertilized eggs in cyprinid fish, a few smelt fish, salmonid fish with thin chorion, European cat-fish and herring by proteolytic enzymes with several modifications.
　Dechorionation are performed by treatment of proteolytic enzymes, such as trypsin, pancreatin, actinase and so on.  In cyprinid species, dechorionation is successively performed by treatment with trypsin solution, containing small amount of urea in some case.  In many case, the treatment should be begun as soon after fertilization as possible.  In goldfish, it takes about 10 min within 4 hours after fertilization, but 2 or 3 hours treatment are required in the embryos after 1 day.
卵膜除去の過程

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Manipulation apparatus

　Denuded eggs without chorion are fragile to manipulate.  Therefore, we handmake special apparatuses for manipulation, such as a dissecting needle, transplant-needle, culture plate and so on.
　Dissecting needle is shown in Figure.  Thin part of Pasteur pipette is melted by fire of fine gas burner and extended manually.  Cut the extended part at appropriate position and melt the tip of it to soften the surface.  For blastoderm transplantation, thinner glass wool is attached to the tip of the dissecting needle with manicure, and cut it leaving 1 to 2 mm long.
　Agar-coated petri dishes are used for manipulation and cultivation of dechorionated eggs, without injuring them.  About 0.8 to 1 % of agar is dissolved in Ringer’s solution at boiling temperature, and poured onto the level petri-dish about 1 mm in thick.  Glass petri dish with flat surface is recommended, because agar is easily removed after experiment.  Dishes can be kept in refrigerator for weeks.  The dish with thick agar-coat is useful at photographing the spherical or motile embryo.  Embryos are able to be hold in the small slit or hole in agar-coat.

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Chromosome set manipulation

　Chromosome set manipulation is useful techniques for improvement of genetic combination, induction of sterile fish and so on.  There are two directions of the treatments, namely reducing and increasing the genome size.  Haploidy is induced for reducing the genome size, and polyploidization is performed for increasing the genome size.  Haploid individuals are induced by fertilization between normal gamete and genetically inactivated gametes.  Polyploid individuals are induced by fertilization followed with the treatment of the second maturation division or the first cleavage.  Hydrostatic pressure is used for inhibition of the second maturation division in salmonid and sturgeon fishes, and temperature treatments in cyprinid fish.

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Blastoderm transplantation

　In cyprinid fish, especially goldfish, operation of blastodisc is easily performed at the blastula stage.  Blastodisc is cut by glass-wool attached to needle, described above, in the agar coated petri-dish.  During operation, embryo is settled on the agar-plate filled with Ringer’s solution containing with 1.6% albumen.  It is important for blastodisc operation to add albumen to Ringer’s solution.  In plain Ringer’s solution without albumen, blastodisc scatters from the cut end during operation, and it takes longer time to heal the wound around the attached ends of transplanted part.
　During operation, embryo should be softly held by fine-forceps or something else not to move.  To prepare the graft, glass-wool knife on the desired point of blastodisc is moved vertically down toward the agar plate, and then, pushing to the surface of agar, is moved slowly horizontally.  Soft agar gel (about 0.8%) is recommended in this operation.  At the same time, the enveloping layer of host blastodisc is removed.  Graft should be transplanted onto the prepared cut end of other blastodisc within one min.  As time go on, adhesion of the cut ends grew gradually becomes difficult.  The cut end does not attached to the surface of blastodisc with enveloping layer.

廃盤移植の過程

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Single cell transplantation (SPT)

　A single cell is picked up the injection needle, hold inside moved to host embryo and inserted to the host embryo.  Transplantation of single cell is very difficult, because picking up, holding in the needle and transplantation of single depend on the many conditions, such as needle (opening size of tip and constriction), injector, water temperature, cell size and adhesion, mental condition and so on. It is highly skillful.
　We use the needle with small constriction in hole size and small opening at the tip.  At first, petri dish filled without agar coating and manipulator with micro-needle are installed under the fluorescent stereo-microsocope.  Dissociated cells and host embryos are scattered in the petri dish.  As the cells adhere to the matrix of petri dish, some coating is required on the surface.  The procedure of single cell transplantation is quite complicated, and, therefore, described as the steps, as follows.

1)     At higher level from the bottom of the petri dish, move the tip of the micro-needle to the center of focus with low magnification.

2)     Move petri dish to the position at the cells distributed.

3)     Check the focus in the cells with high magnification.   

4)     Move down the tip of the needle to the cell level.

5)     Move the tip to the objective cells and suck into the needle with high magnification.

6)     Move the tip to the host embryo with low magnification, then transplanted into blastoderm.

Needles for microinjection and blastomere transplantation

　We use four types of manipulation needles, such as 1) simple pulled needle, 2) needle with constriction, 3) needle which tip was sharpened with large opening, and 4) needle with constriction and small opening.  We make all needles from Drammond micro-caps (50 ml) as material.

プーラーによる胚操作用針の作成

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Blastomere transplantation (ＢＴ)

　Blastomere transplantation at the blastula stage was firstly reported by Lin et al. (1993) in zebrafish.  We modified their procedures of transplantation.  In this operation, glass needle with wide opening at the tip and with constriction is recommended.  The inside of the glass needle is filled with Ringer’s solution and also coated with chicken albumen or yolk of fish egg used, not to adhere with blastomeres.  During operation, donor and host embryos are settled on the agar-plate filled with Ringer’s solution.  Holding the donor embryo with holding needle, the tip of transplantation needle is inserted into the blastodisc.  The donor blastomeres are sucked and collected into the transplantation needle.  When pulled out the tip of the needle from donor blastodisc, collected blastomeres moved slowly into inside and stopped in front of the constriction.  Then the needle is inserted into the host, and donor blastomeres are transplanted.

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