Nucleotide Sequence and Chromosome Mapping of 5S Ribosomal DNA from the Dojo Loach, Misgurnus anguillicaudatus.

There are 2 genetically divergent groups in the dojo loach Misgurnus anguillicaudatus: A and B. Although most wild-type diploids reproduce sexually, clonal diploids (clonal loach) reproduce gynogenetically in certain areas. Clonal loaches produce unreduced isogenic eggs by premeiotic endomitosis, and such diploid eggs develop gynogenetically following activation by the sperm of sympatric wild-type diploids. These clonal loaches have presumably arisen from past hybridization events between 2 different ancestors. The genomic differences between these 2 groups have not been completely elucidated. Thus, new genetic and cytogenetic markers are required to distinguish between these 2 groups. Here, we compared the 5S rDNA region to develop markers for the identification of different dojo loach groups. The nontranscribed sequence (NTS) of the 5S rDNA was highly polymorphic and group-specific. NTSs were found in clades of 2 different groups in clonal loaches. In contrast, we did not find any group-specific sequences in the coding region of the 5S rRNA gene. Sequences were located near the centromere of the short arm of the largest submetacentric chromosomes in groups A and B and clonal loaches. Thus, the 5S rDNA of the dojo loach is conserved at the chromosomal location. Whereas, the sequences of the NTS regions evolved group-specifically in the dojo loach, with the sequences of both groups being conserved in clonal loaches.

FISH Identifies Chromosome Differentiation Between Contemporary Genomes of Wild Types and the Ancestral Genome of Unisexual Clones of Dojo Loach, Misgurnus anguillicaudatus.

In dojo loach (Misgurnus anguillicaudatus), although most wild types are gonochoristic diploids that are genetically differentiated into 2 groups, A and B, clonal lineages appear in certain localities. Clonal loaches have been considered to have hybrid origins between the 2 groups by a series of genetic studies. In this study, using FISH with a newly developed probe (ManDra-A), we identified 26 (1 pair of metacentric and 12 pairs of telocentric chromosomes) of 50 diploid chromosomes in contemporary wild-type group A loach. In contrast, ManDra-A signals were not detected on metacentric chromosomes derived from the ancestral group A of clonal loach. The FISH results clearly showed the presence of certain differentiations in metacentric chromosomes between ancestral and contemporary group A loach. Two-color FISH with ManDra-A and group B-specific ManDra (renamed ManDra-B) probes reconfirmed the hybrid origin of clones by identifying chromosomes from both groups A and B in metaphases. Our results showed the hybrid origin of clonally reproducing fish and the possibility that chromosomal differentiation between ancestral and contemporary fish can affect gametogenesis. In meiotic spermatocytes of sex-reversed clones, ManDra-A, and not ManDra-B, signals were detected in 12 out of 50 bivalents. Thus, the results further support the previous conclusion that clonal gametogenesis was assured by pairing between sister chromosomes duplicated from each ancestral chromosome from group A or B. Our study deepens the knowledge about the association between clonality and hybridity in unisexual vertebrates.

Developmental potential of somatic and germ cells of hybrids between Carassius auratus females and Hemigrammocypris rasborella males.

The cause of hybrid sterility and inviability has not been analyzed in the fin-fish hybrid, although large numbers of hybridizations have been carried out. In this study, we produced allo-diploid hybrids by cross-fertilization between female goldfish (Carassius auratus) and male golden venus chub (Hemigrammocypris rasborella). Inviability of these hybrids was due to breakage of the enveloping layer during epiboly or due to malformation with serious cardiac oedema around the hatching stage. Spontaneous allo-triploid hybrids with two sets of the goldfish genome and one set of the golden venus chub genome developed normally and survived beyond the feeding stage. This improved survival was confirmed by generating heat-shock-induced allo-triploid hybrids that possessed an extra goldfish genome. When inviable allo-diploid hybrid cells were transplanted into goldfish host embryos at the blastula stage, these embryos hatched normally, incorporating the allo-diploid cells. These allo-diploid hybrid cells persisted, and were genetically detected in a 6-month-old fish. In contrast, primordial germ cells taken from allo-diploid hybrids and transplanted into goldfish hosts at the blastula stage had disappeared by 10 days post-fertilization, even under chimeric conditions. In allo-triploid hybrid embryos, germ cells proliferated in the gonad, but had disappeared by 10 weeks post-fertilization. These results showed that while hybrid germ cells are inviable even in chimeric conditions, hybrid somatic cells remain viable.

Roles of maternal wnt8a transcripts in axis formation in zebrafish.

In early zebrafish development, the program for dorsal axis formation begins soon after fertilization. Previous studies suggested that dorsal determinants (DDs) localize to the vegetal pole, and are transported to the dorsal blastomeres in a microtubule-dependent manner. The DDs activate the canonical Wnt pathway and induce dorsal-specific genes that are required for dorsal axis formation. Among wnt-family genes, only the wnt8a mRNA is reported to localize to the vegetal pole in oocytes and to induce the dorsal axis, suggesting that Wnt8a is a candidate DD. Here, to reveal the roles of maternal wnt8a, we generated wnt8a mutants by transcription activator-like effector nucleases (TALENs), and established zygotic, maternal, and maternal zygotic wnt8a mutants by germ-line replacement. Zebrafish wnt8a has two open reading frames (ORF1 and ORF2) that are tandemly located in the genome. Although the zygotic ORF1 or ORF2 wnt8a mutants showed little or no axis-formation defects, the ORF1/2 compound mutants showed antero-dorsalized phenotypes, indicating that ORF1 and ORF2 have redundant roles in ventrolateral and posterior tissue formation. Unexpectedly, the maternal wnt8a ORF1/2 mutants showed no axis-formation defects. The maternal-zygotic wnt8a ORF1/2 mutants showed more severe antero-dorsalized phenotypes than the zygotic mutants. These results indicated that maternal wnt8a is dispensable for the initial dorsal determination, but cooperates with zygotic wnt8a for ventrolateral and posterior tissue formation. Finally, we re-examined the maternal wnt genes and found that Wnt6a is an alternative candidate DD.

Aberrant Meiotic Configurations Cause Sterility in Clone-Origin Triploid and Inter-Group Hybrid Males of the Dojo Loach, Misgurnus anguillicaudatus.

Gonochoristic wild-type dojo loaches (Misgurnus anguillicaudatus) are diploid (2n = 50) and reproduce bisexually. However, sympatric clonal diploids generate unreduced diploid isogenic eggs that develop gynogenetically. Clone-origin triploidy arises following the incorporation of a haploid wild-type sperm nucleus into the diploid egg. Triploid females produce fertile haploid eggs by meiotic hybridogenesis, while triploid males are sterile. Clonal loaches arose from past hybridization event(s) between genetically diverse groups, A and B. Artificial hybrid females between the 2 groups produce unreduced and/or aneuploid eggs, but the hybrid males are sterile. In this study using FISH, we analyzed chromosome pairing in meiotic cells of clone-origin triploid and inter-group hybrid males to clarify the cytogenetic mechanisms underlying the male-specific sterility. We used a repetitive sequence probe to identify group B-derived chromosomes and a 5.8S + 28S rDNA probe to identify pairs of homologous chromosomes. We found that asynapsis and irregular synapsis occur in triploid and hybrid males containing 2 different genomes and that this may cause the formation of sterile germ cells. These results will help us to understand hybrid sterility from the viewpoint of synapsis behavior.

Clonal reproduction assured by sister chromosome pairing in dojo loach, a teleost fish.

Wild-type dojo loach (Misgurnus anguillicaudatus) commonly reproduces bisexually as a gonochoristic diploid (2n = 50), but gynogenetically reproducing clonal diploid lines (2n = 50) exist in certain districts in Japan. Clones have been considered to develop from past hybridization event(s) between two genetically diverse groups, A and B, within the species. Fluorescence in situ hybridization analyses using the repetitive sequence "ManDra" as a probe clearly distinguished 25 chromosomes derived from group B out of a total of 50 diploid chromosomes of the clone, providing strong molecular cytogenetic evidence of its hybrid origin. In meiosis, diploid wild-type showed 25 bivalents, while diploid clones revealed 50 bivalents, indicating the presence of 100 chromosomes. In meiotic chromosome spreads in sex-reversed clonal males, ManDra signals were detected in 25 out of 50 bivalents, and only one out of two bivalents possessing major ribosomal RNA coding regions exhibited two positive ManDra signals. In clonal females, ManDra signals were detected in approximately 25 out of 50 bivalents. Thus, unreduced gametes should be generated by the pairing between sister chromosomes doubled from each ancestral chromosome from the different groups by premeiotic endomitosis. Sister chromosome pairing should assure production of unreduced isogenic clonal gametes due to the absence of the influence of recombination or crossing over.

Intra-ooplasmic injection of a multiple number of sperm to induce androgenesis and polyploidy in the dojo loach Misgurnus anguillicaudatus (Teleostei: Cobitidae).

SummaryPolyspermy was initiated by microinjecting a multiple number of sperm into the activated and dechorionated eggs of dojo loach Misgurnus anguillicaudatus (Teleostei: Cobitidae). A 10-nl sperm suspension from an albino (recessive trait) male (105, 106, 107 or 108 sperm ml -1) was microinjected into eggs from a wild-type female. Although the rates of embryos developing into the blastula stage in the injection group at the highest sperm concentration were similar to that of the control group, the hatching rates of the injection group were much lower. A large proportion of embryos that developed from the injected eggs was haploid and were mosaics containing haploid cells. Most of the haploid and mosaic embryos inherited only paternally derived alleles in the microsatellite markers (i.e. androgenesis was initiated by injecting multiple sperm). In contrast, some haploid embryos contained both paternal and maternal alleles despite haploidy, suggesting that they were mosaics consisting of cells with either paternal or maternal inheritance. The injected eggs displayed diploid, hypotriploid and triploid cells, all of which included both maternally and paternally derived alleles. One albino tetraploid with only paternal alleles was also observed from the injected eggs. These results suggested that part of the sperm microinjected into the ooplasm should form a male pronucleus/pronuclei, which could develop by androgenesis or could fuse with the female pronucleus/pronuclei. Therefore, microinjection of multiple sperm should be considered a potential technique to induce androgenesis and polyploidy.

Chemical and physical guidance of fish spermatozoa into the egg through the micropyle†,‡.

Eggs of teleost fish, unlike those of many other animals, allow sperm entry only at a single site, a narrow canal in the egg's chorion called the micropyle. In some fish (e.g., flounder, herring, and Alaska pollock), the micropyle is a narrow channel in the chorion, with or without a shallow depression around the outer opening of micropyle. In some other fish (e.g., salmon, pufferfish, cod, and medaka), the micropyle is like a funnel with a conical opening. Eggs of all the above fish have a glycoprotein tightly bound to the chorion surface around the micropyle. This glycoprotein directs spermatozoa into the micropylar canal in a Ca2+-dependent manner. This substance, called the micropylar sperm attractant or MISA, increases fertilization efficiency and is essential in herring. In flounder, salmon, and perhaps medaka, fertilization is possible without MISA, but its absence makes fertilization inefficient because most spermatozoa swim over the micropyle without entering it. The mechanism underlying sperm-MISA interactions is yet to be determined, but at least in herring the involvement of Ca2+ and K+ channel proteins, as well as CatSper and adenylyl cyclase, is very likely. In some other fish (e.g., zebrafish, loach, and goldfish), the chorion around the micropyle is deeply indented (e.g., zebrafish and loach) or it has radially or spirally arranged grooves around the outer opening of the micropyle (e.g., goldfish). MISA is absent from the eggs of these fish and sperm entry into micropylar canal seems to be purely physical.

Cryopreservation of zebrafish (Danio rerio) primordial germ cells by vitrification of yolk-intact and yolk-depleted embryos using various cryoprotectant solutions.

The aim of this study was to examine the effects of partial removal of yolk and cryoprotectant mixtures on the viability of cryopreserved primordial germ cells (PGCs) and elucidated the differentiation ability of cryopreserved PGCs in zebrafish. First, dechorionated yolk-intact and yolk-depleted (partially yolk removed) embryos, PGCs of which were labeled with green fluorescence protein (GFP), were vitrified after serial exposures to pretreatment solution (PS) and vitrification solution (VS) that contained ethylene glycol (EG), dimethyl sulfoxide (Me2SO) or propylene glycol at 3 and 5 M, respectively. Although partial removal of yolk improved the viability of cryopreserved PGCs, numbers of PGCs with pseudopodial movement were limited (0­2.6 cells/embryo). Next, yolk-depleted embryos were cryopreserved using mixtures of two types of cryoprotectants. The maximum survival rate of PGCs (81%; 9.6 cells/embryo) was obtained from the yolk-depleted embryos vitrified using PS containing 2 M EG + 1 M Me2SO and VS containing 3 M EG + 2 M Me2SO and 56% (5.3 cells/embryo) of PGCs showed pseudopodial movement. Finally, PGCs recovered from yolk-depleted embryos (wild-type) that were vitrified under the optimum condition were transplanted individually into 236 sterilized recipient blastulae (recessive light-colored). Seven recipients matured and generated progeny with characteristics inherited from the PGC donor. In conclusion, the authors confirmed the beneficial effects of partial removal of yolk on the viability of cryopreserved PGCs and that the viability of the PGCs was improved by using PS and VS that contained two types of cryoprotectants, especially PS containing 2 M EG + 1 M Me2SO and VS containing 3 M EG + 2 M Me2SO, and that recovered PGCs retained ability to differentiate into functional gametes.

Sexual dimorphism of gonadal structure and gene expression in germ cell-deficient loach, a teleost fish.

Germ cell-deficient fish usually develop as phenotypic males. Thus, the presence of germ cells is generally considered to be essential for female gonadal differentiation or the maintenance of ovarian structure. However, little is known of the role of germ cells in the determination of the sexual fate of gonadal somatic cells. We have established an inducible germ cell deficiency system in the loach (Misgurnus anguillicaudatus, Cypriniformes: Cobitidae), a small freshwater fish, using knockdown of the dead end gene with a morpholino antisense oligonucleotide. Interestingly, loach lacking germ cells could develop as either phenotypic males or females, as characterized morphologically by the presence or absence of bony plates in the pectoral fins, respectively. The phenotypic males and females had testicular and ovarian structures, respectively, but lacked germ cells. Gene expression patterns in these male and female germ cell-deficient gonads were essentially the same as those in gonads of normal fish. Our observations indicate that sexually dimorphic gonads can develop in germ cell-deficient loach. In contrast to the situation in other model fish species, the gonadal somatic cells in phenotypic females autonomously differentiated into ovarian tissues and also played a role in the maintenance of gonadal structure. On the basis of our observations, we propose two possible models to explain the role of germ cells in sex determination in fish.

Cold-shock eliminates female nucleus in fertilized eggs to induce androgenesis in the loach (Misgurnus anguillicaudatus), a teleost fish.

BACKGROUND: Androgenesis (all-male inheritance) is generally induced by means of irradiating the eggs to inactivate the maternal genome, followed by fertilization with normal sperm. In fish, the conventional technique for induced androgenesis has been applied for rapid fixation to traits, recovery of cryopreserved genotypes, sex-control, etc. A new method of androgenesis that eliminates the need to irradiate the egg was proposed using the loach, Misgurnus anguillicaudatus (a teleost fish). RESULTS: When the eggs of wild-type females were fertilized with sperm of albino or orange phenotype males and cold-shocked at 0 to 3°C for 60 min duration just after fertilization, generally more than 30% (with a peak of 100%) of the hatched progeny were androgenotes. While a few of them were the normal diploid, most of them turned out to be abnormal haploid. All-male inheritance was verified by the expression of the recessive color trait (albino or orange) and microsatellite genotypes comprising only paternally derived alleles. Nuclear behavior after the cold-shock treatment was traced by microscopic observation of DAPI (4'6-diamidino-2-phenylindole)-stained samples and hematoxylin-eosin stained histological sections, and the extrusion of egg (maternal) nucleus was observed in eggs treated in the optimum timing. CONCLUSION: In this paper, we demonstrate that cold-shock treatment (at 0 and 3°C) of loach eggs for 60 min just after fertilization successfully induces androgenetic haploid development. The most likely mechanism of cold-shock induced androgenesis is an elimination of the egg nucleus together along with the second polar body and subsequent development of a decondensed sperm nucleus or male pronucleus.

Diploid and aneuploid sperm in tetraploid ginbuna, Carassius auratus langsdorfii.

Ginbuna (Carassius auratus langsdorfii (Teleostei: Cyprinidae)) occur in diploid, triploid, and tetraploid forms in wild populations. Diploid females reproduce bisexually, whereas polyploid (triploid and tetraploid) females reproduce gynogenetically with no contribution from sperm nuclei. However, tetraploid males produce diploid sperm. The mechanism responsible for the differences in egg and sperm ploidy has not been elucidated as tetraploid males are rare in wild populations. Here, we aimed to characterize the types of sperm and elucidate the mechanism of spermatogenesis in ginbuna. In the present study, we artificially produced tetraploid males by crossbreeding triploid ginbuna females with diploid goldfish (Carassius auratusauratus) males via accidental incorporation of sperm nuclei. We then examined spermatogenesis to reveal the process by which reduced diploid sperm are generated from tetraploid germ cells. DNA fingerprinting by random amplified polymorphic DNA (RAPD)-PCR indicated that the tetraploid progeny had a paternally derived genome. For the tetraploid male sperm, there were narrow (N-type) and broad (B-type) flow cytometrical histograms. The N-type were determined to be diploid with a low coefficient of variation (CV) by flow cytometry. The B-type were found to be aneuploid (hypodiploid to hexaploid) with a high CV. The head sizes of B-type sperm were variable, whereas those of the N-type sperm were uniform. Computer-assisted sperm analysis (CASA) revealed that both the haploid and diploid B-type sperm were weakly motile compared with the haploid sperm of goldfish and the diploid N-type sperm of tetraploid males. Bivalents and various multivalents were observed in the meiotic configurations of diploid spermatogenesis. In aneuploid spermatogenesis, most of the chromosomes were unpaired univalents and there were very few bivalents. Our findings provide empirical evidence for two different types of spermatogenesis in tetraploid C. a. langsdorfii males. Meiotic synapses might explain the observed differences in the ploidy status of the two sperm types.

Control of Developmental Speed in Zebrafish Embryos Using Different Incubation Temperatures.

The zebrafish is a valuable model organism that is widely used in studies of vertebrate development. In the laboratory, zebrafish embryonic development is normally carried out at 28.5°C. In this study, we sought to determine whether it was possible to modify the speed of embryonic development through the use of short- and long-term variations in incubation temperature. After incubation at 20°C-32°C, most early-stage embryos survived to the epiboly stage, whereas more than half of the embryos died at <20°C or >32°C. The rate of development differed between embryos incubated at the lowest (18°C) and highest (34°C) temperatures: a difference of 60 min was observed at the 2-cell stage and 290 min at the 1k-cell stage. When blastulae that had developed at 28°C were transferred to a temperature lower than 18°C for one or more hours, they developed normally after being returned to the original 28°C. Analyses using green fluorescent protein-buckyball mRNA and in situ hybridization against vasa mRNA showed that primordial germ cells increase under low-temperature culture; this response may be of use for studies involving heterochronic germ cell transplantation. Our study shows that embryonic developmental speed can be slowed, which will be of value for performing time-consuming, complicated, and delicate microsurgical operations.

Production of fertile zebrafish (Danio rerio) possessing germ cells (gametes) originated from primordial germ cells recovered from vitrified embryos.

This study aimed to produce fertile zebrafish (Danio rerio) possessing germ cells (gametes) that originated from cryopreserved primordial germ cells (PGCs). First, to improve the vitrification procedure of PGCs in segmentation stage embryos, dechorionated yolk-intact and yolk-removed embryos, the PGCs of which were labeled with green fluorescent protein, were cooled rapidly after serial exposures to equilibration solution (ES) and vitrification solution (VS), which contained ethylene glycol, DMSO, and sucrose. Yolk removal well prevented ice formation in the embryos during cooling and improved the viability of cryopreserved PGCs. The maximum recovery rate of live PGCs in the yolk-removed embryos vitrified after optimum exposure to ES and VS was estimated to be about 90%, and about 50% of the live PGCs showed pseudopodial movement. Next, to elucidate the ability of cryopreserved PGCs to differentiate into functional gametes, PGCs recovered from the yolk-removed embryos (striped-type) that were vitrified under the optimum exposure to ES and VS were transplanted individually into 218 sterilized recipient blastulae (golden-type). Two days after the transplantation, 7.5% (14/187) of morphologically normal embryos had PGC(s) in the genital ridges. Six (5 males and 1 female) of the 14 recipient embryos developed into mature fish and generated progeny with characteristics inherited from PGC donors. In conclusion, we demonstrated the successful cryopreservation of PGCs by vitrification of yolk-removed embryos and the production of fertile zebrafish possessing germ cells that originated from the PGCs in vitrified embryos.

Hybrid between Danio rerio female and Danio nigrofasciatus male produces aneuploid sperm with limited fertilization capacity.

In the Danio species, interspecific hybridization has been conducted in several combinations. Among them, only the hybrid between a zebrafish (D. rerio) female and a spotted danio (D. nigrofasciatus) male was reported to be fertile. However, beyond these investigations, by means of reproductive biology, gametes of the hybrid have also not been investigated genetically. For this study, we induced a hybrid of the D. rerio female and D. nigrofasciatus male in order to study its developmental capacity, reproductive performance and gametic characteristics. Its hybrid nature was genetically verified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the rhodopsin gene. Almost all the hybrids (36/37) were males, and only one was female. Developing oocytes were observed in the hybrid female, but ovulated eggs have not been obtained thus far. Microscopic observation revealed various head sizes of sperm in the hybrid males. Flow cytometry showed that the hybrid males generated aneuploid sperm with various ploidy levels up to diploidy. In backcrosses between D. rerio females and hybrid males, fertilization rates were significantly lower than the control D. rerio, and most resultant progeny with abnormal appearance exhibited various kinds of aneuploidies ranging from haploidy to triploidy, but only one viable progeny, which survived more than four months, was triploid. This suggested the contribution of fertile diploid sperm of the hybrid male to successful fertilization and development.

Feasibility of cryopreservation of zebrafish (Danio rerio) primordial germ cells by whole embryo freezing.

We investigated the feasibility of cryopreservation of zebrafish (Danio rerio) blastomeres and primordial germ cells (PGCs) by rapid freezing of dechorionated whole embryos at the blastula, gastrula and segmentation stages. Initially we examined the glass-forming properties and embryo toxicities of 5 cryoprotectants: methanol (MeOH), ethylene glycol (EG), dimethyl sulfoxide (DMSO), propylene glycol (PG), and 1,3-butylene glycol (1,3-BG). Embryos at the blastula and gastrula stages had high sensitivities to cryoprotectant toxicities and were fragile against mechanical damage. Thus the segmentation stage embryos, the PGCs of which were visualized by injecting green fluorescence protein-nos1 3'UTR mRNA, were frozen using solutions containing each cryoprotectant at 6 M (first trial) and 2 types of cryoprotectants at 3 M each (second trial). In the first trial, live PGCs were recovered from most of the embryos frozen with EG (about 2 cells/embryo); however, a few embryos had live PGCs when embryos were frozen with other cryoprotectants. In the second trial, a mixture of EG + PG better preserved the viability of PGCs in frozen embryos. Live PGCs were recovered from all embryos frozen with EG + PG (about 3 cells/embryo), and the survival rate of PGCs was estimated to be about 25% based on the number of live PGCs in fresh embryos (about 12 cells/embryo). The present study indicates that we can utilize rapid freezing of dechorionated whole embryos at the segmentation stage for the cryopreservation of PGCs.

Production of Germ-Line Chimeras in Zebrafish.

The induction of germ-line chimerism in fish is a strategy for the reproduction of endangered or genetically valuable fish species. Chimeras can be created by transplanting a single primordial germ cell or multiple blastomeres from a donor into a sterile host embryo. When the host reaches sexual maturity, it will produce donor-originating gametes throughout its reproductive life span. This technique provides unique experimental conditions for basic biology research in model fish species like zebrafish. The success of cell transplantation relies on the effective sterilization of host embryos, the correct identification of developing germ cells, and the synchronization of migratory cues between the host and the transplanted cells. Developments in non-transgenic methods of germ cell ablation and identification have made germ cell transplantation more applicable to use in conservation and aquaculture. In this chapter, we provide a protocol for germ cell labeling by injection of chimeric RNA or FITC-dextran, the sterilization of host embryos using an antisense morpholino oligonucleotide, and two methods for producing germ-line chimeras in zebrafish: single primordial germ cell transplant and blastomere transplant.

Microinjection of Marine Fish Eggs.

Microinjection is a powerful tool for studying embryonic development and analyzing gene functions in fish. This technique was first applied to model species of fish such as zebrafish and medaka whose egg chorions could be removed or softened before microinjection. Recent progress in genome editing using TALEN and CRISPR has opened the opportunity to analyze gene functions in a much wider range of fish including those important to marine aquaculture. Therefore, application of the microinjection technique is also required in these species. However, the characteristics of fish eggs vary widely between species and several technical difficulties need to be overcome in order to use microinjection in a wider range of species. To obtain consistent results with microinjection, an optimal method has to be developed for each target species. In this chapter, we describe the physical characteristics of the eggs of fish species that have been used in microinjection experiments in our laboratory and detail the microinjection system we developed for fish eggs with a hard chorion, such as those of marine species.

Morphological differences in embryos of goldfish (Carassius auratus auratus) under different incubation temperatures.

The goldfish (Carassius auratus auratus) is a useful species for embryonic micromanipulations because of its large egg size and wide temperature tolerance. Here, we describe in detail the rate of development and morphological characteristics of goldfish embryos incubated at temperatures between 10 °C and 30 °C. The cleavage speed increased rapidly as temperature increased. Synchronized cell divisions occurred at 131 min intervals at 10 °C, at 33 min intervals at 20 °C, and at 19 min intervals at 30 °C during the cleavage period. The rate of hatched abnormal embryos significantly increased at temperatures of 26 °C and above, while there was no change in the number of abnormal embryos at temperatures less than 24 °C. Moreover, the blastomeres around the center of the blastodisc rose in the direction of the animal pole at temperatures less than 14 °C. At the lower temperatures, clusters of maternally-supplied germplasm were visualized both at the ends of the first three cleavage furrows and at the border between the lower and upper tiers at the 16- to 32-cell stage, with injection of artificial mRNA and vasa in situ hybridization. This study showed that temperature affects not only developmental speed but also the shape of the blastodisc and the distribution of maternally-supplied materials in the blastodisc. By controlling the temperature, it is possible for researchers to prepare many stages of embryos and shapes of the blastodisc from a single batch of eggs.

Improved Procedure for Induction of the Androgenetic Doubled Haploids in Zebrafish.

Androgenesis is useful for induction of doubled haploids from male genetic resources and contributes to the restoration of individuals from cryopreserved sperm. Here, we determined the suitable conditions for egg in vitro preservation and the suitable dose of UV irradiation for genetic inactivation of the egg nucleus, and established an improved procedure for induction of androgenetic-doubled haploids in zebrafish. The suitable solution for egg preservation was evaluated by the fertilization rate using different types of solutions or conditions. Hank's solution with 0.5% bovine serum albumin (pH8.0) was suitable for the preservation of zebrafish eggs. In addition, we discovered an improvement of fertilization rates by temporal preservation of ovulated eggs in the suitable solution. UV irradiation of eggs at 50-75 mJ/cm2 induced haploid embryos. Microsatellite genotyping using eight loci revealed the paternity and homozygosity of the putative androgenetic doubled haploids. The yield rate of androgenetic doubled haploids, which were induced by UV irradiation and heat shock, ranged from 0.4% to 10.7%.