Reproductive Characteristics and Suitability of Sterile dead end Knockout Nibe Croaker as a Recipient for Intraperitoneal Germ Cell Transplantation.

The use of sterile recipients is crucial for efficiently producing donor-derived offspring through surrogate broodstock technology for practical aquaculture applications. Although knockout (KO) of the dead end (dnd) gene has been used in previous studies as a sterilization method, it has not been reported in marine fish. In this study, nibe croaker was utilized as a model for marine teleosts that produce small pelagic eggs, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system was utilized to produce dnd KO fish. The F1 generation, which carried a nonsense mutation in the dnd gene, was produced by mating founder individuals with wild-type counterparts. Subsequently, the F2 generation was produced by mating the resulting males and females. Among the F2 generations, 24.0% consisted of homozygous KO individuals. Histological analysis revealed that primordial germ cells (PGCs) were present in homozygous KO individuals at 10 days post-hatching (dph), similar to wild-type individuals. However, by 20 dph, PGCs were absent in KO individuals. Furthermore, no germ cells were observed in the gonads of both sexes of homozygous KO individuals at 6 months old, which is the typical maturity age for wild-type individuals of both sexes. In addition, when cryopreserved donor nibe croaker testicular cells were transplanted, only donor-derived offspring were successfully obtained through the spontaneous mating of homozygous KO recipient parents. Results indicate that dnd KO nibe croaker lacks germ cells and can serve as promising recipients, producing only donor-derived gametes as surrogate broodstock.

Fish germ cell cryobanking and transplanting for conservation.

The unprecedented loss of global biodiversity is linked to multiple anthropogenic stressors. New conservation technologies are urgently needed to mitigate this loss. The rights, knowledge and perspectives of Indigenous peoples in biodiversity conservation-including the development and application of new technologies-are increasingly recognised. Advances in germplasm cryopreservation and germ cell transplantation (termed 'broodstock surrogacy') techniques offer exciting tools to preserve biodiversity, but their application has been underappreciated. Here, we use teleost fishes as an exemplar group to outline (1) the power of these techniques to preserve genome-wide genetic diversity, (2) the need to apply a conservation genomic lens when selecting individuals for germplasm cryobanking and broodstock surrogacy and (3) the value of considering the cultural significance of these genomic resources. We conclude by discussing the opportunities and challenges of these techniques for conserving biodiversity in threatened teleost fish and beyond.

The volume and shape of bitterling eggs are more strongly influenced by germ cell autonomy than by the surrounding somatic cells.

There is great variation in the size and shape of teleost eggs from species to species. The size of the teleost egg depends on the amount of yolk accumulated in the egg, which is an important factor directly affecting the survival of hatchlings. Egg shape also contributes significantly to spawning ecology and survival during the prehatching stage. In this study, we used bitterlings, which show a wide variety of egg volumes and shapes, to elucidate whether these factors are determined by germ cells or somatic cells. Reciprocal transplantations of germ cells between two bitterling species revealed that the egg volume was identical to that of the germ cell donor species in both combinations. The egg shape was also very similar to that of the species providing the germ cells. These results suggest that the volume and shape of teleost eggs are greatly influenced by germ cell autonomy.

Determination of dietary essential fatty acids in a deep-sea fish, the splendid alfonsino Beryx splendens: functional characterization of enzymes involved in long-chain polyunsaturated fatty acid biosynthesis.

The splendid alfonsino Beryx splendens is a commercially important deep-sea fish in East Asian countries. Because the wild stock of this species has been declining, there is an urgent need to develop aquaculture systems. In the present study, we investigated the long-chain polyunsaturated fatty acid (LC-PUFA) requirements of B. splendens, which are known as essential dietary components in many carnivorous marine fish species. The fatty acid profiles of the muscles, liver, and stomach contents of B. splendens suggested that it acquires substantial levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from its natural diet. The functional characterization of a fatty acid desaturase (Fads2) and three elongases (Elovl5, Elovl4a, and Elovl4b) from B. splendens confirmed their enzymatic capabilities in LC-PUFA biosynthesis. Fads2 showed Δ6 and Δ8 bifunctional desaturase activities. Elovl5 showed preferential elongase activities toward C18 and C20 PUFA substrates, whereas Elovl4a and Elovl4b showed activities toward various C18-22 substrates. Given that Fads2 showed no Δ5 desaturase activity and no other fads-like sequence was found in the B. splendens genome, EPA and arachidonic acid cannot be synthesized from C18 precursors; hence, they can be categorized as dietary essential fatty acids in B. splendens. EPA can be converted into DHA in B. splendens via the so-called Sprecher pathway. However, given that fads2 is only expressed in the brain, it is unlikely that the capacity of B. splendens to biosynthesize DHA from EPA can fulfill its physiological requirements. These results will be useful to researchers developing B. splendens aquaculture methods.

Production of Germ Cell-Less Rainbow Trout by dead end Gene Knockout and their Use as Recipients for Germ Cell Transplantation.

In germ cell transplantation experiments, the use of sterile recipients that do not produce their own gametes is an important prerequisite. Triploidization and dnd gene knockdown (KD) methods have been widely used to produce sterile fish. However, triploidization does not produce complete sterility in some fish species, and gene KD is labor and time intensive since it requires microinjection into individual fertilized eggs. To overcome these problems, in this study, we generated homozygous mutants of the dead end (dnd) gene in rainbow trout (Oncorhynchus mykiss) using the clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system, analyzed their reproductive capacity, and evaluated their suitability as recipients for germ cell transplantation. By crossing F1 heterozygous mutants produced from founders subjected to genome editing, an F2 generation consisting of approximately 1/4 homozygous knockout mutants (dnd KO) was obtained. The dnd KO hatchlings retained the same number of primordial germ cells (PGCs) as the wild-type (WT) individuals, after which the number gradually decreased. At 1 year of age, germ cells were completely absent in all analyzed individuals. To evaluate the dnd KO individuals as recipients for germ cell transplantation, germ cells prepared from donor individuals were transplanted into the abdominal cavity of dnd KO hatchlings. These cells migrated to the recipient gonads, where they initiated gametogenesis. The mature recipient individuals produced only donor-derived sperm and eggs in equivalent numbers to WT rainbow trout. These results indicate that dnd KO rainbow trout are suitable recipient candidates possessing a high capacity to nurse donor-derived germ cells.

Characterization of a vasa homolog in Mekong giant catfish (Pangasianodon gigas): Potential use as a germ cell marker.

For the long-term preservation of the genetic resources of endangered fish species, a combination of germ cell cryopreservation and transplantation can be an effective technique. To optimize these techniques, it is important to identify undifferentiated germ cells possessing transplantability, such as primordial germ cells, type A spermatogonia (ASGs), and oogonia. In this study, a homolog of vasa cDNA in Mekong giant catfish (MGC-vasa) (Pangasianodon gigas), which is an endangered species inhabiting the Mekong river, was cloned and characterized for use as a putative germ cell marker. Results indicate that MGC-Vasa contained all of the consensus motifs, including the arginine-glycine and arginine-glycine-glycine motifs, as well as the nine conserved motifs belonging to the DEAD-box family of proteins. Results from phylogenetic analysis indicated MGC-vasa also grouped with Vasa and was clearly distinguishable from Pl10 in other teleosts. Results from analysis of abundance of mRNA transcripts using reverse transcription-polymerase chain reaction and in situ hybridization performed on immature Mekong giant catfish testis indicated vasa was present specifically in germ cells, with large abundances of the relevant mRNA in spermatogonia and spermatocytes. Sequence similarity and the specific localization of MGC-vasa in these germ cells suggest that the sequence ascertained in this study was a vasa homolog in Mekong giant catfish. Furthermore, vasa-positive cells were detected in prepared smears of testicular cells, indicating that it may be a useful germ cell marker for enzymatically dissociated cells used for transplantation studies.

Allelic diversification after transposable element exaptation promoted gsdf as the master sex determining gene of sablefish.

Concepts of evolutionary biology suggest that morphological change may occur by rare punctual but rather large changes, or by more steady and gradual transformations. It can therefore be asked whether genetic changes underlying morphological, physiological, and/or behavioral innovations during evolution occur in a punctual manner, whereby a single mutational event has prominent phenotypic consequences, or if many consecutive alterations in the DNA over longer time periods lead to phenotypic divergence. In the marine teleost, sablefish (Anoplopoma fimbria), complementary genomic and genetic studies led to the identification of a sex locus on the Y Chromosome. Further characterization of this locus resulted in identification of the transforming growth factor, beta receptor 1a (tgfbr1a) gene, gonadal somatic cell derived factor (gsdf), as the main candidate for fulfilling the master sex determining (MSD) function. The presence of different X and Y Chromosome copies of this gene indicated that the male heterogametic (XY) system of sex determination in sablefish arose by allelic diversification. The gsdfY gene has a spatio-temporal expression profile characteristic of a male MSD gene. We provide experimental evidence demonstrating a pivotal role of a transposable element (TE) for the divergent function of gsdfY By insertion within the gsdfY promoter region, this TE generated allelic diversification by bringing cis-regulatory modules that led to transcriptional rewiring and thus creation of a new MSD gene. This points out, for the first time in the scenario of MSD gene evolution by allelic diversification, a single, punctual molecular event in the appearance of a new trigger for male development.

Identification of the causative gene of a transparent phenotype of juvenile red sea bream Pagrus major.

Deformities in cultured fish species may be genetic, and identifying causative genes is essential to expand production and maintain farmed animal welfare. We previously reported a genetic deformity in juvenile red sea bream, designated a transparent phenotype. To identify its causative gene, we conducted genome-wide linkage analysis and identified two single nucleotide polymorphisms (SNP) located on LG23 directly linked to the transparent phenotype. The scaffold on which the two SNPs were located contained two candidate genes, duox and duoxa, which are related to thyroid hormone synthesis. Four missense mutations were found in duox and one in duoxa, with that in duoxa showing perfect association with the transparent phenotype. The mutation of duoxa was suggested to affect the transmembrane structure and thyroid-related traits, including an enlarged thyroid gland and immature erythrocytes, and lower thyroxine (T4) concentrations were observed in the transparent phenotype. The transparent phenotype was rescued by T4 immersion. Loss-of-function of duoxa by CRISPR-Cas9 induced the transparent phenotype in zebrafish. Evidence suggests that the transparent phenotype of juvenile red sea bream is caused by the missense mutation of duoxa and that this mutation disrupts thyroid hormone synthesis. The newly identified missense mutation will contribute to effective selective breeding of red sea bream to purge the causative gene of the undesirable phenotype and improve seed production of red sea bream as well as provide basic information of the mechanisms of thyroid hormones and its related diseases in fish and humans.

Production of functional sperm from cryopreserved testicular germ cells following intraperitoneal transplantation into allogeneic surrogate in yellowtail (Seriola quinqueradiata).

The aim of this study was to establish a method for the cryopreservation of spermatogonia of the yellowtail (Seriola quinqueradiata), which is the most commonly farmed fish in Japan. Testicular cells were prepared by enzymatic dissociation of testicular fragments containing an abundance of type A spermatogonia and were added to cryomedium containing dimethyl sulfoxide (DMSO), ethylene glycol, glycerol, or propylene glycol at concentrations of 0.5-2.5 M. The cells were then frozen and stored in liquid nitrogen for 3 days. After thawing, their survival and transplantability were evaluated. Testicular cells were most successfully cryopreserved in 1.0 M DMSO as indicated by survival of 34% of cells. Furthermore, in situ hybridization using the yellowtail vasa probe showed that these recovered cells contained a similar proportion of germ cells to fresh testicular cells before freezing. Transplantation of the recovered cells into the peritoneal cavities of allogeneic larvae resulted in 94% of surviving recipients having donor-derived germ cells in their gonads after 28 days. Sperm were then collected from seven randomly selected recipients once they reached 2 years of age and used to fertilize wild-type eggs, which led to an average of 26% of the first filial (F1) offspring being derived from donor fish, as confirmed through the use of microsatellite markers. Thus, we successfully cryopreserved yellowtail spermatogonia and produced functional sperm via intraperitoneal transplantation into allogeneic recipients.

Disrupted eye and head development in rainbow trout with reduced ultraviolet (sws1) opsin expression.

Visual opsins are proteins expressed by retinal photoreceptors that capture light to begin the process of phototransduction. In vertebrates, the two types of photoreceptors (rods and cones) express one or multiple opsins and are distributed in variable patterns across the retina. Some cones form opsin retinal gradients, as in the mouse, whereas others form more demarcated opsin domains, as in the lattice-like mosaic retinas of teleost fishes. Reduced rod opsin (rh1) expression in mouse, zebrafish, and African clawed frog results in lack of photoreceptor outer segments (i.e., the cilium that houses the opsins) and, in the case of the mouse, to retinal degeneration. The effects of diminished cone opsin expression have only been studied in the mouse where knockout of the short-wavelength sensitive 1 (sws1) opsin leads to ventral retinal cones lacking outer segments, but no retinal degeneration. Here we show that, following CRISPR/Cas9 injections that targeted knockout of the sws1 opsin in rainbow trout, fish with diminished sws1 opsin expression exhibited a variety of developmental defects including head and eye malformations, underdeveloped outer retina, mislocalized opsin expression, cone degeneration, and mosaic irregularity. All photoreceptor types were affected even though sws1 is only expressed in the single cones of wild fish. Our results reveal unprecedented developmental defects associated with diminished cone opsin expression and suggest that visual opsin genes are involved in regulatory processes that precede photoreceptor differentiation.

Cryopreservation and Transplantation of Spermatogonial Stem Cells.

Cryopreservation as a method that enables long-term storage of biological material has long been used for the conservation of valuable zebrafish genetic resources. However, currently, only spermatozoa of zebrafish can be successfully cryopreserved, while protocols for cryopreservation of eggs and embryos have not yet been fully developed. Transplantation of germline stem cells (GSCs) has risen as a favorable method that can bypass the current problem in cryopreservation of female genetic resources and can lead to reconstitution of fish species and lines through surrogate production. Here, we describe essential steps needed for the cryopreservation of spermatogonial stem cells (SSCs) and their utilization in the conservation of zebrafish genetic resources through SSC transplantation and surrogate production.

Flatfishes colonised freshwater environments by acquisition of various DHA biosynthetic pathways.

The colonisation of freshwater environments by marine fishes has historically been considered a result of adaptation to low osmolality. However, most marine fishes cannot synthesise the physiologically indispensable fatty acid, docosahexaenoic acid (DHA), due to incomplete DHA biosynthetic pathways, which must be adapted to survive in freshwater environments where DHA is poor relative to marine environments. By analysing DHA biosynthetic pathways of one marine and three freshwater-dependent species from the flatfish family Achiridae, we revealed that functions of fatty acid metabolising enzymes have uniquely and independently evolved by multi-functionalisation or neofunctionalisation in each freshwater species, such that every functional combination of the enzymes has converged to generate complete and functional DHA biosynthetic pathways. Our results demonstrate the elaborate patchwork of fatty acid metabolism and the importance of acquiring DHA biosynthetic function in order for fish to cross the nutritional barrier at the mouth of rivers and colonise freshwater environments.

Aging- and temperature-related activity of spermatogonial stem cells for germ cell transplantation in medaka.

Spermatogonial transplantation can contribute to developing a novel method of producing seedlings for both aquaculture and biotic conservation. This study's purpose was to investigate aging- and temperature-related changes in the numbers and stem cell functions of type-A spermatogonia (ASG) in the model fish medaka (Oryzias latipes). The ASG numbers in medaka of different ages were quantified via histological observation and enzymatic dissociation of vasa-Gfp medaka testes. The ASG numbers were higher in eight-month-old medaka (maturation) than in four-month-old medaka (the onset of maturation). However, ASG numbers decreased in 18-month-old medaka (senescence). Low water temperature appeared to slow down both testis development and aging processes. To study the effects of aging on ASG stem cell activity, testicular cell suspensions containing GFP-expressed ASG were prepared from vasa-Gfp medaka donors at 4 and 18 months of age and transplanted into recipient hybrid larvae of medaka (O. latipes x O. curvinotus), which provided young stem-cell-niches. The findings revealed no significant differences in ASG colonization rates isolated from medaka of different ages. Each group displayed similar rates of germ-line transmission. Furthermore, water temperature had no significant effects on each ASG's stem cell activity. Taken together, these results indicated that aging and temperature affect ASG numbers. However, ASG isolated from medaka with different ages were transplanted into gonads with a young niche microenvironment, and there was no evidence of donor aging on stem cell activity.

Suitability of hybrid mackerel (Scomber australasicus × S. japonicus) with germ cell-less sterile gonads as a recipient for transplantation of bluefin tuna germ cells.

We aim to establish a small-bodied surrogate broodstock, such as mackerel, which produces functional bluefin tuna gametes by spermatogonial transplantation. When reproductively fertile fish are used as recipients, endogenous gametogenesis outcompetes donor-derived gametogenesis, and recipient fish predominantly produce their gametes. In this study, we assessed fertility of hybrid mackerel, Scomber australasicus × S. japonicus, and its suitability as a recipient for transplantation of bluefin tuna germ cells. Hybrid mackerel were produced by artificially inseminating S. australasicus eggs with S. japonicus spermatozoa. Cellular DNA content and PCR analyses revealed that F1 offspring were diploid carrying both paternal and maternal genomes. Surprisingly, histological observations found no germ cells in hybrid mackerel gonads at 120 days post-hatch (dph), although they were present in the gonad of 30- and 60-dph hybrid mackerel. The frequency of germ cell-less fish was 100% at 120-dph, 63.1% at 1-year-old, and 81.8% at 2-year-old. We also confirmed a lack of expression of germ cell marker (DEAD-box helicase 4, ddx4) in the germ cell-less gonads of hybrid mackerel. By contrast, expression of Sertoli cell marker (gonadal soma-derived growth factor, gsdf) and of Leydig cell marker (steroid 11-beta-hydroxlase, cyp11b1) were clearly detected in hybrid mackerel gonads. Together these results showed that most of the hybrid gonads were germ cell-less sterile, but still possessed supporting cells and steroidogenic cells, both of which are indispensable for nursing donor-derived germ cells. To determine whether hybrid gonads could attract and incorporate donor bluefin tuna germ cells, testicular cells labeled with PKH26 fluorescent dye were intraperitoneally transplanted. Fluorescence observation of hybrid recipients at 14 days post-transplantation revealed that donor cells had been incorporated into the recipient's gonads. This suggests that hybrid mackerel show significant promise for use as a recipient to produce bluefin tuna gametes.

Production of functional eggs and sperm from in vitro-expanded type A spermatogonia in rainbow trout.

Combining cryopreservation of germline stem cells (GSCs) with their subsequent transplantation into recipient fish is a powerful tool for long-term preservation of genetic resources of endangered fishes. However, application of this technique has been limited because endangered species sometimes have small gonads and do not supply enough GSCs to be used for transplantation. This limitation could be overcome by expanding GSCs in vitro, though this has been difficult due to the complexity of reconstructing the gonadal microenvironment that surrounds GSCs. Here, we describe a novel method of in vitro expansion of rainbow trout GSCs using a feeder layer derived from Sertoli cells and a culture medium containing trout plasma. A transplantation assay demonstrated that the in vitro-expanded GSCs exhibited stem cell activity and potency to produce functional eggs, sperm, and eventually healthy offspring. In vitro expansion of GSCs can aid in rescuing fishes that are on the verge of extinction.

Production of Chinese rosy bitterling offspring derived from frozen and vitrified whole testis by spermatogonial transplantation.

Bitterling is a small cyprinid fish facing an increasing risk of extinction owing to habitat destruction and decreasing freshwater mussel population that are used as their spawning substrates. Owing to their large size and high yolk contents, methods for cryopreservation of their eggs or embryos, which is a promising method for long-term preservation of their genetic resources, are still not available. We conducted this study to evaluate the feasibility of gamete production by transplanting cryopreserved testicular cells into germ cell-less recipients that were produced by knockdown of dead end gene. Immature testes isolated from recessive albino Chinese rosy bitterlings were cryopreserved by slow freezing or vitrification. Approximately 3000 slow-frozen or vitrified cells were transplanted into the peritoneal cavity of 4-day-old germ cell-less wild-type Chinese rosy bitterlings. We observed no significant differences in the incorporation rates of the slow-frozen and vitrified cells into the genital ridges of recipients compared with those of freshly prepared cells. When the recipients matured, almost half of the male or female recipients that received freshly prepared, slow-frozen, or vitrified cells produced gametes derived from donor cells, with no significant differences in their fecundity among the 3 groups. Moreover, fertilization of the resulting eggs and sperm produced donor-derived offspring exhibiting the albino phenotype. Therefore, the abovementioned methods could be used as a powerful and practical method for long-term preservation of bitterling genetic resources for biotic conservation.

Preservation of zebrafish genetic resources through testis cryopreservation and spermatogonia transplantation.

Zebrafish is one of the most commonly used model organisms in biomedical, developmental and genetic research. The production of several thousands of transgenic lines is leading to difficulties in maintaining valuable genetic resources as cryopreservation protocols for eggs and embryos are not yet developed. In this study, we utilized testis cryopreservation (through both slow-rate freezing and vitrification) and spermatogonia transplantation as effective methods for long-term storage and line reconstitution in zebrafish. During freezing, utilization of 1.3 M of dimethyl sulfoxide (Me2SO) displayed the highest spermatogonia viability (~60%), while sugar and protein supplementation had no effects. Needle-immersed vitrification also yielded high spermatogonia viability rates (~50%). Both optimal slow-rate freezing and vitrification protocols proved to be reproducible in six tested zebrafish lines after displaying viability rates of >50% in all lines. Both fresh and cryopreserved spermatogonia retained their ability to colonize the recipient gonads after intraperitoneal transplantation of vasa::egfp and actb:yfp spermatogonia into wild-type AB recipient larvae. Colonization rate was significantly higher in dnd-morpholino sterilized recipients than in non-sterilized recipients. Lastly, wild-type recipients produced donor-derived sperm and donor-derived offspring through natural spawning. The method demonstrated in this study can be used for long-term storage of valuable zebrafish genetic resources and for reconstitution of whole zebrafish lines which will greatly improve the current preservation practices.

Enrichment of transplantable germ cells in salmonids using a novel monoclonal antibody by magnetic-activated cell sorting.

In the fish germ cell transplantation system, only type A spermatogonia (ASGs) and oogonia are known to be incorporated into the recipient genital ridges, where they undergo gametogenesis. Therefore, high colonization efficiency can be achieved by enriching undifferentiated germ cells out of whole testicular cells. In this study, we used magnetic-activated cell sorting (MACS) for enriching undifferentiated germ cells of rainbow trout using a monoclonal antibody that recognizes a specific antigen located on the germ cell membrane. We screened the antibodies to be used for MACS by performing immunohistochemistry on rainbow trout gonads. Two antibodies, nos. 172 and 189, showed strong signals for ASGs and oogonia. Next, we performed MACS with antibody no. 172 using gonadal cells isolated from vasa-gfp rainbow trout showing GFP in undifferentiated germ cells. We found that GFP-positive cells are highly enriched in antibody no. 172-positive fractions. Finally, to examine the transplantability of MACS-enriched cells, we intraperitoneally transplanted sorted or unsorted cells into recipient larvae. We observed that transplantability of sorted cells, particularly ovarian cells, were significantly higher than that of unsorted cells. Therefore, MACS with antibody no. 172 could enrich ASGs and oogonia and become a powerful tool to improve transplantation efficiency in salmonids.

A key metabolic gene for recurrent freshwater colonization and radiation in fishes.

Colonization of new ecological niches has triggered large adaptive radiations. Although some lineages have made use of such opportunities, not all do so. The factors causing this variation among lineages are largely unknown. Here, we show that deficiency in docosahexaenoic acid (DHA), an essential ω-3 fatty acid, can constrain freshwater colonization by marine fishes. Our genomic analyses revealed multiple independent duplications of the fatty acid desaturase gene Fads2 in stickleback lineages that subsequently colonized and radiated in freshwater habitats, but not in close relatives that failed to colonize. Transgenic manipulation of Fads2 in marine stickleback increased their ability to synthesize DHA and survive on DHA-deficient diets. Multiple freshwater ray-finned fishes also show a convergent increase in Fads2 copies, indicating its key role in freshwater colonization.

Establishment of novel monoclonal antibodies for identification of type A spermatogonia in teleosts†.

We recently established a germ cell transplantation system in salmonids. Donor germ cells transplanted into the body cavity of recipient embryos migrate toward and are incorporated into the recipient gonad, where they undergo gametogenesis. Among the various types of testicular germ cells, only type A spermatogonia (A-SG) can be incorporated into the recipient gonads. Enriching for A-SG is therefore important for improving the efficiency of germ cell transplantation. To enrich for A-SG, an antibody against a cell surface marker is a convenient and powerful approach used in mammals; however, little is known about cell surface markers for A-SG in fish. To that end, we have produced novel monoclonal antibodies (mAbs) against cell-surface molecules of rainbow trout (Oncorhynchus mykiss) A-SG. We inoculated mice with living A-SG isolated from pvasa-GFP transgenic rainbow trout using GFP-dependent flow cytometry. By fusing lymph node cells of the inoculated mice with myeloma cells, we generated 576 hybridomas. To identify hybridomas that produce mAbs capable of labeling A-SG preferentially and effectively, we screened them using cell ELISA, fluorescence microscopy, and flow cytometry. We thereby identified two mAbs that can label A-SG. By using flow cytometry with these two antibodies, we could enrich for A-SG with transplantability to recipient gonads from amongst total testicular cells. Furthermore, one of these mAbs could also label zebrafish (Danio rerio) spermatogonia. Thus, we expect these monoclonal antibodies to be powerful tools for germ cell biology and biotechnology.