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.

Vitrification of medaka whole testis with a trehalose-containing solution and production of medaka individuals derived from the vitrified cells.

The cryopreservation of teleost eggs and embryos remains challenging, and there are no previous reports that demonstrate successful cryopreservation in medaka (Oryzias latipes). We have reported egg and sperm production, followed by the generation of donor-derived offspring by transplanting vitrified whole testes-derived testicular cells into surrogate fish. The vitrification solutions contained ethylene glycol, sucrose, and ficoll. In this study, we replaced sucrose with trehalose in the vitrification solution and medaka whole testes were vitrified with the solution. The post-vitrification survival (72.8 ± 3.5 %) was markedly improved compared with that achieved using the sucrose-containing solution (44.7 ± 4.2 %). Moreover, we demonstrated the production of eggs, sperm, and donor-derived offspring from testicular cells transplanted into surrogate recipients. The phenotype of donor-derived offspring was identical to that of transplanted testicular cells. These findings suggest that trehalose is effective for the vitrification of medaka whole testis and can be considered an effective and reliable method for the long-term preservation of their genetic resources.

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.

Midgut-derived neuropeptide F controls germline stem cell proliferation in a mating-dependent manner.

Stem cell maintenance is established by neighboring niche cells that promote stem cell self-renewal. However, it is poorly understood how stem cell activity is regulated by systemic, tissue-extrinsic signals in response to environmental cues and changes in physiological status. Here, we show that neuropeptide F (NPF) signaling plays an important role in the pathway regulating mating-induced germline stem cell (GSC) proliferation in the fruit fly Drosophila melanogaster. NPF expressed in enteroendocrine cells (EECs) of the midgut is released in response to the seminal-fluid protein sex peptide (SP) upon mating. This midgut-derived NPF controls mating-induced GSC proliferation via ovarian NPF receptor (NPFR) activity, which modulates bone morphogenetic protein (BMP) signaling levels in GSCs. Our study provides a molecular mechanism that describes how a gut-derived systemic factor couples stem cell behavior to physiological status, such as mating, through interorgan communication.

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.

The unusual rainbow trout sex determination gene hijacked the canonical vertebrate gonadal differentiation pathway.

Evolutionary novelties require rewiring of transcriptional networks and/or the evolution of new gene functions. Sex determination (SD), one of the most plastic evolutionary processes, requires such novelties. Studies on the evolution of vertebrate SD revealed that new master SD genes are generally recruited from genes involved in the downstream SD regulatory genetic network. Only a single exception to this rule is currently known in vertebrates: the intriguing case of the salmonid master SD gene (sdY), which arose from duplication of an immune-related gene. This exception immediately posed the question of how a gene outside from the classical sex differentiation cascade could acquire its function as a male SD gene. Here we show that SdY became integrated in the classical vertebrate sex differentiation cascade by interacting with the Forkhead box domain of the female-determining transcription factor, Foxl2. In the presence of Foxl2, SdY is translocated to the nucleus where the SdY:Foxl2 complex prevents activation of the aromatase (cyp19a1a) promoter in cooperation with Nr5a1 (Sf1). Hence, by blocking a positive loop of regulation needed for the synthesis of estrogens in the early differentiating gonad, SdY disrupts a preset female differentiation pathway, consequently allowing testicular differentiation to proceed. These results also suggest that the evolution of unusual vertebrate master sex determination genes recruited from outside the classical pathway like sdY is strongly constrained by their ability to interact with the canonical gonadal differentiation pathway.

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 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.

Production of donor-derived offspring by allogeneic transplantation of spermatogonia in Chinese rosy bitterling†.

Many bitterling species are facing extinction because of habitat destruction. Since cryopreservation of fish eggs is still not available to date due to their large size and high yolk content, long-term and stable storage of bitterling genetic resources is currently not possible. We recently discovered that cryopreservation of early-stage germ cells is possible in several fish species and that functional gametes derived from the frozen materials can be produced through their transplantation to embryonic recipients. However, bitterlings have uniquely shaped eggs and their embryos are extremely fragile, making it difficult to perform germ cell transplantation. Therefore, as a first step, we conducted intra-species spermatogonial transplantation using recessive albino Chinese rosy bitterling as donors and wild-type Chinese rosy bitterling as recipients to develop a system to convert freezable early-stage germ cells into functional gametes, particularly eggs. Approximately 3000 testicular cells were transplanted into the peritoneal cavity of 4-day-old germ cell-less recipient embryos produced by dead end (dnd)-knockdown. At 6 months, ten male recipients and nine female recipients produced gametes. Mating studies with the opposite sex of recessive albino control fish revealed that six males and three females produced only albino offspring, suggesting that these recipients' endogenous germ cells were completely removed by dnd-knockdown and they produced only donor-derived gametes. Thus, we successfully established a germ cell transplantation system in an iconic endangered teleost, bitterling. The technology established in this study can be directly applied to produce functional gametes of endangered bitterlings using cryopreserved donor cells.

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.

Novel method for mass producing genetically sterile fish from surrogate broodstock via spermatogonial transplantation†.

A stable system for producing sterile domesticated fish is required to prevent genetic contamination to native populations caused by aquaculture escapees. The objective of this study was to develop a system to mass produce stock for aquaculture that is genetically sterile by surrogate broodstock via spermatogonial transplantation (SGTP). We previously discovered that female medaka carrying mutations on the follicle-stimulating hormone receptor (fshr) gene become sterile. In this study, we demonstrated that sterile hybrid recipient females that received spermatogonia isolated from sex-reversed XX males (fshr (-/-)) recovered their fertility and produced only donor-derived fshr (-) X eggs. Natural mating between these females and fshr (-/-) sex-reversed XX males successfully produced large numbers of sterile fshr (-/-) female offspring. In conclusion, we established a new strategy for efficient mass production of sterile fish. This system can be applied to any aquaculture species for which SGTP and methods for producing sterile recipients can be established.

Specific visualization of live type A spermatogonia of Pacific bluefin tuna using fluorescent dye-conjugated antibodies†.

During our previous work toward establishing surrogate broodstock that can produce donor-derived gametes by germ cell transplantation, we found that only type A spermatogonia (ASGs) have the potency to colonize recipient gonads. Therefore, the ability to visualize ASGs specifically would allow the sequential analysis of donor cell behavior in the recipient gonads. Here we produced monoclonal antibodies that could recognize the cell surface antigens of ASGs in Pacific bluefin tuna (Thunnus orientalis), with the aim of visualizing live ASGs. We generated monoclonal antibodies by inoculating Pacific bluefin tuna testicular cells containing ASGs into mice and then screened them using cell-based enzyme-linked immunosorbent assay (ELISA), immunocytochemistry, flow cytometry (FCM), and immunohistochemistry, which resulted in the selection of two antibodies (Nos. 152 and 180) from a pool of 1152 antibodies. We directly labeled these antibodies with fluorescent dye, which allowed ASG-like cells to be visualized in a one-step procedure using immunocytochemistry. Molecular marker analyses against the FCM-sorted fluorescent cells confirmed that ASGs were highly enriched in the antibody-positive fraction. To evaluate the migratory capability of the ASGs, we transplanted visualized cells into the peritoneal cavity of nibe croaker (Nibea mitsukurii) larvae. This resulted in incorporated fluorescent cells labeled with antibody No. 152 being detected in the recipient gonads, suggesting that the visualized ASGs possessed migratory and incorporation capabilities. Thus, the donor germ cell visualization method that was developed in this study will facilitate and simplify Pacific bluefin tuna germ cell transplantation.

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.

Production of functional sperm by subcutaneous auto-grafting of immature testes in rainbow trout.

Sexually mature individuals are indispensable for breeding programs. Salmonids require a long period before reaching sexual maturity, so we aimed to shorten the period required to obtain functional sperm by grafting immature testicular fragments into mature recipients, which we predicted would allow the grafted testicular fragments to skip the long pre-pubertal period. First, we demonstrated successful subcutaneous auto-grafting of testicular fragments in rainbow trout. Unilateral testectomy was performed, and the isolated immature testicular fragment was auto-grafted into the subcutaneous space along the back of recipient fish. The grafted testicular fragments developed synchronously with the recipients' testis remaining in its body cavity, and both eventually produced functional sperm. Next, immature testicular fragments were auto-grafted into the subcutaneous space of sexually mature males. We achieved this, without immune rejection, by isolating and cryopreserving testes from immature fish, and rearing these unilaterally testectomized fish until sexual maturity. The cryopreserved testes were then auto-grafted into the original, now spermiating fish. The grated immature testicular fragments differentiated and produced functional sperm within 5 months after grafting. By combining this grafting method with a technique to avoid immune rejection, we expect to develop a practical method for producing sperm in a shorter period in salmonids.

Stem cell activity of type A spermatogonia is seasonally regulated in rainbow trout.

Spermatogonial stem cells (SSCs) support continuous production of sperm throughout the male's life. However, the biological characteristics of SSCs are poorly understood in animals exhibiting seasonal reproduction, even though most wild animals are seasonal breeders. During the spermiation season in rainbow trout, the lumen of the testes contains only spermatozoa and scattered type A spermatogonia (ASG) along the walls of the testicular lobules. These few remaining ASG, designated "residual ASG," are the only germ cells capable of supporting the next spermatogenesis, suggesting that the residual ASG are true SSCs. However, whether residual ASG can behave as SSCs in any teleost species is unknown. In this study, we attempted to clarify the biological characteristics of SSCs associated with seasonal reproduction in rainbow trout using spermatogonial transplantation. We found that the stem cell activity was clearly regulated seasonally during the annual reproductive cycle. Although the residual ASG exhibited moderate transplantability and colony-forming ability at the beginning of the spermiation season, these parameters decreased dramatically later and remained low until the next spermatogenesis was initiated. Furthermore, no clear correlations were observed between these qualitative changes and previously described morphologic characteristics of ASG or plasma sex steroid levels. Our results suggest that the biological properties of SSC populations in rainbow trout are seasonally regulated by a novel mechanism.

Application of dead end-knockout zebrafish as recipients of germ cell transplantation.

Germ cell transplantation is a promising technology for the propagation of endangered or valuable fishes. In this technique, sterile male and female recipient fish are injected with donor germ cells so they can produce viable gametes derived from the donor cells. The dead end (dnd) gene is involved in the migration of primordial germ cells; therefore, dnd-knockout zebrafish are expected to be germ-cell-free, making them suitable recipients for germ cell transplantation. dnd mutants were produced by microinjecting 2 nl of 10 ng/µl cRNAs encoding zinc finger nucleases against dnd into the blastodisc of zebrafish embryos before the cell- cleavage stage. One of the resulting founder males was mated with a wild-type female, and produced heterozygous mutants in the F1 generation. Mating of these F1 mutants produced an F2 generation with approximately 25% of the clutch being homozygous mutant (dnd-knockout) male, and lacking germ cells (as confirmed by expression analyses of vasa). The resulting dnd-knockout zebrafish males were tested for suitability as germ cell transplantation recipients by intraperitoneal transplantation of testicular cells prepared from vasa-gfp zebrafish. GFP-positive germ cells incorporated into the germ-cell-free gonads of the dnd-knockout recipients matured into functional sperm. Progeny tests revealed that the sperm from these dnd-knockout recipients were derived entirely from donor cells. Thus, we demonstrated that homozygous dnd mutants became germ-cell-free males that are able to nurse donor-derived germ cells.

Hypothermic storage of isolated spermatogonia and oogonia from rainbow trout (Oncorhynchus mykiss).

A growing number of fish species are endangered due to human activities. A short- or long-time preservation of gametes could conserve genetic resources of threatened fish species. The aim of this study was to evaluate a hypothermic condition for short-term preservation of spermatogonia and oogonia cells isolated from immature transgenic rainbow trout, Oncorhynchus mykiss, and to determine the maximum time point for further transplantation. Viability rate of germ cells was investigated after isolation and during storage at 4 °C up to 24 h. Dulbecco's modification of Eagle's medium supplemented with Hepes fetal bovine serum and l-glutamine was used as hypothermic storage media. The results showed that while viability decreased following 24 h storage, the remaining viable cells did not vary morphologically as well as GFP intensity retained similar to those observed in freshly isolated cells. The hypothermal storage study indicated that culture medium is suitable for preserving germ cells in the short periods of time. Simplicity, easily available culture media and low cost provide new insight into hypothermic conditions for preserving and transporting of germ cells for next applied and basic studies.

Generation of juvenile rainbow trout derived from cryopreserved whole ovaries by intraperitoneal transplantation of ovarian germ cells.

Cryopreservation of fish sperm offers the practical applications in the selective breeding and biodiversity conservation. However, because of the lack of cryopreservation methods for fish eggs and embryos, maternally inherited cytoplasmic compartments cannot be successfully preserved. We previously developed an alternative method to derive functional eggs and sperm from cryopreserved whole testis by transplanting testicular cells into female and male recipients. However, if target fish had ovaries, the previous method employing male-derived germ cells would be ineffective. Here, we aimed to generate functional gametes from cryopreserved whole ovaries by transplanting ovarian germ cells into peritoneal cavity of sterile hatchlings. Cryopreservation conditions for rainbow trout ovaries (1.0 M DMSO, 0.1 M trehalose, and 10% egg yolk) were optimized by testing several different cryoprotective agents. Ovarian germ cells from thawed ovaries were intraperitoneally transplanted into allogeneic triploid hatchlings. Transplanted germ cells migrated toward and were incorporated into recipient gonads, where they underwent gametogenesis. Transplantation efficiency of ovarian germ cells remained stable after cryopreservation period up to 1185 days. Although all triploid recipients that did not undergo transplantation were functionally sterile, 5 of 25 female recipients and 7 of 25 male recipients reached sexual maturity at 2.5 years post-transplantation. Inseminating the resultant eggs and sperm generated viable offspring displaying the donor characteristics of orange body color, green fluorescence, and chromosome numbers. This method is thus a breakthrough tool for the conservation of endangered fish species that are crucial to cryopreserve the genetic resources of female fish.