Triploid Recipients Research Articles

Who is the best surrogate for germ stem cell transplantation in fish?

Surrogate broodstock technology in fishes has potential for aquaculture as well as endangered species conservation and propagation. Species with some unfavourable biological characteristics for culturing such as a late maturation or a large body size are ideal candidates for surrogate reproduction using smaller and faster-maturing hosts. One of the general prerequisites for pure donor-derived gamete production is the sterility of the host. Various sterilization methods have been developed and used in fish surrogacy; however, a direct comparison of available methods is missing. Such a knowledge gap hinders choice for the surrogate in various fish species, including those in high commercial demand such as tuna or sturgeons. There is a particular limitation from the point of the live material availability and difficulty to perform a high throughput assessment of different surrogates. Yet, large sturgeons or tuna species are one of the most prominent candidates for surrogacy. Zebrafish (Danio rerio) was utilized in this study as a model species to answer whether and to what extent different sterilization strategies can affect the surrogacy. Germ cell-depleted recipients (produced using knockdown of dead end gene), triploid recipients, and zebrafish x pearl danio (Danio albolineatus) hybrid recipients were tested as they represent the most frequently used types of surrogates. Spermatogonia isolated from the vas::EGFP transgenic strain were intraperitoneally transplanted into swim-up 5 day old zebrafish. Colonization rate of transplanted germ cells, survival, gonadal development, and reproductive output of the fish were analysed. Germ cell-depleted recipients with normal somatic cells were identified as the most convenient among tested sterilization methods considering adult germline chimera rate and their reproductive output. The present study provides a significant aid for selecting suitable surrogates in fishes.

Production of juvenile masu salmon (Oncorhynchus masou) from spermatogonia-derived sperm and oogonia-derived eggs via intraperitoneal transplantation of immature germ cells

No effective cryopreservation technique exists for fish eggs and embryos; thus, the cryopreservation of germ cells (spermatogonia or oogonia) and subsequent generation of eggs and sperm would be an alternative solution for the long-term preservation of piscine genetic resources. Nevertheless, in our previous study using rainbow trout, we showed that recipients transplanted with XY spermatogonia or XX oogonia produced unnatural sex-biased F1 offspring. To overcome these obstacles, we transplanted immature germ cells (XX oogonia or XY spermatogonia; frozen for 33 days) into the body cavities of triploid hatchlings, and the transplanted germ cells possessed a high capacity for differentiating into eggs and sperm in the ovaries and testes of recipients. Approximately 30% of triploid recipients receiving frozen germ cells generated normal salmon that displayed the donor-derived black body color phenotype, although all triploid salmon not receiving transplants were functionally sterile. Furthermore, F1 offspring obtained from insemination of the oogonia-derived eggs and spermatogonia-derived sperm show a normal sex ratio of 1:1 (female:male). Thus, this method presented a critical technique for practical conservation projects for other teleost fish species and masu salmon.

Production of tiger puffer Takifugu rubripes from cryopreserved testicular germ cells using surrogate broodstock technology

Tiger puffer Takifugu rubripes is one of most important fish in aquaculture. Recently, there has been progress in the production of tiger puffer aquaculture strains carrying economically desirable genetic traits. Cryopreservation techniques for the long-term preservation of their germ cells may reduce the risk of losing valuable genetic resources and the costs associated with maintenance of broodstock; however, cryopreservation methods for eggs and embryos, which contain both nuclear DNA and maternally inherited cytoplasmic compartments, have not yet been developed. In this study, we developed a methodology to produce functional gametes derived from cryopreserved testicular germ cells. Testes collected from tiger puffer were cryopreserved by a slow-freezing method, and the cryomedium for tiger puffer testis was optimized by testing several different cryoprotectants. Testicular germ cells prepared from frozen-thawed testes cryopreserved for at least two months were intraperitoneally transplanted into diploid or triploid hatchlings of grass puffer T. alboplumbeus. Transplanted germ cells migrated toward and were incorporated into recipient gonads. At 10 months post transplantation, 36.4% and 64.2% of diploid and triploid recipients, respectively, produced donor-derived sperm. In female recipients aged two years, production of donor-derived eggs was confirmed in one of 62 (1.6%) triploid recipients but not in diploid recipients. Insemination with the resultant sperm and eggs from triploid recipients generated viable offspring that originated from transplanted tiger puffer germ cells. Survival and growth potential of the offspring generated by the cross of both male and female triploid recipients were also comparable to those of control tiger puffer. This method is thus a breakthrough tool for the preservation of valuable genetic resources of tiger puffer and would help to efficiently manage broodstock with desirable genetic traits.

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.

Production of functionally sterile triploid Nibe croaker Nibea mitsukurii induced by cold-shock treatment with special emphasis on triploid aptitude as surrogate broodstock

Among the pelagic egg spawning marine teleosts, spermatogonial transplantation techniques were first developed in the Nibe croaker Nibea mitsukurii; however, production of donor-derived offspring had not yet been achieved. In salmonids, when triploid individuals were used as recipients, the resulting surrogate broodstock produced only donor-derived gametes, as the triploids were incapable of producing their own gametes. Therefore, in the present study, we determined the optimal parameters for cold-shock inducing triploidy through the suppression of meiosis II resulting in retention of the second polar body in fertilized Nibe croaker eggs. It was determined that cold-shock treatment, which was carried out at 10°C for a period of 15min beginning 5min after fertilization, following pre-shock treatment at 22°C achieved a 100% triploidization rate as well as hatching rates of which were high enough for practical application in the pelagic egg spawning marine teleost (16.4±10.4%). Average chromosome numbers of cold-shock treated larvae counted using mitotic figures found on the fin membrane of 1day post-hatch larvae were 67.3±0.7, which was approximately 1.5 times larger than that of control larvae (46.8±1.1), suggesting that triploidy had been induced. No differences in survival rates of hatched larvae during the rotifer-feeding stage existed between treated and non-treated groups. Gonadosomatic index and gametogenesis were impaired in both sexes of triploid Nibe croaker. Although triploids produced small amounts of gametes, no progeny from crosses between triploids and diploids survived later than 24h subsequent to hatching. These results indicated that the triploid Nibe croaker developed in this study could be effectively used as surrogate recipients in spermatogonial transplantation. Statement of relevanceIn intraperitoneal germ-cell transplantation techniques in salmonids, donor-derived diploid germ cells proliferated and differentiated within triploid recipient gonads, and the surrogate recipients produced only donor-derived gametes, which were capable of generating viable offspring when used in fertilization. To date, germ-cell transplantation techniques have been developed for use in perciforms, including sciaenids, however, triploid recipients have not yet been utilized in marine fishes. In this study, triploid Nibe croaker was produced at high triploidization rates (~100%) using cold-shock treatments. Analysis of the gonadal development and gametogenesis of triploid Nibe croaker in two consecutive spawning seasons revealed that the triploid Nibe croaker was functionally sterile in both sexes and that the triploids would be suitable for use as surrogate recipients.

Efficient production of donor-derived gametes from triploid recipients following intra-peritoneal germ cell transplantation into a marine teleost, Nibe croaker (Nibea mitsukurii)

Intra-peritoneal spermatogonial transplantation could become important in the production of allogenic or xenogenic donor-derived offspring from surrogate parent fish in commercial aquaculture. However, the rates at which surrogates with chimeric germ-lines are produced and contribute donor-derived gametes and offspring to subsequent generations must be improved for this technology to become practical, especially for marine fishes. Here, we performed intra-peritoneal spermatogonial transplantation into triploid Nibe croaker Nibea mitsukurii to evaluate the suitability of functionally sterile triploids as surrogates. Donor testicular cells were collected from hemizygous pHSC-GFP transgenic (gfp/−) individuals and then transplanted into the peritoneal cavities of 12-day-old larvae (diploid or triploid). By 6months after transplantation, 37% (male) and 29% (female) of triploid recipients had produced gfp-positive, donor-derived gametes; these rates were 7- and 4-fold higher, respectively, than the rates for diploid male and female recipients, respectively. Sperm and eggs from triploid recipients were artificially fertilized with eggs or sperm, respectively, from wild-type fish; approximately 50% of the resulting offspring were gfp-positive, suggesting that nearly all of offspring originated from donor-derived gametes that had descended from transplanted gfp/− germ cells. The fecundity and spawning frequency of surrogate triploid recipients were not significantly different to those of non-transplanted diploids, and the triploid recipients produced subsequent donor-derived offspring for at least two spawning seasons. Our results demonstrated that use of functionally sterile triploid recipients improved the percentage of recipients that produce donor-derived gametes. Further, the use of triploid recipients resulted in exclusive production of donor-derived gametes in this marine teleost. Statement of relevanceIntra-peritoneal spermatogonial transplantation using allogenic triploid recipients was first demonstrated in marine teleosts. In addition to efficient and exclusive donor-derived gametogenesis, we found that use of functionally sterile triploid recipients improved the percentage of recipients that produce donor-derived gametes. Consequently, triploid surrogates were more suitable than diploid surrogates and that use of triploid surrogates may accelerate the application of surrogate broodstock technology in aquaculture-targeted, genetically valuable, or endangered marine teleosts.

Production of germ cell‐deficient salmonids by dead end gene knockdown, and their use as recipients for germ cell transplantation

We previously established a spermatogonial transplantation model in fish using triploid recipients. Although triploid salmonids are sterile, they carry a limited number of immature triploid germ cells that potentially compete with the donor-derived germ cells for their niche. We therefore assessed the biological characteristics of germ cell-deficient gonads in rainbow trout for their suitability as recipients for germ cell transplantation in this study. Antisense morpholino oligonucleotides against the dead end gene were microinjected into the fertilized eggs of rainbow trout to eliminate endogenous germ cells, leaving only their supporting cells. Unlike similar approaches performed in zebrafish and medaka, these germ cell-deficient rainbow trout did not show a male-biased sex ratio. Approximately 30,000 spermatogonia were then transplanted into the body cavities of both germ cell-deficient and control recipients. The donor-derived germ cells showed significantly higher proliferation in the gonads of germ cell-deficient recipients than those in the gonads of the control recipients. Finally, the applicability of the germ cell-deficient recipients for xenogeneic transplantation was evaluated by transplanting rainbow trout spermatogonia into germ cell-deficient masu salmon recipients. The resulting recipient salmon matured normally and produced trout gametes, and early survival of the resulting trout offspring was as high as that of the control offspring. Thus, dead end-knockdown salmonids appear to be ideal recipients for the intraperitoneal transplantation of spermatogonia.

Production of viable trout offspring derived from frozen whole fish.

Long-term preservation of fish fertility is essential for the conservation of endangered fishes. However, cryopreservation techniques for fish oocytes and embryos have not yet been developed. In the present study, functional eggs and sperm were derived from whole rainbow trout that had been frozen in a freezer and stored without the aid of exogenous cryoprotectants. Type A spermatogonia retrieved from frozen-thawed whole trout remained viable after freezing duration up to 1,113 days. Long-term-frozen trout spermatogonia that were intraperitoneally transplanted into triploid salmon hatchlings migrated toward the recipient gonads, where they were incorporated, and proliferated rapidly. Although all triploid recipients that did not undergo transplantation were functionally sterile, 2 of 12 female recipients and 4 of 13 male recipients reached sexual maturity. Eggs and sperm obtained from the salmon recipients were capable of producing donor-derived trout offspring. This methodology is thus a convenient emergency tool for the preservation of endangered fishes.

Production of genetically diversified fish seeds using spermatogonial transplantation

Abstract Large-scale release of hatchery-produced seeds has been conducted in order to restore worldwide fishery production; however, concerns exist regarding the genetic effects of hatchery stock on wild fish populations, due to the reduced genetic variation often associated with hatchery-reared fish. Therefore, it is important that fish seeds used in stock enhancement possess sufficient genetic diversity to mitigate their genetic impact on wild fish populations. To promote genetic diversity of artificial seeds, seed production should be performed using a sufficiently large broodstock; however, maintenance of large broodstocks requires significant investments with respect to space, labor, and other associated costs. In order to circumvent the need for these investments, the present study proposed a means of producing gametes that possess a large amount of genetic diversity using only a small number of surrogate-broodstock through spermatogonial transplantation. We previously established spermatogonial transplantation methodology capable of producing functional eggs and sperm derived from donor-type A spermatogonia (ASGs). It was expected that if ASGs isolated from testes of different donor individuals were mixed prior to their transplantation into the peritoneal cavity of a single recipient, the resulting recipient would produce gametes derived from several donor individuals. To determine the feasibility of this scenario, mixed donor ASGs derived from vasa-GFP, vasa-DsRed and hsc-GFP transgenic rainbow trout (Oncorhynchus mykiss) were transplanted into triploid recipients. At 20 days post-transplantation, approximately 80% of recipients possessed donor ASGs derived from all three strains within their gonads and mean numbers of incorporated ASGs derived from each donor strain were nearly equal. At 100 days post-transplantation, incorporated donor ASGs proliferated within the gonads of both male and female recipients, and some had differentiated into oocytes within recipient ovaries. Furthermore, in order to ascertain whether incorporated donor ASGs derived from the three strains could differentiate into functional gametes, gametes collected from mature male and female recipients were used in artificial insemination with gametes obtained from wild-type fish of the opposite sex. Phenotypic analysis of F1 offspring revealed that functional sperm and eggs derived from three donor strains were successfully produced in the gonads of individual recipients. These results suggested that individual recipients that received ASGs from multiple donors were capable of producing gametes with different genetic backgrounds. Therefore, it was concluded that the method of spermatogonial transplantation outlined in the present study could serve as a novel and efficient method of producing fish seeds with increased genetic diversity for use in stock enhancement.

Generation of functional eggs and sperm from cryopreserved whole testes

The conservation of endangered fish is of critical importance. Cryobanking could provide an effective backup measure for use in conjunction with the conservation of natural populations; however, methodology for cryopreservation of fish eggs and embryos has not yet been developed. The present study established a methodology capable of deriving functional eggs and sperm from frozen type A spermatogonia (ASGs). Whole testes taken from rainbow trout were slowly frozen in a cryomedium, and the viability of ASGs within these testes did not decrease over a 728-d freezing period. Frozen-thawed ASGs that were intraperitoneally transplanted into sterile triploid hatchlings migrated toward, and were incorporated into, recipient genital ridges. Transplantability of ASGs did not decrease after as much as 939 d of cryopreservation. Nearly half of triploid recipients produced functional eggs or sperm derived from the frozen ASGs and displayed high fecundity. Fertilization of resultant gametes resulted in the successful production of normal, frozen ASG-derived offspring. Feasibility and simplicity of this methodology will call for an immediate application for real conservation of endangered wild salmonids.

Graft-versus-host reaction (GVHR) in clonal amago salmon, Oncorhynchus rhodurus

The graft-versus-host reaction (GVHR) was demonstrated in a salmonid model system of clonal diploid and triploid amago salmon. Triploid operculum grafts on clonal diploid evoked an acute rejection within 12 days. Grafts exchanged among triploid amago salmon exhibited prolonged survival for 18 days. In contrast, diploid grafts on triploid, and allografts among clonal diploid amago salmon were accepted. A typical GVHR was induced in triploid recipients by intraperitonal injection of head kidney cells from sensitised diploid donors. The clinical signs of graft-versus-host disease (GVHD) were observed in the recipients after 1 week of cell injection as a loss of appetite and appearance of solid faeces, followed by haemorrhage, local swelling of ventral skin and an enlarged spleen. Three of six fish died within 1 month. Water temperature and frequency of sensitisation are critical to induce GVHR. Diploid donors had to be sensitised three times at 20 °C to induce the typical GVHR. GVHR was most effectively induced by head kidney cells, followed by peripheral blood leucocytes (PBL) and spleen cells. Ploidy analysis by flow cytometry revealed that the donor head kidney cells greatly increased in the recipient liver, head kidney and spleen, and reached the peak after 9 days of donor cell injection. The results in the present study are quite similar to the findings in ginbuna and ginbuna-gold fish hybrid system, suggesting the presence of T cells in salmonid as well as cyprinid fish.