Donor-derived Gametes Research Articles

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.

Targeted mutagenesis in chicken using CRISPR/Cas9 system.

The CRISPR/Cas9 system is a simple and powerful tool for genome editing in various organisms including livestock animals. However, the system has not been applied to poultry because of the difficulty in accessing their zygotes. Here we report the implementation of CRISPR/Cas9-mediated gene targeting in chickens. Two egg white genes, ovalbumin and ovomucoid, were efficiently (>90%) mutagenized in cultured chicken primordial germ cells (PGCs) by transfection of circular plasmids encoding Cas9, a single guide RNA, and a gene encoding drug resistance, followed by transient antibiotic selection. We transplanted CRISPR-induced mutant-ovomucoid PGCs into recipient chicken embryos and established three germline chimeric roosters (G0). All of the roosters had donor-derived mutant-ovomucoid spermatozoa, and the two with a high transmission rate of donor-derived gametes produced heterozygous mutant ovomucoid chickens as about half of their donor-derived offspring in the next generation (G1). Furthermore, we generated ovomucoid homozygous mutant offspring (G2) by crossing the G1 mutant chickens. Taken together, these results demonstrate that the CRISPR/Cas9 system is a simple and effective gene-targeting method in chickens.

Assessment of yellowtail kingfish (Seriola lalandi) as a surrogate host for the production of southern bluefin tuna (Thunnus maccoyii) seed via spermatogonial germ cell transplantation.

Germ cell transplantation is an innovative technology for the production of interspecies surrogates, capable of facilitating easier and more economical management of large-bodied broodstock, such as the bluefin tuna. The present study explored the suitability of yellowtail kingfish (Seriola lalandi) as a surrogate host for transplanted southern bluefin tuna (Thunnus maccoyii) spermatogonial cells to produce tuna donor-derived gametes upon sexual maturity. Germ cell populations in testes of donor T. maccoyii males were described using basic histology and the molecular markers vasa and dead-end genes. The peripheral area of the testis was found to contain the highest proportions of dead-end-expressing transplantable Type A spermatogonia. T. maccoyii Type A spermatogonia-enriched preparations were transplanted into the coelomic cavity of 6-10-day-old post-hatch S. lalandi larvae. Fluorescence microscopy and polymerase chain reaction analysis detected the presence of tuna cells in the gonads of the transplanted kingfish fingerlings at 18, 28, 39 and 75 days after transplantation, indicating that the transplanted cells migrated to the genital ridge and had colonised the developing gonad. T. maccoyii germ cell-derived DNA or RNA was not detected at later stages, suggesting that the donor cells were not maintained in the hosts' gonads.

GnRHa-induced spawning of the Eastern little tuna (Euthynnus affinis) in a 70-m3 land-based tank

Abstract A suitable recipient species is essential for efficient production of donor-derived gametes. With its relatively small body size, short generation time, and physiological and genetic similarities to Pacific bluefin tuna (PBT; Thunnus orientalis), Eastern little tuna (ELT, Euthynnus affinis) is a suitable candidate recipient species for spermatogonial transplantation. To provide a stable source of recipient larvae for spermatogonial transplantation, in the present study we developed a hormone administration technique for reliable seed production in captive ELT. In June 2009, 32 wild-caught 1-year-old ELTSwere transferred to a 70-m3 land-based tank at our facility (situated at 34°97′ N, 139°76′ E) and reared in the tank for 13 months. Fourteen of the fish (43.8%) survived until July 2010. During the 2010 spawning season (August–October), no spontaneous spawning was observed. To induce spawning, gonadotropin-releasing hormone agonist (GnRHa) was administrated to 9 of 14 ELT broodstocks (5 females and 4 males) in September 2010. The first spawning was observed at 3 days post-implantation, and continual spawning events were observed over the following 8 days. The number of eggs collected per day ranged from 50,000 to 170,000. The mean (± SD) fertilization and hatching percentages were 87 ± 12% and 30 ± 19%, respectively. Parentage assignment by DNA genotyping revealed that 3 of the 5 implanted females produced viable offspring, and one of these females participated in up to nine consecutive spawning events. For the second spawning induction trial, twelve 2-year-old ELTS (6 males and 6 females) were maintained in a net pen before the trial and were implanted with GnRHa pellets in August 2011 when they were transferred to the same tank used for the first trial. Production of fertilized eggs was observed continually for 11 days after GnRHa administration. The mean (± SD) number of hatched larvae per day was 138,059 ± 115,352, and the mean (± SD) fertilization and hatching percentages were 61 ± 42% and 42 ± 33%, respectively. Thus, GnRHa administration is effective for inducing spawning in ELT maintained in captivity and leads to a more reliable production of ELT seed. The reproduction enhancement techniques developed in the present study may ultimately facilitate development of a surrogate broodstock technology for PBT.

Surrogate production of eggs and sperm by intrapapillary transplantation of germ cells in cytoablated adult fish.

Germ cell transplantation (GCT) is a promising assisted reproductive technology for the conservation and propagation of endangered and valuable genetic resources. In teleost fish, GCT in adult gonads has been achieved only in male recipients, limiting greatly the usefulness of this technique in situations where both sexes need equal and timely attention for conservation and/or propagation. Here we describe a simplified GCT approach that ultimately leads to production of donor-derived eggs and sperm in considerably short time. Donor germ cells isolated from young pejerrey Odontesthes bonariensis (Atherinopsidae) were transplanted non-surgically through the genital papilla into the sexually mature gonads of Patagonian pejerrey O. hatcheri recipients whose gonads have been depleted of endogenous GCs by heat (26°C) and chemical treatment (four doses of Busulfan at 30 mg/kg and 40 mg/kg for females and males, respectively). Transplanted spermatogonial and oogonial cells were able to recolonize the recipients' gonads and produce functional donor origin eggs and sperm within 7 months from the GCT. We confirmed the presence of donor-derived gametes by PCR in 17% and 5% of the surrogate O. hatcheri fathers and mothers, respectively. The crosses between surrogate fathers and O. bonariensis mothers yielded 12.6–39.7% pure O. bonariensis and that between a surrogate mother and an O. bonariensis father yielded 52.2% pure O. bonariensis offspring. Our findings confirm that transplantation of germ cells into sexually competent adult fish by non-surgical methods allows the production of functional donor-derived eggs and sperm in a considerably short time. The methods described here could play a vital role in conservation and rapid propagation of endangered fish genetic resources.

The Pacific bluefin tuna (Thunnus orientalis) dead end gene is suitable as a specific molecular marker of type A spermatogonia

We developed a spermatogonial transplantation technique to produce donor-derived gametes in surrogate fish. Our ultimate aim is to establish surrogate broodstock that can produce bluefin tuna. We previously determined that only type A spermatogonia (ASG) could colonize recipient gonads in salmonids. Therefore, it is necessary to develop a precise molecular marker that can distinguish ASG in order to develop efficient spermatogonial transplantation methods. In this study, the Pacific bluefin tuna (Thunnus orientalis) dead end (BFTdnd) gene was identified as a specific marker for ASG. In situ hybridization and RT-PCR analysis with various types of spermatogenic cell populations captured by laser microdissection revealed that localization of BFTdnd mRNA was restricted to ASG, and not detected in other differentiated spermatogenic cells. In order to determine if BFTdnd can be used as a molecular marker to identify germ cells with high transplantability, transplantation of dissociated testicular cells isolated from juvenile, immature, and mature Pacific bluefin tuna, which have different proportions of dnd-positive ASG, were performed using chub mackerel as the surrogate recipient species. Colonization of transplanted donor germ cells was only successful with testicular cells from immature Pacific Bluefin tuna, which contained higher proportions of dnd-positive ASG than juvenile and mature fish. Thus, BFTdnd is a useful tool for identifying highly transplantable ASG for spermatogonial transplantation.

Gonadal Development and Fertility of Triploid Grass Puffer Takifugu niphobles Induced by Cold Shock Treatment

Tiger puffer Takifugu rubripes is one of the most valuable fish species in Japan; however, there has not been much progress in their selective breeding until recently despite their potential in aquaculture. Their long generation time and the large body size of their broodstock make breeding difficult. Recently, we made a surrogate broodstock, which produced gametes of different species in salmonids. Therefore, by using closely related recipients, which have small body sizes and short generation times, it is possible to accelerate breeding of the tiger puffer. Thus, we considered the grass puffer Takifugu niphobles, which has a short generation time and a small maturation size, as a potential recipient for gamete production of the tiger puffer. Furthermore, if sterile triploid individuals are used as recipients, the resulting surrogate broodstock would produce only donor-derived gametes. Therefore, we examined conditions for inducing triploidy by suppressing meiosis II to retain the second polar body in grass puffer. We found that cold shock treatment, which is 5°C for 30min starting from 5min after fertilization, is optimal to obtain high triploidization and hatching rates. Although the resulting triploid grass puffers produced small amounts of gametes in both sexes, the offspring derived from the gametes could not live for over 3days. Furthermore, we found that triploid grass puffer showed normal plasma sex steroid levels compared with diploids. These are important characteristics of triploid grass puffer as surrogate recipients used for germ cell transplantation.

Germline Replacement by Transfer of Primordial Germ Cells into Partially Sterilized Embryos in the Chicken1

We report a novel technique for almost complete replacement of the recipient germline with donor germ cells in the chicken. Busulfan solubilized in a sustained-release emulsion was injected into the yolk of fertile eggs before incubation. A dose of 100 microg was found to provide the best outcome in terms of reducing the number of endogenous primordial germ cells (PGCs) in embryonic gonads (0.6% of control numbers) and hatchability (36.4%). This was applied for preparing partially sterilized embryos to serve as recipients for the transfer of exogenous PGCs. Immunohistochemical analysis showed that the proportion of donor PGCs in busulfan-treated embryos was significantly higher than in controls (98.6% vs. 6.4%). Genetic cross-test analysis revealed that the germline transmission rate in busulfan-treated chickens was significantly higher than in controls (99.5% vs. 6.0%). Of 11 chimeras, 7 produced only donor-derived progenies, suggesting that these produced only donor-derived gametes in the recipient's gonads. This novel germline replacement technique provides a powerful tool for studying germline differentiation, for generating transgenic individuals, and for conserving genetic resources in birds.

Germ Cell Transplantation Using Sexually Competent Fish: An Approach for Rapid Propagation of Endangered and Valuable Germlines

The transplantation of germ cells into adult recipient gonads is a tool with wide applications in animal breeding and conservation of valuable and/or endangered species; it also provides a means for basic studies involving germ cell (GC) proliferation and differentiation. Here we describe the establishment of a working model for xenogeneic germ cell transplantation (GCT) in sexually competent fish. Spermatogonial cells isolated from juveniles of one species, the pejerrey Odontesthes bonariensis (Atherinopsidae), were surgically transplanted into the gonads of sexually mature Patagonian pejerrey O. hatcheri, which have been partially depleted of endogenous GCs by a combination of Busulfan (40 mg/kg) and high water temperature (25°C) treatments. The observation of the donor cells' behavior showed that transplanted spermatogonial cells were able to recolonize the recipients' gonads and resume spermatogenesis within 6 months from the GCT. The presence of donor-derived gametes was confirmed by PCR in 20% of the surrogate O. hatcheri fathers at 6 months and crosses with O. bonariensis mothers produced hybrids and pure O. bonariensis, with donor-derived germline transmission rates of 1.2–13.3%. These findings indicate that transplantation of spermatogonial cells into sexually competent fish can shorten considerably the production time of donor-derived gametes and offspring and could play a vital role in germline conservation and propagation of valued and/or endangered fish species.

Production of quail ( Coturnix japonica) germline chimeras by transfer of gonadal primordial germ cells into recipient embryos

The possibility of producing quail germline chimeras by the transfer of gonadal primordial germ cells (gPGCs) into recipient embryos was investigated. Japanese quail of the black (D: homozygous for the autosomal incomplete dominant gene D) and wild-type plumage (WP: d + / d + ) strains were used as donors and recipients, respectively. Gonadal cells were retrieved from the gonads of 5-day-old D embryos, and gPGCs were enriched by magnetism-activated cell sorting. Fresh (noncultured) gPGCs or those isolated after culture for 3 days with gonadal stromal cells present in the mixed cell population were introduced into the dorsal aorta of 2-day-old recipient WP embryos. Hatchability of the recipient embryos was 23.7% (31/131) and 34.4% (31/90) for those transfused with cultured or noncultured gPGCs, respectively. Of the hatched quail, 28 acquired sexual maturity; among these animals, 7.1% (1/14) and 21.4% (3/14) of those that received cultured or noncultured gPGCs, respectively, were proved to be germline chimeras. The percentage of germline transmission to the donor-derived gametes in the chimeras that received cultured and noncultured gPGCs were 1.9 and 2.2–4.7%, respectively. In conclusion, quail gPGCs retrieved from 5-day-old embryos were thus transmitted in the germline after their transfer to quail embryos of a different strain. This property of the gPGCs was not adversely affected by culture for up to 3 days.

Primordial germ cell: a novel tool for fish bioengineering

If in vitro-cultured cells could be converted into individual fish, they would have numerous applications in the field of genome biology and biotechnology. In spite of the benefits, however, no such system is presently available. Because primordial germ cells (PGCs) have the potential to be converted into individual fish via maturation and fertilization processes, we chose them to be the starting material for our system. As the first step, we visualized live PGCs in rainbow trout. Because the vasa transcript is restricted to the germ cell lineage, its regulatory regions are activated only in PGCs. Therefore, we produced transgenic strains carrying the green fluorescent protein (GFP) gene driven by the vasa gene regulatory regions. The resulting transgenic embryos showed green fluorescence specifically in PGCs. As the second step, the GFP-labeled PGCs were purified by flow cytometry. The genital ridges isolated from the transgenic embryos were dissociated by trypsin and sorted into GFP-positive and GFP-negative cells. The GFP-positive cells possessed morphological characteristics typical of PGCs. In addition, the vasa gene was expressed only in the GFP-positive cells, confirming that they were PGCs. To obtain functional gametes derived from isolated PGCs, a technique for converting PGCs into eggs and sperm is necessary. For this purpose, we developed a method for transplanting PGCs into developing embryos in order to incorporate them into the germ cell lineage of the recipient embryos. Approximately 10 PGCs isolated from newly hatched embryos were transplanted into the peritoneal cavity of recipient hatchlings. The transplanted PGCs actively migrated towards, and were finally incorporated into, the genital ridge. The PGCs that settled in the genital ridges of the recipient embryos proliferated, started meiosis, and differentiated into eggs and sperm in synchrony with the germ cells of allogenic recipient embryos. Further, the donor-derived gametes produced normal progenies through fertilization. These techniques, in combination with in vitro culture, genetic modification, and cryopreservation of PGCs, are expected to have numerous applications in the field of fish bioengineering.

Irradiation of fish embryos prior to blastomere transfer boosts the colonisation of their gonads by donor-derived gametes.

Blastomere transplantation into fish blastula embryos results in somatic chimeras, which generally provide null or a small proportion of gametes derived from the donor. This may partly explain why none of the ES-like cell lines established from fish embryos has contributed to the germline of chimeras when transplanted at the blastula stage. Here, we report that a moderate gamma-irradiation of recipient embryos, followed by transplantation of dispersed blastomeres, considerably enhances the proportion of donor-derived gametes (53% versus 5% in average). In fish, the resulting protocol should maximise the pluripotency level measured in vivo for embryonic cell lines and for cultured germ cells.

Developmental origin of avian primordial germ cells and its unique differentiation in the gonads of mixed-sex chimeras.

Developmental origin of avian primordial germ cells and its unique differentiation in the gonads of mixed-sex chimeras.

The developmental origin of primordial germ cells and the transmission of the donor-derived gametes in mixed-sex germline chimeras to the offspring in the chicken.

A novel system has been developed to determine the origin and development of primordial germ cells (PGCs) in avian embryos directly. Approximately 700 cells were removed from the center of the area pellucida, the outer of the area pellucida, and the area opaca of the stage X blastoderm (Eyal-Giladi and Kochav, 1976; Dev Biol 49:321-337). When the cells were removed from the center of the area pellucida, the mean number of circulating PGCs per 1 microliter of blood was significantly decreased to 13 (P < 0.05) in the embryo at stage 15 (Hamburger and Hamilton, 1951: J Morphol 88:49-92) as compared to intact embryos of 51. When the removed recipient cells from the center of the area pellucida were replenished with 500 donor cells, no reduction in the PGC number was observed. The removal of cells from the outer of area pellucida or from the area opaca had no effect on the number of PGCs. When another set of the manipulated embryos were cultured ex vivo to hatching and reared to sexual maturity, the absence of germ cells and the degeneration of seminiferous tubules were observed in resulting chickens derived from the blastoderm from which the cells were removed from the center of the area pellucida. Chimeric embryos produced by the male donor cells and the female recipient contained the female-derived cells at 97.2% in the whole embryo and 94.3% in the erythrocytes at 5 days of incubation. At 5-7 days of incubation, masculinization was observed in about one half of the mixed-sex embryos. The proportions of the female-derived cells in the whole embryo and in the erythrocytes were 76.5% and 80.2% at 7 days to 55.7% and 62.5% at 10 days of incubation, respectively. When the chimeras reached their sexual maturity, they were test mated to assess donor contribution to their germline. Five of six male chimeras (83%) and three of five female chimeras (60%) from male donor cells and a female recipient embryo from which 700 cells at the center of area pellucida were removed were germline chimeras. Three of the five male germline chimeras (60%) and one of the three female germline chimeras (33%) transmitted exclusively (100%) donor-derived gametes into the offspring. When embryonic cells were removed from the outer of area pellucida or area opaca, regardless of the sex combination of the donor and the recipient, the transmission of the donor-derived gametes was essentially null. The findings in the present studies demonstrated, both in vivo and in vitro, that the PGCs originate in the central part of the area pellucida and that the developmental fate to germ cell (PGCs) had been destined at stage X blastoderm in chickens.