Production Of Donor-derived Offspring Research Articles

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.

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.

070 Production of donor-derived offspring by allogenic transplantation of cryopreserved spermatogonia in medaka

Although numerous inbred lines and endangered wild populations of medaka exist, the only method currently available for preserving the resources is to rear live individuals, as protocols for cryopreservation of fish eggs or embryos have not yet been successfully established due to their large size. However, rearing live individuals is always associated with the risk of mutation due to mobile elements, and the loss of parent fish through infectious disease or accidents related to rearing conditions and equipment. Our group developed a novel method named surrogate broodstock technology. By transplanting donor germ cells into recipient fish of another species, the donor germ cells mature into sperm or oocytes in recipient gonads (Takeuchi et al., 2004; Okutsu et al., 2006; Yoshizaki et al., 2011). More importantly, the germ cells cryoporeserved can be differentiated into functional oocytes (Okutsu et al., 2007; Lee et al., 2013). In this study, we aimed to cryopreserve spermatogonia that can be differentiated into both sperm and eggs. When medaka spermatogonia were dehydrated with hypertonic solutions at 25°C, the survival was decreased in 0.5 M sucrose solution. However, survival rates were improved, when dehydration was carried out at 0°C. Toxicity assay of cryoprotectant (−10%, 25°C) revealed that propylene glycol (PG) was the least toxic, while ethylene glycol (EG) proved less toxic than glycerol and Me2SO. Based on these results, vitrification solutions containing ficoll and sucrose were based with either PG or EG and the testes were vitrified after exposure to them at 0°C. Although survival of cells treated with PG was low, survival rate of 44.9% was obtained using vitrification solution containing EG. Thus, vitrification method of medaka whole testis includes spermatogonia was established. When vitrified spermatogonia from vasa-GFP transgenic strain (body color: orange) were transplanted into larvae of non-transgenic strain (body color: black), female recipients produced oocytes showing green fluorescence. In their gonads, however, there were recipient-derived oocytes together with donor-derived green fluorescent oocytes. Since recipients producing gametes derived only from transplanted donor germ cells are desirable, infertile triploids were used as the recipients in the next experiment. Triploid medaka was prepared by placing fertilized eggs at 41°C water for 5 min (Naruse et al., 1985). When cryopreserved spermatogonia were transplanted into triploid larvae, the mature recipients produced only sperm and eggs showing green fluorescence, which is the donor-derived characteristic. Moreover, the body colors of the offspring obtained by mating the recipient males and females were orange (100%, 1169/1169), suggesting that all of them were derived from cryopreserved spermatogonia. Thus, we succeeded to produce the donor-derived offspring by allogenic transplantation of cryopreserved spermatogonia in medaka. These techniques will facilitate the establishment of a stable and reliable system for preserving valuable medaka strains semi-permanently. Source of funding: Grant-in-Aid for Research Fellow of the Japan Society for the Promotion of Science. Conflict of interest: None declared. sekishinsuke@hotmail.com

Production of Donor-Derived Offspring by Allogeneic Transplantation of Spermatogonia in the Yellowtail (Seriola quinqueradiata)1

Although the yellowtail (Seriola quinqueradiata) is the fish most commonly farmed in Japan, breeding of this species has not yet started. This is primarily due to the lack of sufficiently sophisticated methods for manipulating gametogenesis, which makes it difficult to collect gametes from specific dams and sires. If it were possible to produce large numbers of surrogate fish by transplanting germ cells isolated from donor individuals harboring desirable genetic traits, then the probability of acquiring gametes carrying the donor-derived haplotype would increase, and breeding programs involving this species might increase as a result. As a first step, we established a method for the allogeneic transplantation of yellowtail spermatogonia and the production of donor-derived offspring. Donor cells were collected from immature (10-month-old) yellowtail males with testes containing abundant type A spermatogonia, labeled with PKH26 fluorescent dye, and transferred into the peritoneal cavities of 8-day-old larvae. Fluorescence observation at 28 days post-transplantation revealed that PKH26-labeled cells were incorporated into recipients' gonads. To assess whether donor-derived spermatogonia could differentiate into functional gametes in the allogeneic recipient gonads, gametes collected from nine male and four female adult recipients were fertilized with wild-type eggs and milt. Analysis of microsatellite DNA markers confirmed that some of the first filial (F(1)) offspring were derived from donor fish, with the average contribution of donor-derived F(1) offspring being 66% and the maximum reaching 99%. These findings confirmed that our method was effective for transplanting yellowtail spermatogonia into allogeneic larvae to produce donor-derived offspring.

Production of donor-derived offspring after ovarian transplantation between Muscovy (Cairina moschata) and Pekin (Anas platyrhynchos) ducks

Transplantation of ovarian tissue between different breeds of chickens or Japanese quail can result in normal egg production and give rise to donor-derived offspring with a high frequency. The feasibility of interspecific ovarian transplantation was evaluated by transplanting ovarian tissue from Muscovy ducks to Pekin ducks just after hatch, with subsequent treatment of the recipients with an immunosuppressant. Among the 8 mature Pekin ducks that had received ovarian tissue from Muscovy ducklings, 2 produced fertile eggs when mated with Muscovy drakes, and one of these produced only Muscovy ducks, which were derived from the transplanted Muscovy ovary. The other produced mule ducks that were hybrids of the host Pekin duck and the Muscovy drake. Further development of interspecific transplantation of ovaries may provide a simple method for the conservation and propagation of endangered birds.

Production of Donor-Derived Offspring from Cryopreserved Ovarian Tissue in Japanese Quail (Coturnix japonica)1

Cryopreservation of avian ova and embryos is challenging because of the yolky structure of the egg. As an alternative, with the development of effective cryopreservation protocols, ovarian tissue cryopreservation could be used for cryobanking for birds. Pieces of ovarian tissue of week-old Japanese quail (Coturnix japonica) were frozen at 0.5 degrees C/min in a programmable freezer or vitrified by immersion in liquid nitrogen. Straws containing slow-frozen samples were thawed in ice water, and vitrified samples were removed from the vials and transferred into sucrose, with the concentration lowered in sequence at room temperature. Cell viability of tissue was estimated by trypan blue assay, and tissue histology was examined by light microscopy. Frozen-thawed or vitrified-warmed tissue from WB (recessive plumage color) chicks was transplanted into week-old ovariectomized QO (wild-type plumage) chicks, with some chicks receiving fresh tissue as a control group. At sexual maturity, QO recipients were mated to WB males, and the production of WB offspring demonstrated successful cryopreservation and transplantation. Donor-derived offspring were obtained from the ovarian tissue that had been cryopreserved by either slow-freezing or vitrification. The vitrification protocol used in this study showed better outcomes at each level of evaluation. This study demonstrated that the function of ovarian tissue in avian species can be successfully preserved at subzero temperatures and recovered by transplantation. The vitrification protocol is recommended because of high efficiency and overall simplicity.

Production of donor-derived offspring by transfer of primordial germ cells in Japanese quail.

We transfused concentrated primordial germ cells (PGCs) of the black strain (D: homozygous for the autosomal incomplete dominant gene, D) of quail into the embryos of the wild-type plumage strain (WP: d+/d+) of quail. The recipient quail were raised until sexual maturity and a progeny test of the putative germline chimeras was performed to examine the donor gamete-derived offspring (D/d+). Thirty-one percent (36/115) of the transfused quail hatched and 21 (13 females and 8 males) of them reached maturity. Five females and 2 males were germline chimeras producing donor gamete-derived offspring. Transmission rates of the donor derived gametes in the chimeric females and males were 1.8-8.3% and 2.6-63.0%, respectively. Germline chimeric and the other putative chimeric males were also test-mated with females from the sex-linked imperfect albino strain (AL: d+/d+, al/W, where al indicates the sex-linked imperfect albino gene on the Z chromosome, and W indicates the W chromosome) for autosexing of W-bearing spermatozoa: No albino offspring were born.