RESUMO
Zebra finch is a unique model for behavioral, neural, and genomic studies of vocal learning. Several transgenic zebra finches have been produced, although the germline transmission efficiencies are reportedly low. Recently, there have been attempts to produce germline chimeras using primordial germ cells (PGCs). However, this has been hampered by difficulties associated with the manipulation of the small eggs and the fact that the zebra finch is an altricial species that requires parental care after birth, unlike precocial chickens. Consequently, it is difficult to transplant PGCs into embryos and maintain the chimeras. Here, we developed a busulfan-mediated system for transplantation of PGCs into adult testes, to produce germline chimeras with an improved germline transmission capacity. We established microsomal glutathione-S-transferase II (MGSTII)-overexpressing PGCs that are resistant to busulfan, which induces germ cell-specific cytotoxicity, and transplanted them into testes rendered temporarily infertile by busulfan. The recipients were given a second dose of busulfan to deplete endogenous germ cells and enrich the transplanted cells, and donor cell-derived spermatogenesis was accomplished. This method requires fewer recipients due to higher survival rates, and there is no need to wait for maturation of the founders, which is required when transplanting PGCs into embryos. These results are expected to improve transgenic zebra finch production.
Assuntos
Galinhas , Aves Canoras , Masculino , Animais , Galinhas/genética , Testículo , Bussulfano/farmacologia , Células Germinativas/transplante , Animais Geneticamente ModificadosRESUMO
In germ cell transplantation experiments, the use of sterile recipients that do not produce their own gametes is an important prerequisite. Triploidization and dnd gene knockdown (KD) methods have been widely used to produce sterile fish. However, triploidization does not produce complete sterility in some fish species, and gene KD is labor and time intensive since it requires microinjection into individual fertilized eggs. To overcome these problems, in this study, we generated homozygous mutants of the dead end (dnd) gene in rainbow trout (Oncorhynchus mykiss) using the clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system, analyzed their reproductive capacity, and evaluated their suitability as recipients for germ cell transplantation. By crossing F1 heterozygous mutants produced from founders subjected to genome editing, an F2 generation consisting of approximately 1/4 homozygous knockout mutants (dnd KO) was obtained. The dnd KO hatchlings retained the same number of primordial germ cells (PGCs) as the wild-type (WT) individuals, after which the number gradually decreased. At 1 year of age, germ cells were completely absent in all analyzed individuals. To evaluate the dnd KO individuals as recipients for germ cell transplantation, germ cells prepared from donor individuals were transplanted into the abdominal cavity of dnd KO hatchlings. These cells migrated to the recipient gonads, where they initiated gametogenesis. The mature recipient individuals produced only donor-derived sperm and eggs in equivalent numbers to WT rainbow trout. These results indicate that dnd KO rainbow trout are suitable recipient candidates possessing a high capacity to nurse donor-derived germ cells.
Assuntos
Infertilidade , Oncorhynchus mykiss , Animais , Transplante de Células/métodos , Técnicas de Inativação de Genes , Células Germinativas/transplante , Gônadas , Oncorhynchus mykiss/genéticaRESUMO
The purpose of this review is to educate the reader on the role that cryopreservation has played and continues to play in the ever-evolving field of assisted reproductive technologies, specifically in clinical human fertility treatment. We discuss the science behind the cryopreservation methods and investigated some of the major considerations that any clinic or cryobank faces in terms of risks and liabilities, physical challenges that accompany the constantly growing collection of cryopreserved specimens, and what this means on the ethical and legal front. Finally, we take a glimpse in the future to explore what may be on the horizon for the preservation of gametes and reproductive tissues.
Assuntos
Criopreservação/métodos , Preservação da Fertilidade/métodos , Técnicas de Reprodução Assistida , Criopreservação/tendências , Preservação da Fertilidade/tendências , Células Germinativas/fisiologia , Células Germinativas/transplante , Humanos , Técnicas de Reprodução Assistida/tendências , VitrificaçãoRESUMO
The allogeneic transplantation of primordial germ cells (PGCs) derived from somatic cells overcomes the limitation of avian cloning. Here, we transdifferentiate chicken embryo fibroblasts (CEFs) from black feathered Langshan chickens to PGCs and transplant them into White Plymouth Rock chicken embryos to produce viable offspring with characteristics inherited from the donor. We express Oct4/Sox2/Nanog/Lin28A (OSNL) to reprogram CEFs to induced pluripotent stem cells (iPSCs), which are further induced to differentiate into PGCs by BMP4/BMP8b/EGF. DNA demethylation, histone acetylation and glycolytic activation elevate the iPSC induction efficiency, while histone acetylation and glycolytic inhibition facilitate PGCs formation. The induced PGCs (iPGCs) are transplanted into the recipients, which are self-crossed to produce 189/509 somatic cells derived chicken with the donor's characteristics. Microsatellite analysis and genome sequencing confirm the inheritance of genetic information from the donor. Thus, we demonstrate the feasibility of avian cloning from somatic cells.
Assuntos
Transdiferenciação Celular/genética , Clonagem de Organismos/métodos , Células Germinativas/transplante , Células-Tronco Pluripotentes Induzidas/fisiologia , Criação de Animais Domésticos/métodos , Animais , Proteína Morfogenética Óssea 4/genética , Células Cultivadas , Embrião de Galinha/citologia , Galinhas , Fator de Crescimento Epidérmico/genética , Estudos de Viabilidade , Fibroblastos/fisiologia , Células Germinativas/fisiologia , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição SOXB1/genética , Transplante Homólogo/métodosRESUMO
Murine animal models from genetically modified pluripotent stem cells (PSCs) are essential for functional genomics and biomedical research, which require germline transmission for the establishment of colonies. However, the quality of PSCs, and donor-host cell competition in chimeras often present strong barriers for germline transmission. Here, we report efficient germline transmission of recalcitrant PSCs via blastocyst complementation, a method to compensate for missing tissues or organs in genetically modified animals via blastocyst injection of PSCs. We show that blastocysts from germline-deficient Prdm14 knockout rats provide a niche for the development of gametes originating entirely from the donor PSCs without any detriment to somatic development. We demonstrate the potential of this approach by creating PSC-derived Pax2/Pax8 double mutant anephric rats, and rescuing germline transmission of a PSC carrying a mouse artificial chromosome. Furthermore, we generate mouse PSC-derived functional spermatids in rats, which provides a proof-of-principle for the generation of xenogenic gametes in vivo. We believe this approach will become a useful system for generating PSC-derived germ cells in the future.
Assuntos
Blastocisto/metabolismo , Proteínas de Ligação a DNA/deficiência , Células Germinativas/fisiologia , Proteínas de Ligação a RNA/genética , Espermátides/metabolismo , Fatores de Transcrição/deficiência , Animais , Blastocisto/patologia , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias , Feminino , Técnicas de Inativação de Genes , Engenharia Genética , Células Germinativas/transplante , Masculino , Camundongos , Modelos Animais , Células-Tronco Pluripotentes , Ratos , Fatores de Transcrição/genética , TranscriptomaRESUMO
Sexually transmitted infections are of major concern to reproductive specialists. Heading the list are human immunodeï¬ciency virus types 1 and 2 and hepatitis B and C viruses. These pathogens, which may cause incurable chronic infections, can be transmitted through assisted reproductive technologies and from infected mothers to the fetus or newborn. This document replaces the document of the same name last published in 2013 (Fertil Steril 2013;99:340-6).
Assuntos
Comitês Consultivos/normas , Autoenxertos/virologia , Células Germinativas/virologia , Infertilidade Feminina/virologia , Doenças Virais Sexualmente Transmissíveis/transmissão , Feminino , Células Germinativas/transplante , Humanos , Infertilidade Feminina/terapia , Fatores de Risco , Doenças Virais Sexualmente Transmissíveis/prevenção & controleRESUMO
In animals, spermatogonial transplantation in sterile adult males is widely developed; however, despite its utility, ovarian germ cell transplantation is not well developed. We previously showed that the interspecific hybrid offspring of sciaenid was a suitable model for germ cell transplantation studies as they have germ cell-less gonads. However, all these gonads have testis-like characteristics. Here, we tested whether triploidization in hybrid embryos could result in germ cell-less ovary development. Gonadal structure dimorphism and sex-specific gene expression patterns were examined in 6-month-old triploid hybrids (3nHybs). Thirty-one percent of 3nHybs had germ cell-less gonads with an ovarian cavity. cyp19a1a and foxl2, ovarian differentiation-related genes, were expressed in these gonads, whereas dmrt1 and vasa were not expressed, suggesting ovary-like germ cell-less gonad development. Some (26%) 3nHybs had testis-like germ cell-less gonads. Ovarian germ cells collected from homozygous green fluorescent protein (GFP) transgenic blue drum (BD) (Nibea mitsukurii) were transplanted into 6-month-old 3nHybs gonads via the urogenital papilla or oviduct. After 9 months, the recipients were crossed with wild type BD. Among the six 3nHyb recipients that survived, one female and one male produced fertile eggs and motile sperm carrying gfp-specific DNA sequences. Progeny tests revealed that all F1 offspring possessed gfp-specific DNA sequences, suggesting that these recipients produced only donor-derived eggs or sperm. Histological observation confirmed donor-derived gametogenesis in the 3nHyb recipients' gonads. Overall, triploidization reduces male-biased sex differentiation in germ cell-less gonads. We report, for the first time, donor-derived egg production in an animal via direct ovarian germ cell transplantation into a germ cell-less ovary.
Assuntos
Peixes/genética , Peixes/fisiologia , Células Germinativas/transplante , Gônadas/citologia , Triploidia , Animais , Animais Geneticamente Modificados , Aromatase/genética , Aromatase/metabolismo , Temperatura Baixa , RNA Helicases DEAD-box , Embrião não Mamífero , Feminino , Proteína Forkhead Box L2/genética , Proteína Forkhead Box L2/metabolismo , Regulação da Expressão Gênica , Masculino , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Fish populations continue to decline globally, signalling the need for new initiatives to conserve endangered species. Over the past two decades, with advances in our understanding of fish germ line biology, new exsitu management strategies for fish genetics and reproduction have focused on the use of germ line cells. The development of germ cell transplantation techniques for the purposes of propagating fish species, most commonly farmed species such as salmonids, has been gaining interest among conservation scientists as a means of regenerating endangered species. Previously, exsitu conservation methods in fish have been restricted to the cryopreservation of gametes or maintaining captive breeding colonies, both of which face significant challenges that have restricted their widespread implementation. However, advances in germ cell transplantation techniques have made its application in endangered species tangible. Using this approach, it is possible to preserve the genetics of fish species at any stage in their reproductive cycle regardless of sexual maturity or the limitations of brief annual spawning periods. Combining cryopreservation and germ cell transplantation will greatly expand our ability to preserve functional genetic samples from threatened species, to secure fish biodiversity and to produce new individuals to enhance or restore native populations.
Assuntos
Aquicultura , Criopreservação/veterinária , Espécies em Perigo de Extinção , Peixes/fisiologia , Células Germinativas/transplante , Reprodução , Técnicas de Reprodução Assistida/veterinária , Animais , Feminino , Peixes/genética , Masculino , Densidade DemográficaRESUMO
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.
Assuntos
Cruzamentos Genéticos , Células Germinativas/citologia , Células Germinativas/transplante , Gônadas/metabolismo , Hibridização Genética , Infertilidade/genética , Atum/genética , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Reprodução , Testículo/citologia , Testículo/metabolismo , Testículo/transplanteRESUMO
The ovarian follicle pool size is limited; it decreases with age and following germ cell-damaging chemo- or radiation therapies. Due to a trend of delaying child-bearing age in the modern society, it is important to investigate the possibility to maintain the follicle reserve for middle-aged women and cancer-bearing patients subject to therapies. Earlier studies demonstrated the important role of the mammalian targets of the rapamycin (MTOR) signaling pathway in the activation of primordial follicles and suggested that treatment with the MTOR inhibitor rapamycin could maintain the follicle pool in rodents. Here, we confirmed the ability of rapamycin treatment for 3 weeks to suppress primordial follicle development and to maintain follicle pool size in mice. We further demonstrated that the developmental potential of oocytes was not affected by rapamycin treatment and the effect of rapamycin to decrease initial follicle recruitment is reversible. Using human ovarian cortical fragments grafted into immune-deficient mice, we demonstrated the ability of rapamycin to suppress follicle growth from the primordial stage. Our studies provide the basis for further studies on the possibility of using MTOR inhibitors to maintain follicle reserve in middle-aged women and cancer patients before/during germ cell-damaging therapies.
Assuntos
Oócitos/efeitos dos fármacos , Oogênese/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Reserva Ovariana/efeitos dos fármacos , Ovário/transplante , Sirolimo/farmacologia , Envelhecimento/fisiologia , Animais , Feminino , Células Germinativas/efeitos dos fármacos , Células Germinativas/metabolismo , Células Germinativas/transplante , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Pessoa de Meia-Idade , Oócitos/fisiologia , Oogênese/fisiologia , Folículo Ovariano/fisiologia , Ovário/efeitos dos fármacos , Ovário/metabolismo , GravidezRESUMO
Germline genome editing (GGE) holds the potential to mitigate or even eliminate human heritable genetic disease, but also carries genuine risks if not appropriately regulated and performed. It also raises fears in some quarters of apocalyptic scenarios of designer babies that could radically change human reproduction. Clinical need and the availability of alternatives are key considerations in the ensuing ethical debate. Writing from the perspective of a fertility clinic, we offer a realistic projection of the demand for GGE. We lay out a framework proposing that GGE, hereditary genetic disorders, and in vitro fertilization are fundamentally entwined concepts. We note that the need for GGE to cure heritable genetic disease is typically grossly overestimated, mainly due to the underappreciated role of preimplantation genetic testing. However, we might still find applications for GGE in the correction of chromosomal abnormalities in early embryos, but techniques for that purpose do not yet exist.
Assuntos
Fertilização in vitro/tendências , Edição de Genes/ética , Engenharia Genética/ética , Feminino , Fertilização in vitro/ética , Engenharia Genética/tendências , Testes Genéticos , Células Germinativas/transplante , Humanos , Gravidez , Diagnóstico Pré-Implantação/ética , Técnicas de Reprodução Assistida/ética , Técnicas de Reprodução Assistida/tendênciasRESUMO
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.
Assuntos
Animais Geneticamente Modificados , Anticorpos Monoclonais/química , Células Germinativas , Separação Imunomagnética , Oncorhynchus mykiss , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Feminino , Células Germinativas/citologia , Células Germinativas/transplante , Masculino , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/metabolismoRESUMO
We report for the first time, a comparison of two approaches for artificially induced triploidy in zebrafish (Danio rerio) using cold shock and heat shock treatments. Of the two methods, heat shock treatment proved more effective with a triploid production rate of 100% in particular females. Subsequently, triploid zebrafish larvae were used as recipients for intraperitoneal transplantation of ovarian and testicular cells originating from vas:EGFP strain in order to verify their suitability for surrogate reproduction. Production of donor-derived sperm was achieved in 23% of testicular cell recipients and 16% of ovarian cell recipients, indicating the suitability of triploids as surrogate hosts for germ cell transplantation. Success of the transplantation was confirmed by positive GFP signal detected in gonads of dissected fish and stripped sperm. Germline transmission was confirmed by fertilization tests followed by PCR analysis of embryos with GFP specific primers. Reproductive success of germline chimera triploids evaluated as fertilization rate and progeny development was comparable to control groups.
Assuntos
Engenharia Genética/veterinária , Células Germinativas/transplante , Triploidia , Peixe-Zebra/genética , Animais , Cruzamento/métodos , Feminino , Citometria de Fluxo , Engenharia Genética/métodos , Masculino , TemperaturaRESUMO
Surrogate propagation is a systematic approach to producing donor-derived gametes using germline chimeras. In fish, the use of germline chimeras to study the development of germ cells was first conducted in the 1990s in the model fish species medaka (Oryzias latipes) and zebrafish (Danio rerio). More recently, surrogate propagation has been actively investigated as a means of efficient gamete production not only in model fish species but also in aquaculture species and endangered species. Surrogate propagation has the following components: combination of the donor and host species, host sterilization, donor cell preparation, transplantation of germ cells, and gametogenesis and gamete production in surrogate fish. In this review, we first provide a general overview of previous studies related to germ cell transplantation and the methodologies developed for different species, and consider how these have been applied in practice. Second, we consider the development of primordial germ cells in fish embryos, particularly the molecular biological approaches used for the visualization of germ cells and sterilization of host embryos. Finally, we discuss sex control in germline chimeras, which may be a key component of the use of surrogate production in aquaculture. We focus on techniques to produce sterile fish, as these are crucial to the exclusive production of donor gametes in a surrogate host. The advantages and disadvantages of various aspects of surrogate propagation are discussed, the potential use of surrogate propagation as a seedling production system is considered, and future perspectives for aquaculture are suggested.
Assuntos
Peixes/embriologia , Células Germinativas/transplante , Animais , Cruzamento/métodos , Movimento Celular , Proliferação de Células , QuimeraRESUMO
Gene targeting in mammals has revolutionized the study of complex diseases, involving the interaction of multiple genes, cells, and organ systems. In cancer, genetically engineered mouse models deciphered biological principles by integrating molecular mechanisms, cellular processes, and environmental signals. Major advances in manipulative mouse genetics are currently emerging from breakthroughs in gene editing, which open new avenues for rapid model generation. Here, we review recent developments in engineering CRISPR mouse models of cancer. We describe engineering strategies, including germline manipulation of zygotes or embryonic stem cells, direct in vivo somatic gene editing, and ex vivo targeting of cellular transplant models. We also discuss promises and limitations of the expanding spectrum of CRISPR applications, ranging from engineering of simple mutations over complex genomic rearrangements to gene and epigenome regulation. Fast and scalable in vivo CRISPR methodologies pave the way for a new phase of functional cancer genomics.
Assuntos
Sistemas CRISPR-Cas/genética , Engenharia Genética , Terapia Genética , Neoplasias/genética , Animais , Modelos Animais de Doenças , Células-Tronco Embrionárias/metabolismo , Epigenoma/genética , Edição de Genes , Células Germinativas/metabolismo , Células Germinativas/transplante , Humanos , Camundongos , Mutação , Neoplasias/patologiaRESUMO
PURPOSE: To heighten awareness of the potential legal and financial burdens faced by those providing cryopreservation storage services of embryos and gametes in light of recent lawsuits involving inadvertent thawing of specimens. METHODS: Case law review of US legal databases and courthouse dockets with a focus on lawsuits against reproductive endocrinologists and cryostorage facilities offering cryopreservation. Emphasis was placed on court decisions, awarded damages, and legal and media coverage related to cryostorage failure events. RESULTS: Lawsuits pertaining to two notable ongoing cases of cryostorage failure that occurred at fertility clinics in the US in 2018 were reviewed. Media coverage of these events and plaintiff and defense attorney strategies were evaluated. Legal documents from previous, similar cryostorage failures were also reviewed. Common claims in cryostorage system failures include breach of contract and negligent handling of property. Facilities offering cryostorage services are vulnerable to significant burden, legally and financially, if they are to experience a storage system failure. CONCLUSION: Providing cryostorage services is not without significant financial risk. Inadvertent thawing of specimens can lead to high damage awards against cryostorage facilities and those individuals linked to a cryostorage failure event. Because monetary damages can surpass insurance policy limits, those providing cryostorage services should be aware of plaintiff attorney strategies, common legal defenses, and basic asset protection principles to safeguard themselves if ever faced with these situations. Facilities should also carry out regular maintenance and safety checks on equipment and alarm structures to deter such events.
Assuntos
Criopreservação , Imperícia/legislação & jurisprudência , Técnicas de Reprodução Assistida/legislação & jurisprudência , Manejo de Espécimes , Feminino , Células Germinativas/transplante , Humanos , Masculino , Técnicas de Reprodução Assistida/economiaRESUMO
Successful derivation and cultivation of primordial germ cells (PGCs) opened the way to efficient transgenesis and genome editing in the chicken. Furthermore, implantation of male PGCs from non-chicken galliform species into the chicken embryos resulted in cross-species germline chimeras and viable offspring. We have recently improved the PGC technology by demonstrating that chicken male PGCs transplanted into the testes of adult cockerel recipients mature into functional sperms. However, the availability of this orthotopic transplantation for cross-species transfer remains to be explored. Here we tested the capacity of genetically distant male PGCs to mature in the microenvironment of adult testes. We derived PGCs from the Chinese black-bone Silkie and transplanted them into infertile White Leghorn cockerels. Within 15-18 weeks after transplantation, we observed restoration of spermatogenesis in recipient cockerels and production of healthy progeny derived from the transplanted PGCs. Our findings also indicate the possibility of cross-species orthotopic transplantation of PGCs. Thus, our results might contribute to the preservation of endangered avian species and maintaining the genetic variability of the domestic chicken.
Assuntos
Galinhas , Quimera/genética , Conservação dos Recursos Naturais , Células Germinativas/transplante , Espermatozoides/citologia , Animais , Cruzamento/métodos , Células Cultivadas , Embrião de Galinha , Galinhas/classificação , Galinhas/genética , Conservação dos Recursos Naturais/métodos , Cruzamentos Genéticos , Espécies em Perigo de Extinção , Preservação da Fertilidade/métodos , Preservação da Fertilidade/veterinária , Masculino , Espermatogênese/fisiologia , Espermatozoides/transplante , Testículo/citologia , Transplante Heterólogo/veterináriaRESUMO
Germ cell transplantation technology has played a critical role in germline modification and preservation of genetic resources. Several germ cell transplantation systems have been developed, including sperm, oocyte, or germline stem cell transplantation systems in mammals. Meanwhile, in avian species, this has mostly relied on primordial germ cell (PGC) transplantation for efficient germline transmission. In this chapter, we describe how to isolate PGCs from avian embryos and produce germline chimeras through transplantation of donor PGCs to recipient embryos.
Assuntos
Transplante de Células , Galinhas , Células Germinativas/citologia , Células Germinativas/transplante , Animais , Movimento Celular , Separação Celular/métodos , Transplante de Células/métodos , Cruzamentos Genéticos , Embrião não Mamífero , Imunofluorescência , Imuno-Histoquímica , Separação Imunomagnética/métodosRESUMO
The induction of germ-line chimerism in fish is a strategy for the reproduction of endangered or genetically valuable fish species. Chimeras can be created by transplanting a single primordial germ cell or multiple blastomeres from a donor into a sterile host embryo. When the host reaches sexual maturity, it will produce donor-originating gametes throughout its reproductive life span. This technique provides unique experimental conditions for basic biology research in model fish species like zebrafish. The success of cell transplantation relies on the effective sterilization of host embryos, the correct identification of developing germ cells, and the synchronization of migratory cues between the host and the transplanted cells. Developments in non-transgenic methods of germ cell ablation and identification have made germ cell transplantation more applicable to use in conservation and aquaculture. In this chapter, we provide a protocol for germ cell labeling by injection of chimeric RNA or FITC-dextran, the sterilization of host embryos using an antisense morpholino oligonucleotide, and two methods for producing germ-line chimeras in zebrafish: single primordial germ cell transplant and blastomere transplant.
Assuntos
Desenvolvimento Embrionário/genética , Células Germinativas/citologia , Células Germinativas/metabolismo , Peixe-Zebra/genética , Animais , Blastômeros/citologia , Blastômeros/metabolismo , Blástula , Técnicas de Cultura Embrionária , Embrião não Mamífero , Células Germinativas/transplante , Microinjeções/métodos , ReproduçãoRESUMO
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.