Migration Of Primordial Germ Cells Research Articles

Primordial germ cell in teleost fish with special references to its specification and migration

Summary Germ-line chimeras provide a valuable approach to surrogate propagation in poultry farming and aquaculture. The appropriate combinations of donor germ cells and host individuals enable the production of eggs and sperm and of offspring from strains with a high commercial value or valuable genotype via an easily reared species or strain. Primordial germ cells (PGCs) are one of the candidate cell types that can be used in the production of germ-line chimeras. The model teleost species, zebrafish, has been used to study specification, differentiation and migration of PGCs. However, it is important that the properties of PGCs are also determined for fish species used in aquaculture. Recent technical developments together with improvements in embryo manipulation techniques have enabled analyses of the differentiation of PGCs and their migration to the genital ridge. Here, we report the characteristic properties of PGCs from several teleost species and investigate their behavior in germ-line chimeras using fluorescently-tagged PGCs.

An Evolutionarily Conserved Arginine Is Essential for Tre1 G Protein-Coupled Receptor Function During Germ Cell Migration in Drosophila melanogaster

BackgroundG protein-coupled receptors (GPCRs) play central roles in mediating cellular responses to environmental signals leading to changes in cell physiology and behaviors, including cell migration. Numerous clinical pathologies including metastasis, an invasive form of cell migration, have been linked to abnormal GPCR signaling. While the structures of some GPCRs have been defined, the in vivo roles of conserved amino acid residues and their relationships to receptor function are not fully understood. Trapped in endoderm 1 (Tre1) is an orphan receptor of the rhodopsin class that is necessary for primordial germ cell migration in Drosophila melanogaster embryos. In this study, we employ molecular genetic approaches to identify residues in Tre1 that are critical to its functions in germ cell migration.Methodology/Principal FindingsFirst, we show that the previously reported scattershot mutation is an allele of tre1. The scattershot allele results in an in-frame deletion of 8 amino acids at the junction of the third transmembrane domain and the second intracellular loop of Tre1 that dramatically impairs the function of this GPCR in germ cell migration. To further refine the molecular basis for this phenotype, we assayed the effects of single amino acid substitutions in transgenic animals and determined that the arginine within the evolutionarily conserved E/N/DRY motif is critical for receptor function in mediating germ cell migration within an intact developing embryo.Conclusions/SignificanceThese structure-function studies of GPCR signaling in native contexts will inform future studies into the basic biology of this large and clinically important family of receptors.

A crucial role for lipid phosphorylation in WntD-mediated primordial germ cell migration

A crucial role for lipid phosphorylation in WntD-mediated primordial germ cell migration

Nanog Regulates Primordial Germ Cell Migration Through Cxcr4b

Gonadal development in vertebrates depends on the early determination of primordial germ cells (PGCs) and their correct migration to the sites where the gonads develop. Several genes have been implicated in PGC specification and migration in vertebrates. Additionally, some of the genes associated with pluripotency, such as Oct4 and Nanog, are expressed in PGCs and gonads, suggesting a role for these genes in maintaining pluripotency of the germ lineage, which may be considered the only cell type that perpetually maintains stemness properties. Here, we report that medaka Nanog (Ol-Nanog) is expressed in the developing PGCs. Depletion of Ol-Nanog protein causes aberrant migration of PGCs and inhibits expression of Cxcr4b in PGCs, where it normally serves as the receptor of Sdf1a to guide PGC migration. Moreover, chromatin immunoprecipitation analysis demonstrates that Ol-Nanog protein binds to the promoter region of Cxcr4b, suggesting a direct regulation of Cxcr4b by Ol-Nanog. Simultaneous overexpression of Cxcr4b mRNA and depletion of Ol-Nanog protein in PGCs rescues the migration defective phenotype induced by a loss of Ol-Nanog, whereas overexpression of Sdf1a, the ligand for Cxcr4b, does not restore proper PGC migration. These results indicate that Ol-Nanog mediates PGC migration by regulating Cxcr4b expression.

Molecular characterization of isolated from murine adult tissues very small embryonic/epiblast like stem cells (VSELs).

Pluripotent very small embryonic/epiblast derived stem cells (VSELs) as we hypothesize are deposited at begin of gastrulation in developing tissues and play an important role as backup population of pluripotent stem cells (PSCs) for tissue committed stem cells (TCSCs). We envision that during steady state conditions these cells may be involved in tissue rejuvenation and in processes of regeneration/repair after organ injuries. Molecular analysis of adult bone marrow (BM)-derived purified VSELs revealed that they i) express pluripotent stem cells markers e.g., Oct4, Nanog, Klf-4, SSEA-1 ii) share several markers characteristic for epiblast as well as migratory primordial germ cells (PGCs), and iii) possess a unique pattern of genomic imprinting (e.g., erasure of differently methylated regions at Igf2-H19 and Rasgrf1 loci and hypermethylation at KCNQ1 and Igf2R loci). This supports that VSELs are related to epiblast-derived migrating PGC-like cells and, despite their pluripotent stem cell character, changes in the epigenetic signature of imprinted genes keep these cells quiescent in adult tissues and prevent them from teratoma formation. In contrast epigenetic changes/mutations that lead to activation of imprinted genes could potentially lead to tumor formation by these cells. Mounting evidence accumulates that perturbation of expression of imprinted genes is a common phenomenon observed in developing tumors.

The chemokine receptor CXCR7 is expressed on lymphatic endothelial cells during renal allograft rejection

CXCR7 is an atypical receptor for the chemokines CXCL11 and CXCL12, which were found to be involved in animal models of allograft injury. We studied the expression of CXCR7 and its ligands in human kidneys by first quantifying the mRNA in 53 renal allograft biopsies. Receptor and ligand mRNAs were expressed in renal allografts, with a significant induction of CXCL11 and CXCL12 in biopsies showing borderline lesions and acute rejection. Immunohistochemical analysis for CXCR7 was performed in a series of 64 indication and 24 protocol biopsies. The indication biopsies included 46 acute rejections, 6 with interstitial fibrosis and tubular atrophy, and 12 pretransplant biopsies as controls. In control biopsies, CXCR7 protein was found on smooth muscle and on endothelial cells of a small number of peritubular vessels. The number of CXCR7-positive vessels was increased in acute rejection and, using double immunofluorescence labeling, a subset of these CXCR7-positive endothelial cells were identified as lymphatic vessels. Both CXCR7-positive blood and lymphatic vessels increased during allograft rejection. We found that CXCR7 is present in both blood and lymphatic endothelial cells in human renal allografts. Whether its presence modulates the formation of chemokine gradients and the recruitment of inflammatory cells will require further experimental studies.

Zebrafish dead end possesses ATPase activity that is required for primordial germ cell development

Zebrafish dead end (dnd) mRNA is specifically expressed in primordial germ cells (PGCs) and is required for PGC migration and survival. Previous studies have shown that zebrafish Dnd functions by protecting the 3'UTRs of nanos1 and TDRD7 from miR-430b-mediated RNA deadenylation. In this work, we demonstrate that zebrafish Dnd protein possesses Mg(2+)-dependent ATPase activity that is required for PGC formation. Michaelis-Menten analysis revealed that the ATPase has a k(cat) of 0.632 +/- 0.036/min under optimal conditions, and mapping studies using Dnd truncates showed that ATPase resides in the last 91 aa of the Dnd C terminus. Internal deletion and point mutagenesis analysis of this region were used to identify key amino acids required for ATPase activity. Rescue experiments conducted by injecting mRNAs encoding the Dnd ATPase mutants into embryos in which the endogenous dnd expression was inhibited demonstrated that the ATPase activity is required for normal zebrafish PGC survival. Real-time PCR analysis showed that the expression of PGC markers nanos1 and TDRD7 but not vasa were down-regulated when dnd mutant proteins lacking ATPase were expressed in the rescued embryos, indicating that the Dnd ATPase is involved in protecting nanos1 and TDRD7 transcripts.

Perturbation of Notch/Suppressor of Hairless pathway disturbs migration of primordial germ cells in Xenopus embryo

Primordial germ cells (PGCs) in Xenopus embryo are specified in the endodermal cell mass and migrate dorsally toward the future gonads. The role of the signal mediated by Notch and Suppressor of Hairless [Su(H)] was analyzed on the migrating PGCs at the tailbud stage. X-Notch-1 and X-Delta-1 are expressed in the migrating PGCs and surrounding endodermal cells, whereas X-Delta-2 and X-Serrate-1 are expressed preferentially in the PGCs. Suppression and constitutive activation of the Notch/Su(H) signaling in the whole endoderm region or selectively in the PGCs resulted in an increase in ectopic PGCs located in lateral or ventral regions. Knocking down of the Notch ligands by morpholino oligonucleotides revealed that X-Delta-2 was indispensable for the correct PGC migration. The ectopic PGCs seemed to have lost their motility in the Notch/Su(H) signal-manipulated embryos. Our results suggest that a cell-to-cell interaction via the Notch/Su(H) pathway has a significant role in the PGC migration by regulating cell motility.

Inter-species transplantation and migration of primordial germ cells in cyprinid fish

Primordial germ cells (PGCs) are the only cells in developing embryos that can transmit genetic information to the next generation. PGCs therefore have considerable potential value for gene banking and cryopreservation, particularly via production of donor gametes using germ-line chimeras. In some animal species, including teleost fish, the feasibility of using PGC transplantation to obtain donor-derived offspring, within and between species, has been demonstrated. Successful use of PGC transplantation to produce germ-line chimeras is absolutely dependent on the migration of the transplanted cells from the site of transplantation to the host gonadal region. Here, we induced germ-line chimeras between teleost species using two different protocols: blastomere transplantation and single PGC transplantation. We evaluated the methods using the rate of successful migration of transplanted PGCs to the gonadal region of the host embryo. First, we transplanted blastomeres from zebrafish, pearl danio, goldfish, or loach into blastula-stage zebrafish embryos. Some somatic cells, derived from donor blastomeres, were co-transplanted with the PGCs and formed aggregates in the host embryos; a low efficiency of PGC transfer was achieved. Second, a single PGC from the donor species was transplanted into a zebrafish embryo. In all inter-species combinations, the donor PGC migrated toward the gonadal region of the host embryo at a comparatively high rate, regardless of the phylogenetic relationship of the donor and host species. These transplantation experiments showed that the mechanism of PGC migration is highly conserved beyond the family barrier in fish and that transplantation of a single PGC is an efficient method for producing inter-species germ-line chimeras.

Influence of Microgravity on the Concentration of Circulating Primordial Germ Cells in Silky Chicken Offspring

This study was performed to examine the implications of microgravity on circulating primordial germ cells (PGCs) of the offspring of silky chickens at stages 13 to 17. China's ShenZhou-3 unmanned spaceship was launched on March 25, 2002, at 22: 00 Beijing time (14: 00GMT). The spaceship carried nine fertilized silky chicken eggs (F0) to test the reliability of the life-support system in the space environment. One female and two male chickens were born from these eggs. The three chickens mated naturally and F1 fertilized eggs were collected. Blood was collected from the dorsal aorta or the marginal vein of the embryos at stages 13 to 17, and the number of circulating PGCs of F1 offspring was counted. A similar experimental protocol was performed for the control group (C1 and C2 group). No differences were observed except at stage 15, when the F1 offspring of the flight group (F0 group) showed higher PGC concentrations than the other treatment groups. These results indicated that microgravity may have little effect on the migration and concentration of PGCs in F1 offspring, perhaps because the flight chickens were raised to maturity on Earth under a gravity of 1×g and had sufficient time to recover. Thus, microgravity appeared to have little effect on the PGC concentrations of F1 offspring of silky chickens during circulating stages 13 to 17.

Migration and Accumulation of Primordial Germ Cells of Early Embryo in Quail

In this research, we have identified primordial germ cells (PGCs) in quail embryo using Quail Hemangioblastic Lineage (QH1) monoclonal antibody analysis. Quail PGCs originated from the opaca of unincubated blastodisc, and then transferred to the pellucida and the germinal crescent. At 27 hours post-incubation, a few PGCs first appeared in blood vessels of the pellucida, where many PGCs accumulated at 36 hours post-incubation. The PGCs scattered or clustered from head to omphalo mesenteric and mainly settled down in the mesenchymal blood vessels of head at 45 hours post-incubation. The size of PGCs population increased significantly (P<0.05) from stage XII (12.8±4.82 µm) to primitive streak stage (106.7±8.74 µm) and from Head process stage (95.8±19.74 µm) to tenth somite stage (199.4±19.97 µm). It is concluded that the PGCs scattered in the head area before migration to the germinal crescent and distributed randomly in both gland. The number of PGCs varied at different stages with two peaks, primitive streak stage (18 hours post-incubation) and tenth somite (36 hours post-incubation).

Mechanisms guiding primordial germ cell migration: strategies from different organisms.

The regulated migration of cells is essential for development and tissue homeostasis, and aberrant cell migration can lead to an impaired immune response and the progression of cancer. Primordial germ cells (PGCs), precursors to sperm and eggs, have to migrate across the embryo to reach somatic gonadal precursors, where they carry out their function. Studies of model organisms have revealed that, despite important differences, several features of PGC migration are conserved. PGCs require an intrinsic motility programme and external guidance cues to survive and successfully migrate. Proper guidance involves both attractive and repulsive cues and is mediated by protein and lipid signalling.

In vitro germ cell differentiation during sex differentiation in a teleost fish

To clarify the sexually dimorphic mechanisms of gonadal sex differentiation, we established an in vitro culture system for gonadal sex differentiation using the teleost fish Oreochromis niloticus. In vivo, the entry of germ cells into meiosis occurs around 35 days after hatching (dah) in XX gonads, whereas in XY gonads, meiotic cells became differentiated around 85 dah. In our in vitro culture system using gonads from young fry at 23 dah, the meiotic cells in the XX gonads appeared after 21 days of culture. In contrast, in the XY gonads, no meiotic cells were detected after 21 days. These results indicate that germ cell differentiation in this culture system progresses in a manner similar to that in vivo. To identify the gene products that are involved in the entry of germ cells into meiosis or in the arrest of germ cells at the gonial stage of gonadal sex differentiation, we performed subtractive hybridization screening with this in vitro culture system. From the screening process, we identified the female-related gene, FR-3, which is a homolog of zebrafish nanos-related gene (nos). The nos gene was expressed after gonadal formation around 35 dah in XX gonads, but not in XY gonads. In situ hybridization indicated that nos is expressed in oogenic meiotic cells, but not in spermatogenic meiotic cells. Further examination revealed that nos was expressed in oogenic meiotic cells after gonadal formation, specifically in teleost fish. Together, nos may be also involved in oogenic meiosis, with the exception of primordial germ cell migration.

Genetic and Epigenetic Studies Reveal That Murine Oct-4+ Very Small Embryonic/Epiblast-Like Stem Cells (VSELs) Present in Adult Tissues Share Several Similarities/Markers with Epiblast-Derived Migratory Primordial Germ Cells (PGCs).

Abstract 2521Poster Board II-498Recently, we identified a population of pluripotent VSELs in murine adult bone marrow (BM; Leukemia 2006:20;857). Compared with other adult stem cells (SCs), VSELs show unique epigenetic features including: i) open chromatin structures in the promoter of Oct-4 and Nanog; and ii) parent-of-origin-specific reprogramming of genomic imprinting. These features explain the pluripotent embryonic-like nature and quiescent status of these cells, respectively (Leukemia 2009:In press), and indicate their relation to an epiblast/germ-line pluripotent (P)SC population. To better understand the developmental origin of VSELs, we examined gene expression profiles and the epigenetic status of epiblast and germ-line related genes in these cells. We employed real time quantitative PCR (RQ-PCR) to evaluate gene expression, a bisulfite sequencing strategy to evaluate DNA methylation, and chromatin immunoprecipitation (ChIP) to elucidate histone codes in genes of interest. VSELs were isolated from murine BM by multiparameter fluorescence-activated cell sorter (FACS) as a population of Sca+lin−CD45− along with Sca+lin−CD45+ hematopoietic (H)SCs and BM mononuclear cells (MNCs). We noticed that VSELs, similarly to epiblast PSCs (EpiSCs), highly express the stemness genes (e.g., Oct-4, Nanog, Sox2, Klf4) and epiblast markers (Gbx2, Fgf5, Nodal). However, the Rex1 gene is expressed at a lower level compared to the murine ESC-D3 line. Moreover, VSELs also highly express the Stella, Blimp1, Dnd1, and Nanos3, which are developmental regulators during specification in the proximal epiblast of PGCs. Accordingly, the Stella promoter in VSELs was partially demethylated and highly enriched for transcriptionally active histones (acetylated H3, trimethylated lysine4 of H3) while being simultaneously less enriched for repressive ones (dimethylated lysine9 and trimethylated lysine27 of H3). In particular, we noticed that VSELs resemble migratory PGCs. To support this notion, VSELs: i) express several markers of migratory PGCs (Dppa2, Dppa4, Mvh); ii) do not express post-migratory PGCs genes (Dazl, Sycp3); and iii) have reprogrammed DNA demethylation in the repetitive sequence (LINE1) and the promoters of Mvh, Dazal, and Sycp3. Finally, VSELs express less the transcripts for cMyc, Stat3, Snai, and Ecat1, which are expressed in early specified PGCs. In conclusion, our previous data showing reprogramming of genomic imprinting in VSELs with the present gene expression profile and epigenetic studies strongly supports VSELs developmentally originating from epiblast-derived germ-line SCs (PGCs), particularly migratory PGCs. We believe VSELs are deposited during embryogenesis in the adult tissues as a backup for tissue-committed SCs and that epigenetic reprogramming tightly controls their proliferative potential. Thus, identification of mechanisms that control and modify the epigenetic marks in VSELs will be crucial for developing more powerful strategies to “unleash the power” of these cells and employ them in regenerative medicine. Disclosures:No relevant conflicts of interest to declare.

Prenylation-deficient G protein gamma subunits disrupt GPCR signaling in the zebrafish

Prenylation of G protein gamma (γ) subunits is necessary for the membrane localization of heterotrimeric G proteins and for functional heterotrimeric G protein coupled receptor (GPCR) signaling. To evaluate GPCR signaling pathways during development, we injected zebrafish embryos with mRNAs encoding Gγ subunits mutated so that they can no longer be prenylated. Low-level expression of these prenylation-deficient Gγ subunits driven either ubiquitously or specifically in the primordial germ cells (PGCs) disrupts GPCR signaling and manifests as a PGC migration defect. This disruption results in a reduction of calcium accumulation in the protrusions of migrating PGCs and a failure of PGCs to directionally migrate. When co-expressed with a prenylation-deficient Gγ, 8 of the 17 wildtype Gγ isoforms individually confer the ability to restore calcium accumulation and directional migration. These results suggest that while the Gγ subunits possess the ability to interact with G Beta (β) proteins, only a subset of wildtype Gγ proteins are stable within PGCs and can interact with key signaling components necessary for PGC migration. This in vivo study highlights the functional redundancy of these signaling components and demonstrates that prenylation-deficient Gγ subunits are an effective tool to investigate the roles of GPCR signaling events during vertebrate development.

Analysis of SDF-1/CXCR4 signaling in primordial germ cell migration and survival or differentiation in Xenopus laevis

Directional migration of primordial germ cells (PGCs) toward future gonads is a common feature in many animals. In zebrafish, mouse and chicken, SDF-1/CXCR4 chemokine signaling has been shown to have an important role in PGC migration. In Xenopus, SDF-1 is expressed in several regions in embryos including dorsal mesoderm, the target region that PGCs migrate to. CXCR4 is known to be expressed in PGCs. This relationship is consistent with that of more well-known animals. Here, we present experiments that examine whether chemokine signaling is involved in PGC migration of Xenopus. We investigate: (1) Whether injection of antisense morpholino oligos (MOs) for CXCR4 mRNA into vegetal blastomere containing the germ plasm or the precursor of PGCs disturbs the migration of PGCs? (2) Whether injection of exogenous CXCR4 mRNA together with MOs can restore the knockdown phenotype? (3) Whether the migratory behavior of PGCs is disturbed by the specific expression of mutant CXCR4 mRNA or SDF-1 mRNA in PGCs? We find that the knockdown of CXCR4 or the expression of mutant CXCR4 in PGCs leads to a decrease in the PGC number of the genital ridges, and that the ectopic expression of SDF-1 in PGCs leads to a decrease in the PGC number of the genital ridges and an increase in the ectopic PGC number. These results suggest that SDF-1/CXCR4 chemokine signaling is involved in the migration and survival or in the differentiation of PGCs in Xenopus.

The expression of a novel cxcr4 gene in Xenopus embryo.

The aim of the present work was to identify a homologue of zebrafish cxcr4b in Xenopus, which could be involved in primordial germ cell (PGC) guidance migration. Following a BLAST analysis, the clone gi 27519681, homologous to the zebrafish gene z-cxcr4b, was identified, inserted into pCMV-SPORT6 plasmid and cloned in Escherichia coli. Embryonic expression of x-cxcr4b was analyzed by RT-PCR. X-cxcr4b was weakly expressed maternally but sharply increased after the mid-blastula transition (MBT), declining significantly at stage 45 when PGCs migration is complete. In contrast, RT-PCR of isolated presumptive PGCs showed strong maternal expression at stage 8, which decreased by stage 10 post-MBT and was not detected at stage 14. Whole mount in situ hybridization of x-cxcr4b mRNA showed that this gene is expressed in neural and haematopoietic tissues, and should be linked to important processes during embryonic development of these organs. Although weak staining could be seen in some samples within the anterior endoderm, expression of x-cxcr4b was never coincident with that of Xpat mRNA, which labels PGCs restricted to the posterior endoderm. Therefore, maternal x-cxcr4b is specifically downregulated within PGCs at pre-migratory stages while it is expressed in other tissues.

Migration and Proliferation of Intact and Genetically Modified Primordial Germ Cells and the Generation of a Transgenic Chicken1

This study evaluated gonadal migration and postmigratory proliferation of intact and genetically modified chicken primordial germ cells (PGCs). A randomized, controlled trial was conducted with the gonadal population of PGCs and transgenic chicken production as major parameters. PGCs (0, 90, 900, 1800, or 3000 cells) were transferred into 53-h-old embryos. The percentage of PGCs migrating on Day 6 of development was highest (35.8%) following the transfer of 900 PGCs and did not change with increases in transferred PGCs. The number of migrating PGCs gradually increased (P = 0.0001) as the number of transferred PGCs was increased. Gonadal migration was detected after the transfer of intact and genetically modified PGCs, but prominent decreases in PGC migration (from 21.9% to 0.38%) and chimera ratio (from 0.4 to 0.007) occurred with genetically modified PGCs. However, subsequent vigorous proliferation of the modified PGCs (3.67-fold increase from transferred number) led to the derivation of a germline chimera and produced a transgenic hatchling. In conclusion, the number of migrating PGCs increased as the number of transferred cells increased. Vigorous proliferation after transfer compensated for the decreased migration capacity of genetically modified PGCs and resulted in the production of a transgenic chicken.

Germ Cell Depletion During Embryogenesis Does Not Affect Subsequent Gonadal Differentiation in Goldfish.

In mammals, the germ line is essential for early folliculogenesis, and thus germ cell deficient animals fail to maintain ovarian differentiation. However, in teleosts, the roles of germ cells in the regulation of sex determination and differentiation are less well understood. Recent reports suggest that germ cell numbers influence ovary fate determination in zebrafish. Fish that lack germ cells or have only a single transplanted primordial germ cell (PGC) develop as phenotypic males. However, as sex determination and gonadal differentiation differ among teleost species, this phenomenon may be restricted to zebrafish or juvenile undifferentiated gonochoristic species. Here, we examined whether germ cells are necessary for gonadal differentiation in goldfish, a gonochoristic species that possesses XX-XY genetic sex determination. Our first step was to isolate the dead end (dnd) gene, which is necessary for the proper migration and survival of PGCs, and to design an anti-sense morpholino (MO) to this gene. Then, we compared germ cell development at 5 months in uninjected controls and in fish derived from embryos injected with dnd-MO at the 1-4 cell stage. In contrast to zebrafish, goldfish without germ cells developed either an ovary or a testis. Two types of gonad were identified: one was flat, wide and transparent, the other was narrow and string-like. Histological analysis showed the former to be an "empty ovary" with a clear ovarian cavity, while the latter was an "gempty testis" with a tubular structure. Thus, germ cells are not essential for gonadal fate determination in goldfish. In a subsequent experiment, we tested whether function was restored to these empty gonads in germline chimeras produced by transplanting a single PGC. Donor PGCs from embryos with an albino phenotype were labeled using gfp-nos1 3'UTR mRNA injected at the 1-4 cell stage. The embryos were enzymatically dissociated at the early somite stage and a single PGC was transplanted into each host embryo (wild type phenotype), which had been injected with dnd-MO. These germline chimeras developed either an ovary or a testis with a fully mature gonad on one side and an empty gonad on the other, showing the typical features of single PGC-transplanted chimeras. Cross breeding with male chimeric and female albino fish yielded 100% albino offspring, indicating that complete germ line replacement had occurred and the PGC-transplanted chimeras had recovered gonadal function. These findings suggest that, in goldfish, phenotypic sex is determined by somatic cells that express the relevant genes under genetic sex control rather than an interaction between germ cells and somatic cells. The generation of germline chimeras provides a valuable approach to investigating the contribution of germ cells to sex determination and differentiation, gonad formation and gametogenesis in teleosts, because of the ability to completely replace the germ line. (platform)

Isolation of Adult Gonadal Stem Cells from Coturnix japonica and Characterization of Embryonic Gonadal Stem Cells in Gallus gallus for Xenotransfer.

Interspecific (xeno) transfer of embryonic primordial germ cells (PGCs) from quail (Coturnix japonica) to chicken (Gallus gallus) has produced germ cell chimeras that generate viable donor-derived germ cells. The practical application of this technique for the preservation of endangered species requires the use of gonadal stem cells (GSCs) recovered from adult birds at necropsy. Using the quail to chicken model, we examined the feasibility and efficacy of isolating adult GSCs for xenotransfer into host embryos. In this study, GSCs from adult quail testes were quantified by flow cytometry using antibodies against GSC-specific antigens: stage-specific embryonic antigen-1 (SSEA1; 88%), stage-specific embryonic antigen-4 (SSEA4; <1%), CD9; <1%, epithelial membrane antigen-1 (EMA1; <1%), integrin alpha-6 (alpha6; <1%), and integrin beta-1 (beta1; 3%). The high percentage of SSEA1 positive cells indicates that this antigen is too widely expressed to be a differential stem cell marker for adult testicular cells. The percentage of beta1 positive cells suggests that this marker is more suitable for stem cell identification in this model. GSC populations were then enriched by magnetic activated cell sorting (MACS). Following MACS, the proportion of testicular cells expressing stem cell antigens increased: SSEA1, 1.1 fold; SSEA4, 40.3; EMA1, 70.1; CD9, 209.7: alpha6, 114.6; beta1, 23.4. Finally, we quantified the expression of stem cell markers in the embryonic gonad of 11-day incubated chicken embryos to determine the characteristics of control, non-chimeric host tissue for comparison to post-xenotransfer chimeric gonads. In the male embryonic gonad, we found a greater number of SSEA1 and β1 antigen expressing cells and low expression levels of the other antigens tested (SSEA4, CD9, EMA1, alpha6). We found a similar pattern in the female embryonic gonad, with a SSEA1 and β1 expressed at higher levels than the other antigens tested. Ideally, we aim to transplant a GSC enriched population coincident with endogenous PGC migration that will best integrate into the host gonad and outnumber, thus supplant, the endogenous PGCs. The quail to chicken germ cell chimera using adult gonadal tissue is a model for the development of xenotransfer of endangered avian GSCs. With this technique, GSC rescued from genetically important or endangered species may survive to produce functional gametes in the gonads of non-endangered hosts. (poster)