Direct in vitro propagation of avian germ cells from an embryonic gonad biorepository.

Direct introduction of cryopreserved embryonic gonadal germ cells (GGC) into a sterile chicken surrogate host to reconstitute a chicken breed has been demonstrated as a feasible approach for preserving and utilizing chicken genetic resources. This method is highly efficient using male gonads; however, a large number of frozen female embryonic gonads is needed to provide sufficient purified GGC for the generation of fertile surrogate female hosts. Applying this method to indigenous chicken breeds and other bird species is difficult due to small flock numbers and poor egg production in each egg laying cycle. Propagating germ cells from the frozen gonadal tissues may be a solution for the biobanking of these birds. Here, we describe a simplified method for culture of GGC from frozen embryonic 9.5 d gonads. At this developmental stage, the germ cells are autonomously shed into medium, yielding hundreds to thousands of mitosis-competent germ cells. The resulting cultures of GGC have over 90% purity, uniformly express SSEA-1 and DAZL antigens and can re-colonize recipient's gonads. The GGC recovery rate from frozen gonads are 42% to 100%, depending on length of cryopreservation and the breed or line of chickens. Entire chicken embryos can also be directly cryopreserved for later gonadal isolation and culture. This storage method is a supplementary approach to safeguard local indigenous chicken breeds bearing valuable genetic traits and should be applicable to the biobanking of many bird species.

Innovations in poultry reproduction using cryopreserved avian germ cells.

Meat and eggs from chicken are the major source of animal protein for the human population. The cryopreservation of poultry species is needed to guarantee sustainable production. Here, we describe the existing cryopreservation technologies for avian reproductive cells using embryonic germ cells, spermatozoa and ovarian tissues. We outline strategies to reconstitute chicken breeds from their cryopreserved embryonic germ cells using surrogate hosts and discuss the perspectives for genetic conservation and reconstitution of chicken and wild avian species using surrogate host animals.

Enhanced germline stem cell longevity in Drosophila diapause.

In many species including humans, aging reduces female fertility. Intriguingly, some animals preserve fertility longer under specific environmental conditions. For example, at low temperature and short day-length, Drosophila melanogaster enters a state called adult reproductive diapause. As in other stressful conditions, ovarian development arrests at the yolk uptake checkpoint; however, mechanisms underlying fertility preservation and post-diapause recovery are largely unknown. Here, we report that diapause causes more complete arrest than other stresses yet preserves greater recovery potential. During dormancy, germline stem cells (GSCs) incur DNA damage, activate p53 and Chk2, and divide less. Despite reduced niche signaling, germline precursor cells do not differentiate. GSCs adopt an atypical, suspended state connected to their daughters. Post-diapause recovery of niche signaling and resumption of division contribute to restoring GSCs. Mimicking one feature of quiescence, reduced juvenile hormone production, enhanced GSC longevity in non-diapausing flies. Thus, diapause mechanisms provide approaches to GSC longevity enhancement.

DNase II mediates a parthanatos-like developmental cell death pathway in Drosophila primordial germ cells.

During Drosophila embryonic development, cell death eliminates 30% of the primordial germ cells (PGCs). Inhibiting apoptosis does not prevent PGC death, suggesting a divergence from the conventional apoptotic program. Here, we demonstrate that PGCs normally activate an intrinsic alternative cell death (ACD) pathway mediated by DNase II release from lysosomes, leading to nuclear translocation and subsequent DNA double-strand breaks (DSBs). DSBs activate the DNA damage-sensing enzyme, Poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) and the ATR/Chk1 branch of the DNA damage response. PARP-1 and DNase II engage in a positive feedback amplification loop mediated by the release of PAR polymers from the nucleus and the nuclear accumulation of DNase II in an AIF- and CypA-dependent manner, ultimately resulting in PGC death. Given the anatomical and molecular similarities with an ACD pathway called parthanatos, these findings reveal a parthanatos-like cell death pathway active during Drosophila development.

Reconstitution of prospermatogonial specification in vitro from human induced pluripotent stem cells.

Establishment of spermatogonia throughout the fetal and postnatal period is essential for production of spermatozoa and male fertility. Here, we establish a protocol for in vitro reconstitution of human prospermatogonial specification whereby human primordial germ cell (PGC)-like cells differentiated from human induced pluripotent stem cells are further induced into M-prospermatogonia-like cells and T1 prospermatogonia-like cells (T1LCs) using long-term cultured xenogeneic reconstituted testes. Single cell RNA-sequencing is used to delineate the lineage trajectory leading to T1LCs, which closely resemble human T1-prospermatogonia in vivo and exhibit gene expression related to spermatogenesis and diminished proliferation, a hallmark of quiescent T1 prospermatogonia. Notably, this system enables us to visualize the dynamic and stage-specific regulation of transposable elements during human prospermatogonial specification. Together, our findings pave the way for understanding and reconstructing human male germline development in vitro.

Collectively stabilizing and orienting posterior migratory forces disperses cell clusters in vivo.

Individual cells detach from cohesive ensembles during development and can inappropriately separate in disease. Although much is known about how cells separate from epithelia, it remains unclear how cells disperse from clusters lacking apical-basal polarity, a hallmark of advanced epithelial cancers. Here, using live imaging of the developmental migration program of Drosophila primordial germ cells (PGCs), we show that cluster dispersal is accomplished by stabilizing and orienting migratory forces. PGCs utilize a G protein coupled receptor (GPCR), Tre1, to guide front-back migratory polarity radially from the cluster toward the endoderm. Posteriorly positioned myosin-dependent contractile forces pull on cell-cell contacts until cells release. Tre1 mutant cells migrate randomly with transient enrichment of the force machinery but fail to separate, indicating a temporal contractile force threshold for detachment. E-cadherin is retained on the cell surface during cell separation and augmenting cell-cell adhesion does not impede detachment. Notably, coordinated migration improves cluster dispersal efficiency by stabilizing cell-cell interfaces and facilitating symmetric pulling. We demonstrate that guidance of inherent migratory forces is sufficient to disperse cell clusters under physiological settings and present a paradigm for how such events could occur across development and disease.

Proteomic and metabolomic analyses uncover sex-specific regulatory pathways in mouse fetal germline differentiation†.

Regulatory mechanisms of germline differentiation have generally been explained via the function of signaling pathways, transcription factors, and epigenetic regulation; however, little is known regarding proteomic and metabolomic regulation and their contribution to germ cell development. Here, we conducted integrated proteomic and metabolomic analyses of fetal germ cells in mice on embryonic day (E)13.5 and E18.5 and demonstrate sex- and developmental stage-dependent changes in these processes. In male germ cells, RNA processing, translation, oxidative phosphorylation, and nucleotide synthesis are dominant in E13.5 and then decline until E18.5, which corresponds to the prolonged cell division and more enhanced hyper-transcription/translation in male primordial germ cells and their subsequent repression. Tricarboxylic acid cycle and one-carbon pathway are consistently upregulated in fetal male germ cells, suggesting their involvement in epigenetic changes preceding in males. Increased protein stability and oxidative phosphorylation during female germ cell differentiation suggests an upregulation of aerobic energy metabolism, which likely contributes to the proteostasis required for oocyte maturation in subsequent stages. The features elucidated in this study shed light on the unrevealed mechanisms of germ cell development.

Origin and function of the yolk sac in primate embryogenesis.

Human embryogenesis is hallmarked by two phases of yolk sac development. The primate hypoblast gives rise to a transient primary yolk sac, which is rapidly superseded by a secondary yolk sac during gastrulation. Moreover, primate embryos form extraembryonic mesoderm prior to gastrulation, in contrast to mouse. The function of the primary yolk sac and the origin of extraembryonic mesoderm remain unclear. Here, we hypothesise that the hypoblast-derived primary yolk sac serves as a source for early extraembryonic mesoderm, which is supplemented with mesoderm from the gastrulating embryo. We discuss the intricate relationship between the yolk sac and the primate embryo and highlight the pivotal role of the yolk sac as a multifunctional hub for haematopoiesis, germ cell development and nutritional supply.

Germline genome protection: implications for gamete quality and germ cell tumorigenesis.

BACKGROUND: Germ cells have a unique and critical role as the conduit for hereditary information and therefore employ multiple strategies to protect genomic integrity and avoid mutations. Unlike somatic cells, which often respond to DNA damage by arresting the cell cycle and conducting DNA repair, germ cells as well as long-lived pluripotent stem cells typically avoid the use of error-prone repair mechanisms and favor apoptosis, reducing the risk of genetic alterations. Testicular germ cell tumors, the most common cancers of young men, arise from pre-natal germ cells. OBJECTIVES: To summarize the current understanding of DNA damage response mechanisms in pre-meiotic germ cells and to discuss how they impact both the origins of testicular germ cell tumors and their remarkable responsiveness to genotoxic chemotherapy. MATERIALS AND METHODS: We conducted a review of literature gathered from PubMed regarding the DNA damage response properties of testicular germ cell tumors and the germ cells from which they arise, as well as the influence of these mechanisms on therapeutic responses by testicular germ cell tumors. RESULTS AND DISCUSSION: This review provides a comprehensive evaluation of how the developmental origins of male germ cells and their inherent germ cell-like DNA damage response directly impact the development and therapeutic sensitivity of testicular germ cell tumors. CONCLUSIONS: The DNA damage response of germ cells directly impacts the development and therapeutic sensitivity of testicular germ cell tumors. Recent advances in the study of primordial germ cells, post-natal mitotically dividing germ cells, and pluripotent stem cells will allow for new investigations into the initiation, progression, and treatment of testicular germ cell tumors.

Enhancement of chicken primordial germ cell in vitro maintenance using an automated cell image analyser.

Primordial germ cells (PGCs) were isolated from blood samples of chicken embryos. We established four PGC lines: two males (FS-ZZ-101, GFP-ZZ-4ZP) and two females (FS-ZW-111, GFP-ZW-5ZP). We could not detect a significant difference in the marker expression profile, but there was a remarkable difference between the proliferation rates of these PGC lines. We monitored the number of PGCs throughout a three-day period using a high-content screening cell imaging and analysing system (HCS). We compared three different initial cell concentrations in the wells: ~1000 cells (1×, ~4000 (4× and ~8000 (8×. For the GFPZW- 5ZP, FS-ZZ-101 and FS-ZW-111 PGC lines the lowest doubling time was observed at 4× concentration, while for GFP-ZZ-4ZP we found the lowest doubling time at 1× concentration. At 8× initial concentration, the growth rate was high during the first two days for all cell lines, but this was followed by the appearance of cell aggregates decreasing the cell growth rate. We could conclude that the difference in proliferation rate could mainly be attributed to genotypic variation in the established PGC lines, but external factors such as cell concentration and quality of the culture medium also affect the growth rate of PGCs.

Regeneration of the germline in the annelid Capitella teleta.

The germline is essential for sexual reproduction and survival of the species. In many metazoans, the developmental potential to generate a distinct germline is segregated from somatic cell lineages early in embryogenesis, suggesting that the unique features of the germline must be established from its onset. Previous studies suggest that germ cells cannot regenerate once removed from the embryo, but few animals have been experimentally tested. We investigated the ability of the germline to regenerate in a lophotrochozoan, the segmented worm Capitella teleta, which has a stereotyped cell lineage program by deleting the germline precursor (cell 3D) in early stage embryos using an infrared laser. Larvae and juveniles resulting from germline deletions were examined for presence of multipotent progenitor cells (MPCs), stem cells that form the germ cells and somatic stem cells. In contrast to control deletions of a non-germline macromere, most larvae resulting from deletion of cell 3D lacked MPCs as assayed by expression of germline markers CapI-vasa, CapI-nanos and Ct-piwi1, but showed persistent expression of these markers in the somatic posterior growth zone. However, approximately 13% of experimental larvae had MPCs, indicative of some germline regeneration. In contrast, by two weeks post-metamorphosis, all juveniles resulting from deletion of cell 3D had MPCs, as detected by CapI-vasa expression. Furthermore, when raised to adulthood, most animals developed reproductive structures and were fertile. In another set of deletions, both the D quadrant mesodermal and germline progenitors were removed. These juveniles also regenerated MPCs. Surprisingly, this deletion caused substantial ectopic expression of CapI-vasa and CapI-nanos in other larval tissues. Our results indicate that C. teleta can regenerate the germline following removal of the germline progenitors in the early embryo. The dramatic difference in ability to regenerate the germline between the larval and adult stages suggests that there are two distinct compensation events at two phases of the life cycle: a regulative event in the early stage larva and a stem cell transition event after metamorphosis, when the animals are capable of substantial body regeneration.

C-X-C chemokine receptor type 4 (CXCR4) is a key receptor for chicken primordial germ cell migration.

In mammals, germ cells originate outside of the developing gonads and follow a unique migration pattern through the embryonic tissue toward the genital ridges. Many studies have attempted to identify critical receptors and factors involved in germ cell migration. However, relatively few reports exist on germ cell receptors and chemokines that are involved in germ cell migration in avian species. In the present study, we investigated the specific migratory function of C-X-C chemokine receptor type 4 (CXCR4) in chicken primordial germ cells (PGCs). We induced loss-of-function via a frameshift mutation in the CXCR4 gene in chicken PGCs using clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR/Cas9) genome editing. The migratory capacity of CXCR4 knockout PGCs was significantly reduced in vivo after transplantation into recipient embryos. However, CXCR4-expressing somatic cell lines, such as chicken DT40 and DF1, failed to migrate into the developing gonads, suggesting that another key factor(s) is necessary for targeting and settlement of PGCs into the genital ridges. In conclusion, we show that CXCR4 plays a critical role in the migration of chicken germ cells.

Basic fibroblast growth factor is critical to reprogramming buffalo (Bubalus bubalis) primordial germ cells into embryonic germ stem cell-like cells.

Primordial germ cells (PGCs) are destined to form gametes in vivo, and they can be reprogrammed into pluripotent embryonic germ (EG) cells in vitro. Buffalo PGC have been reported to be reprogrammed into EG-like cells, but the identities of the major signaling pathways and culture media involved in this derivation remain unclear. Here, the effects of basic fibroblast growth factor (bFGF) and downstream signaling pathways on the reprogramming of buffalo PGCs into EG-like cells were investigated. Results showed bFGF to be critical to buffalo PGCs to dedifferentiate into EG-like cells (20 ng/mL is optimal) with many characteristics of pluripotent stem cells, including alkaline phosphatase (AP) activity, expression of pluripotency marker genes such as OCT4, NANOG, SOX2, SSEA-1, CDH1, and TRA-1-81, and the capacity to differentiate into all three embryonic germ layers. After chemically inhibiting pathways or components downstream of bFGF, data showed that inhibition of the PI3K/AKT pathway led to significantly lower EG cell derivation, while inhibition of P53 activity resulted in an efficiency of EG cell derivation comparable to that in the presence of bFGF. These results suggest that the role of bFGF in PGC-derived EG-like cell generation is mainly due to the activation of the PI3K/AKT/P53 pathway, in particular, the inhibition of P53 function.

High-throughput analysis reveals novel maternal germline RNAs crucial for primordial germ cell preservation and proper migration.

During oogenesis, hundreds of maternal RNAs are selectively localized to the animal or vegetal pole, including determinants of somatic and germline fates. Although microarray analysis has identified localized determinants, it is not comprehensive and is limited to known transcripts. Here, we utilized high-throughput RNA-sequencing analysis to comprehensively interrogate animal and vegetal pole RNAs in the fully grown Xenopus laevis oocyte. We identified 411 (198 annotated) and 27 (15 annotated) enriched mRNAs at the vegetal and animal pole, respectively. Ninety were novel mRNAs over 4-fold enriched at the vegetal pole and six were over 10-fold enriched at the animal pole. Unlike mRNAs, microRNAs were not asymmetrically distributed. Whole-mount in situ hybridization confirmed that all 17 selected mRNAs were localized. Biological function and network analysis of vegetally enriched transcripts identified protein-modifying enzymes, receptors, ligands, RNA-binding proteins, transcription factors and co-factors with five defining hubs linking 47 genes in a network. Initial functional studies of maternal vegetally localized mRNAs show that sox7 plays a novel and important role in primordial germ cell (PGC) development and that ephrinB1 (efnb1) is required for proper PGC migration. We propose potential pathways operating at the vegetal pole that highlight where future investigations might be most fruitful.

C. elegans GLP-1/Notch activates transcription in a probability gradient across the germline stem cell pool.

C. elegans Notch signaling maintains a pool of germline stem cells within their single-celled mesenchymal niche. Here we investigate the Notch transcriptional response in germline stem cells using single-molecule fluorescence in situ hybridization coupled with automated, high-throughput quantitation. This approach allows us to distinguish Notch-dependent nascent transcripts in the nucleus from mature mRNAs in the cytoplasm. We find that Notch-dependent active transcription sites occur in a probabilistic fashion and, unexpectedly, do so in a steep gradient across the stem cell pool. Yet these graded nuclear sites create a nearly uniform field of mRNAs that extends beyond the region of transcriptional activation. Therefore, active transcription sites provide a precise view of where the Notch-dependent transcriptional complex is productively engaged. Our findings offer a new window into the Notch transcriptional response and demonstrate the importance of assaying nascent transcripts at active transcription sites as a readout for canonical signaling.

Cholesterol induces proliferation of chicken primordial germ cells.

Primordial germ cells (PGCs) are the precursors of sperm and eggs and may serve as suitable cells for use in research in developmental biology and transgenic animals. However, the long-term propagation of PGCs in vitro has so far been plagued by the loss of their germ cell characteristics. This is largely because of the scarcity of knowledge concerning cell division and proliferation in these cells and the poor optimization of the culture medium. The sonic hedgehog (SHH) signaling pathway is involved in proliferation of many types of cells, but little is known about its role in chicken PGCs. The results of the current study indicate that the proliferation of chicken PGCs increases significantly when cholesterol, a molecule that facilitates the trafficking of HH ligands, is supplemented in the culture medium. This effect was attenuated when an SHH antagonist, cyclopamine was added, suggesting the involvement of SHH signaling in this process. The characterization of PGCs treated with cholesterol has shown that these cells express germ-cell-related markers and retain their capability to colonize the embryonic gonad after re-introduction to vasculature of stage-15 HH embryos, indicating that proliferation of PGCs induced by cholesterol does not alter the germ cell characteristics of these cells.

Single-cell RNA sequencing: revealing human pre-implantation development, pluripotency and germline development.

Early human development is a dynamic, heterogeneous, complex and multidimensional process. During the first week, the single-cell zygote undergoes eight to nine rounds of cell division generating the multicellular blastocyst, which consists of hundreds of cells forming spatially organized embryonic and extra-embryonic tissues. At the level of transcription, degradation of maternal RNA commences at around the two-cell stage, coinciding with embryonic genome activation. Although numerous efforts have recently focused on delineating this process in humans, many questions still remain as thorough investigation has been limited by ethical issues, scarce availability of human embryos and the presence of minute amounts of DNA and RNA. In vitro cultures of embryonic stem cells provide some insight into early human development, but such studies have been confounded by analysis on a population level failing to appreciate cellular heterogeneity. Recent technical developments in single-cell RNA sequencing have provided a novel and powerful tool to explore the early human embryo in a systematic manner. In this review, we will discuss the advantages and disadvantages of the techniques utilized to specifically investigate human development and consider how the technology has yielded new insights into pre-implantation development, embryonic stem cells and the establishment of the germ line.

The Grooved Rodent Incisor Recapitulates Rudimentary Teeth Characteristics of Ancestral Mammals.

It is known from the paleontology studies of eutherian mammals that incisor numbers were reduced during evolution. The evolutionary lost incisors may remain as vestigial structures at embryonic stages. The recapitulation of the incisor patterns among mammalian species will potentially uncover the mechanisms underlying the phenotypic transition of incisors during evolution. Here, we showed that a minute tooth formed in the presumptive groove region of the gerbil upper incisor at the early developmental stages, during which multiple epithelial swellings and Shh transcription domains spatiotemporally appeared in the dental epithelium, suggests the existence of vestigial dental primordia. Interestingly, when we trimmed the surrounding mesenchyme from incisor tooth germs at or before the bud stage prior to ex vivo culture, the explants developed different incisor phenotypes ranging from triplicated incisors, duplicated incisors, to Lagomorpha-like incisors, corresponding to the incisor patterns in the eutherian mammals. These results imply that the phenotypic transition of incisors during evolution, as well as the achievement of ultimate incisors in adults, arose from differential integrations of primordia. However, when the incisor tooth germ was trimmed at the cap stage, a grooved incisor developed similar to the normal condition. Furthermore, the incisor tooth germ developed a small but smooth incisor after the additional removal of the minute tooth and a lateral rudiment. These results suggest that multiple dental primordia integrated before the cap stage, with the labial primordia contributing to the labial face of the functional incisor. The minute tooth that occupied the boundary of the 2 labial primordia might be implicated in the groove formation. This study sheds light on how rudiments incorporate into functional organs and aids the understanding of incisor evolution.

Dead end1 is an essential partner of NANOS2 for selective binding of target RNAs in male germ cell development.

RNA-binding proteins (RBPs) play important roles for generating various cell types in many developmental processes, including eggs and sperms. Nanos is widely known as an evolutionarily conserved RNA-binding protein implicated in germ cell development. Mouse NANOS2 interacts directly with the CCR4-NOT (CNOT) deadenylase complex, resulting in the suppression of specific RNAs. However, the mechanisms involved in target specificity remain elusive. We show that another RBP, Dead end1 (DND1), directly interacts with NANOS2 to load unique RNAs into the CNOT complex. This interaction is mediated by the zinc finger domain of NANOS2, which is essential for its association with target RNAs. In addition, the conditional deletion of DND1 causes the disruption of male germ cell differentiation similar to that observed in Nanos2-KO mice. Thus, DND1 is an essential partner for NANOS2 that leads to the degradation of specific RNAs. We also present the first evidence that the zinc finger domain of Nanos acts as a protein-interacting domain for another RBP, providing a novel insight into Nanos-mediated germ cell development.

NUP50 is necessary for the survival of primordial germ cells in mouse embryos.

Nucleoporin 50 kDa (NUP50), a component of the nuclear pore complex, is highly expressed in male germ cells, but its role in germ cells is largely unknown. In this study, we analyzed the expression and function of NUP50 during the embryonic development of germ cells using NUP50-deficient mice. NUP50 was expressed in germ cells of both sexes at embryonic day 15.5 (E15.5), E13.5, and E12.5. In addition, NUP50 expression was also detected in primordial germ cells (PGCs) migrating into the genital ridges at E9.5. The gonads of Nup50-/- embryos of both sexes contained few PGCs at both E11.5 and E12.5 and no developing germ cells at E15.5. The migratory PGCs in Nup50-/- embryos at E9.5 showed increased apoptosis but a normal rate of proliferation, resulting in the progressive loss of germ cells at later stages. Taken together, these results suggest that NUP50 plays an essential role in the survival of PGCs during embryonic development.