Proliferation Of Primordial Germ Cells Research Articles

Diaph1 knockout inhibits mouse primordial germ cell proliferation and affects gonadal development

BackgroundExploring the molecular mechanisms of primordial germ cell (PGC) migration and the involvement of gonadal somatic cells in gonad development is valuable for comprehending the origins and potential treatments of reproductive-related diseases.MethodsDiaphanous related formin 1 (Diaph1, also known as mDia1) was screened by analyzing publicly available datasets (ATAC-seq, DNase-seq, and RNA-seq). Subsequently, the CRISPR-Cas9 technology was used to construct Diaph1 knockout mice to investigate the role of Diaph1 in gonad development.ResultsBased on data from public databases, a differentially expressed gene Diaph1, was identified in the migration of mouse PGC. Additionally, the number of PGCs was significantly reduced in Diaph1 knockout mice compared to wild type mice, and the expression levels of genes related to proliferation (Dicer1, Mcm9), adhesion (E-cadherin, Cdh1), and migration (Cxcr4, Hmgcr, Dazl) were significantly decreased. Diaph1 knockout also inhibited Leydig cell proliferation and induced apoptosis in the testis, as well as granulosa cell apoptosis in the ovary. Moreover, the sperm count in the epididymal region and the count of ovarian follicles were significantly reduced in Diaph1 knockout mice, resulting in decreased fertility, concomitant with lowered levels of serum testosterone and estradiol. Further research found that in Diaph1 knockout mice, the key enzymes involved in testosterone synthesis (CYP11A1, 3β-HSD) were decreased in Leydig cells, and the estradiol-associated factor (FSH receptor, AMH) in granulosa cells were also downregulated.ConclusionsOverall, our findings indicate that the knockout of Diaph1 can disrupt the expression of factors that regulate sex hormone production, leading to impaired secretion of sex hormones, ultimately resulting in damage to reproductive function. These results provide a new perspective on the molecular mechanisms underlying PGC migration and gonadal development, and offer valuable insights for further research on the causes, diagnosis, and treatment of related diseases.

Depletion of placental brain-derived neurotrophic factor (BDNF) is attributed to premature ovarian insufficiency (POI) in mice offspring

IntroductionPremature ovarian insufficiency (POI) is one of the causes of female infertility. Unexplained POI is increasingly affecting women in their reproductive years. However, the etiology of POI is diverse and remains elusive. We and others have shown that brain-derived neurotrophic factor (BDNF) plays an important role in adult ovarian function. Here, we report on a novel role of BDNF in the Developmental Origins of POI.MethodsPlacental BDNF knockout mice were created using CRISPR/CAS9. Homozygous knockout (cKO(HO)) mice didn’t survive, while heterozygous knockout (cKO(HE)) mice did. BDNF reduction in cKO(HE) mice was confirmed via immunohistochemistry and Western blots. Ovaries were collected from cKO(HE) mice at various ages, analyzing ovarian metrics, FSH expression, and litter sizes. In one-month-old mice, oocyte numbers were assessed using super-ovulation, and oocyte gene expression was analyzed with smart RNAseq. Ovaries of P7 mice were studied with SEM, and gene expression was confirmed with RT-qPCR. Alkaline phosphatase staining at E11.5 and immunofluorescence for cyclinD1 assessed germ cell number and cell proliferation.ResultscKO(HE) mice had decreased ovarian function and litter size in adulthood. They were insensitive to ovulation induction drugs manifested by lower oocyte release after superovulation in one-month-old cKO(HE) mice. The transcriptome and SEM results indicate that mitochondria-mediated cell death or aging might occur in cKO(HE) ovaries. Decreased placental BDNF led to diminished primordial germ cell proliferation at E11.5 and ovarian reserve which may underlie POI in adulthood.ConclusionThe current results showed decreased placental BDNF diminished primordial germ cell proliferation in female fetuses during pregnancy and POI in adulthood. Our findings can provide insights into understanding the underlying mechanisms of POI.

P38 MAPK as a gatekeeper of reprogramming in mouse migratory primordial germ cells.

Mammalian germ cells are derived from primordial germ cells (PGCs) and ensure species continuity through generations. Unlike irreversible committed mature germ cells, migratory PGCs exhibit a latent pluripotency characterized by the ability to derive embryonic germ cells (EGCs) and form teratoma. Here, we show that inhibition of p38 mitogen-activated protein kinase (MAPK) by chemical compounds in mouse migratory PGCs enables derivation of chemically induced Embryonic Germ-like Cells (cEGLCs) that do not require conventional growth factors like LIF and FGF2/Activin-A, and possess unique naïve pluripotent-like characteristics with epiblast features and chimera formation potential. Furthermore, cEGLCs are regulated by a unique PI3K-Akt signaling pathway, distinct from conventional naïve pluripotent stem cells described previously. Consistent with this notion, we show by performing ex vivo analysis that inhibition of p38 MAPK in organ culture supports the survival and proliferation of PGCs and also potentially reprograms PGCs to acquire indefinite proliferative capabilities, marking these cells as putative teratoma-producing cells. These findings highlight the utility of our ex vivo model in mimicking in vivo teratoma formation, thereby providing valuable insights into the cellular mechanisms underlying tumorigenesis. Taken together, our research underscores a key role of p38 MAPK in germ cell development, maintaining proper cell fate by preventing unscheduled pluripotency and teratoma formation with a balance between proliferation and differentiation.

Non-invasive dsRNA delivery via feeding for effective gene silencing in teleost fish: A novel approach in the study of gene function analysis

RNA interference (RNAi) is a powerful technique for gene silencing, involving post-transcriptional regulation of gene expression. Successful application of RNAi has been demonstrated in various organisms like nematodes, insects, and oysters by ingesting double-stranded RNA (dsRNA)-expressing bacteria. In this study, we attempted a non-invasive feeding method in the marine economy teleost fish, Sebastes schlegelii. To validate the feasibility of this approach in S. schlegelii, we utilized rotifers, Artemia nauplii, and commercial pellet feed as vectors to deliver Escherichia coli strain HT115 engineered to express dsRNA, targeting dead end (dnd) gene, known to be important for primordial germ cells (PGCs) migration and proliferation in fish. Our experimental results consistently showed that knockdown of the dnd gene effectively inhibited the development of PGCs in S. schlegelii, resulting in gonadal dysplasia and even sex reversal. This study marks a significant advancement in understanding gene function in teleost fish, laying the groundwork for future large-scale research in this field.

Novel compound heterozygous variants in FANCI cause premature ovarian insufficiency.

Premature ovarian insufficiency (POI) is a common reproductive aging disorder due to a dramatic decline of ovarian function before 40 years of age. Accumulating evidence reveals that genetic defects, particularly those related to DNA damage response, are a crucial contributing factor to POI. We have demonstrated that the functional Fanconi anemia (FA) pathway maintains the rapid proliferation of primordial germ cells to establish a sufficient reproductive reserve by counteracting replication stress, but the clinical implications of this function in human ovarian function remain to be established. Here, we screened the FANCI gene, which encodes a key component for FA pathway activation, in our whole-exome sequencing database of 1030 patients with idiopathic POI, and identified two pairs of novel compound heterozygous variants, c.[97C > T];[1865C > T] and c.[158-2A > G];[c.959A > G], in two POI patients, respectively. The missense variants did not alter FANCI protein expression and nuclear localization, apart from the variant c.158-2A > G causing abnormal splicing and leading to a truncated mutant p.(S54Pfs*5). Furthermore, the four variants all diminished FANCD2 ubiquitination levels and increased DNA damage under replication stress, suggesting that the FANCI variants impaired FA pathway activation and replication stress response. This study first links replication stress response defects with the pathogenesis of human POI, providing a new insight into the essential roles of the FA genes in ovarian function.

Ten-eleven translocation 1 mediating DNA demethylation regulates the proliferation of chicken primordial germ cells through the activation of Wnt4/β-catenin signaling pathway.

The objective of this study was to investigate the regulation relationship of Teneleven translocation 1 (Tet1) in DNA demethylation and the proliferation of primordial germ cells (PGCs) in chickens. siRNA targeting Tet1 was used to transiently knockdown the expression of Tet1 in chicken PGCs, and the genomic DNA methylation status was measured. The proliferation of chicken PGCs was detected by flow cytometry analysis and cell counting kit-8 assay when activation or inhibition of Wnt4/β-catenin signaling pathway. And the level of DNA methylation and hisotne methylation was also tested. Results revealed that knockdown of Tet1 inhibited the proliferation of chicken PGCs and downregulated the mRNA expression of Cyclin D1 and cyclin-dependent kinase 6 (CDK6), as well as pluripotency-associated genes (Nanog, PouV, and Sox2). Flow cytometry analysis confirmed that the population of PGCs in Tet1 knockdown group displayed a significant decrease in the proportion of S and G2 phase cells, which meant that there were less PGCs entered the mitosis process than that of control. Furthermore, Tet1 knockdown delayed the entrance to G1/S phase and this inhibition was rescued by treated with BIO. Consistent with these findings, Wnt/β-catenin signaling was inactivated in Tet1 knockdown PGCs, leading to aberrant proliferation. Further analysis showed that the methylation of the whole genome increased significantly after Tet1 downregulation, while hydroxymethylation obviously declined. Meanwhile, the level of H3K27me3 was upregulated and H3K9me2 was downregulated in Tet1 knockdown PGCs, which was achieved by regulating Wnt/β-catenin signaling pathway. These results suggested that the self-renewal of chicken PGCs and the maintenance of their characteristics were regulated by Tet1 mediating DNA demethylation through the activation of Wnt4/β-catenin signaling pathway.

Effects of Insulin on Proliferation, Apoptosis, and Ferroptosis in Primordial Germ Cells via PI3K-AKT-mTOR Signaling Pathway.

Primordial germ cells (PGCs) are essential for the genetic modification, resource conservation, and recovery of endangered breeds in chickens and need to remain viable and proliferative in vitro. Therefore, there is an urgent need to elucidate the functions of the influencing factors and their regulatory mechanisms. In this study, PGCs collected from Rugao yellow chicken embryonic eggs at Day 5.5 were cultured in media containing 0, 5, 10, 20, 50, and 100 μg/mL insulin. The results showed that insulin regulates cell proliferation in PGCs in a dose-dependent way, with an optimal dose of 10 μg/mL. Insulin mediates the mRNA expression of cell cycle-, apoptosis-, and ferroptosis-related genes. Insulin at 50 μg/mL and 100 μg/mL slowed down the proliferation with elevated ion content and GSH/oxidized glutathione (GSSG) in PGCs compared to 10 μg/mL. In addition, insulin activates the PI3K/AKT/mTOR pathway dose dependently. Collectively, this study demonstrates that insulin reduces apoptosis and ferroptosis and enhances cell proliferation in a dose-dependent manner via the PI3K-AKT-mTOR signaling pathway in PGCs, providing a new addition to the theory of the regulatory role of the growth and proliferation of PGC in vitro cultures.

First study on repeatable culture of primordial germ cells from various embryonic regions with giant feeder cells in Japanese quail (Coturnix japonica)

Japanese quail (JQ, Coturnix japonica) is a farmed animal with a high economic value and has been used extensively as an avian model for research. Germline chimera production based on cryopreserved primordial germ cells (PGCs) is possible for conservation management of quail breeds as successful isolation has been reported of PGCs from their blood and gonads. However, the repeatable cultivation protocol has not been elucidated yet, which has hindered technological development. The current study characterized cultivation of pregonadal PGCs isolated from embryonic parts; embryonic blood (cPGCs), whole embryonic tissues (tPGCs), parts of tail buds (tbPGCs), and a mixture of blood and tail bud tissues (ctbPGCs). The results showed that the cultivation system required the presence of specific embryonic cells to act as a feeder for JQ-PGCs and that such a system facilitated more successful cultivation, as shown by the percentages of isolation and cultivation in tbPGCs (100%, 100%, respectively), tPGCs (60%, 55%, respectively), and ctbPGCs (60%, 30%, respectively), but not in cPGCs (0%) cultured on a mitomycin-treated JQ feeder cell-line. Once the co-culture system had been established, the PGCs could be propagated for at least 5 months. These PGCs expressed germ cell-specific markers (DAZL and CVH) and could colonize embryonic gonads. Conclusively, the isolation of pregonadal PGCs and their long-term cultivation in vitro requires a unique embryonic cell, giant cell feeder, that is indispensable for the proliferation of PGCs. Characterization of cell signaling sustaining a mutual interaction between the PGCs and the specific feeder cells will elucidate a superior environment for in vitro cultivation, as well as support the minimal transfer of used xenobiotics in chimera production.

FAAP100 is required for the resolution of transcription-replication conflicts in primordial germ cells

BackgroundThe maintenance of genome stability in primordial germ cells (PGCs) is crucial for the faithful transmission of genetic information and the establishment of reproductive reserve. Numerous studies in recent decades have linked the Fanconi anemia (FA) pathway with fertility, particularly PGC development. However, the role of FAAP100, an essential component of the FA core complex, in germ cell development is unexplored.ResultsWe find that FAAP100 plays an essential role in R-loop resolution and replication fork protection to counteract transcription-replication conflicts (TRCs) during mouse PGC proliferation. FAAP100 deletion leads to FA pathway inactivation, increases TRCs as well as cotranscriptional R-loops, and contributes to the collapse of replication forks and the generation of DNA damage. Then, the activated p53 signaling pathway triggers PGC proliferation defects, ultimately resulting in insufficient establishment of reproductive reserve in both sexes of mice.ConclusionsOur findings suggest that FAAP100 is required for the resolution of TRCs in PGCs to safeguard their genome stability.

Niche cells regulate primordial germ cell quiescence in response to basement membrane signaling.

Stem cell quiescence, proliferation and differentiation are controlled by interactions with niche cells and a specialized extracellular matrix called basement membrane (BM). Direct interactions with adjacent BM are known to regulate stem cell quiescence; however, it is less clear how niche BM relays signals to stem cells that it does not contact. Here, we examine how niche BM regulates Caenorhabditis elegans primordial germ cells (PGCs). BM regulates PGC quiescence even though PGCs are enwrapped by somatic niche cells and do not contact the BM; this can be demonstrated by depleting laminin, which causes normally quiescent embryonic PGCs to proliferate. We show that following laminin depletion, niche cells relay proliferation-inducing signals from the gonadal BM to PGCs via integrin receptors. Disrupting the BM proteoglycan perlecan blocks PGC proliferation when laminin is depleted, indicating that laminin functions to inhibit a proliferation-inducing signal originating from perlecan. Reducing perlecan levels in fed larvae hampers germline growth, suggesting that BM signals regulate germ cell proliferation under physiological conditions. Our results reveal how BM signals can regulate stem cell quiescence indirectly, by activating niche cell integrin receptors.

Fance deficiency inhibits primordial germ cell proliferation associated with transcription–replication conflicts accumulate and DNA repair defects

Fanconi anemia (FA) gene mutations are critical components in the genetic etiology of premature ovarian insufficiency (POI). Fance−/− mice detected meiotic arrest of primordial germ cells (PGCs) as early as embryonic day (E) 13.5 and exhibited decreased ovarian reserve after birth. However, the mechanism of Fance defect leading to dysgenesis of PGCs is unclear. We aimed to explore the effect of Fance defects on mitotic proliferation of PGCs. Combined with transcriptomic sequencing and validation, we examined the effect of Fance defects on cell cycle, transcription–replication conflicts (TRCs), and multiple DNA repair pathways in PGCs during active DNA replication at E11.5 and E12.5. Results showed Fance defects cause decreased numbers of PGCs during rapid mitosis at E11.5 and E12.5. Mitotic cell cycle progression of Fance−/− PGCs was blocked at E11.5 and E12.5, shown by decreased cell proportions in S and G2 phases and increased cell proportions in M phase. RNA-seq suggested the mechanisms involved in DNA replication and repair. We found Fance−/− PGCs accumulate TRCs during active DNA replication at E11.5 and E12.5. Fance−/− PGCs down-regulate multiple DNA repair pathways at E11.5 and E12.5 including the FA pathway, homologous recombination (HR) pathway, and base excision repair (BER) pathway. In conclusion, Fance defect impaired the mitotic proliferation of PGCs leading to rapidly decreased numbers and abnormal cell cycle distribution. Proliferation inhibition of Fance−/− PGCs was associated with accumulated TRCs and down-regulation of FA, HR, BER pathways. These provided a theoretical basis for identifying the inherited etiology and guiding potential fertility management for POI.

NRF1 promotes primordial germ cell development, proliferation and survival.

Primordial germ cells (PGCs) are the germline precursors that give rise to oocytes and sperm, ensuring the continuation of life. While the PGC specification is extensively studied, it remains elusive how the PGC population is sustained and expanded after they migrate to embryonic gonads before birth. This study demonstrates that NRF1, a known regulator for mitochondrial metabolism, plays critical roles in post-migrating PGC development. We show that NRF1 protein level gradually increases in post-migrating PGCs during embryonic development. Conditional Nrf1 knockout from embryonic germ cells leads to impaired PGC proliferation and survival. In addition, NRF1 may also actively drive PGC derivation from pluripotent stem cells. Using whole genome transcriptome profiling and ChIP-seq analyses, we further reveal that NRF1 directly regulates key signalling molecules in PGC formation, transcription factors in proliferation and cell cycle and enzymes in mitochondrial metabolism. Overall, our findings highlight an essential requirement of NRF1 in regulating a broad transcriptional network to support post-migrating PGC development both in vitro and in vivo.

Coordination of canonical and noncanonical Hedgehog signalling pathways mediated by WDR11 during primordial germ cell development

WDR11, a gene associated with Kallmann syndrome, is important in reproductive system development but molecular understanding of its action remains incomplete. We previously reported that Wdr11-deficient embryos exhibit defective ciliogenesis and developmental defects associated with Hedgehog (HH) signalling. Here we demonstrate that WDR11 is required for primordial germ cell (PGC) development, regulating canonical and noncanonical HH signalling in parallel. Loss of WDR11 disrupts PGC motility and proliferation driven by the cilia-independent, PTCH2/GAS1-dependent noncanonical HH pathway. WDR11 modulates the growth of somatic cells surrounding PGCs by regulating the cilia-dependent, PTCH1/BOC-dependent canonical HH pathway. We reveal that PTCH1/BOC or PTCH2/GAS1 receptor context dictates SMO localisation inside or outside of cilia, respectively, and loss of WDR11 affects the signalling responses of SMO in both situations. We show that GAS1 is induced by PTCH2-specific HH signalling, which is lost in the absence of WDR11. We also provide evidence supporting a role for WDR11 in ciliogenesis through regulation of anterograde intraflagellar transport potentially via its interaction with IFT20. Since WDR11 is a target of noncanonical SMO signalling, WDR11 represents a novel mechanism by which noncanonical and canonical HH signals communicate and cooperate.

LIN28 is essential for the maintenance of chicken primordial germ cells

Understanding the mechanism of stem cell maintenance underlies the establishment of long-term and mass culture methods for stem cells that are fundamental for clinical and agricultural applications. In this study, we use chicken primordial germ cell (PGC) as a model to elucidate the molecular mechanisms underlying stem cell maintenance. The PGC is a useful experimental model because it is readily gene-manipulatable and easy to test gene function in vivo using transplantation. Previous studies to establish a long-term culture system have shown that secreted factors such as FGF2 are required to maintain the self-renewal capability of PGC. On the other hand, we know little about intracellular regulators responsible for PGC maintenance. Among representative stem cell factors, we focus on RNA-binding factors LIN28A and LIN28B as possible central regulators for the gene regulatory network essential to PGC maintenance. By taking advantage of the CRISPR/Cas9-mediated gene editing and a clonal culture technique, we find that both LIN28A and LIN28B regulate the proliferation of PGC in vitro. We further showed that colonization efficiency of grafted PGC at the genital ridges, rudiments for the gonads, of chicken embryos were significantly decreased by knockout (KO) of LIN28A or LIN28B. Of note, overexpression of human LIN28 in LIN28-KO PGC was sufficient to restore the low colonization rates, suggesting that LIN28 plays a key role in PGC colonization at the gonads. Transcriptomic analyses of LIN28-KO PGC reveal that several genes related to mesenchymal traits are upregulated, including EGR1, a transcription factor that promotes the differentiation of mesodermal tissues. Finally, we show that the forced expression of human EGR1 deteriorates replication activity and colonization efficiency of PGCs. Taken together, this work demonstrates that LIN28 maintains self-renewal of PGC by suppressing the expression of differentiation genes including EGR1.

The constitutively active pSMAD2/3 relatively improves the proliferation of chicken primordial germ cells

AbstractIn many multicellular organisms, mature gametes originate from primordial germ cells (PGCs). Improvements in the culture of PGCs are important not only for developmental biology research, but also for preserving endangered species, and for genome editing and transgenic animal technologies. SMAD2/3 appear to be powerful regulators of gene expression; however, their potential positive impact on the regulation of PGC proliferation has not been taken into consideration. Here, the effect of TGF‐β signaling as the upstream activator of SMAD2/3 transcription factors was evaluated on chicken PGCs' proliferation. For this, chicken PGCs at stages 26−28 Hamburger−Hamilton were obtained from the embryonic gonadal regions and cultured on different feeders or feeder‐free substrates. The results showed that TGF‐β signaling agonists (IDE1 and Activin‐A) improved PGC proliferation to some extent while treatment with SB431542, the antagonist of TGF‐β, disrupted PGCs' proliferation. However, the transfection of PGCs with constitutively active SMAD2/3 (SMAD2/3CA) resulted in improved PGC proliferation for more than 5 weeks. The results confirmed the interactions between overexpressed SMAD2/3CA and pluripotency‐associated genes NANOG, OCT4, and SOX2. According to the results, the application of SMAD2/3CA could represent a step toward achieving an efficient expansion of avian PGCs.

The Synchronized Progression from Mitosis to Meiosis in Female Primordial Germ Cells between Layers and Broilers.

Layer and broiler hens show a dramatic difference in the volume and frequency of egg production. However, it is unclear whether the intrinsic competency of oocyte generation is also different between the two types of chicken. All oocytes were derived from the primordial germ cells (PGC) in the developing embryo, and female PGC proliferation (mitosis) and the subsequent differentiation (meiosis) determine the ultimate ovarian pool of germ cells available for future ovulation. In this study, we systematically compared the cellular phenotype and gene expression patterns during PGC mitosis (embryonic day 10, E10) and meiosis (E14) between female layers and broilers to determine whether the early germ cell development is also subjected to the selective breeding of egg production traits. We found that PGCs from E10 showed much higher activity in cell propagation and were enriched in cell proliferation signaling pathways than PGCs from E14 in both types of chicken. A common set of genes, namely insulin-like growth factor 2 (IGF2) and E2F transcription factor 4 (E2F4), were identified as the major regulators of cell proliferation in E10 PGCs of both strains. In addition, we found that E14 PGCs from both strains showed an equal ability to initiate meiosis, which was associated with the upregulation of key genes for meiotic initiation. The intrinsic cellular dynamics during the transition from proliferation to differentiation of female germ cells were conserved between layers and broilers. Hence, we surmise that other non-cell autonomous mechanisms involved in germ-somatic cell interactions would contribute to the divergence of egg production performance between layers and broilers.

UBE2T resolves transcription-replication conflicts and protects common fragile sites in primordial germ cells.

The proper development of primordial germ cells (PGCs) is an essential prerequisite for gametogenesis and mammalian fertility. The Fanconi anemia (FA) pathway functions in maintaining the development of PGCs. FANCT/UBE2T serves as an E2 ubiquitin-conjugating enzyme that ubiquitylates the FANCD2-FANCI complex to activate the FA pathway, but its role in the development of PGCs is not clear. In this study, we found that Ube2t knockout mice showed defects in PGC proliferation, leading to severe loss of germ cells after birth. Deletion of UBE2T exacerbated DNA damage and triggered the activation of the p53 pathway. We further demonstrated that UBE2T counteracted transcription-replication conflicts by resolving R-loops and stabilizing replication forks, and also protected common fragile sites by resolving R-loops in large genes and promoting mitotic DNA synthesis to maintain the genome stability of PGCs. Overall, these results provide new insights into the function and regulatory mechanisms of the FA pathway ensuring normal development of PGCs.

Comparative single-cell transcriptomic analysis reveals differences in signaling pathways in gonadal primordial germ cells between chicken (Gallus gallus) and zebra finch (Taeniopygia guttata).

Primordial germ cells (PGCs) have been used in avian genetic resource conservation and transgenic animal production. Despite their potential applications to numerous avian taxa facing extinction due to habitat loss and degradation, research has largely focused on poultry, such as chickens, in part owing to the difficulty in obtaining intact PGCs from other species. Recently, phenotypic differences between PGCs of chicken and zebra finch, a wild bird with vocal learning, in early embryonic development have been reported. In this study, we used advanced single-cell RNA sequencing (scRNA-seq) technology to evaluate zebra finch and chicken PGCs and surrounding cells, and to identify species-specific characteristics. We constructed single-cell transcriptome landscapes of chicken gonadal PGCs for a comparison with previously reported scRNA-seq data for zebra finch. We identified interspecific differences in several signaling pathways in gonadal PGCs and somatic cells. In particular, NODAL and insulin signaling pathway activity levels were higher in zebra finch than in chickens, whereas activity levels of the downstream FGF signaling pathway, involved in the proliferation of chicken PGCs, were higher in chickens. This study is the first cross-species single-cell transcriptomic analysis targeting birds, revealing differences in germ cell development between phylogenetically distant Galliformes and Passeriformes. Our results provide a basis for understanding the reproductive physiology of avian germ cells and for utilizing PGCs in the restoration of endangered birds and the production of transgenic birds.

DAZL regulates proliferation of human primordial germ cells by direct binding to precursor miRNAs and enhances DICER processing activity.

Understanding the molecular and cellular mechanisms of human primordial germ cells (hPGCs) is essential in studying infertility and germ cell tumorigenesis. Many RNA-binding proteins (RBPs) and non-coding RNAs are specifically expressed and functional during hPGC developments. However, the roles and regulatory mechanisms of these RBPs and non-coding RNAs, such as microRNAs (miRNAs), in hPGCs remain elusive. In this study, we reported a new regulatory function of DAZL, a germ cell-specific RBP, in miRNA biogenesis and cell proliferation. First, DAZL co-localized with miRNA let-7a in human PGCs and up-regulated the levels of >100 mature miRNAs, including eight out of nine let-7 family, miR21, miR22, miR125, miR10 and miR199. Purified DAZL directly bound to the loops of precursor miRNAs with sequence specificity of GUU. The binding of DAZL to the precursor miRNA increased the maturation of miRNA by enhancing the cleavage activity of DICER. Furthermore, cell proliferation assay and cell cycle analysis confirmed that DAZL inhibited the proliferation of in vitro PGCs by promoting the maturation of these miRNAs. Evidently, the mature miRNAs up-regulated by DAZL silenced cell proliferation regulators including TRIM71. Moreover, DAZL inhibited germline tumor cell proliferation and teratoma formation. These results demonstrate that DAZL regulates hPGC proliferation by enhancing miRNA processing.

Initiative on Avian Primordial Germ Cell Cryobanking in Thailand.

Background: Biobanking the reproductive tissues or cells of animals preserves the genetic and reproductive ability of the species in long-term storage and promotes sharing of reproductive materials. In avian species, the primordial germ cell (PGC) is one of the most promising reproductive cells to be preserved in biobanks, due to self-renewal properties and direct access to the germ line mediated by PGC transfer. Methods: To conserve the genetic resource of local chicken breeds that are of conservation importance, we systematically isolated two types of pregonadal PGCs from chicken embryos-circulating and tissue PGCs. PGCs of individual embryos were separately isolated, cultured, and cryopreserved. Characteristics of cultured PGCs are described and evaluated. Results: The efficiency of PGC isolation from individual embryos was 98.9% (660/667). In most cases, both matching circulating and tissue PGC lines were isolated from the same embryo (68.2%, 450/660), whereas the remaining lines were from a single source, being either tissue (30.6%, 202/660) or circulating (1.2%, 8/660). Efficient PGC isolation and proliferation can be expected in cultures of circulating PGCs (68.7% and 64.3%, respectively) and tissue PGCs (97.8% and 80.7%, respectively). Following cryopreservation, recovered cells sustained PGC identities including expression of chicken vasa homolog and deleted in azoospermia-like proteins and migration ability to recipient embryonic gonads. Culture conditions equally supported proliferation of circulating and tissue PGCs from both sexes. Combining tissue PGC culture in the regimen prevented 30.3% loss of PGC cultures in the case where circulating PGC culture was ineffective. Cultured circulating and tissue PGCs were similar in morphology, but optimal culture characteristics were different. Conclusion: We applied the approach of PGC isolation from blood and tissue origins on a wide scale and demonstrated its efficiency for biobanking chicken PGCs. The workflow can be operated effectively almost year-round in a tropical climate. It was also described in ample and practical details, which are suitable for adoption or optimization in other conditions.