The derivation of germ-line competent avian primordial germ cells establishes a cell-based model system for the investigation of germ cell differentiation and the production of genetically modified ...animals. Current methods to modify primordial germ cells using DNA or retroviral vectors are inefficient and prone to epigenetic silencing. Here, we validate the use of transposable elements for the genetic manipulation of primordial germ cells. We demonstrate that chicken primordial germ cells can be modified in vitro using transposable elements. Both piggyBac and Tol2 transposons efficiently transpose primordial germ cells. Tol2 transposon integration sites were spread throughout both the macro- and microchromosomes of the chicken genome and were more prevalent in gene transcriptional units and intronic regions, consistent with transposon integrations observed in other species. We determined that the presence of insulator elements was not required for reporter gene expression from the integrated transposon. We further demonstrate that a gene-trap cassette carried in the Tol2 transposon can trap and mutate endogenous transcripts in primordial germ cells. Finally, we observed that modified primordial germ cells form functional gametes as demonstrated by the generation of transgenic offspring that correctly expressed a reporter gene carried in the transposon. Transposable elements are therefore efficient vectors for the genetic manipulation of primordial germ cells and the chicken genome.
Avian primordial germ cells (PGCs) have significant potential to be used as a cell-based system for the study and preservation of avian germplasm, and the genetic modification of the avian genome. It ...was previously reported that PGCs from chicken embryos can be propagated in culture and contribute to the germ cell lineage of host birds.
We confirm these results by demonstrating that PGCs from a different layer breed of chickens can be propagated for extended periods in vitro. We demonstrate that intracellular signalling through PI3K and MEK is necessary for PGC growth. We carried out an initial characterisation of these cells. We find that cultured PGCs contain large lipid vacuoles, are glycogen rich, and express the stem cell marker, SSEA-1. These cells also express the germ cell-specific proteins CVH and CDH. Unexpectedly, using RT-PCR we show that cultured PGCs express the pluripotency genes c-Myc, cKlf4, cPouV, cSox2, and cNanog. Finally, we demonstrate that the cultured PGCs will migrate to and colonise the forming gonad of host embryos. Male PGCs will colonise the female gonad and enter meiosis, but are lost from the gonad during sexual development. In male hosts, cultured PGCs form functional gametes as demonstrated by the generation of viable offspring.
The establishment of in vitro cultures of germline competent avian PGCs offers a unique system for the study of early germ cell differentiation and also a comparative system for mammalian germ cell development. Primary PGC lines will form the basis of an alternative technique for the preservation of avian germplasm and will be a valuable tool for transgenic technology, with both research and industrial applications.
Conventional dendritic cells (cDCs) are antigen-presenting cells (APCs) that play a central role in linking innate and adaptive immunity. cDCs have been well described in a number of different ...mammalian species, but remain poorly characterised in the chicken. In this study, we use previously described chicken cDC specific reagents, a novel gene-edited chicken line and single-cell RNA sequencing (scRNAseq) to characterise chicken splenic cDCs. In contrast to mammals, scRNAseq analysis indicates that the chicken spleen contains a single, chemokine receptor XCR1 expressing, cDC subset. By sexual maturity the XCR1
cDC population is the most abundant mononuclear phagocyte cell subset in the chicken spleen. scRNAseq analysis revealed substantial heterogeneity within the chicken splenic XCR1
cDC population. Immature MHC class II (MHCII)
XCR1
cDCs expressed a range of viral resistance genes. Maturation to MHCII
XCR1
cDCs was associated with reduced expression of anti-viral gene expression and increased expression of genes related to antigen presentation via the MHCII and cross-presentation pathways. To visualise and transiently ablate chicken XCR1
cDCs
, we generated
-iCaspase9-RFP chickens using a CRISPR-Cas9 knockin transgenesis approach to precisely edit the
locus, replacing the XCR1 coding region with genes for a fluorescent protein (TagRFP), and inducible Caspase 9. After inducible ablation, the chicken spleen is initially repopulated by immature CD1.1
XCR1
cDCs. XCR1
cDCs are abundant in the splenic red pulp, in close association with CD8
T-cells. Knockout of XCR1 prevented this clustering of cDCs with CD8
T-cells. Taken together these data indicate a conserved role for chicken and mammalian XCR1
cDCs in driving CD8
T-cells responses.
Disruption of human fetal testis development is widely accepted to underlie testicular germ cell cancer (TGCC) origin and additional disorders within testicular dysgenesis syndrome (TDS). However, ...the mechanisms for the development of testicular dysgenesis in humans are unclear. We used ex vivo culture and xenograft approaches to investigate the importance of Nodal and Activin signaling in human fetal testis development. Inhibition of Nodal, and to some extent Activin, signaling disrupted seminiferous cord formation, abolished AMH expression, reduced androgen secretion, and decreased gonocyte numbers. Subsequent xenografting of testicular tissue rescued the disruptive effects on seminiferous cords and somatic cells but not germ cell effects. Stimulation of Nodal signaling increased the number of germ cells expressing pluripotency factors, and these persisted after xenografting. Our findings suggest a key role for Nodal signaling in the regulation of gonocyte differentiation and early human testis development with implications for the understanding of TGCC and TDS origin.
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•Nodal and Activin signaling was manipulated during human testicular development•Inhibiting Nodal/Activin signaling results in testicular dysgenesis and gonocyte loss•Effects on somatic cell lineages and testicular morphology are rescued with time•Nodal pathway stimulation results in persisting gonocytes resembling TGCC precursors
Jørgensen et al. determine the role of Nodal signaling in human fetal testis development using ex vivo culture and xenografting approaches. They provide insights into the involvement of Nodal signaling in seminiferous cord formation and the regulation of pluripotency factor expression in fetal gonocytes, with implications for the development of testicular cancer.
Testicular germ cell cancer (TGCC) develops from pre-malignant germ neoplasia in situ (GCNIS) cells. GCNIS originates from fetal gonocytes (POU5F1
/MAGE-A4
), which fail to differentiate to ...pre-spermatogonia (POU5F1
/MAGE-A4
) and undergo malignant transformation. Gankyrin is an oncogene which has been shown to prevent POU5F1 degradation and specifically interact with MAGE-A4 in hepatocellular carcinoma (HCC) cells. We aimed to investigate the role of Gankyrin in progression from gonocyte to pre-invasive GCNIS and subsequent invasive TGCC.
We determined Gankyrin expression in human fetal testicular tissue (gestational weeks 9-20; n = 38), human adult testicular tissue with active spermatogenesis (n = 9), human testicular tissue with germ cell maturation delay (n = 4), testicular tissue from patients with pre-invasive GCNIS (n = 6), and invasive TGCC including seminoma (n = 6) and teratoma (n = 7). Functional analysis was performed in-vitro by siRNA knock-down of Gankyrin in the NTera2 cells (derived from embryonal carcinoma).
Germ cell expression of Gankyrin was restricted to a sub-population of prespermatogonia in human fetal testes. Nuclear Gankyrin was also expressed in GCNIS cells of childhood and adult pre-invasive TGCC patients, and in GCNIS from seminoma and non-seminoma patients. Cytoplasmic expression was observed in seminoma tumour cells and NTera2 cells. Gankyrin knock-down in NTera2 cells resulted in an increase in apoptosis mediated via the TP53 pathway, whilst POU5F1 expression was unaffected. Furthermore, Gankyrin knock-down in NTera2 cells increased cisplatin sensitivity with an increase in cell death (13%, p < 0.05) following Gankyrin knock-down, when compared to cisplatin treatment alone, likely via BAX and FAS. Our results demonstrate that Gankyrin expression changes in germ cells during normal transition from gonocyte to prespermatogonia. In addition, changes in Gankyrin localisation are associated with progression of pre-invasive GCNIS to invasive TGCC. Furthermore, we found that Gankyrin is involved in the regulation of NTera2 cell survival and that a reduction in Gankyrin expression can modulate cisplatin sensitivity.
These results suggest that manipulation of Gankyrin expression may reduce the cisplatin dose required for the treatment of TGCC, with benefits in reducing dose-dependent side effects of chemotherapy. Further studies are required in order to assess the effects of modulating Gankyrin on GCNIS/TGCC using in vivo models.
Abstract Background Androgen signalling is essential for masculinisation of the male fetus. Failure of masculinisation during fetal life can result in disorders of sex development. Masculinisation is ...mediated partly by ligand binding to the luteinising hormone/human choriogonadotropin receptor (LHCGR). In rodents, the initial stages of masculinisation are gonadotropin independent, though whether this also occurs in human beings remains unknown because accurate timing and cell-type localisation of LHCGR protein expression in human fetal testis remains unresolved. We aimed to characterise the spatiotemportal expression of LHCGR in human fetal testis. Methods Human fetal testis tissue (n=12, 8–20 weeks' gestation) was obtained from elective terminations after informed patient consent. We used a combination of RT-PCR for LHGCR and Leydig cells (marker 3β hydroxysteroid dehydrogenase 3βHSD) and immunofluorescent co-localisation for LHCGR, Leydig cell steroidogenesis (3βHSD), and endothelial cells (markers CD31 and von Willebrand factor) to determine the exact timing and cellular location of LHCGR mRNA and protein in the human fetal testis. The study received ethics approval. Findings LHCGR mRNA was detected from 9 weeks' gestation, but protein was not detectable until 12 weeks'. Expression of 3βHSD (indicating active steroidogenesis) was detected in the first trimester (10 weeks') preceding the expression of LHCGR. LHCGR did not co-localise with the Leydig cell marker 3βHSD during the early second trimester and only became consistently co-localised at 20 weeks' gestation. Before expression in fetal Leydig cells, LHCGR was restricted to endothelial cells (demonstrated by co-localisation with CD31 and von Willebrand factor). Interpretation These results indicate that gonadotropin-mediated signalling through the LHCGR in human fetal Leydig cells is not established until later than previously described, suggesting a period of gonadotropin insensitivity during masculinisation in fetal life. Future functional studies will determine the mechanisms involved in androgen signalling in the human fetal testis. The present findings are important for understanding the pathogenesis of disorders of sex development in man. Funding Wellcome Trust.
Focal dysgenesis is a consistent feature of testicular dysgenesis syndrome (TDS) in humans. Rodent studies show that perturbation of androgens (e.g. following phthalate exposure) during a fetal ...masculinisation programming window (MPW) predisposes to a TDS phenotype. This study aimed to determine whether dissociation and reconstitution of rat fetal testis tissue during the MPW can be used to model and manipulate seminiferous cord development, including induction of focal dysgenesis, as described in TDS. Dissociated fetal rat testes were xenotransplanted subcutaneously into recipient mice for 4 weeks. Transplanted mice were treated with vehicle or di-n-butyl-phthalate (DBP, a plasticising chemical known to induce testicular dysgenesis in vivo in rats). Testosterone production by the transplants was measured in recipient mice and immunofluorescence was performed on the retrieved transplants to identify features consistent with focal testicular dysgenesis. Re-aggregation of rat fetal testis tissue xenotransplants during the MPW results in reconstitution of seminiferous cords. Features of focal testicular dysgenesis were present in re-aggregated testis, including ectopic Sertoli cells and intratubular Leydig cells (ITLCs). DBP exposure of recipient mice reduced androgen-dependent seminal vesicle weight (8.3 vs 26.7 mg; p < 0.05), but did not enhance features of focal dysgenesis including number of ITLCs (0.07 vs 0.10 cells/mm
; p > 0.05). We conclude that seminiferous cord reformation during the MPW results in development of focal dysgenesis. The system may be used to separate specific effects (e.g. androgen suppression) of individual chemical exposures from other mechanisms that may be conserved in TDS.
Conventional dendritic cells (cDC) are bone marrow‐derived immune cells that play a central role in linking innate and adaptive immunity. cDCs efficiently uptake, process and present antigen to naïve ...T cells, driving clonal expansion of antigen‐specific T‐cell responses. In chicken, vital reagents are lacking for the efficient and precise identification of cDCs. In this study, we have developed several novel reagents for the identification and characterization of chicken cDCs. Chicken FLT3 cDNA was cloned and a monoclonal antibody to cell surface FLT3 was generated. This antibody identified a distinct FLT3HI splenic subset which lack expression of signature markers for B cells, T cells or monocyte/macrophages. By combining anti‐FLT3 and CSF1R‐eGFP transgenic expression, three major populations within the mononuclear phagocyte system were identified in the spleen. The cDC1 subset of mammalian cDCs express the chemokine receptor XCR1. To characterize chicken cDCs, a synthetic chicken chemokine (C motif) ligand (XCL1) peptide conjugated to Alexa Fluor 647 was developed (XCL1AF647). Flow cytometry staining of XCL1AF647 on splenocytes showed that all chicken FLT3HI cells exclusively express XCR1, supporting the hypothesis that this population comprises bona fide chicken cDCs. Further analysis revealed that chicken cDCs expressed CSF1R but lacked the expression of CSF2R. Collectively, the cell surface phenotypes of chicken cDCs were partially conserved with mammalian XCR1+ cDC1, with distinct differences in CSF1R and CSF2R expression compared with mammalian orthologues. These original reagents allow the efficient identification of chicken cDCs to investigate their important roles in the chicken immunity and diseases.
We have developed tools to specifically identify chicken conventional dendritic cells (cDCs). Chicken cDCs are express high levels of FLT3 and XCR1. While the resemble the mammalian cDC1 subset, significant differences exist.
Most common male reproductive disorders are linked to lower testosterone exposure in fetal life, although the factors responsible for suppressing fetal testosterone remain largely unknown. Protracted ...use of acetaminophen during pregnancy is associated with increased risk of cryptorchidism in sons, but effects on fetal testosterone production have not been demonstrated. We used a validated xenograft model to expose human fetal testes to clinically relevant doses and regimens of acetaminophen. Exposure to a therapeutic dose of acetaminophen for 7 days significantly reduced plasma testosterone (45% reduction; P = 0.025) and seminal vesicle weight (a biomarker of androgen exposure; 18% reduction; P = 0.005) in castrate host mice bearing human fetal testis xenografts, whereas acetaminophen exposure for just 1 day did not alter either parameter. Plasma acetaminophen concentrations (at 1 hour after the final dose) in exposed host mice were substantially below those reported in humans after a therapeutic oral dose. Subsequent in utero exposure studies in rats indicated that the acetaminophen-induced reduction in testosterone likely results from reduced expression of key steroidogenic enzymes (Cyp11a1, Cyp17a1). Our results suggest that protracted use of acetaminophen (1 week) may suppress fetal testosterone production, which could have adverse consequences. Further studies are required to establish the dose-response and treatment-duration relationships to delineate the maximum dose and treatment period without this adverse effect.
Analgesic exposure during pregnancy may affect aspects of fetal gonadal development that are targeted by endocrine disruptors.
We investigated whether therapeutically relevant doses of acetaminophen ...and ibuprofen affect germ cell (GC) development in human fetal testes/ovaries using
and xenograft approaches.
First-trimester human fetal testes/ovaries were cultured and exposed to acetaminophen or ibuprofen (7 d). Second-trimester human fetal testes were xenografted into mice and exposed to acetaminophen (1 or 7 d), or ibuprofen (7 d). To determine mechanism of action, a human GC tumor–derived cell line (NTera2) exhibiting fetal GC characteristics was used in addition to
and
rat models.
Gonocyte (TFAP2C
) number was reduced relative to controls in first-trimester human fetal testes exposed in vitro to acetaminophen (-28%) or ibuprofen (-22%) and also in ovaries exposed to acetaminophen (-43%) or ibuprofen (-49%). Acetaminophen exposure reduced gonocyte number by 17% and 30% in xenografted second-trimester human fetal testes after treatment of host mice for 1 or 7 d, respectively. NTera2 cell number was reduced following exposure to either analgesic or prostaglandin E
(PGE
) receptor antagonists, whereas PGE
agonists prevented acetaminophen-induced reduction in NTera2 cell number. Expression of GC pluripotency genes, and genes that regulate DNA/histone methylation, also differed from controls following analgesic and PGE
receptor antagonist exposures. Gene expression changes were observed in rat fetal testis/ovary cultures and after in vivo acetaminophen exposure of pregnant rats. For example, expression of the epigenetic regulator
, was increased following exposure to acetaminophen in human NTera2 cells, rat fetal testis/ovary cultures, and in fetal testes and ovaries after in vivo exposure of pregnant rats, indicating translatability across experimental models and species.
Our results demonstrate evidence of PGE
-mediated effects of acetaminophen and ibuprofen on GC/NTera2 cells, which raises concerns about analgesic use during human pregnancy that warrant further investigation. https://doi.org/10.1289/EHP2307.
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