Primordial Germ Cell Specification Research Articles

The gonadal niche safeguards human fetal germline cell development following maternal SARS-CoV-2 infection

During pregnancy, germline development is vital for maintaining the continuation of species. Recent studies have shown increased pregnancy risks in COVID-19 patients at the perinatal stage. However, the potential consequence of infection for reproductive quality in developing fetuses remains unclear. Here, we analyze the transcriptome and DNA methylome of the fetal germline following maternal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We find that infection at early gestational age, a critical period of human primordial germ cell specification and epigenetic reprogramming, trivially affects fetal germ cell (FGC) development. Additionally, FGC-niche communications are not compromised by maternal infection. Strikingly, both general and SARS-CoV-2-specific immune pathways are greatly activated in gonadal niche cells to protect FGCs from maternal infection. Notably, there occurs an "in advance" development tendency in FGCs after maternal infection. Our study provides insights into the impacts of maternal SARS-CoV-2 infection on fetal germline development and serves as potential clinical guidance for future pandemics.

Generation of induced pluripotent stem cells from Bornean orangutans.

Introduction: Orangutans, classified under the Pongo genus, are an endangered non-human primate (NHP) species. Derivation of induced pluripotent stem cells (iPSCs) represents a promising avenue for conserving the genetic resources of these animals. Earlier studies focused on deriving orangutan iPSCs (o-iPSCs) from Sumatran orangutans (Pongo abelii). To date, no reports specifically target the other Critically Endangered species in the Pongo genus, the Bornean orangutans (Pongo pygmaeus). Methods: Using Sendai virus-mediated Yamanaka factor-based reprogramming of peripheral blood mononuclear cells to generate iPSCs (bo-iPSCs) from a female captive Bornean orangutan. In this study, we evaluate the colony morphology, pluripotent markers, X chromosome activation status, and transcriptomic profile of the bo-iPSCs to demonstrate the pluripotency of iPSCs from Bornean orangutans. Results: The bo-iPSCs were successfully derived from Bornean orangutans, using Sendai virus-mediated Yamanaka factor-based reprogramming of peripheral blood mononuclear cells. When a modified 4i/L/A (m4i/L/A) culture system was applied to activate the WNT signaling pathway in these bo-iPSCs, the derived cells (m-bo-iPSCs) manifested characteristics akin to human naive pluripotent stem cells, including high expression levels of KLF17, DNMT3L, and DPPA3/5, as well as the X chromosome reactivation. Comparative RNA-seq analysis positioned the m-bo-iPSCs between human naive and formative pluripotent states. Furthermore, the m-bo-iPSCs express differentiation capacity into all three germlines, evidenced by controlled in vitro embryoid body formation assay. Discussion: Our work establishes a novel approach to preserve the genetic diversity of endangered Bornean orangutans while offering insights into primate stem cell pluripotency. In the future, derivation of the primordial germ cell-like cells (PGCLCs) from m-bo-iPSCs is needed to demonstrate the further specific application in species preservation and broaden the knowledge of primordial germ cell specification across species.

Germ plasm dynamics during oogenesis and early embryonic development in Xenopus and zebrafish.

Specification of the germline and its segregation from the soma mark one of the most crucial events in the lifetime of an organism. In different organisms, this specification can occur through either inheritance or inductive mechanisms. In species such as Xenopus and zebrafish, the specification of primordial germ cells relies on the inheritance of maternal germline determinants that are synthesized and sequestered in the germ plasm during oogenesis. In this review, we discuss the formation of the germ plasm, how germline determinants are recruited into the germ plasm during oogenesis, and the dynamics of the germ plasm during oogenesis and early embryonic development.

The spatiotemporal pattern of glypican coordinates primordial germ cell differentiation with ovary development

The establishment, proliferation, and differentiation of stem cells are coordinated with organ development and regulated by the signals in the microenvironment. Prior to gonad formation, how primordial germ cells (PGC) differentiate spatiotemporally to coordinate with gonadogenesis is unclear. In adult ovary, drosophila extracellular glypican Dally in germline stem cell (GSC) niche promotes BMP signaling to inhibit germline differentiation. Here we investigated the relation between the fate of PGC and the spatiotemporal pattern of glypican during ovary development. We found that Dally in ovarian soma assisted BMP signaling to prevent PGC from precocious differentiation. Dally's presence raises the "hurdle" for ecdysone peaks to eventually remove the transcription factor Kr and de-repress pro-differentiation factor, temporally postponing PGC differentiation until GSC niche establishment. The spatiotemporal glypican in somatic matrix assists PGC to integrate the ovarian local BMP and organismal steroid signals that coordinate PGC's program with organ/body development to maximize reproductive potential.

Divergent Roles of KLF4 During Primordial Germ Cell Fate Induction from Human Embryonic Stem Cells.

As a core transcriptional factor regulating pluripotency, Krüppel-like factor 4 (KLF4) has gained much attention in the field of stem cells during the past decades. However, few research have focused on the function of KLF4 during human primordial germ cell (PGC) specification. Here, we induced human PGC-like cells (hPGCLCs) from human embryonic stem cells (hESCs) and the derived hPGCLCs upregulated PGC-related genes, like SOX17, BLIMP1, TFAP2C, NANOS3, and the naïve pluripotency gene KLF4. The KLF4-knockout hESCs formed typical multicellular colonies with clear borders, expressed pluripotency genes, such as NANOG, OCT4, and SOX2, and exhibited no differences in proliferation capacity compared with wild type hESCs. Notably, KLF4 deletion in hESCs did not influence the induction of PGCLCs in vitro. In contrast, overexpression of KLF4 during PGC induction process inhibited the efficiency of PGCLC formation from hESCs in vitro. Overexpression of KLF4 may regenerate the naïve ground state in hESCs and results in repression for PGC specification. Thus, KLF4 could be a downstream target of human PGC program and the upregulation of KLF4 is prepared for late stage of germline development.

Epigenetic regulation limits competence of pluripotent stem cell-derived oocytes.

Recent studies have reported the differentiation of pluripotent cells into oocytes in vitro. However, the developmental competence of in vitro-generated oocytes remains low. Here, we perform a comprehensive comparison of mouse germ cell development invitro over all culture steps versus in vivo with the goal to understand mechanisms underlying poor oocyte quality. We show that the in vitro differentiation of primordial germ cells to growing oocytes and subsequent follicle growth is critical for competence for preimplantation development. Systematic transcriptome analysis of single oocytes that were subjected to different culture steps identifies genes that are normally upregulated during oocyte growth to be susceptible for misregulation during in vitro oogenesis. Many misregulated genes are Polycomb targets. Deregulation of Polycomb repression is therefore a key cause and the earliest defect known in in vitro oocyte differentiation. Conversely, structurally normal in vitro-derived oocytes fail at zygotic genome activation and show abnormal acquisition of 5-hydroxymethylcytosine on maternal chromosomes. Our data identify epigenetic regulation at an early stage of oogenesis limiting developmental competence and suggest opportunities for future improvements.

DMRT1 regulates human germline commitment

Germline commitment following primordial germ cell (PGC) specification during early human development establishes an epigenetic programme and competence for gametogenesis. Here we follow the progression of nascent PGC-like cells derived from human embryonic stem cells in vitro. We show that switching from BMP signalling for PGC specification to Activin A and retinoic acid resulted in DMRT1 and CDH5 expression, the indicators of migratory PGCs in vivo. Moreover, the induction of DMRT1 and SOX17 in PGC-like cells promoted epigenetic resetting with striking global enrichment of 5-hydroxymethylcytosine and locus-specific loss of 5-methylcytosine at DMRT1 binding sites and the expression of DAZL representing DNA methylation-sensitive genes, a hallmark of the germline commitment programme. We provide insight into the unique role of DMRT1 in germline development for advances in human germ cell biology and in vitro gametogenesis.

Induced differentiation of primordial germ cell like cells from SOX9+ porcine skin derived stem cells

Understanding the mechanisms behind porcine primordial germ cell like cells (pPGCLCs) development, differentiation, and gametogenesis is crucial in the treatment of infertility. In this study, SOX9+ skin derived stem cells (SOX9+ SDSCs) were isolated from fetal porcine skin and a high-purity SOX9+ SDSCs population was obtained. The SOX9+ SDSCs were induced to transdifferentiate into PGCLCs during 8 days of cultured. The results of RNA-seq, western blot and immunofluorescence staining verified SDSCs have the potential to transdifferentiate into PGCLCs from aspects of transcription factor activation, germ layer differentiation, energy metabolism, and epigenetic changes. Both adherent and suspended cells were collected. The adherent cells were found to be very similar to early porcine primordial germ cells (pPGCs). The suspended cells resembled late stage pPGCs and had a potential to enter meiotic process. This SDSCs culture-induced in vitro model is expected to provide suitable donor cells for stem cell transplantation in the future.

Complete human day 14 post-implantation embryo models from naive ES cells

The ability to study human post-implantation development remains limited owing to ethical and technical challenges associated with intrauterine development after implantation1. Embryo-like models with spatially organized morphogenesis and structure of all defining embryonic and extra-embryonic tissues of the post-implantation human conceptus (that is, the embryonic disc, the bilaminar disc, the yolk sac, the chorionic sac and the surrounding trophoblast layer) remain lacking1,2. Mouse naive embryonic stem cells have recently been shown to give rise to embryonic and extra-embryonic stem cells capable of self-assembling into post-gastrulation structured stem-cell-based embryo models with spatially organized morphogenesis (called SEMs)3. Here we extend those findings to humans using only genetically unmodified human naive embryonic stem cells (cultured in human enhanced naive stem cell medium conditions)4. Such human fully integrated and complete SEMs recapitulate the organization of nearly all known lineages and compartments of post-implantation human embryos, including the epiblast, the hypoblast, the extra-embryonic mesoderm and the trophoblast layer surrounding the latter compartments. These human complete SEMs demonstrated developmental growth dynamics that resemble key hallmarks of post-implantation stage embryogenesis up to 13–14 days after fertilization (Carnegie stage 6a). These include embryonic disc and bilaminar disc formation, epiblast lumenogenesis, polarized amniogenesis, anterior–posterior symmetry breaking, primordial germ-cell specification, polarized yolk sac with visceral and parietal endoderm formation, extra-embryonic mesoderm expansion that defines a chorionic cavity and a connecting stalk, and a trophoblast-surrounding compartment demonstrating syncytium and lacunae formation. This SEM platform will probably enable the experimental investigation of previously inaccessible windows of human early post implantation up to peri-gastrulation development.

The people behind the papers - Christopher Cooke and Naomi Moris.

Primordial germ cells (PGCs) are the precursors of gametes, and a presumptive PGC-like cell (PGCLC) population was recently identified in mouse gastruloids. A new paper in Development investigates this population in detail and offers new insight into the signals that contribute to PGC specification and maturation. To hear more about the story, we caught up with first author Christopher Cooke and corresponding author Naomi Moris, Group Leader at the Francis Crick Institute, UK.

Mammal Reproductive Homeobox (Rhox) Genes: An Update of Their Involvement in Reproduction and Development.

The reproductive homeobox on the X chromosome (RHOX) genes were first identified in the mouse during the 1990s and have a crucial role in reproduction. In various transcription factors with a key regulatory role, the homeobox sequence encodes a "homeodomain" DNA-binding motif. In the mouse, there are three clusters of Rhox genes (α, β, and γ) on the X chromosome. Each cluster shows temporal and/or quantitative collinearity, which regulates the progression of the embryonic development process. Although the RHOX family is conserved in mammals, the interspecies differences in the number of RHOX genes and pseudogenes testifies to a rich evolutionary history with several relatively recent events. In the mouse, Rhox genes are mainly expressed in reproductive tissues, and several have a role in the differentiation of primordial germ cells (Rhox1, Rhox6, and Rhox10) and in spermatogenesis (Rhox1, Rhox8, and Rhox13). Despite the lack of detailed data on human RHOX, these genes appear to be involved in the formation of germ cells because they are predominantly expressed during the early (RHOXF1) and late (RHOXF2/F2B) stages of germ cell development. Furthermore, the few variants identified to date are thought to induce or predispose to impaired spermatogenesis and severe oligozoospermia or azoospermia. In the future, research on the pathophysiology of the human RHOX genes is likely to confirm the essential role of this family in the reproductive process and might help us to better understand the various causes of infertility and characterize the associated human phenotypes.

Porcine Germ Cells Phenotype during Embryonic and Adult Development.

Primordial germ cells (PGCs) are the precursors of gametes. Due to their importance for the formation and reproduction of an organism, understanding the mechanisms and pathways of PGCs and the differences between males and females is essential. However, there is little research in domestic animals, e.g., swine, regarding the epigenetic and pluripotency profiles of PGCs during development. This study analyzed the expression of epigenetic and various pluripotent and germline markers associated with the development and differentiation of PGCs in porcine (pPGCs), aiming to understand the different gene expression profiles between the genders. The analysis of gonads at different gestational periods (from 24 to 35 days post fertilization (dpf) and in adults) was evaluated by immunofluorescence and RT-qPCR and showed phenotypic differences between the gonads of male and female embryos. In addition, the pPGCs were positive for OCT4 and VASA; some cells were H3k27me3 positive in male embryos and adult testes. In adults, the cells of the testes were positive for germline markers, as confirmed by gene expression analysis. The results may contribute to understanding the pPGC pathways during reproductive development, while also contributing to the knowledge needed to generate mature gametes in vitro.

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.

The dynamic expression of SOX17 in germ cells from human female foetus and adult ovaries after specification.

SOX17 has been identified as a critical factor in specification of human primordial germ cells, but whether SOX17 regulates development of germ cells after sex differentiation is poorly understood. We collected specimens of gonadal ridge from an embryo (n=1), and ovaries of foetuses (n=23) and adults (n=3). Germ cells were labelled with SOX17, VASA (classic germ cells marker), phosphohistone H3 (PHH3, mitosis marker) and synaptonemal complex protein 3 (SCP3, meiosis marker). SOX17 was detected in both cytoplasm and nucleus of oogonia and oocytes of primordial and primary follicles from 15 to 28 gestational weeks (GW). However, it was exclusively expressed in cytoplasm of oogonia at 7 GW, and in nucleus of oocytes in secondary follicles. Co-expression rates of SOX17 in VASA+ germ cells ranged from 81.29% to 97.81% in foetuses. Co-staining rates of SOX17 and PHH3 or SCP3 were 0%-34% and 0%-57%, respectively. Interestingly, we distinguished a subpopulation of SOX17+VASA- germ cells in fetal ovaries. These cells clustered in the cortex and could be co-stained with the mitosis marker PHH3 but not the meiosis marker SCP3. The dynamic expression of SOX17 was detected in human female germ cells. We discovered a population of SOX17+ VASA- germ cells clustering at the cortex of ovaries. We could not find a relationship between mitosis or meiosis and SOX17 or VASA staining in germ cells. Our findings provide insight into the potential role of SOX17 involving germ cells maturation after specification, although the mechanism is unclear and needs further investigation.

From in vivo to in vitro: exploring the key molecular and cellular aspects of human female gametogenesis.

Human oogenesis is a highly complex and not yet fully understood process due to ethical and technological barriers that limit studies in the field. In this context, replicating female gametogenesis in vitro would not only provide a solution for some infertility problems, but also be an excellent study model to better understand the biological mechanisms that determine the formation of the female germline. In this review, we explore the main cellular and molecular aspects involved in human oogenesis and folliculogenesis in vivo, from the specification of primordial germ cells (PGCs) to the formation of the mature oocyte. We also sought to describe the important bidirectional relationship between the germ cell and the follicular somatic cells. Finally, we address the main advances and different methodologies used in the search for obtaining cells of the female germline in vitro.

Origin and segregation of the human germline.

Human germline-soma segregation occurs during weeks 2-3 in gastrulating embryos. Although direct studies are hindered, here, we investigate the dynamics of human primordial germ cell (PGCs) specification using in vitro models with temporally resolved single-cell transcriptomics and in-depth characterisation using in vivo datasets from human and nonhuman primates, including a 3D marmoset reference atlas. We elucidate the molecular signature for the transient gain of competence for germ cell fate during peri-implantation epiblast development. Furthermore, we show that both the PGCs and amnion arise from transcriptionally similar TFAP2A-positive progenitors at the posterior end of the embryo. Notably, genetic loss of function experiments shows that TFAP2A is crucial for initiating the PGC fate without detectably affecting the amnion and is subsequently replaced by TFAP2C as an essential component of the genetic network for PGC fate. Accordingly, amniotic cells continue to emerge from the progenitors in the posterior epiblast, but importantly, this is also a source of nascent PGCs.

The embryonic origin of primordial germ cells in the tardigrade Hypsibius exemplaris.

Primordial germ cells (PGCs) give rise to gametes - cells necessary for the propagation and fertility of diverse organisms. Current understanding of PGC development is limited to the small number of organisms whose PGCs have been identified and studied. Expanding the field to include little-studied taxa and emerging model organisms is important to understand the full breadth of the evolution of PGC development. In the phylum Tardigrada, no early cell lineages have been identified to date using molecular markers. This includes the PGC lineage. Here, we describe PGC development in the model tardigrade Hypsibius exemplaris. The four earliest-internalizing cells (EICs) exhibit PGC-like behavior and nuclear morphology. The location of the EICs is enriched for mRNAs of conserved PGC markers wiwi1 (water bear piwi 1) and vasa. At early stages, both wiwi1 and vasa mRNAs are detectable uniformly in embryos, which suggests that these mRNAs do not serve as localized determinants for PGC specification. Only later are wiwi1 and vasa enriched in the EICs. Finally, we traced the cells that give rise to the four PGCs. Our results reveal the embryonic origin of the PGCs of H.exemplaris and provide the first molecular characterization of an early cell lineage in the tardigrade phylum. We anticipate that these observations will serve as a basis for characterizing the mechanisms of PGC development in this animal.

Derivation of new pluripotent stem cells from human extended pluripotent stem cells with formative features and trophectoderm potential.

Previous studies have demonstrated the existence of intermediate stem cells, which have been successfully obtained from human naive pluripotent stem cells (PSCs) and peri-implantation embryos. However, it is not known whether human extended pluripotent stem cells (hEPSCs) can be directly induced into intermediate stem cells. Moreover, the ability of extra-embryonic lineage differentiation in intermediate stem cells has not been verified. In this issue, we transformed hEPSCs into a kind of novel intermediate pluripotent stem cell resembling embryonic days 8-9 (E8-E9) epiblasts and proved its feature of formative epiblasts. We engineered hEPSCs from primed hPSCs under N2B27-LCDM (N2B27 plus Lif, CHIR, DiH and MiH) conditions. Then, we added Activin A, FGF and XAV939 to modulate signalling pathways related to early humans' embryogenesis. We performed RNA-seq and CUT&Tag analysis to compare with AF9-hPSCs from different pluripotency stages of hPSCs. Trophectoderm (TE), primordial germ cells-like cells (PGCLC) and endoderm, mesoderm, and neural ectoderm induction were conducted by specific small molecules and proteins. AF9-hPSCs transcription resembled that of E8-E9 peri-implantation epiblasts. Signalling pathway responsiveness and histone methylation further revealed their formative pluripotency. Additionally, AF9-hPSCs responded directly to primordial germ cells (PGCs) specification and three germ layer differentiation signals in vitro. Moreover, AF9-hPSCs could differentiate into the TE lineage. Therefore, AF9-hPSCs represented an E8-E9 formative pluripotency state between naïve and primed pluripotency, opening new avenues for studying human pluripotency development during embryogenesis.

Examination of the effects of Nanos gene knockdown on fecundity and reproductive modes in the brine shrimp, Artemia parthenogenetica

Artemia is not only a crucial live food source in fish and crustacean larviculture but also an ideal experimental organism for biological studies. Female Artemia subjects release either diapause-encysted embryos (cysts) via oviparous reproduction or nauplii via ovoviviparous reproduction. As a maternal effector gene, nanos is involved in the differentiation and migration of primordial germ cells. In the present study, the open reading frame (ORF) of the nanos gene of Artemia parthenogenetica was screened and analyzed. Nanos cDNA contained a 933-bp ORF, encoding 310 amino acids with a conserved zinc-finger motif. The expression patterns of nanos in various tissues and during embryo development in Artemia were analyzed by qRT-PCR. The results showed that nanos was specifically expressed in the ovary, with the highest expression being observed during early ovary development. Moreover, nanos knockdown induced by dsRNA injection significantly decreased the gene and protein expression levels of nanos. In addition, knockdown of nanos resulted in an obvious shift from ovoviviparity to oviparity and a reduced number of offspring, corresponding to irregular oocyte shape, cell membrane damage, and cytoplasmic vacuolization. Our study suggests that nanos not only regulates the ovary development and reproductive performance of Artemia but also play a critical role in determining its reproductive mode. This study will improve our knowledge of the role of nanos in the reproductive process of Artemia and the molecular mechanisms underlying reproduction in this shrimp.

Observation of the Expression of Dazl and Vasa Suggests that Chinese Soft-shelled Turtle Use the Inductive Mode of Primordial Germ Cell Specification

The origin, migration and differentiation of primordial germ cells (PGCs) have always been an important issue in developmental biology. At present, studies on the specification of PGCs in amniotes mainly focus on birds and mammals. Turtles are reptiles known as "living fossils", and their sex determination is influenced by both genetic and environmental factors, making them an ideal model animal for the study of vertebrate sex differentiation. However, the study of PGCs specification in turtles is very limited. Here we reported using RNA probes for the germ cell-specific markers Dazl and Vasa to examine growing oocytes, early embryos and adult ovaries in the Chinese soft-shelled turtle. The results shows that the expression of Dazl and Vasa could not be detected in the growing oocytes and early embryos, and the specification of germline begins around E7, indicating that the Chinese soft-shelled turtle do not contain germ plasm and belongs to the inductive specification.