Primordial Germ Cell-like Cells Research Articles

Quantitative Dynamics of Chromatin Remodeling during Germ Cell Specification from Mouse Embryonic Stem Cells

Germ cell specification is accompanied by epigenetic remodeling, the scale and specificity of whichare unclear. Here, we quantitatively delineate chromatin dynamics during induction of mouse embryonic stem cells (ESCs) to epiblast-like cells (EpiLCs) and from there into primordial germ cell-like cells (PGCLCs), revealing large-scale reorganization of chromatin signatures including H3K27me3 and H3K9me2 patterns. EpiLCs contain abundant bivalent gene promoters characterized by low H3K27me3, indicating a state primed for differentiation. PGCLCs initially lose H3K4me3 from many bivalent genes but subsequently regain this mark with concomitant upregulation of H3K27me3, particularly at developmental regulatory genes. PGCLCs progressively lose H3K9me2, including at lamina-associated perinuclear heterochromatin, resulting in changes in nuclear architecture. T recruits H3K27ac to activate BLIMP1 and early mesodermal programs during PGCLC specification, which is followed by BLIMP1-mediated repression of a broad range of targets, possibly through recruitment and spreading of H3K27me3. These findings provide a foundation for reconstructing regulatory networks of the germline epigenome.

Stem cells derived from human first-trimester umbilical cord have the potential to differentiate into oocyte-like cells in vitro

Compared to stem cells derived from human term umbilical cord, stem cells derived from human first-trimester umbilical cord (hFTUC) exhibit a significantly greater proliferative potential, and more efficiency in terms of their in vitro differentiation. In the present study, we investigated whether hFTUC-derived stem cells are able to differentiate into germ cells. The hFTUC-derived stem cells were first isolated, expanded and then cultured in differentiation medium containing human follicular fluid, follicle-stimulating hormone (FSH)/luteinizing hormone (LH) and estradiol for 24 days. During the period of induction, a subpopulation of the cultured cells appeared that had a morphological resemblance to primordial germ cells (PGCs) and cumulus-oocyte complex (COC)-like cells, and oocyte-like cells (OLCs). The PGC-like cells expressed specific markers indicative of germ cell formation such as octamer-binding transcription factor 4 (OCT4), stage-specific embryonic antigen 1 (SSEA1), B lymphocyte-induced maturation protein-1 (BLIMP1), PR domain containing 14 (PRDM14), transcription factor AP-2 gamma (TFAP2C), VASA, STELLA, deleted in azoospermia-like (DAZL) and interferon-induced transmembrane protein 3 (IFITM3). The OLCs, which contained a single germinal vesicle, expressed oocyte-specific markers, such as synaptonemal complex protein 3 (SCP3), growth/differentiation factor-9 (GDF9), GDF9B and zona pellucida (ZP)1, ZP2 and ZP3. The COC-like cells secreted estradiol, vascular endothelial growth factor and leukemia inhibitory factor. Thus, our findings suggest that hFTUC-derived stem cells have an intrinsic ability to differentiate into OLCs, which may provide an in vitro model for the identification of factors involved in germ cell formation and differentiation.

Human primordial germ cell commitment in vitro associates with a unique PRDM14 expression profile.

Primordial germ cells (PGCs) develop only into sperm and oocytes in vivo. The molecular mechanisms underlying human PGC specification are poorly understood due to inaccessibility of cell materials and lack of in vitro models for tracking the earliest stages of germ cell development. Here, we describe a defined and stepwise differentiation system for inducing pre-migratory PGC-like cells (PGCLCs) from human pluripotent stem cells (PSCs). In response to cytokines, PSCs differentiate first into a heterogeneous mesoderm-like cell population and then into PGCLCs, which exhibit minimal PRDM14 expression. PGC specification in humans is similar to the murine process, with the sequential activation of mesodermal and PGC genes, and the suppression of neural induction and of de novo DNA methylation, suggesting that human PGC formation is induced via epigenesis, the process of germ cell specification via inductive signals from surrounding somatic cells. This study demonstrates that PGC commitment in humans shares key features with that of the mouse, but also highlights key differences, including transcriptional regulation during the early stage of human PGC development (3–6 weeks). A more comprehensive understanding of human germ cell development may lead to methodology for successfully generating PSC-derived gametes for reproductive medicine.

The generation of sex cells.

Primordial germ cells (PGCs) are the earliest population of germ cells established during embryonic development and constitute the beginning of the totipotent state. A recent study provides a new protocol for the efficient generation of PGC-like cells from human embryonic stem cells, providing an in vitro platform to study human PGC differentiation and specification.

Exogenous supplementation of Activin A enhances germ cell differentiation of human embryonic stem cells†

Human embryonic stem cells (hESCs) derived in the presence of Activin A (ActA) demonstrate an increased differentiation propensity toward the germ cell lineage. In addition, mouse epiblast stem cells and mouse epiblast-like cells are poised toward germ cell differentiation and are derived in the presence of ActA. We therefore investigated whether supplementation with ActA enhances in vitro hESC differentiation toward germ cell lineage. ActA up-regulated early primordial germ cell (PGC) genes STELLA/DPPA3 (developmental pluripotency associated 3) and tyrosine kinase receptor cKIT in both ActA-derived and standard-derived hESCs indicating its role in priming hESCs toward the PGC lineage. Indeed, ActA plus bone morphogenic protein 4 (BMP4) strongly increased germ cell differentiation potential of hESCs based on the high expression of late PGC markers DAZL (deleted in azoospermia-like) and VASA/DDX4 (DEAD-box polypeptide 4) at mRNA and protein level. Hence, the combination of ActA with BMP4 provides an additional boost for hESCs to develop into postmigratory germ cells. Together with increased VASA expression in the presence of ActA and BMP4, we also observed up-regulation of endoderm-specific genes GATA4 (GATA binding protein 4) and GATA6. Finally, we were able to further mature these in vitro-derived PGC-like cells (PGCLCs) by culturing them in in vitro maturation (IVM) medium, resulting in the formation of germ cell-like clusters and induction of meiotic gene expression. In conclusion, we demonstrate for the first time a synergism between ActA and BMP4 in facilitating germ cell-directed differentiation of hESCs, which is enhanced by extended culture in IVM medium, as shown by cytoplasmic VASA-expressing PGCLCs. We propose a novel relationship between the endoderm and germ cell lineage during hESC differentiation.

Mechanism and Reconstitution In Vitro of Germ Cell Development in Mammals.

The germ cell lineage creates new individuals, perpetuating/diversifying the genetic and epigenetic information across generations. Based on the knowledge obtained through investigations into the mechanisms of germ cell specification and development in mice, we have succeeded in precisely reconstituting the specification and subsequent development of germ cells in culture in both males and females: Embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) are induced into epiblast-like cells (EpiLCs) and then into primordial germ cell-like cells (PGCLCs), which robustly contribute to spermatogenesis and oogenesis and to fertile offspring. This in vitro mouse PGC specification/development system has led to the elucidation of signaling, transcriptional, and epigenetic regulation during germ cell development in a detailed fashion. More recently, based on this system, we and others have demonstrated the induction of human PGCLCs from human ESCs/iPSCs, creating an opportunity for understanding the mechanism of human germ cell development in vitro.

Generation of Oocytes from Mouse ES/iPS Cells

Abstract: One of the paramount goals in reproductive biology is to produce functional oocytes in culture through a series of differentiation processes that accurately mimic those in vivo. Such a culture system would provide a larger number of oocytes than those available in vivo, which would be of help in the elucidation of mechanisms underlying germ cell development. All germ cells originate from a small group of primordial germ cells (PGCs) that segregate from somatic cell lineages at an early developmental stage. Recently, we developed a culture system in which embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) differentiate into PGC-like cells (PGCLCs) through an epiblast-like cell population. PGCLCs are capable of differentiating into functional oocytes when transplanted into the ovarian bursa with E12.5 gonadal somatic cells. In this review, we introduce our differentiation method and discuss possible applications of this culture system.

In vitro generation of zebrafish PGC-like cells.

The possibility of generating primordial germ cells (PGCs) in vitro from noncommitted embryonic cells represents an extremely useful tool in current research. Primordial germ cell in vitro differentiation has been successfully reported in mammals. However, contrary to fish, PGC specification in mammals is an inductive mechanism. This study is the first to date to describe a rapid method for PGC in vitro differentiation in teleosts. Primordial germ cell-like cells were characterized by several lines of evidence, including gene expression, cell complexity, size, and image analysis for the quantification of fluorescence under vasa promoter. Moreover, differentiated cells were able to colonize the genital ridge after transplantation. Differentiation treatments increased the number of PGCs in culture, causing differentiation of cells rather than inducing their proliferation. These results open up the possibility of differentiating genetically modified embryonic cells to PGC-like cells to ensure their transmission to the progeny and could be crucial for an in-depth understanding of germline differentiation in teleosts.

Induction of primordial germ cell-like cells from mouse embryonic stem cells by ERK signal inhibition.

Primordial germ cells (PGCs) are embryonic germ cell precursors. Specification of PGCs occurs under the influence of mesodermal induction signaling during in vivo gastrulation. Although bone morphogenetic proteins and Wnt signaling play pivotal roles in both mesodermal and PGC specification, the signal regulating PGC specification remains unknown. Coculture of mouse embryonic stem cells (ESCs) with OP9 feeder cells induces mesodermal differentiation in vitro. Using this mesodermal differentiation system, we demonstrated that PGC-like cells were efficiently induced from mouse ESCs by extracellular signal-regulated kinase (ERK) signaling inhibition. Inhibition of ERK signaling by a MAPK/ERK kinase (MEK) inhibitor upregulated germ cell marker genes but downregulated mesodermal genes. In addition, the PGC-like cells showed downregulation of DNA methylation and formed pluripotent stem cell colonies upon treatment with retinoic acid. These results show that inhibition of ERK signaling suppresses mesodermal differentiation but activates germline differentiation program in this mesodermal differentiation system. Our findings provide a new insight into the signaling networks regulating PGC specification.

BMP4 promotes SSEA-1(+) hUC-MSC differentiation into male germ-like cells in vitro.

Recent studies have demonstrated that primordial germ cells (PGC) can be differentiated from human umbilical cord mesenchymal stem cells (hUC-MSCs), and embryonic stem cells (ESCs) in vitro. Nevertheless, efficiencies were low and unstable. Here, whether hUC-MSCs can be induced to differentiate into germ-like cells with the aid of bone morphogenetic protein (BMP4) was investigated. Human umbilical cord mesenchymal stem cells were freshly isolated and cultured with BMP4. SSEA-1(+/-) cells were purified using magnetic-activated cell sorting (MACS) from the hUC-MSCs, and further induced with BMP4. Quantitative real-time PCR (qRT-PCR) and immunofluorescence analysis were used to determine PGC and germ-like cell-specific markers. Human umbilical cord mesenchymal stem cells differentiated into SSEA-1(+) spherical PGC-like cells efficiently with 12.5 ng/ml BMP4. qRT-PCR and immunofluorescence analysis demonstrated that SSEA-1(+) cells expressed higher levels of PGC-specific markers than SSEA-1(-) cells. Furthermore, SSEA-1(+) cells were induced with BMP4 to differentiate into STRA8, SCP3, DMRT1 and PLZF-positive male germ-like cells, and some sperm-like cells were obtained by 7-14 days after induction. These results suggest that SSEA-1(+) hUC-MSCs can differentiate into male germ-like cells in the presence of BMP4. This study provides an efficient protocol to study germ-cell development using hUC-MSCs.

Induction of dedifferentiated male mouse adipose stromal vascular fraction cells to primordial germ cell-like cells.

The adipose stromal vascular fraction (SVF) contains abundant mesenchymal stem cell populations that have a limited ability to self-renew and differentiate. Male mouse adipose SVF cells were dedifferentiated by reprogramming factors (c-Myc, Oct4, Sox2, and Klf4) to form embryonic stem cell-like cells (ESCLCs), which upgraded their limited differentiation potential. The ESCLCs were induced to differentiate toward epiblast-like cells (EpiLCs) and primordial germ cell-like cells (PGCLCs) by culturing in media supplied with activin A and BMP-4, respectively. The derived ESCLCs possess embryonic stem cell features and can automatically form embryonic bodies. After culture in EpiLC induction medium for 2-3 days, ESCLCs formed flattened epithelial structures that were different from their original water drop-like colonies, and the expression of pluripotency-related genes decreased. When the cells that had been cultured in EpiLC induction medium for 2 days were isolated and cultured in PGCLC induction medium for 4-6 days, they formed typical water drop-like colonies again. Moreover, expression of the pluripotency-related genes and the primordial germ cell (PGC) specification-related genes increased. During progression from ESCLCs toward EpiLCs and PGCLCs, the levels of histone methylases H3K9me2 and H3K27me3 kept changing, which resembled those seen in PGC specification. The derived PGCLCs expressed SSEA-1, Blimp-1, and Stella. Furthermore, methylation of Igf2r and Snrpn was retained, but H19 and Kcnq1ot1 methylation levels were slightly reduced compared to non-PGCLCs, suggesting that the derived PGCLCs may have initiated the process of imprint erasure.

Formation and cultivation of medaka primordial germ cells

Primordial germ cell (PGC) formation is pivotal for fertility. Mammalian PGCs are epigenetically induced without the need for maternal factors and can also be derived in culture from pluripotent stem cells. In egg-laying animals such as Drosophila and zebrafish, PGCs are specified by maternal germ plasm factors without the need for inducing factors. In these organisms, PGC formation and cultivation in vitro from indeterminate embryonic cells have not been possible. Here, we report PGC formation and cultivation in vitro from blastomeres dissociated from midblastula embryos (MBEs) of the fish medaka (Oryzias latipes). PGCs were identified by using germ-cell-specific green fluorescent protein (GFP) expression from a transgene under the control of the vasa promoter. Embryo perturbation was exploited to study PGC formation in vivo, and dissociated MBE cells were cultivated under various conditions to study PGC formation in vitro. Perturbation of somatic development did not prevent PGC formation in live embryos. Dissociated MBE blastomeres formed PGCs in the absence of normal somatic structures and of known inducing factors. Most importantly, under culture conditions conducive to stem cell derivation, some dissociated MBE blastomeres produced GFP-positive PGC-like cells. These GFP-positive cells contained genuine PGCs, as they expressed PGC markers and migrated into the embryonic gonad to generate germline chimeras. Our data thus provide evidence for PGC preformation in medaka and demonstrate, for the first time, that PGC formation and derivation can be obtained in culture from early embryos of medaka as a lower vertebrate model.

Making gametes from pluripotent stem cells--a promising role for very small embryonic-like stem cells.

The urge to have one’s own biological child supersedes any desire in life. Several options have been used to obtain gametes including pluripotent stem cells (embryonic ES and induced pluripotent iPS stem cells); gonadal stem cells (spermatogonial SSCs, ovarian OSCs stem cells), bone marrow, mesenchymal cells and fetal skin. However, the field poses a huge challenge including inefficient existing protocols for differentiation, epigenetic and genetic changes associated with extensive in vitro manipulation and also ethical/regulatory constraints. A tremendous leap in the field occurred using mouse ES and iPS cells wherein they were first differentiated into epiblast-like cells and then primordial germ cell-like cells. These on further development produced sperm, oocytes and live offspring (had associated genetic problems). Evidently differentiating pluripotent stem cells into primordial germ cells (PGCs) remains a major bottleneck. Against this backdrop, we propose that a novel population of pluripotent stem cells termed very small embryonic-like stem cells (VSELs) may serve as an alternative, potential source of autologus gametes, keeping in mind that they are indeed PGCs surviving in adult mammalian ovaries and testes. Both VSELs and PGCs are pluripotent, relatively quiescent because of epigenetic modifications of parentally imprinted genes loci like Igf2-H19 and KCNQ1p57, share several markers like Stella, Fragilis, Mvh, Dppa2, Dppa4, Sall4, Blimp1 and functional receptors. VSELs are localized in the basement membrane of seminiferous tubules in testis and in the ovary surface epithelium. Ovarian stem cells from mouse, rabbit, sheep, marmoset and humans (menopausal women and those with premature ovarian failure) spontaneously differentiate into oocyte-like structures in vitro with no additional requirement of growth factors. Thus a more pragmatic option to obtain autologus gametes may be the pluripotent VSELs and if we could manipulate them in vivo – existing ethical and epigenetic/genetic concerns associated with in vitro culture may also be minimized. The field of oncofertility may undergo a sea-change and existing strategies of cryopreservation of gametes and gonadal tissue for fertility preservation in cancer patients will necessitate a revision. However, first the scientific community needs to arrive at a consensus about VSELs in the gonads and then work towards exploiting their potential.

Culture Conditions for Maintain Propagation, Long-term Survival and Germline Transmission of Chicken Primordial Germ Cell-Like Cells

Culture Conditions for Maintain Propagation, Long-term Survival and Germline Transmission of Chicken Primordial Germ Cell-Like Cells

Generation of male germ cells from mouse induced pluripotent stem cells in vitro

Germ cells are the only cell type that passes genetic information to the next generation. In most metazoan species, primordial germ cells (PGCs) were induced from epiblasts by signals from the neighboring tissues. In vitro derivation of germ cells from the pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) and induced PSCs (iPSCs) are of great values for the treatment of infertility, for animal breeding, and for studying the mechanism of germ cell development. Although the derivations of male germ cells from PSCs have been previously reported, most of the studies failed to conduct the induction in a well-controlled and highly efficient manner. Here, we report the derivation of induced PGC-like cells (iPGCLCs) from mouse iPSCs via induced epiblast-like cells (iEpiLCs) as being monitored by the expression of enhanced green fluorescent protein gene under the control of the promoter of stimulated by retinoic acid 8 (Stra8-EGFP). The identity of iPGCLCs was characterized by examining the expression of multiple marker genes as well as by the recovery of spermatogenesis after they were transplanted to the testis of infertile W/Wv mice. Furthermore, iPGCLCs were either induced to germline stem cell-like cells (iGSCLCs) or reverted back to embryonic germ cell-like cells (iEGCLCs). In conclusion, we have established an efficient procedure for inducing iPSCs into iPGCLCs that can be further expanded and induced to more developed germ cells. This work indicates that the technology of in vitro germ cell induction is becoming more sophisticated and can be further improved.

Germ lineage properties in the urochordate Botryllus schlosseri – From markers to temporal niches

The primordial germ cells (PGCs) in the colonial urochordate Botryllus schlosseri are sequestered in late embryonic stage. PGC-like populations, located at any blastogenic stage in specific niches, inside modules with curtailed lifespan, survive throughout the life of the colony by repeated weekly migration to newly formed buds. This cyclical migration and the lack of specific markers for PGC-like populations are obstacles to the study on PGCs. For that purpose, we isolated the Botryllus DDX1 (BS-DDX1) and characterized it by normal expression patterns and by specific siRNA knockdown experiments. Expression of BS-DDX1 concurrent with BS-Vasa, γ-H2AX, BS-cadherin and phospho-Smad1/5/8, demarcate PGC cells from soma cells and from more differentiated germ cells lineages, which enabled the detection of additional putative transient niches in zooids. Employing BS-cadherin siRNA knockdown, retinoic acid (RA) administration or β-estradiol administration affirmed the BS-Vasa+BS-DDX1+BS-cadherin+γ-H2AX+phospho-Smad1/5/8+ population as the B. schlosseri PGC-like cells. By striving to understand the PGC-like cells trafficking between transient niches along blastogenic cycles, CM-DiI-stained PGC-like enriched populations from late blastogenic stage D zooids were injected into genetically matched colonial ramets at blastogenic stages A or C and their fates were observed for 9 days. Based on the accumulated data, we conceived a novel network of several transient and short lived ‘germ line niches’ that preserve PGCs homeostasis, protecting these cells from the weekly astogenic senescence processes, thus enabling the survival of the PGCs throughout the organism's life.

Influence of Activin A Supplementation During Human Embryonic Stem Cell Derivation on Germ Cell Differentiation Potential

Human embryonic stem cells (hESCs) are more similar to "primed" mouse epiblast stem cells (mEpiSCs). mEpiSCs, which are derived in Activin A, show an increased propensity to form primordial germ cell (PGC)-like cells in response to bone morphogenic protein 4 (BMP4). Hence, we hypothesized that hESCs derived in the presence of Activin A may be more competent in differentiating towards PGC-like cells after supplementation with BMP4 compared to standard hESC lines. We were able to successfully derive two hESC lines in the presence of Activin A, which were pluripotent and showed higher base levels of STELLA and cKIT compared to standard hESC lines derived without Activin A addition. Furthermore, upon differentiation as embryoid bodies in the presence of BMP4, we observed upregulation of VASA at day 7, both at the transcript and protein level compared to standard hESC lines, which appeared to take longer time for PGC specification. Unlike other hESC lines, nuclear pSMAD2/3 presence confirmed that Activin signalling was switched on in Activin A-derived hESC lines. They were also responsive to BMP4 based on nuclear detection of pSMAD1/5/8 and showed endodermal differentiation as a result of GATA-6 expression. Hence, our results provide novel insights into the impact of hESC derivation in the presence of Activin A and its subsequent influence on germ cell differentiation potential in vitro.

Transcription Factor TFAP2C Regulates Major Programs Required for Murine Fetal Germ Cell Maintenance and Haploinsufficiency Predisposes to Teratomas in Male Mice

Maintenance and maturation of primordial germ cells is controlled by complex genetic and epigenetic cascades, and disturbances in this network lead to either infertility or malignant aberration. Transcription factor TFAP2C has been described to be essential for primordial germ cell maintenance and to be upregulated in several human germ cell cancers. Using global gene expression profiling, we identified genes deregulated upon loss of Tfap2c in embryonic stem cells and primordial germ cell-like cells. We show that loss of Tfap2c affects many aspects of the genetic network regulating germ cell biology, such as downregulation of maturation markers and induction of markers indicative for somatic differentiation, cell cycle, epigenetic remodeling and pluripotency. Chromatin-immunoprecipitation analyses demonstrated binding of TFAP2C to regulatory regions of deregulated genes (Sfrp1, Dmrt1, Nanos3, c-Kit, Cdk6, Cdkn1a, Fgf4, Klf4, Dnmt3b and Dnmt3l) suggesting that these genes are direct transcriptional targets of TFAP2C in primordial germ cells. Since Tfap2c deficient primordial germ cell-like cells display cancer related deregulations in epigenetic remodeling, cell cycle and pluripotency control, the Tfap2c-knockout allele was bred onto 129S2/Sv genetic background. There, mice heterozygous for Tfap2c develop with high incidence germ cell cancer resembling human pediatric germ cell tumors. Precursor lesions can be observed as early as E16.5 in developing testes displaying persisting expression of pluripotency markers. We further demonstrate that mice with a heterozygous deletion of the TFAP2C target gene Nanos3 are also prone to develop teratomas. These data highlight TFAP2C as a critical and dose-sensitive regulator of germ cell fate.

Induction of mouse germ-cell fate by transcription factors in vitro

The germ-cell lineage ensures the continuity of life through the generation of male and female gametes, which unite to form a totipotent zygote. We have previously demonstrated that, by using cytokines, embryonic stem cells and induced pluripotent stem cells can be induced into epiblast-like cells (EpiLCs) and then into primordial germ cell (PGC)-like cells with the capacity for both spermatogenesis and oogenesis, creating an opportunity for understanding and regulating mammalian germ-cell development in both sexes in vitro. Here we show that, without cytokines, simultaneous overexpression of three transcription factors, Blimp1 (also known as Prdm1), Prdm14 and Tfap2c (also known as AP2γ), directs EpiLCs, but not embryonic stem cells, swiftly and efficiently into a PGC state. Notably, Prdm14 alone, but not Blimp1 or Tfap2c, suffices for the induction of the PGC state in EpiLCs. The transcription-factor-induced PGC state, irrespective of the transcription factors used, reconstitutes key transcriptome and epigenetic reprogramming in PGCs, but bypasses a mesodermal program that accompanies PGC or PGC-like-cell specification by cytokines including bone morphogenetic protein 4. Notably, the transcription-factor-induced PGC-like cells contribute to spermatogenesis and fertile offspring. Our findings provide a new insight into the transcriptional logic for PGC specification, and create a foundation for the transcription-factor-based reconstitution and regulation of mammalian gametogenesis.

Gonadogenesis and expression pattern of the vasa gene in the sea cucumber Apostichopus japonicus during early development

Vasa has been extensively used as a germ-line marker to trace the origin and migration pathway of primordial germ cells (PGCs) in many organisms, but little work has been reported on vasa genes and the origin of PGCs in holothurians. Using in situ hybridization and immunohistochemistry, vasa mRNA and protein of the sea cucumber Apostichopus japonicus (Aj-vasa) was detected in the cytoplasm of the unfertilized egg and was equally distributed in the cytoplasm of early embryos, from the two-cell embryo to the blastula, indicating that Aj-vasa mRNA is maternally supplied. Later, expression of both Aj-vasa mRNA and protein centralizes gradually in newly organized structures from blastula to five-tentacle larva, and then is restricted to PGC-like cells of the original gonad in juveniles with 0.1-cm body length. The structure of the gonad develops further from a simple tubular gonad in 0.5-cm-length juveniles to a branched gonad in 3-cm-length juveniles. Our findings showed that the maternal supply of the vasa gene products in A. japonicus is different from that in sea urchin Strongylocentrotus purpuratus, of echinoderm, and suggested that the specialization of PGCs is an epigenesis mechanism in A. japonicus.