Migration Of Primordial Germ Cells Research Articles

Research progress of nanog gene in fish.

Nanog is a crucial regulatory factor in maintaining the self-renewal and pluripotency of embryonic stem cells. It is involved in various biological processes, such as early embryonic development, cell reprogramming, cell cycle regulation, the proliferation and migration of primordial germ cells. While research on this gene has primarily focused on mammals, there has been a growing interest in studying nanog in fish. However, there is a notable lack of comprehensive reviews regarding this gene in fish, which is essential for guiding future research. This review aims to provide a thorough summary of the gene's structure, expression patterns, functions and regulatory mechanisms in fish. The findings suggest that nanog probably has both conserved and divergent functions in regulating cell pluripotency, early embryonic development, and germ cell development in teleosts compared to other species, including mammals. These insights lay the foundation for future research and applications of the nanog gene, providing a new perspective for understanding the evolution and conserved charactristics of teleost nanog.

Comprehensive profiling of migratory primordial germ cells reveals niche-specific differences in non-canonical Wnt and Nodal-Lefty signaling in anterior vs posterior migrants.

Mammalian primordial germ cells (PGCs) migrate asynchronously through the embryonic hindgut and dorsal mesentery to reach the gonads. We previously found that interaction with different somatic niches regulates PGC proliferation along the migration route. To characterize transcriptional heterogeneity of migrating PGCs and their niches, we performed single-cell RNA sequencing of 13,262 mouse PGCs and 7,868 surrounding somatic cells during migration (E9.5, E10.5, E11.5) and in anterior versus posterior locations to enrich for leading and lagging migrants. Analysis of PGCs by position revealed dynamic gene expression changes between faster or earlier migrants in the anterior and slower or later migrants in the posterior at E9.5; these differences include migration-associated actin polymerization machinery and epigenetic reprogramming-associated genes. We furthermore identified changes in signaling with various somatic niches, notably strengthened interactions with hindgut epithelium via non-canonical WNT (ncWNT) in posterior PGCs compared to anterior. Reanalysis of a previously published dataset suggests that ncWNT signaling from the hindgut epithelium to early migratory PGCs is conserved in humans. Trajectory inference methods identified putative differentiation trajectories linking cell states across timepoints and from posterior to anterior in our mouse dataset. At E9.5, we mainly observed differences in cell adhesion and actin cytoskeletal dynamics between E9.5 posterior and anterior migrants. At E10.5, we observed divergent gene expression patterns between putative differentiation trajectories from posterior to anterior including Nodal signaling response genes Lefty1, Lefty2, and Pycr2 and reprogramming factors Dnmt1, Prc1, and Tet1. At E10.5, we experimentally validated anterior migrant-specific Lefty1/2 upregulation via whole-mount immunofluorescence staining for LEFTY1/2 proteins, suggesting that elevated autocrine Nodal signaling accompanies the late stages of PGC migration. Together, this positional and temporal atlas of mouse PGCs supports the idea that niche interactions along the migratory route elicit changes in proliferation, actin dynamics, pluripotency, and epigenetic reprogramming.

Maternal dnd1 is essential for migration and maintenance of PGCs in Nile tilapia at larval stage

As a germ-cell-specific maternal mRNA and RNA binding protein, dead end (dnd1) plays an important role in migration and maintenance of primordial germ cells (PGCs), and therefore, disruption of dnd1 results in no germ cells. However, the effect and duration of maternal dnd1 on PGCs in zygote mutants have not been reported yet. In the present study, CRISPR/Cas9 was used to mutate dnd1 in tilapia. Supported by the maternal dnd1 mRNA, germ cells of the dnd1 homozygous mutants proliferated and migrated normally before 7 dpf (days post fertilization). After the degradation of maternal dnd1 mRNA, significant downregulation of piwil1, piwil2, nanos2, nanos3 and vasa mRNA, and blocked migration of germ cells were observed at 14 dpf. In dnd1 zygote mutants, the number of germ cells was decreased significantly compared with wild type (WT) at 25 dpf, and no germ cell was observed at 30 dpf. Consistently, no expression of vasa and nanos3 was detected in the adult dnd1 mutants by qPCR. Interestingly, Cyp19a1a/cyp19a1a and foxl2 were expressed in dnd1-deficient XX gonads, while Cyp11c1/cyp11c1 and dmrt1 were expressed in dnd1-deficient XY gonads as in WT gonads. Correspondingly, both serum E2 level in XX and 11-KT level in XY fish did not differ significantly between dnd1 mutant and WT fish. Taken together, the present study demonstrates that disruption of dnd1 lead to progressive loss of germ cells, while the somatic cells are not affected in tilapia. The sexual fate of gonads is determined by the genetic sex instead of germ cell number, which makes tilapia a potential surrogate model for germ cell transplantation.

Adamts9 is required for the development of primary ovarian follicles and maintenance of female sex in zebrafish†.

Previous studies have suggested that adamts9 (a disintegrin and metalloprotease with thrombospondin type-1 motifs, member 9), an extracellular matrix (ECM) metalloprotease, participates in primordial germ cell (PGC) migration and is necessary for female fertility. In this study, we found that adamts9 knockout (KO) led to reduced body size, and female-to-male sex conversion in late juvenile or adult zebrafish; however, primary sex determination was not affected in early juveniles of adamts9 KO. Overfeeding and lowering the rearing density rescued growth defects in female adamts9 KO fish but did not rescue defects in ovarian development in adamts9 KO. Delayed PGC proliferation, significantly reduced number and size of Stage IB follicles (equivalent to primary follicles) in early juveniles of adamts9 KO, and arrested development at Stage IB follicles in mid- or late-juveniles of adamts9 KO are likely causes of female infertility and sex conversion. Via RNAseq, we found significant enrichment of differentially expressed genes involved in ECM organization during sexual maturation in ovaries of wildtype fish; and significant dysregulation of these genes in adamts9 KO ovaries. RNAseq analysis also showed enrichment of inflammatory transcriptomic signatures in adult ovaries of these adamts9 KO. Taken together, our results indicate that adamts9 is critical for development of primary ovarian follicles and maintenance of female sex, and loss of adamts9 leads to defects in ovarian follicle development, female infertility, and sex conversion in late juveniles and mature adults. These results show that the ECM and extracellular metalloproteases play major roles in maintaining ovarian follicle development in zebrafish.

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.

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.

The journey of a generation: advances and promises in the study of primordial germ cell migration.

The germline provides the genetic and non-genetic information that passes from one generation to the next. Given this important role in species propagation, egg and sperm precursors, called primordial germ cells (PGCs), are one of the first cell types specified during embryogenesis. In fact, PGCs form well before the bipotential somatic gonad is specified. This common feature of germline development necessitates that PGCs migrate through many tissues to reach the somatic gonad. During their journey, PGCs must respond to select environmental cues while ignoring others in a dynamically developing embryo. The complex multi-tissue, combinatorial nature of PGC migration is an excellent model for understanding how cells navigate complex environments in vivo. Here, we discuss recent findings on the migratory path, the somatic cells that shepherd PGCs, the guidance cues somatic cells provide, and the PGC response to these cues to reach the gonad and establish the germline pool for future generations. We end by discussing the fate of wayward PGCs that fail to reach the gonad in diverse species. Collectively, this field is poised to yield important insights into emerging reproductive technologies.

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.

Abstract 2022 Elucidating the Role of Phospholipase C in Primordial Germ Cell Migration

Abstract 2022 Elucidating the Role of Phospholipase C in Primordial Germ Cell Migration

SDF-1/CXCR4 signal is involved in the induction of Primordial Germ Cell migration in a model marine fish, Japanese anchovy (Engraulis japonicus)

Primordial germ cells (PGCs) are pivotal for gonadal development and reproductive success. Though artificial induction of sterility by targeting PGCs are gaining popularity due to its advantages in fish surrogacy and biodiversity management, it is often skill and time intensive. In this study, we have focused on understanding the role of PGCs and the chemotactic SDF-1/CXCR4 signaling on gonad development of Japanese anchovy (JA, Engraulis japonicus), an upcoming marine model organism with eco-commercial values, with an aim to develop a novel, easy, and versatile gonad sterilization method. Our data showed that PGC migration related genes, i.e., sdf-1a, sdf-1b, cxcr4a, cxcr4b and vasa, are phylogenetically closer relatives of respective herring (Clupea harengus) and zebrafish (Danio rerio) homolog. Subsequently, PGC marking and live tracing experiments confirmed that PGC migration in JA initiates from 16 hours post fertilization (hpf) followed by PGC settlement in the gonadal ridge at 44 hpf. We found that overexpression of zebrafish sdf-1a mRNA in the germ cell suppresses cxcr4a and increases cxcr4b transcription at 8 hpf, dose dependently disrupts PGC migration at 24–48 hpf, induces PGC death and upregulates sdf-1b at 5 days post hatching. 48 h of immersion treatment with CXCR4 antagonist (AMD3100, Abcam) also accelerated PGC mismigration and pushed the PGC away from gonadal ridge in a dose responsive manner, and further when grown to adulthood caused germ cell less gonad formation in some individuals. Cumulatively, our data, for the first time, suggests that JA PGC migration is largely regulated by SDF1/CXCR4 signaling, and modulation of this signaling has strong potential for sterile, germ cell less gonad preparation at a mass scale. However, further in-depth analysis is pertinent to apply this methodology in marine fish species to successfully catapult Japanese anchovy into a true marine fish model.

Modeling the SDF-1/CXCR4 protein using advanced artificial intelligence and antagonist screening for Japanese anchovy.

SDF-1/CXCR4 chemokine signaling are indispensable for cell migration, especially the Primordial Germ Cell (PGC) migration towards the gonadal ridge during early development. We earlier found that this signaling is largely conserved in the Japanese anchovy (Engraulis japonicus, EJ), and a mere treatment of CXCR4 antagonist, AMD3100, leads to germ cell depletion and thereafter gonad sterilization. However, the effect of AMD3100 was limited. So, in this research, we scouted for CXCR4 antagonist with higher potency by employing advanced artificial intelligence deep learning-based computer simulations. Three potential candidates, AMD3465, WZ811, and LY2510924, were selected and in vivo validation was conducted using Japanese anchovy embryos. We found that seven transmembrane motif of EJ CXCR4a and EJ CXCR4b were extremely similar with human homolog while the CXCR4 chemokine receptor N terminal (PF12109, essential for SDF-1 binding) was missing in EJ CXCR4b. 3D protein analysis and cavity search predicted the cavity in EJ CXCR4a to be five times larger (6,307ų) than that in EJ CXCR4b (1,241ų). Docking analysis demonstrated lower binding energy of AMD3100 and AMD3465 to EJ CXCR4a (Vina score -9.6) and EJ CXCR4b (Vina score -8.8), respectively. Furthermore, we observed significant PGC mismigration in microinjected AMD3465 treated groups at 10, 100 and 1 × 105nM concentration in 48h post fertilized embryos. The other three antagonists showed various degrees of PGC dispersion, but no significant effect compared to their solvent control at tested concentrations was observed. Cumulatively, our results suggests that AMD3645 might be a better candidate for abnormal PGC migration in Japanese anchovy and warrants further investigation.

Germ cell migration: Unexpected role of juvenile hormone before juvenile stages

Juvenile hormone is best known for its role in maintaining juvenile-stage insects in their immature states during postembryonic development. A new study finds an unexpected role for this signaling lipid in guiding primordial germ cell migration during embryogenesis- possibly an ancestral function of isoprenoid signaling molecules.

Juvenile hormones direct primordial germ cell migration to the embryonic gonad

Germ cells are essential to sexual reproduction. Across the animal kingdom, extracellular signaling isoprenoids, such as retinoic acids (RAs) in vertebrates and juvenile hormones (JHs) in invertebrates, facilitate multiple processes in reproduction. Here we investigated the role of these potent signaling molecules in embryonic germ cell development, using JHs in Drosophila melanogaster as a model system. In contrast to their established endocrine roles during larval and adult germline development, we found that JH signaling acts locally during embryonic development. Using an invivo biosensor, we observed active JH signaling first within and near primordial germ cells (PGCs) as they migrate to the developing gonad. Through invivo and invitro assays, we determined that JHs are both necessary and sufficient for PGC migration. Analysis into the mechanisms of this newly uncovered paracrine JH function revealed that PGC migration was compromised when JHs were decreased or increased, suggesting that specific titers or spatiotemporal JH dynamics are required for robust PGC colonization of the gonad. Compromised PGC migration can impair fertility and cause germ cell tumors in many species, including humans. In mammals, retinoids have many roles in development and reproduction. We found that like JHs in Drosophila, RA was sufficient to impact mouse PGC migration invitro. Together, our study reveals a previously unanticipated role of isoprenoids as local effectors of pre-gonadal PGC development and suggests a broadly shared mechanism in PGC migration.

Spermatogonial depletion and a spermatogenesis defect in the Dp(16)1Yey mouse model of Down syndrome

In brief Little is known about the mechanism responsible for male infertility in Down syndrome. Duplication of Mmu16 chromosome (an orthologue of Hsa21, bearing Dyrk1A) in Dp(16)1Yey mice is responsible for impaired spermatogenesis through spermatogonial depletion and hypogonadotropic hypogonadism. Abstract Down syndrome (DS), or trisomy 21, is the most common chromosomal disorder in humans. Men with DS are infertile. The DYRK1A gene on Hsa21 is involved in several features of DS. Overexpression of the homolog dyrk1A disrupts primordial germ cell migration in zebrafish, and overexpression of Dyrk1A impairs gonadotropic axis function and the early stages of spermatogenesis in the mouse. Other genes on Hsa21 might be involved in the pathogenesis of infertility in DS. We investigated the Dp(16)1Yey mouse model of DS, which features segmental duplication of chromosome Mmu16 (orthologous to a large part of Hsa21 and carrying Dyrk1A and 112 other genes). Using an immunohistochemical assay for the spermatogonial marker STRA8, we observed spermatogonial depletion in the Dp(16)1Yey mouse. This was correlated with low mRNA expression of GFRα1 (a marker of the self-renewal stem cell pool) in an RT-qPCR assay and low protein expression of PLZF (a marker of differentiating stem cells) in a slot-blot assay. Spermatogenesis was present but impaired, with a low sperm count, low protamine-1 expression, a low testis weight, and a low seminiferous tubule diameter. Low circulating luteinizing hormone and follicle-stimulating hormone levels and an elevated testis anti-Müllerian hormone level (as measured in ELISAs) revealed the presence of hypogonadotropic hypogonadism. The Dp(16)1Yey mouse model of DS recapitulates observations made in zebrafish and mice overexpressing DYRK1A homologs. The presence of an excess of Mmu16 material perturbs spermatogenesis and the gonadotropic axis. More generally, DYRK1A’s role in human infertility (outside DS) remains to be characterized.

Traceability of primordial germ cells in three neotropical fish species aiming genetic conservation actions.

Primordial germ cells (PGCs) are embryonic pluripotent cells that can differentiate into spermatogonia and oogonia, and therefore, PGCs are a genetic source for germplasm conservation through cryobanking and the generation of germline chimeras. The knowledge of PGC migration routes is essential for transplantation studies. In this work, the mRNA synthesized from the ddx4 3'UTR sequence of Pseudopimelodus mangurus, in fusion with gfp or dsred, was microinjected into zygotes of three neotropical species (P. mangurus, Astyanax altiparanae, and Prochilodus lineatus) for PGC labeling. Visualization of labeled PGCs was achieved by fluorescence microscopy during embryonic development. In addition, ddx4 and dnd1 expressions were evaluated during embryonic development, larvae, and adult tissues of P. mangurus, to validate their use as a PGC marker. As a result, the effective identification of presumptive PGCs was obtained. DsRed-positive PGC of P. mangurus was observed in the hatching stage, GFP-positive PGC of A. altiparanae in the gastrula stage, and GFP-positive PGCs from P. lineatus were identified at the segmentation stage, with representative labeling percentages of 29% and 16% in A. altiparanae and P. lineatus, respectively. The expression of ddx4 and dnd1 of P. mangurus confirmed the specificity of these genes in germ cells. These results point to the functionality of the P. mangurus ddx4 3'UTR sequence as a PGC marker, demonstrating that PGC labeling was more efficient in A. altiparanae and P. lineatus. The procedures used to identify PGCs in P. mangurus consolidate the first step for generating germinal chimeras as a conservation action of P. mangurus.

The Distribution of the Germ Plasm Gene <i>germes</i> among Anurans

The germes gene is a marker of germ plasm and primordial germ cells (PGC) described in the African clawed frog Xenopus laevis. It is known that overexpression of its mutant form negatively affects the formation and migration of PGC. However, until now it was not known how widely this gene is represented in animals of different phylogenetic groups. In this work, we performed bioinformatic analysis of genomic and transcriptome sequences of animals with germ plasm. It turned out that germes homologs are present only in representatives of the genera Xenopus and Hymenochirus of the family Pipidae (order Anura). The obtained results were confirmed by RT-PCR analysis of the expression of germes orthologs in the ovaries of six representatives of different Anura families. Phylogenetic analysis of cloned sequences of germes homologs suggests the appearance of this gene in the ancestors of Pipidae and its secondary loss in the genus Pseudohymenochirus. It is also identified that the amino acid sequences of the functional domains of the Germes protein are rather conservative.

The mitochondrial protein Sod2 is important for the migration, maintenance, and fitness of germ cells.

To maintain a range of cellular functions and to ensure cell survival, cells must control their levels of reactive oxygen species (ROS). The main source of these molecules is the mitochondrial respiration machinery, and the first line of defense against these toxic substances is the mitochondrial enzyme superoxide dismutase 2 (Sod2). Thus, investigating early expression patterns and functions of this protein is critical for understanding how an organism develops ways to protect itself against ROS and enhance tissue fitness. Here, we report on expression pattern and function of zebrafish Sod2, focusing on the role of the protein in migration and maintenance of primordial germ cells during early embryonic development. We provide evidence that Sod2 is involved in purifying selection of vertebrate germ cells, which can contribute to the fitness of the organism in the following generations.

Hedgehog signaling guides migration of primordial germ cells to the Drosophila somatic gonad.

In addition to inducing nonautonomous specification of cell fate in both Drosophila and vertebrates, the Hedgehog pathway guides cell migration in a variety of different tissues. Although its role in axon guidance in the vertebrate nervous system is widely recognized, its role in guiding the migratory path of primordial germ cells (PGCs) from the outside surface of the Drosophila embryo through the midgut and mesoderm to the SGPs (somatic gonadal precursors) has been controversial. Here we present new experiments demonstrating (1) that Hh produced by mesodermal cells guides PGC migration, (2) that HMG CoenzymeA reductase (Hmgcr) potentiates guidance signals emanating from the SGPs, functioning upstream of hh and of 2 Hh pathway genes important for Hh-containing cytonemes, and (3) that factors required in Hh receiving cells in other contexts function in PGCs to help direct migration toward the SGPs. We also compare the data reported by 4 different laboratories that have studied the role of the Hh pathway in guiding PGC migration.

Dynamic and broad expression of adamts9 in developing and adult zebrafish.

Previous studies showed that Adamts9 is involved in multiple functions including ovulation, spine formation, primordial germ cell migration, and development of primary ovarian follicles in animals. However, systemic examination and high-resolution analyses of adamts9 expression are missing due to lack of a sensitive reporter assay. In the present study, we created a new transgenic zebrafish reporter line Tg(adamts9:EGFP) and assayed its expression in various tissues and cells during development and in adults at high-resolution using confocal imaging. Reporter expression was validated with real-time quantitative PCR, whole mount in situ hybridization, and immunohistochemistry for endogenous adamts9. Strong expression of the adamts9:EGFP transgene was found in a wide range of adult and embryonic zebrafish tissues/cells including ovaries, testes, brains, eyes, pectoral fins, intestine, skin, gill, muscle, and heart; while lower expression was observed in the liver and growing ovarian follicles (stages II and III). Our results of a broad and dynamic expression pattern for this evolutionary conserved metalloprotease suggest involvement of adamts9 in the development and physiological functions of various tissues in animals.

Histological demonstration of gonad development in the banana shrimp, Fenneropenaeus merguiensis

The genetic diversity of the banana shrimp Fenneropenaeus merguiensis is at risk because of over-harvesting, which consequently reduces food security. This endangerment is exacerbated because this species is not commonly cultivated by farmers. Overall, these factors necessitate conservation of this shrimp species across its natural habitat. Information on the migration of primordial germ cells (PGCs) to form a gonad is essential for shrimp preservation techniques such as broodstock preparation, sex differentiation, and germ cell transplantation. In this study, histological analysis and in situ hybridization of vasa expression (from embryo to testis development) were used to demonstrate the movement of PGCs. Hematoxylin and Eosin staining and in situ hybridization with the VASA probe revealed that the PGCs migrated retrogradely along the midgut, colonizing the area between the hepatopancreas and heart, a region that becomes the genital ridge in the postlarval stage. External sexual organs appeared at approximately 4 months of age. Through real-time PCR, the expression of the vasa gene was detected early on postlarval day 7, whereas its abundant expression was detected in the ovaries and testes of adult shrimp. This study could help with the identification and monitoring of PGCs or spermatogonia in banana shrimp and facilitate the implementation of other germ cell-relevant techniques in the future.