Migration Of Primordial Germ Cells Research Articles

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 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.

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.

Human DND1-RRM2 forms a non-canonical domain-swapped dimer.

RNA Recognition Motifs (RRMs) being the most abundant RNA binding domain in eukaryotes, is a major player in cellular regulation. Human DND1 (dead end protein homolog1) also known as microRNA-mediated repression inhibitor 1 is an RNA binding protein containing two RRMs in tandem. It is essential for migration and viability of primordial germ cells during development, its complete loss causes lethality, and mutations in the gene are associated with perturbed cellular regulation and eventually cancer. Using x-ray crystallography we have determined 2.3 Å structure of the human DND1 RRM2 domain. The structure revealed an interesting non-canonical RRM fold. This is maintained by the formation of a 3D domain-swapped dimer between β1 and β4 strands across protomers. We have delineated the structural basis of the stable domain-swapped dimer formation using the residue level dynamics of protein explored by NMR spectroscopy and MD simulations. Our results indicate that monomer to dimer switch is governed by several determinants such as hinge loop residues, its length, hydrophobicity, concentration of protein, disulphide bond, and so forth. Several variations in the canonical βαββαβ topology have been observed so far. However, this is the first report of domain swapping in the RRM domain, which may increase the binding surface area and allow this protein to exhibit multifaceted role during post-transcriptional regulation. Our structural and dynamics studies substantiate major determinants and molecular basis for domain-swapped dimerization observed in the RRM domain that could allow varied roles for its RRMs.

Histogenesis of intracranial germ cell tumors: primordial germ cell vs. embryonic stem cell.

Intracranial germ cell tumor (iGCT) is a rare disorder and often occurs during childhood and adolescence. iGCTs are frequently localized in pineal region and hypothalamic-neurohypophyseal axis (HNA). In spite of well-established clinical and pathological entity, histogenesis of iGCTs remains unsettled. Current theories of histogenesis of iGCTs include germ cell theory (from primordial germ cells (PGCs) of aberrant migration) and stem cell theory (transformed embryonic stem (ES) cells). In order to comprehend the histogenesis, we revisit the origin, migration, and fate of the human PGCs, and their transformation processes to iGCT. In "germ cell theory," transformation of ectopic PGCs to iGCT is complex and involves multiple transcription factors. Germinoma is derived from ectopic PGCs and is considered a prototype of all GCTs. Non-germinomatous germ cell tumors (NGGCTs) develop from more differentiated counterparts of embryonic and extra-embryonic tissues. However, there is a distinct genomic/epigenomic landscape between germinoma and NGGCT. ES cells transformed from ectopic PGCs through molecular dysregulation or de-differentiation may become the source of iGCT. "Stem cell theory" is transformation of endogenous ES cells or primitive neural stem cell to iGCTs. It supports histological diversity of NGGCTs because of ES cell's pluripotency. However, neural stem cells are abundantly present along the subependymal zone; therefore, it does not explain why iGCTs almost exclusively occur in pineal and HNA locations. Also, the vast difference of methylation status between germinoma and NGGCT makes it difficult to theorize all iGCTs derive from the common cellular linage. Transformation of PGCs to ES cells is the most logical mechanism for histogenesis of iGCT. However, its detail remains an enigma and needs further investigations.

Conditions for transplantation of primordial germ cells in the yellowtail tetra, Astyanax altiparanae.

Biotechniques, including surrogate propagation derived from primordial germ cell (PGC) transplantation, are valuable tools for the reconstitution of endangered fish species. Although promising, there are no previous studies reporting such approaches using neotropical fish species. The aim of this study was to establish germline chimeras in neotropical fish by using the yellowtail tetra Astyanax altiparanae as a model species of the order Characiformes. Germline chimeras were obtained after transplantation of PGCs cultivated under different conditions: saline medium and supplemented with DMEM, amino acids, vitamins, glutamine, pyruvate, and fetal bovine serum, and subsequently transplanted into A. altiparanae triploids and triploid hybrids from the cross between A. altiparanae (♀) and A. fasciatus (♂). The results indicate ectopic migration in host embryos after transplantation of PGCs cultivated in saline medium. However, PGCs cultivated in supplemented medium migrated to the region of the gonadal ridge in 4.5% of triploid and 19.3% in triploid hybrid. In addition, the higher expression of dnd1, ddx4 and dazl genes was found in PGCs cultivated in supplemented culture medium. This indicates that the culture medium influences the maintenance and development of the cultivated cells. The expression levels of nanos and cxcr4b (related to the differentiation and migration of PGCs) were decreased in PGCs from the supplemented culture medium, supporting the results of ectopic migration. This is the first study to report the transplantation of PGCs to obtain germline chimera in neotropical species. The establishment of micromanipulation procedures in a model neotropical species will open new insights for the conservation of endangered species.

The anticancer effects of Metformin in the male germ tumor SEM-1 cell line are mediated by HMGA1.

Germ cell tumors (GCTs) are the most common type of cancer in young men. These tumors usually originate from the testis, but they can occasionally develop from extragonadal sites probably due to primordial germ cells (PGCs) migration errors. Cisplatin-based chemotherapy is usually effective for male GCTs, but the risk of toxicity is high and new therapeutic strategies are needed. Although Metformin (Met) has been widely studied as a potential cancer treatment over the past decades, there is limited evidence to support its use in treating male GCTs. Additionally, the mechanism by which it acts on tumor cells is still not entirely understood. SEM-1 cells, a newly established human cell line of extragonadal origin, were treated with Met. Cell viability was studied by MTT assay, while cell migration and invasion were studied by the wound healing assay and the transwell assay, respectively. The effect of Met on 3D spheroid formation was determined by seeding SEM-1 cells in appropriate cell suspension culture conditions, and cell cycle was characterized by flow cytometry. Factors involved in PGCs migration and GCT invasion, such as IGFBP1, IGF1R, MMP-11 and c-Kit, together with cyclin D1 (a key regulator of cell cycle progression), and the upstream factor, HMGA1, were determined by immunoblots. Treatment of SEM-1 cells with Met resulted in a potent and dose-dependent reduction of cell proliferation, as evidenced by decreased nuclear abundance of cyclin D1 and cell cycle arrest in G1 phase. Also, Met prevented the formation of 3D spheroids, and blocked cell migration and invasion by reducing the expression of IGFBP1, IGF1R and MMP-11. Both, IGFBP1 and MMP-11 are under control of HMGA1, a chromatin-associated protein that is involved in the regulation of important oncogenic, metabolic and embryological processes. Intriguingly, an early reduction in the nuclear abundance of HMGA1 occurred in SEM-1 cells treated with Met. Our results document the antiproliferative and antimigratory effects of Met in SEM-1 cells, providing new insights into the potential treatments for male GCTs. The anticancer properties of Met in SEM-1 cells are likely related to its ability to interfere with HMGA1 and downstream targets, including cyclin D1, the IGFs system, and MMP-11.

Tetraspanin Cd9b plays a role in fertility in zebrafish.

In mice, CD9 expression on the egg is required for efficient sperm-egg fusion and no effects on ovulation or male fertility are observed in CD9 null animals. Here we show that cd9b knockout zebrafish also appear to have fertility defects. In contrast to mice, fewer eggs were laid by cd9b knockout zebrafish pairs and, of the eggs laid, a lower percentage were fertilised. These effects could not be linked to primordial germ cell numbers or migration as these were not altered in the cd9b mutants. The decrease in egg numbers could be rescued by exchanging either cd9b knockout partner, male or female, for a wildtype partner. However, the fertilisation defect was only rescued by crossing a cd9b knockout female with a wildtype male. To exclude effects of mating behaviour we analysed clutch size and fertilisation using in vitro fertilisation techniques. Number of eggs and fertilisation rates were significantly reduced in the cd9b mutants suggesting the fertility defects are not solely due to courtship behaviours. Our results indicate that CD9 plays a more complex role in fish fertility than in mammals, with effects in both males and females.