Fetal Germ Cell Development Research Articles

How the extra X chromosome impairs the development of male fetal germ cells.

The dosage of X-linked genes is accurately regulated with the development of fetal germ cells (FGCs)1,2. How aberrant dosage of X-linked genes impairs FGC development in humans remains poorly understood. FGCs of patients with Klinefelter syndrome (KS), who have an extra X chromosome, provide natural models for addressing this issue3. Here we demonstrate that most human FGCs in KS are arrested at an early stage, characterized by the upregulation of genes related to pluripotency, the WNT pathway and the TGF-β pathway, along with the downregulation of genes involved in FGC differentiation. The limited KS FGCs that are capable of reaching the late stage remain relatively naive. X chromosomes are not inactivated and the dosage of X-linked genes is excessive in KS FGCs. X-linked genes dominate the differentially expressed genes and are enriched in critical biological processes associated with the developmental delay of KS FGCs. Moreover, aberrant interactions between Sertoli cells and FGCs disrupt the migration of late FGCs to the basement membrane in KS. Notably, inhibition of the TGF-β pathway improves the differentiation of KS FGCs. Our findings elucidate how the extra X chromosome impairs the development of male FGCs and reveal the initial molecular events preceding germ cell loss in KS.

Control of epigenomic landscape and development of fetal male germ cells through L-serine metabolism

Sex-specific metabolic characteristics emerge in the mouse germ line after reaching the genital ridges around embryonic day 10.5, coinciding with sexual differentiation. However, the impact of such metabolic characteristics on germ cell development remains unclear. In this study, we observed the specific upregulation in male fetal germ cells of D-3-phosphoglycerate dehydrogenase (PHGDH), the primary enzyme in the serine-glycine-one-carbon metabolism, along with an increase in a downstream metabolite, S-adenosylmethionine (SAM), crucial for protein and nucleic acid methylation. Inhibiting PHGDH in fetal testes resulted in reduced SAM levels in germ cells, accompanied by increases in the number of mouse vasa homolog (MVH/VASA)-positive germ cells and the promyelocytic leukemia zinc finger (PLZF)-positive undifferentiated spermatogonia ratio. Furthermore, PHGDH inhibition led to a decrease in the methylation of histone H3 and DNA, resulting in aberrations in gene expression profiles. In summary, our findings underscore the significant role of certain metabolic mechanisms in the development of male germ cells.

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.

The Impact of Activin A on Fetal Gonocytes: Chronic Versus Acute Exposure Outcomes.

Activin A, a TGFβ superfamily member, is important for normal testis development through its actions on Sertoli cell development. Our analyses of altered activin A mouse models indicated gonocyte abnormalities, implicating activin A as a key determinant of early germline formation. Whether it acts directly or indirectly on germ cells is not understood. In humans, the fetal testis may be exposed to abnormally elevated activin A levels during preeclampsia, maternal infections, or following ingestion of certain medications. We hypothesized that this may impact fetal testis development and ultimately affect adult fertility. Germ cells from two mouse models of altered activin bioactivity were analysed. RNA-Seq of gonocytes purified from E13.5 and E15.5 Inhba KO mice (activin A subunit knockout) identified 46 and 44 differentially expressed genes (DEGs) respectively, and 45 in the E13.5 Inha KO (inhibin alpha subunit knockout; increased activin A) gonocytes. To discern direct effects of altered activin bioactivity on germline transcripts, isolated E13.5 gonocytes were cultured for 24h with activin A or with the activin/Nodal/TGFβ inhibitor, SB431542. Gonocytes responded directly to altered signalling, with activin A promoting a more differentiated transcript profile (increased differentiation markers Dnmt3l, Nanos2 and Piwil4; decreased early germ cell markers Kit and Tdgf1), while SB431542 had a reciprocal effect (decreased Nanos2 and Piwil4; increased Kit). To delineate direct and indirect effects of activin A exposure on gonocytes, whole testes were cultured 48h with activin A or SB431542 and collected for histological and transcript analyses, or EdU added at the end of culture to measure germ and Sertoli cell proliferation using flow cytometry. Activin increased, and SB431542 decreased, Sertoli cell proliferation. SB431542-exposure resulted in germ cells escaping mitotic arrest. Analysis of FACS-isolated gonocytes following whole testis culture showed SB431542 increased the early germ cell marker Kit, however there was a general reduction in the impact of altered activin A bioavailability in the normal somatic cell environment. This multifaceted approach identifies a capacity for activin A to directly influence fetal germ cell development, highlighting the potential for altered activin A levels in utero to increase the risk of testicular pathologies that arise from impaired germline maturation.

Eicosanoid Biosynthesis in Male Reproductive Development: Effects of Perinatal Exposure to NSAIDs and Analgesic Drugs.

Increasing rates of infertility associated with declining sperm counts and quality, as well as increasing rates of testicular cancer are contemporary issues in the United States and abroad. These conditions are part of the Testicular Dysgenesis Syndrome, which includes a variety of male reproductive disorders hypothesized to share a common origin based on disrupted testicular development during fetal and neonatal stages of life. Male reproductive development is a highly regulated and complex process that relies on an intricate coordination between germ, Leydig, and Sertoli cells as well as other supporting cell types, to ensure proper spermatogenesis, testicular immune privilege, and endocrine function. The eicosanoid system has been reported to be involved in the regulation of fetal and neonatal germ cell development as well as overall testicular homeostasis. Moreover, non-steroidal anti-inflammatory drugs (NSAIDs) and analgesics with abilities to block eicosanoid synthesis by targeting either or both isoforms of cyclooxygenase enzymes, have been found to adversely affect male reproductive development. This review will explore the current body of knowledge on the involvement of the eicosanoid system in male reproductive development, as well as discuss adverse effects of NSAIDs and analgesic drugs administered perinatally, focusing on toxicities reported in the testis and on major testicular cell types. Rodent and epidemiological studies will be corroborated by findings in invertebrate models for a comprehensive report of the state of the field, and to add to our understanding of the potential long-term effects of NSAID and analgesic drug administration in infants.

AZFa candidate gene UTY and its X homologue UTX are expressed in human germ cells.

SummaryThe Ubiquitous Transcribed Y (UTY a.k.a. KDM6C) AZFa candidate gene on the human Y chromosome and its paralog on the X chromosome, UTX (a.k.a. KDM6A), encode a histone lysine demethylase removing chromatin H3K27 methylation marks at genes transcriptional start sites for activation. Both proteins harbour the conserved Jumonji C (JmjC) domain, functional in chromatin metabolism, and an extended N-terminal tetratricopeptide repeat (TPR) block involved in specific protein interactions. Specific antisera for human UTY and UTX proteins were developed to distinguish the expression of both proteins in human germ cells by immunohistochemical experiments on appropriate tissue sections. In the male germ line, UTY was expressed in the fraction of A spermatogonia located at the basal membrane, probably including spermatogonia stem cells. UTX expression was more spread in all spermatogonia and in early spermatids. In female germ line, UTX expression was found in the primordial germ cells of the ovary. UTY was also expressed during fetal male germ cell development, whereas UTX expression was visible only at distinct gestation weeks. Based on these results and the conserved neighboured location of UTY and DDX3Y in Yq11 found in mammals of distinct lineages, we conclude that UTY, such as DDX3Y, is part of the Azoospermia factor a (AZFa) locus functioning in human spermatogonia to support the balance of their proliferation-differentiation rate before meiosis. Comparable UTY and DDX3Y expression was also found in gonadoblastoma and dysgerminoma cells found in germ cell nests of the dysgenetic gonads of individuals with disorders of sexual development and a Y chromosome in karyotype (DSD-XY). This confirms that AZFa overlaps with GBY, the Gonadoblastoma susceptibility Y locus, and includes the UTY gene.Lay summaryAZFa Y genes are involved in human male germ cells development and support gonadoblastoma (germ cell tumour precursor cells) in the aberrant germ cells of the gonads of females with genetic disorders of sexual development. The AZFa UTY gene on the male Y chromosome is equivalent to UTX on the female X chromosome. These genes are involved in removing gene regulators to enable activation of other genes (i.e. removal of histone methylation known as epigenetic modifications). We wanted to learn the function of UTY and UTX in developing sperm and eggs in human tissues and developed specific antibodies to detect both proteins made by these genes. Both UTY and UTX proteins were detected in adult and fetal sperm precursor cells (spermatogonia). UTX was detected in egg precursor cells (primordial germ cells). UTY was detected in gonadoblastoma and dysgerminoma tumour cells (germ cell tumours originating from genetic disorders of sexual development due to having a Y chromosome). Based on our study, we conclude that UTY is not only part of AZFa, but also of GBY the overlapping gonadoblastoma susceptibility Y region.

Dissecting the epigenomic dynamics of human fetal germ cell development at single-cell resolution

Proper development of fetal germ cells (FGCs) is vital for the precise transmission of genetic and epigenetic information through generations. The transcriptional landscapes of human FGC development have been revealed; however, the epigenetic reprogramming process of FGCs remains elusive. Here, we profiled the genome-wide DNA methylation and chromatin accessibility of human FGCs at different phases as well as gonadal niche cells at single-cell resolution. First, we found that DNA methylation levels of FGCs changed in a temporal manner, whereas FGCs at different phases in the same embryo exhibited comparable DNA methylation levels and patterns. Second, we revealed the phase-specific chromatin accessibility signatures at the promoter regions of a large set of critical transcription factors and signaling pathway genes. We also identified potential distal regulatory elements including enhancers in FGCs. Third, compared with other hominid-specific retrotransposons, SVA_D might have a broad spectrum of binding capacity for transcription factors, including SOX15 and SOX17. Finally, using an in vitro culture system of human FGCs, we showed that the BMP signaling pathway promoted the cell proliferation of FGCs, and regulated the WNT signaling pathway by orchestrating the chromatin accessibility of its ligand genes. Our single-cell epigenomic atlas and functional assays provide valuable insights for understanding the strongly heterogeneous, unsynchronized, yet highly robust nature of human germ cell development.

Recent advances in mammalian reproductive biology.

Reproductive biology is a uniquely important topic since it is about germ cells, which are central for transmitting genetic information from generation to generation. In this review, we discuss recent advances in mammalian germ cell development, including preimplantation development, fetal germ cell development and postnatal development of oocytes and sperm. We also discuss the etiologies of female and male infertility and describe the emerging technologies for studying reproductive biology such as gene editing and single-cell technologies.

Maternal vitamin C regulates reprogramming of DNA methylation and germline development.

Development is often assumed to be hardwired in the genome, but several lines of evidence indicate that it is susceptible to environmental modulation with potential long-term consequences, including in mammals1,2. The embryonic germline is of particular interest because of the potential for intergenerational epigenetic effects. The mammalian germline undergoes extensive DNA demethylation3-7 that occurs in large part by passive dilution of methylation over successive cell divisions, accompanied by active DNA demethylation by TET enzymes3,8-10. TET activity has been shown to be modulated by nutrients and metabolites, such as vitamin C11-15. Here we show that maternal vitamin C is required for proper DNA demethylation and the development of female fetal germ cells in a mouse model. Maternal vitamin C deficiency does not affect overall embryonic development but leads to reduced numbers of germ cells, delayed meiosis and reduced fecundity in adult offspring. The transcriptome of germ cells from vitamin-C-deficient embryos is remarkably similar to that of embryos carrying a null mutation in Tet1. Vitamin C deficiency leads to an aberrant DNA methylation profile that includes incomplete demethylation of key regulators of meiosis and transposable elements. These findings reveal that deficiency in vitamin C during gestation partially recapitulates loss of TET1, and provide a potential intergenerational mechanism for adjusting fecundity to environmental conditions.

Retinoic acid signaling in ovarian folliculogenesis and steroidogenesis

Retinoids are essential for reproduction. Most research has focused on the role of retinoic acid signaling in the regulation of meiosis during early fetal germ cell development. However, less attention has been paid to the possible effects of retinoic acid signaling in adult female gonads. Retinoic acid, its receptors, and the key enzymes required for retinoic acid synthesis are expressed in the ovaries and they are involved in the regulation of folliculogenesis and steroidogenesis. Exposure to compounds that can interfere with normal retinoic acid signaling is associated with adverse ovarian outcomes, including altered steroidogenesis and reduction in indicators of ovarian reserve in women and laboratory animal models. These observations call for more attention to retinoids as regulators of adult ovarian physiology and as possible targets of endocrine disruption by environmental chemicals. In this review, we summarize the current knowledge of retinoids in folliculogenesis and steroidogenesis in post-pubertal mammalian ovaries.

Effect of environmental and pharmaceutical exposures on fetal testis development and function: a systematic review of human experimental data.

BACKGROUNDOverall, the incidence of male reproductive disorders has increased in recent decades. Testicular development during fetal life is crucial for subsequent male reproductive function. Non-genomic factors such as environmental chemicals, pharmaceuticals and lifestyle have been proposed to impact on human fetal testicular development resulting in subsequent effects on male reproductive health. Whilst experimental studies using animal models have provided support for this hypothesis, more recently a number of experimental studies using human tissues and cells have begun to translate these findings to determine direct human relevance.OBJECTIVE AND RATIONALEThe objective of this systematic review was to provide a comprehensive description of the evidence for effects of prenatal exposure(s) on human fetal testis development and function. We present the effects of environmental, pharmaceutical and lifestyle factors in experimental systems involving exposure of human fetal testis tissues and cells. Comparison is made with existing epidemiological data primarily derived from a recent meta-analysis.SEARCH METHODSFor identification of experimental studies, PubMed and EMBASE were searched for articles published in English between 01/01/1966 and 13/07/2018 using search terms including ‘endocrine disruptor’, ‘human’, ‘fetal’, ‘testis’, ‘germ cells’, ‘testosterone’ and related search terms. s were screened for selection of full-text articles for further interrogation. Epidemiological studies involving exposure to the same agents were extracted from a recent systematic review and meta-analysis. Additional studies were identified through screening of bibliographies of full-texts of articles identified through the initial searches.OUTCOMESA total of 25 experimental studies and 44 epidemiological studies were included. Consistent effects of analgesic and phthalate exposure on human fetal germ cell development are demonstrated in experimental models, correlating with evidence from epidemiological studies and animal models. Furthermore, analgesic-induced reduction in fetal testosterone production, which predisposes to the development of male reproductive disorders, has been reported in studies involving human tissues, which also supports data from animal and epidemiological studies. However, whilst reduced testosterone production has been demonstrated in animal studies following exposure(s) to a variety of environmental chemicals including phthalates and bisphenol A, these effects are not reproduced in experimental approaches using human fetal testis tissues.WIDER IMPLICATIONSDirect experimental evidence for effects of prenatal exposure(s) on human fetal testis development and function exists. However, for many exposures the data is limited. The increasing use of human-relevant models systems in which to determine the effects of environmental exposure(s) (including mixed exposures) on development and function of human tissues should form an important part of the process for assessment of such exposures by regulatory bodies to take account of animal–human differences in susceptibility.

Gestational nutrition 1: alterations to gestational nutrition can increase indicators of fertility in sheep.

The aim of this study was to examine the relationships between gestational nutrition, fetal ovarian development and offspring fertility in female sheep and to highlight the potential mechanisms underlying these relationships. Adult sheep (n = 79) were fed either a maintenance or 0.6 of maintenance plane of nutrition for the first 55 days of gestation and thereafter fed ad libitum. Fetuses were collected for analysis at days 55 and 75 of gestation. Female offspring were monitored from birth until 19 months of age. Effects of restricted nutrition were observed on maternal plasma concentrations of progesterone, creatinine, albumin and Ca2+ at day 55 and creatinine at day 75. Concentrations of metabolic factors and steroid hormones in day 75 fetal plasma were not affected by the restricted maternal plane of nutrition. At day 55 of gestation, fetal ovarian germ cell development was not affected by maternal plane of nutrition. At day 75 of gestation ovaries from fetuses whose dams were exposed to restricted nutrition contained more germ cells but had lower germ cell proliferation rates than controls. For female offspring at 8 months of age, the dams gestational plane of nutrition did not affect the onset of puberty, ovulation rate (OR) and antral follicle counts (AFC). At 19 months of age, ewes from dams exposed to the restricted plane of gestational nutrition had higher OR, AFC and progesterone concentrations while concentrations of FSH were lower. In conclusion, while effects on fertility per se are yet to be determined, a reduced maternal plane of gestational nutrition can improve indicators of fertility in female offspring.

Uncomfortable Uncertainty: Do OTC Analgesics Disrupt Fetal Germ Cell Development?

Uncomfortable Uncertainty: Do OTC Analgesics Disrupt Fetal Germ Cell Development?

Apoptosis Is a Demanding Selective Tool During the Development of Fetal Male Germ Cells.

Apoptosis is widely known to play a major role on diseases related to male infertility. Diseases of the male genital tract as defective spermatogenesis, decreased sperm motility, sperm DNA fragmentation, testicular torsion, varicocele and immunological infertility are strongly related to apoptotic cell death. Apoptosis must not be considered only as a fail on germ cell physiology or a secondary effect of certain pathologies and exogenous hazardous agents. Apoptosis orchestrates correct function and development of the male germ cell from the early embryonic stages of gonadal differentiation to the fertilization. In this review we have tried to address a reading frame of the main knowledge about apoptosis in male germ cell development. Focussing on mechanisms concerning cellular apoptosis, which are independent of exogenous stimuli, we aimed to highlight that apoptosis is a selective instrument that guarantees the delivery of genetic message to offspring.

Loss of Glis3 causes dysregulation of retrotransposon silencing and germ cell demise in fetal mouse testis

Fetal germ cell development is regulated by an elaborate combination of cell-extrinsic and cell-intrinsic signals. Here we identify a novel role for the Krüppel-like transcription factor Gli-Similar 3 (Glis3) in male germ cell development in the mouse embryos. Glis3 is expressed in male germ cells during the brief window of time prior to initiation of piRNA-dependent retrotransposon surveillance. Disruption of Glis3 function led to a widespread reduction in retrotransposon silencing factors, aberrant retrotransposon expression and pronounced germ cell loss. Experimental induction of precocious Glis3 expression in vivo before its normal expression resulted in premature expression of several piRNA pathway members, suggesting that GLIS3 is necessary for the activation of the retrotransposon silencing programs. Our findings reveal an unexpected role for GLIS3 in the development of male germ cells and point to a central role for GLIS3 in the control of retrotransposon silencing in the fetal germline.

Effects of Exposure to Acetaminophen and Ibuprofen on Fetal Germ Cell Development in Both Sexes in Rodent and Human Using Multiple Experimental Systems.

Background:Analgesic exposure during pregnancy may affect aspects of fetal gonadal development that are targeted by endocrine disruptors.Objectives:We investigated whether therapeutically relevant doses of acetaminophen and ibuprofen affect germ cell (GC) development in human fetal testes/ovaries using in vitro and xenograft approaches.Methods:First-trimester human fetal testes/ovaries were cultured and exposed to acetaminophen or ibuprofen (7 d). Second-trimester human fetal testes were xenografted into mice and exposed to acetaminophen (1 or 7 d), or ibuprofen (7 d). To determine mechanism of action, a human GC tumor–derived cell line (NTera2) exhibiting fetal GC characteristics was used in addition to in vitro and in vivo rat models.Results and Discussion:Gonocyte () number was reduced relative to controls in first-trimester human fetal testes exposed in vitro to acetaminophen (–28%) or ibuprofen (–22%) and also in ovaries exposed to acetaminophen (–43%) or ibuprofen (–49%). Acetaminophen exposure reduced gonocyte number by 17% and 30% in xenografted second-trimester human fetal testes after treatment of host mice for 1 or 7 d, respectively. NTera2 cell number was reduced following exposure to either analgesic or prostaglandin () receptor antagonists, whereas agonists prevented acetaminophen-induced reduction in NTera2 cell number. Expression of GC pluripotency genes, and genes that regulate DNA/histone methylation, also differed from controls following analgesic and receptor antagonist exposures. Gene expression changes were observed in rat fetal testis/ovary cultures and after in vivo acetaminophen exposure of pregnant rats. For example, expression of the epigenetic regulator TET1, was increased following exposure to acetaminophen in human NTera2 cells, rat fetal testis/ovary cultures, and in fetal testes and ovaries after in vivo exposure of pregnant rats, indicating translatability across experimental models and species.Conclusions:Our results demonstrate evidence of effects of acetaminophen and ibuprofen on GC/NTera2 cells, which raises concerns about analgesic use during human pregnancy that warrant further investigation. https://doi.org/10.1289/EHP2307

Mutations causing specific arrests in the development of mouse primordial germ cells and gonocytes.

This review focuses on those mouse mutations that cause an effect on the morphology, viability, and/or behavior of primordial germ cells (PGCs) and gonocytes at specific steps of their fetal development up to the start of spermatogenesis, a few days after birth. To restrict the area covered, mice with mutations that cause abnormal hormone levels or mutations of genes not expressed in germ cells that secondarily cause spermatogenic problems are not discussed. To make our literature search as comprehensive as possible, Pubmed was searched for "(primordial germ cells OR prospermatogonia OR prespermatogonia OR gonocytes OR spermatogonia or meiosis or spermiogenesis or spermatogenesis) AND mouse AND (knockout or mutant or transgenic)." This search started at 2003 as mutants created earlier were already retrieved for a previous review. The resulting citations were then further selected for complete or partial arrests at the level of PGCs and/or gonocytes. Fifty-nine protein coding genes and two miRNA coding genes were found that arrest the development of PGCs and gonocytes at specific steps providing a better insight into the regulation of the development of these cells. As to be expected, often problems in fetal germ cell development have an effect on the fertility of the mice at adulthood.

Regulation of fetal male germ cell development by members of the TGFβ superfamily

There is now substantial evidence that members of the transforming growth factor-β (TGFβ family) regulate germ cell development in the mouse fetal testis. Correct development of germ cells during fetal life is critical for establishment of effective spermatogenesis and for avoiding the formation of testicular germ cell cancer in later life. Here we consider the evidence for involvement of various TGFβ family members, attempt to reconcile discrepancies and clarify what we believe to be the likely in vivo roles of these factors.

Single-Cell RNA-Seq Analysis Maps Development of Human Germline Cells and Gonadal Niche Interactions

Human fetal germ cells (FGCs) are precursors to sperm and eggs and are crucial for maintenance of the species. However, the developmental trajectories and heterogeneity of human FGCs remain largely unknown. Here we performed single-cell RNA-seq analysis of over 2,000 FGCs and their gonadal niche cells in female and male human embryos spanning several developmental stages. We found that female FGCs undergo four distinct sequential phases characterized by mitosis, retinoic acid signaling, meiotic prophase, and oogenesis. Male FGCs develop through stages of migration, mitosis, and cell-cycle arrest. Individual embryos of both sexes simultaneously contain several subpopulations, highlighting the asynchronous and heterogeneous nature of FGC development. Moreover, we observed reciprocal signaling interactions between FGCs and their gonadal niche cells, including activation of the bone morphogenic protein (BMP) and Notch signaling pathways. Our work provides key insights into the crucial features of human FGCs during their highly ordered mitotic, meiotic, and gametogenetic processes invivo.

Mice Lacking Hbp1 Function Are Viable and Fertile.

Fetal germ cell development is tightly regulated by the somatic cell environment, and is characterised by cell cycle states that differ between XY and XX gonads. In the testis, gonocytes enter G1/G0 arrest from 12.5 days post coitum (dpc) in mice and maintain cell cycle arrest until after birth. Failure to correctly maintain G1/G0 arrest can result in loss of germ cells or, conversely, germ cell tumours. High mobility group box containing transcription factor 1 (HBP1) is a transcription factor that was previously identified in fetal male germ cells at the time of embryonic cell cycle arrest. In somatic cells, HBP1 is classified as a tumour suppressor protein, known to regulate proliferation and senescence. We therefore investigated the possible role of HBP1 in the initiation and maintenance of fetal germ cell G1/G0 arrest using the mouse model. We identified two splice variants of Hbp1, both of which are expressed in XY and XX fetal gonads, but only one of which is localised to the nucleus in in vitro assays. To investigate Hbp1 loss of function, we used embryonic stem (ES) cells carrying a Genetrap mutation for Hbp1 to generate mice lacking Hbp1 function. We found that Hbp1-genetrap mouse mutant germ cells proliferated correctly throughout development, and adult males were viable and fertile. Multiple Hbp1-LacZ reporter mouse lines were generated, unexpectedly revealing Hbp1 embryonic expression in hair follicles, eye and limbs. Lastly, in a model of defective germ cell G1/G0 arrest, the Rb1-knockout model, we found no evidence for Hbp1 mis-regulation, suggesting that the reported RB1-HBP1 interaction is not critical in the germline, despite co-expression.