Isolation and Culture Techniques for Fetal Mouse Germ Cells.

The fetal gonad contains a great variety of differentiating cell populations, of which germ cells make up a relatively small percentage. In order to study germ cell-specific gene and protein expression, as well as determine direct effects of signaling molecules, it is necessary to prepare enriched populations of germ cells and maintain them in culture for several hours to multiple days. The protocols in this chapter are designed to provide a guide for the isolation or enrichment of primordial germ cells (from 9.5 days post coitum (dpc) to 18.5 dpc) by flow cytometry (Subheading 3.1) or magnetic sorting (Subheading 3.2), followed by feeder-free primary germ cell culture (Subheading 3.3).

Variants in SART3 cause a spliceosomopathy characterised by failure of testis development and neuronal defects.

Squamous cell carcinoma antigen recognized by T cells 3 (SART3) is an RNA-binding protein with numerous biological functions including recycling small nuclear RNAs to the spliceosome. Here, we identify recessive variants in SART3 in nine individuals presenting with intellectual disability, global developmental delay and a subset of brain anomalies, together with gonadal dysgenesis in 46,XY individuals. Knockdown of the Drosophila orthologue of SART3 reveals a conserved role in testicular and neuronal development. Human induced pluripotent stem cells carrying patient variants in SART3 show disruption to multiple signalling pathways, upregulation of spliceosome components and demonstrate aberrant gonadal and neuronal differentiation in vitro. Collectively, these findings suggest that bi-allelic SART3 variants underlie a spliceosomopathy which we tentatively propose be termed INDYGON syndrome (Intellectual disability, Neurodevelopmental defects and Developmental delay with 46,XY GONadal dysgenesis). Our findings will enable additional diagnoses and improved outcomes for individuals born with this condition.

Generation and characterization of a Ddx4-iCre transgenic line for deletion in the germline beginning at genital ridge colonization.

Germline-specific Cre lines are useful for analyses of primordial germ cell, spermatogonial and oogonial development, but also for whole-body deletions when transmitted through subsequent generations. Several germ cell specific Cre mouse strains exist, with various degrees of specificity, efficiency, and temporal activation. Here, we describe the CRISPR/Cas9 targeted insertion of an improved Cre (iCre) sequence in-frame at the 3' end of the Ddx4 locus to generate the Ddx4-P2A-iCre allele. Our functional assessment of this new allele, designated Ddx4iCreJoBo , reveals that Cre activity begins in PGCs from at least E10.5, and that it achieves higher efficiency for early gonadal (E10.5-12.5) germline deletion when compared to the inducible Oct4CreERT2 line. We found the Ddx4iCreJoBo allele to be hypomorphic for Ddx4 expression and homozygous males, but not females, were infertile. Using two reporter lines (R26RLacZ and R26RtdTomato ) and a floxed gene of interest (Criptoflox ) we found ectopic activity in multiple organs; global recombination (a common feature of germline Cre alleles) varies from 10 to 100%, depending on the particular floxed allele. There is a strong maternal effect, and therefore it is preferable for Ddx4iCreJoBo to be inherited from the male parent if ubiquitous deletion is not desired. With these limitations considered, we describe the Ddx4iCreJoBo line as useful for germline studies in which early gonadal deletion is required.

AOP Key Event Relationship report: Linking decreased retinoic acid levels with disrupted meiosis in developing oocytes.

The Adverse Outcome Pathway (AOP) concept is an emerging tool in regulatory toxicology that uses simplified descriptions to show cause-effect relationships between stressors and toxicity outcomes in intact organisms. The AOP structure is a modular framework, with Key Event Relationships (KERs) representing the unit of causal relationship based on existing knowledge, describing the connection between two Key Events. Because KERs are the only unit to support inference it has been argued recently that KERs should be recognized as the core building blocks of knowledge assembly within the AOP-Knowledge Base. Herein, we present a first case to support this proposal and provide a full description of a KER linking decreased all-trans retinoic acid (atRA) levels in developing ovaries with disrupted meiotic entry of oogonia. We outline the evidence to support a role for atRA in inducing meiosis in oogonia across mammals; this is important because elements of the RA synthesis/degradation pathway are recognized targets for numerous environmental chemicals. The KER we describe will be used to support an intended AOP linking inhibition of the atRA producing ALDH1A enzymes with reduced fertility in women.

Instructing Mouse Germ Cells to Adopt a Female Fate.

BACKGROUND: Germ cells are critical for the survival of our species. They are the only cells that undergo meiosis - the reductive form of cell division that is necessary for genetic reassortment of chromosomes and production of the haploid gametes, the sperm and eggs. Remarkably, the initial female/male fate decision in fetal germ cells does not depend on whether they are chromosomally XX or XY; rather, initial sexual fate is imposed by influences from the surrounding tissue. In mammals, the female germline is particularly precious: despite recent suggestions that germline stem cells exist in the ovary, it is still generally accepted that the ovarian reserve is finite, and its size is dependant on germ cells of the fetal ovary initiating meiosis in a timely manner. SUMMARY: Prior to 2006, evidence suggested that gonadal germ cells initiate meiotic prophase I by default, but more recent data support a key role for the signalling molecule retinoic acid (RA) in instructing female germ cell fate. Newer findings also support a key meiosis-inducing role for another signalling molecule, bone morphogenic protein (BMP). Nonetheless, many questions remain. KEY MESSAGES: Here, we review knowledge thus far regarding extrinsic and intrinsic determinants of a female germ cell fate, focusing on the mouse model.

Deletion of NFIX results in defective progression through meiosis within the mouse testis†.

Members of the nuclear factor I (NFI) family are key regulators of stem cell biology during development, with well-documented roles for NFIA, NFIB, and NFIX in a variety of developing tissues, including brain, muscle, and lung. Given the central role these factors play in stem cell biology, we posited that they may be pivotal for spermatogonial stem cells or further developing spermatogonia during testicular development. Surprisingly, in stark contrast to other developing organ systems where NFI members are co-expressed, these NFI family members show discrete patterns of expression within the seminiferous tubules. Sertoli cells (spermatogenic supporting cells) express NFIA, spermatocytes express NFIX, round spermatids express NFIB, and peritubular myoid cells express each of these three family members. Further analysis of NFIX expression during the cycle of the seminiferous epithelium revealed expression not in spermatogonia, as we anticipated, but in spermatocytes. These data suggested a potential role for NFIX in spermatogenesis. To investigate, we analyzed mice with constitutive deletion of Nfix (Nfix-null). Assessment of germ cells in the postnatal day 20 (P20) testes of Nfix-null mice revealed that spermatocytes initiate meiosis, but zygotene stage spermatocytes display structural defects in the synaptonemal complex, and increased instances of unrepaired DNA double-strand breaks. Many developing spermatocytes in the Nfix-null testis exhibited multinucleation. As a result of these defects, spermatogenesis is blocked at early diplotene and very few round spermatids are produced. Collectively, these novel data establish the global requirement for NFIX in correct meiotic progression during the first wave of spermatogenesis.

Two ovarian candidate enhancers, identified by time series enhancer RNA analyses, harbor rare genetic variations identified in ovarian insufficiency.

The genetic regulation of ovarian development remains largely unclear. Indeed, in most cases of impaired ovarian development-such as 46,XX disorders of sex development (DSD) without SRY, and premature ovarian insufficiency (POI)-the genetic causes have not been identified, and the vast majority of disease-associated sequence variants could lie within non-coding regulatory sequences. In this study, we aimed to identify enhancers of five ovarian genes known to play key roles in early ovarian development, basing our analysis on the expression of enhancer derived transcripts (eRNAs), which are considered to characterize active enhancers. Temporal expression profile changes in mouse WT1-positive ovarian cells were obtained from cap analysis of gene expression at E13.5, E16.5 and P0. We compared the chronological expression profiles of ovarian-specific eRNA with expression profiles for each of the ovarian-specific genes, yielding two candidate sequences for enhancers of Wnt4 and Rspo1. Both sequences are conserved between mouse and human, and we confirmed their enhancer activities using transient expression assays in murine granulosa cells. Furthermore, by sequencing the region in patients with impaired ovarian development in 24 patients, such as POI, gonadal dysgenesis and 46,XX DSD, we identified rare single nucleotide variants in both sequences. Our results demonstrate that combined analysis of the temporal expression profiles of eRNA and mRNA of target genes presents a powerful tool for locating cis-element enhancers, and a means of identifying disease-associated sequence variants that lie within non-coding regulatory sequences, thus advancing an important unmet need in forward human genetics.

WDR62 is required for centriole duplication in spermatogenesis and manchette removal in spermiogenesis.

WDR62 is a scaffold protein involved in centriole duplication and spindle assembly during mitosis. Mutations in WDR62 can cause primary microcephaly and premature ovarian insufficiency. We have generated a genetrap mouse model deficient in WDR62 and characterised the developmental effects of WDR62 deficiency during meiosis in the testis. We have found that WDR62 deficiency leads to centriole underduplication in the spermatocytes due to reduced or delayed CEP63 accumulation in the pericentriolar matrix. This resulted in prolonged metaphase that led to apoptosis. Round spermatids that inherited a pair of centrioles progressed through spermiogenesis, however, manchette removal was delayed in WDR62 deficient spermatids due to delayed Katanin p80 accumulation in the manchette, thus producing misshapen spermatid heads with elongated manchettes. In mice, WDR62 deficiency resembles oligoasthenoteratospermia, a common form of subfertility in men that is characterised by low sperm counts, poor motility and abnormal morphology. Therefore, proper WDR62 function is necessary for timely spermatogenesis and spermiogenesis during male reproduction.

Identification of regulatory elements required for Stra8 expression in fetal ovarian germ cells of the mouse.

In mice, the entry of germ cells into meiosis crucially depends on the expression of stimulated by retinoic acid gene 8 (Stra8). Stra8 is expressed specifically in pre-meiotic germ cells of females and males, at fetal and postnatal stages, respectively, but the mechanistic details of its spatiotemporal regulation are yet to be defined. In particular, there has been considerable debate regarding whether retinoic acid is required, in vivo, to initiate Stra8 expression in the mouse fetal ovary. We show that the distinctive anterior-to-posterior pattern of Stra8 initiation, characteristic of germ cells in the fetal ovary, is faithfully recapitulated when 2.9 kb of the Stra8 promoter is used to drive eGFP expression. Using in vitro transfection assays of cutdown and mutant constructs, we identified two functional retinoic acid responsive elements (RAREs) within this 2.9 kb regulatory element. We also show that the transcription factor DMRT1 enhances Stra8 expression, but only in the presence of RA and the most proximal RARE. Finally, we used CRISPR/Cas9-mediated targeted mutation studies to demonstrate that both RAREs are required for optimal Stra8 expression levels in vivo.

CRIPTO and miR-371a-3p Are Serum Biomarkers of Testicular Germ Cell Tumors and Are Detected in Seminal Plasma from Azoospermic Males.

miR-371a-3p is currently the most informative reported biomarker for germ cell tumors (GCTs). Another developmental-related biomarker, CRIPTO, is involved in the regulation of pluripotency and germ cell fate commitment. We aimed to assess the value of CRIPTO as a diagnostic and prognostic biomarker of testicular GCTs (TGCTs) and also to assess its presence in seminal plasma samples, compared with miR-371a-3p. In total, 217 and 94 serum/seminal plasma samples were analyzed. CRIPTO was quantified using ELISA and miR-371a-3p using bead-based isolation followed by RT-qPCR. Methylation profiling (EPIC array) for the CRIPTO promoter region was undertaken in 35 TGCT tissues plus four (T)GCT cell lines. Significantly higher CRIPTO concentration was found in sera of non-seminomas compared to controls (p = 0.0297), and in stage II/III disease compared to stage I (p = 0.0052, p = 0.0097). CRIPTO concentration was significantly positively correlated with miR-371a-3p levels in serum (r = 0.16) and seminal plasma (r = 0.40). CRIPTO/miR-371a-3p levels were significantly higher in seminal plasma controls when compared to serum controls (p = 0.0001, p < 0.0001). CRIPTO/miR-371a-3p were detected both in normospermic and azoospermic males, and levels were higher in TGCTs compared to GCNIS-only. We have provided the largest dataset of evaluation of CRIPTO in serum and seminal plasma of GCTs, showing its potential value as a biomarker of the disease.

Developmental biology meets toxicology: contributing reproductive mechanisms to build adverse outcome pathways.

An adverse outcome pathway (AOP) is a simplified description of the sequence of mechanistic events that lead to a particular toxicological effect, from initial trigger to adverse outcome. Although designed to inform regulatory risk assessors, the AOP framework also provides a platform for innovative collaborations between experts from relevant research fields and the regulatory community. The underpinning for any AOP is basic knowledge about molecular and developmental processes; such knowledge can only be attained by solid bioscientific research. Starting with this fundamental knowledge, the objective is to devise novel testing strategies that focus on key events in a causative pathway. It is anticipated that such a knowledge-based approach will ultimately alleviate many of the burdens associated with classical chemical testing strategies that typically involve large-scale animal toxicity regimens. This hails from the notion that a solid understanding of the underlying mechanisms will allow the development and use of alternative test methods, including both in vitro and in silico approaches. This review is specifically targeted at professionals working in bioscientific fields, such as developmental and reproductive biology, and aims to (i) inform on the existence of the AOP framework and (ii) encourage new cross-disciplinary collaborations. It is hoped that fundamental biological knowledge can thus be better exploited for applied purposes: firstly, an improved understanding of how our perpetual exposure to environmental chemicals is causing human reproductive disease and, secondly, new approaches to screen for harmful chemicals more efficiently. This is not an instructional manual on how to create AOPs; rather, we discuss how to harness fundamental knowledge from the biosciences to assist regulatory toxicologists in their efforts to protect humans against chemicals that harm human reproductive development and function.

A simple, web-based repository for the management, access and analysis of micrographic images.

Microscopy is advancing at a rapid pace, enabling high-speed, high-resolution analyses to be conducted in a wide range of cellular contexts. For example, the capacity to quickly capture high-resolution images from multiple optical sections over multiple channels with confocal microscopy has allowed researchers to gain deeper understanding of tissue morphology via techniques such as three-dimensional rendering, as have more recent advances such as lattice light sheet microscopy and superresolution structured illumination microscopy. With this, though, comes the challenge of storing, curating, analysing and sharing data. While there are ways in which this has been attempted previously, few approaches have provided a central repository in which all of these different aspects of microscopy can be seamlessly integrated. Here, we describe a web-based storage and analysis platform called Microndata, that enables relatively straightforward storage, annotation, tracking, analysis and multi-user access to micrographs. This easy to use tool will simplify and harmonise laboratory work flows, and, importantly, will provide a central storage repository that is readily accessed, even after the researcher responsible for capturing the images has left the laboratory. Microndata is open-source software, available at http://www.microndata.net/.

Author Correction: Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Sexually dimorphic germ cell identity in mammals.

Germ cells are the stem cells of the species. Thus, it is critical that we have a good understanding of how they are specified, how the somatic cells instruct and support them, how they commit to one or other sex, and how they ultimately develop into functional gametes. Here, we focus on specifics of how sexual fate is determined during fetal life. Because the majority of relevant experimental work has been done using the mouse model, we focus on that species. We review evidence regarding the identity of instructive signals from the somatic cells, and the molecular responses that occur in germ cells in response to those extrinsic signals. In this way we aim to clarify progress to date regarding the mechanisms underlying the mitotic to meiosis switch in germ cells of the fetal ovary, and those involved in adopting and securing male fate in germ cells of the fetal testis.

Nr5a1 suppression during the murine fetal period optimizes ovarian development by fine-tuning Notch signaling.

The nuclear receptor NR5A1 is equally expressed and required for development of the gonadal primordia of both sexes, but, after sex determination, it is upregulated in XY testes and downregulated in XX ovaries. We have recently demonstrated, in mice, that this downregulation is mediated by forkhead box L2 (FOXL2) and hypothesized that adequate suppression of Nr5a1 is essential for normal ovarian development. Further, analysis of human patients with disorders/differences of sex development suggests that overexpression of NR5A1 can result in XX (ovo)testicular development. Here, we tested the role of Nr5a1 by overexpression in fetal gonads using a Wt1-BAC (bacterial artificial chromosome) transgene system. Enforced Nr5a1 expression compromised ovarian development in 46,XX mice, resulting in late-onset infertility, but did not induce (ovo)testis differentiation. The phenotype was similar to that of XX mice lacking Notch signaling. The expression level of Notch2 was significantly reduced in Nr5a1 transgenic mice, and the ovarian phenotype was almost completely rescued by in utero treatment with a NOTCH2 agonist. We conclude that suppression of Nr5a1 during the fetal period optimizes ovarian development by fine-tuning Notch signaling.

Gene editing of the multi-copy H2A.B gene and its importance for fertility.

BACKGROUND: Altering the biochemical makeup of chromatin by the incorporation of histone variants during development represents a key mechanism in regulating gene expression. The histone variant H2A.B, H2A.B.3 in mice, appeared late in evolution and is most highly expressed in the testis. In the mouse, it is encoded by three different genes. H2A.B expression is spatially and temporally regulated during spermatogenesis being most highly expressed in the haploid round spermatid stage. Active genes gain H2A.B where it directly interacts with polymerase II and RNA processing factors within splicing speckles. However, the importance of H2A.B for gene expression and fertility are unknown. RESULTS: Here, we report the first mouse knockout of this histone variant and its effects on fertility, nuclear organization, and gene expression. In view of the controversy related to the generation of off-target mutations by gene editing approaches, we test the specificity of TALENs by disrupting the H2A.B multi-copy gene family using only one pair of TALENs. We show that TALENs do display a high level of specificity since no off-target mutations are detected by bioinformatics analyses of exome sequences obtained from three consecutive generations of knockout mice and by Sanger DNA sequencing. Male H2A.B.3 knockout mice are subfertile and display an increase in the proportion of abnormal sperm and clogged seminiferous tubules. Significantly, a loss of proper RNA Pol II targeting to distinct transcription-splicing territories and changes to pre-mRNA splicing are observed. CONCLUSION: We have produced the first H2A.B knockout mouse using the TALEN approach.

Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9.

Disorders of sex development (DSDs) are conditions affecting development of the gonads or genitalia. Variants in two key genes, SRY and its target SOX9, are an established cause of 46,XY DSD, but the genetic basis of many DSDs remains unknown. SRY-mediated SOX9 upregulation in the early gonad is crucial for testis development, yet the regulatory elements underlying this have not been identified in humans. Here, we identified four DSD patients with overlapping duplications or deletions upstream of SOX9. Bioinformatic analysis identified three putative enhancers for SOX9 that responded to different combinations of testis-specific regulators. All three enhancers showed synergistic activity and together drive SOX9 in the testis. This is the first study to identify SOX9 enhancers that, when duplicated or deleted, result in 46,XX or 46,XY sex reversal, respectively. These enhancers provide a hitherto missing link by which SRY activates SOX9 in humans, and establish SOX9 enhancer mutations as a significant cause of DSD.

Retinoic Acid Antagonizes Testis Development in Mice.

Mammalian sex determination depends on a complex interplay of signals that promote the bipotential fetal gonad to develop as either a testis or an ovary, but the details are incompletely understood. Here, we investigated whether removal of the signaling molecule retinoic acid (RA) by the degradative enzyme CYP26B1 is necessary for proper development of somatic cells of the testes. Gonadal organ culture experiments suggested that RA promotes expression of some ovarian markers and suppresses expression of some testicular markers, acting downstream of Sox9. XY Cyp26b1-null embryos, in which endogenous RA is not degraded, develop mild ovotestes, but more important, steroidogenesis is impaired and the reproductive tract feminized. Experiments involving purified gonadal cells showed that these effects are independent of germ cells and suggest the direct involvement of the orphan nuclear receptor DAX1. Our results reveal that active removal of endogenous RA is required for normal testis development in the mouse.

Prolonged prenatal hypoxia selectively disrupts collecting duct patterning and postnatal function in male mouse offspring.

KEY POINTS: In the present study, we investigated whether hypoxia during late pregnancy impairs kidney development in mouse offspring, and also whether this has long-lasting consequences affecting kidney function in adulthood. Hypoxia disrupted growth of the kidney, particularly the collecting duct network, in juvenile male offspring. By mid-late adulthood, these mice developed early signs of kidney disease, notably a compromised response to water deprivation. Female offspring showed no obvious signs of impaired kidney development and did not develop kidney disease, suggesting an underlying protection mechanism from the hypoxia insult. These results help us better understand the long-lasting impact of gestational hypoxia on kidney development and the increased risk of chronic kidney disease. ABSTRACT: Prenatal hypoxia is a common perturbation to arise during pregnancy, and can lead to adverse health outcomes in later life. The long-lasting impact of prenatal hypoxia on postnatal kidney development and maturation of the renal tubules, particularly the collecting duct system, is relatively unknown. In the present study, we used a model of moderate chronic maternal hypoxia throughout late gestation (12% O2 exposure from embryonic day 14.5 until birth). Histological analyses revealed marked changes in the tubular architecture of male hypoxia-exposed neonates as early as postnatal day 7, with disrupted medullary development and altered expression of Ctnnb1 and Crabp2 (encoding a retinoic acid binding protein). Kidneys of the RARElacZ line offspring exposed to hypoxia showed reduced ß-galactosidase activity, indicating reduced retinoic acid-directed transcriptional activation. Wild-type male mice exposed to hypoxia had an early decline in urine concentrating capacity, evident at 4 months of age. At 12 months of age, hypoxia-exposed male mice displayed a compromised response to a water deprivation challenge, which was was correlated with an altered cellular composition of the collecting duct and diminished expression of aquaporin 2. There were no differences in the tubular structures or urine concentrating capacity between the control and hypoxia-exposed female offspring at any age. The findings of the present study suggest that prenatal hypoxia selectively disrupts collecting duct patterning through altered Wnt/ß-catenin and retinoic acid signalling and this results in impaired function in male mouse offspring in later life.