MCM8 interacts with DDX5 to promote R-loop resolution.

MCM8 has emerged as a core gene in reproductive aging and is crucial for meiotic homologous recombination repair. It also safeguards genome stability by coordinating the replication stress response during mitosis, but its function in mitotic germ cells remains elusive. Here we found that disabling MCM8 in mice resulted in proliferation defects of primordial germ cells (PGCs) and ultimately impaired fertility. We further demonstrated that MCM8 interacted with two known helicases DDX5 and DHX9, and loss of MCM8 led to R-loop accumulation by reducing the retention of these helicases at R-loops, thus inducing genome instability. Cells expressing premature ovarian insufficiency-causative mutants of MCM8 with decreased interaction with DDX5 displayed increased R-loop levels. These results show MCM8 interacts with R-loop-resolving factors to prevent R-loop-induced DNA damage, which may contribute to the maintenance of genome integrity of PGCs and reproductive reserve establishment. Our findings thus reveal an essential role for MCM8 in PGC development and improve our understanding of reproductive aging caused by genome instability in mitotic germ cells.

Sexually dimorphic DNA damage responses and mutation avoidance in the mouse germline.

Germ cells specified during fetal development form the foundation of the mammalian germline. These primordial germ cells (PGCs) undergo rapid proliferation, yet the germline is highly refractory to mutation accumulation compared with somatic cells. Importantly, while the presence of endogenous or exogenous DNA damage has the potential to impact PGCs, there is little known about how these cells respond to stressors. To better understand the DNA damage response (DDR) in these cells, we exposed pregnant mice to ionizing radiation (IR) at specific gestational time points and assessed the DDR in PGCs. Our results show that PGCs prior to sex determination lack a G1 cell cycle checkpoint. Additionally, the response to IR-induced DNA damage differs between female and male PGCs post-sex determination. IR of female PGCs caused uncoupling of germ cell differentiation and meiotic initiation, while male PGCs exhibited repression of piRNA metabolism and transposon derepression. We also used whole-genome single-cell DNA sequencing to reveal that genetic rescue of DNA repair-deficient germ cells (Fancm-/- ) leads to increased mutation incidence and biases. Importantly, our work uncovers novel insights into how PGCs exposed to DNA damage can become developmentally defective, leaving only those genetically fit cells to establish the adult germline.

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.

Controlled X-chromosome dynamics defines meiotic potential of female mouse in vitro germ cells.

The mammalian germline is characterized by extensive epigenetic reprogramming during its development into functional eggs and sperm. Specifically, the epigenome requires resetting before parental marks can be established and transmitted to the next generation. In the female germline, X-chromosome inactivation and reactivation are among the most prominent epigenetic reprogramming events, yet very little is known about their kinetics and biological function. Here, we investigate X-inactivation and reactivation dynamics using a tailor-made in vitro system of primordial germ cell-like cell (PGCLC) differentiation from mouse embryonic stem cells. We find that X-inactivation in PGCLCs in vitro and in germ cell-competent epiblast cells in vivo is moderate compared to somatic cells, and frequently characterized by escaping genes. X-inactivation is followed by step-wise X-reactivation, which is mostly completed during meiotic prophase I. Furthermore, we find that PGCLCs which fail to undergo X-inactivation or reactivate too rapidly display impaired meiotic potential. Thus, our data reveal fine-tuned X-chromosome remodelling as a critical feature of female germ cell development towards meiosis and oogenesis.

The developmental dynamics of the human male germline.

Male germ cells undergo a complex sequence of developmental events throughout fetal and postnatal life that culminate in the formation of haploid gametes: the spermatozoa. Errors in these processes result in infertility and congenital abnormalities in offspring. Male germ cell development starts when pluripotent cells undergo specification to sexually uncommitted primordial germ cells, which act as precursors of both oocytes and spermatozoa. Male-specific development subsequently occurs in the fetal testes, resulting in the formation of spermatogonial stem cells: the foundational stem cells responsible for lifelong generation of spermatozoa. Although deciphering such developmental processes is challenging in humans, recent studies using various models and single-cell sequencing approaches have shed new insight into human male germ cell development. Here, we provide an overview of cellular, signaling and epigenetic cascades of events accompanying male gametogenesis, highlighting conserved features and the differences between humans and other model organisms.

Cellular remodeling and JAK inhibition promote zygotic gene expression in the Ciona germline.

Transcription control is a major determinant of cell fate decisions in somatic tissues. By contrast, early germline fate specification in numerous vertebrate and invertebrate species relies extensively on RNA-level regulation, exerted on asymmetrically inherited maternal supplies, with little-to-no zygotic transcription. However delayed, a maternal-to-zygotic transition is nevertheless poised to complete the deployment of pre-gametic programs in the germline. Here, we focus on early germline specification in the tunicate Ciona to study zygotic genome activation. We first demonstrate that a peculiar cellular remodeling event excludes localized postplasmic Pem-1 mRNA, which encodes the general inhibitor of transcription. Subsequently, zygotic transcription begins in Pem-1-negative primordial germ cells (PGCs), as revealed by histochemical detection of elongating RNA Polymerase II, and nascent Mef2 transcripts. In addition, we uncover a provisional antagonism between JAK and MEK/BMPRI/GSK3 signaling, which controls the onset of zygotic gene expression, following cellular remodeling of PGCs. We propose a 2-step model for the onset of zygotic transcription in the Ciona germline and discuss the significance of germ plasm dislocation and remodeling in the context of developmental fate specification.

Dual role of Ovol2 on the germ cell lineage segregation during gastrulation in mouse embryogenesis.

In mammals, primordial germ cells (PGCs), the origin of the germ line, are specified from the epiblast at the posterior region where gastrulation simultaneously occurs, yet the functional relationship between PGC specification and gastrulation remains unclear. Here, we show that OVOL2, a transcription factor conserved across the animal kingdom, balances these major developmental processes by repressing the epithelial-to-mesenchymal transition (EMT) that drives gastrulation and the upregulation of genes associated with PGC specification. Ovol2a, a splice variant encoding a repressor domain, directly regulates EMT-related genes and, consequently, induces re-acquisition of potential pluripotency during PGC specification, whereas Ovol2b, another splice variant missing the repressor domain, directly upregulates genes associated with PGC specification. Taken together, these results elucidate the molecular mechanism underlying allocation of the germ line among epiblast cells differentiating into somatic cells through gastrulation. This article has an associated 'The people behind the papers' interview.

Specialized germline P-bodies are required to specify germ cell fate in Caenorhabditis elegans embryos.

In animals with germ plasm, specification of the germline involves 'germ granules', cytoplasmic condensates that enrich maternal transcripts in the germline founder cells. In Caenorhabditis elegans embryos, P granules enrich maternal transcripts, but surprisingly P granules are not essential for germ cell fate specification. Here, we describe a second condensate in the C. elegans germ plasm. Like canonical P-bodies found in somatic cells, 'germline P-bodies' contain regulators of mRNA decapping and deadenylation and, in addition, the intrinsically-disordered proteins MEG-1 and MEG-2 and the TIS11-family RNA-binding protein POS-1. Embryos lacking meg-1 and meg-2 do not stabilize P-body components, misregulate POS-1 targets, mis-specify the germline founder cell and do not develop a germline. Our findings suggest that specification of the germ line involves at least two distinct condensates that independently enrich and regulate maternal mRNAs in the germline founder cells. This article has an associated 'The people behind the papers' interview.

Transcription-replication conflicts in primordial germ cells necessitate the Fanconi anemia pathway to safeguard genome stability.

Preserving a high degree of genome integrity and stability in germ cells is of utmost importance for reproduction and species propagation. However, the regulatory mechanisms of maintaining genome stability in the developing primordial germ cells (PGCs), in which rapid proliferation is coupled with global hypertranscription, remain largely unknown. Here, we find that mouse PGCs encounter a constitutively high frequency of transcription-replication conflicts (TRCs), which lead to R-loop accumulation and impose endogenous replication stress on PGCs. We further demonstrate that the Fanconi anemia (FA) pathway is activated by TRCs and has a central role in the coordination between replication and transcription in the rapidly proliferating PGCs, as disabling the FA pathway leads to TRC and R-loop accumulation, replication fork destabilization, increased DNA damage, dramatic loss of mitotically dividing mouse PGCs, and consequent sterility of both sexes. Overall, our findings uncover the unique source and resolving mechanism of endogenous replication stress during PGC proliferation, provide a biological explanation for reproductive defects in individuals with FA, and improve our understanding of the monitoring strategies for genome stability during germ cell development.

From primordial germ cells to spermatids in Caenorhabditis elegans.

Development of a syncytial germline for gamete formation requires complex regulation of cytokinesis and cytoplasmic remodeling. Recently, several uncovered cellular events have been investigated in the Caenorhabditis elegans (C. elegans) germline. In these cellular processes, the factors involved in contractility are highly conserved with those of mitosis and meiosis. However, the underlying regulatory mechanisms are far more complicated than previously thought, likely due to the single syncytial germline structure. In this review, we highlight how the proteins involved in contractility ensure faithful cell division in different cellular contexts and how they contribute to maintaining intercellular bridge stability. In addition, we discuss the current understanding of the cellular events of cytokinesis and cytoplasmic remodeling during the development of the C. elegans germline, including progenitor germ cells, germ cells, and spermatocytes. Comparisons are made with relevant systems in Drosophila melanogaster (D. melanogaster) and other animal models.

Fetal germ cell development in humans, a link with infertility.

Gametes are cells that have the unique ability to give rise to new individuals as well as transmit (epi)genetic information across generations. Generation of functionally competent gametes, oocytes and sperm cells, depends to some extent on several fundamental processes that occur during fetal development. Direct studies on human fetal germ cells remain hindered by ethical considerations and inaccessibility to human fetal material. Therefore, the majority of our current knowledge of germ cell development still comes from an invaluable body of research performed using different mammalian species. During the last decade, our understanding of human fetal germ cells has increased due to the successful use of human pluripotent stem cells to model aspects of human early gametogenesis and advancements on single-cell omics. Together, this has contributed to determine the cell types and associated molecular signatures in the developing human gonads. In this review, we will put in perspective the knowledge obtained from several mammalian models (mouse, monkey, pig). Moreover, we will discuss the main events during human fetal (female) early gametogenesis and how the dysregulation of this highly complex and lengthy process can link to infertility later in life.

Paternal source of germ plasm determinants in the viviparous teleost, Gambusia holbrooki; dads do matter.

The identity of germ cells, the progenitors of life, is thought to be acquired by two modes; either by maternal signals (preformed) or induced de novo from pluripotent cells (epigenesis) in the developing embryos. However, paternal roles seem enshrouded or completely overlooked in this fundamental biological process. Hence, we investigated the presence of germplasm transcripts in the sperm of Gambusia holbrooki, a live-bearing fish, demonstrating their presence and suggesting paternal contributions. Interestingly, not all germplasm markers were present (nanos1 and tdrd6) in the sperm, but some were conspicuous (dazl, dnd-α, piwi II, and vasa), indicating that the latter is required for establishing germ cell identity in the progeny, with a possible parent-specific role. Furthermore, there were also spatial differences in the distribution of these determinants, suggesting additional roles in sperm physiology and/or fertility. Our results support the hypothesis that dads also play a vital role in establishing the germ cell identity, especially in G. holbrooki, which shares elements of both preformation and induction modes of germline determination. This, coupled with its life history traits, makes G. holbrooki an excellent system for dissecting evolutionary relationships between the two germline determination modes, their underpinning mechanisms and ultimately the perpetuity of life.

Germ Granule Evolution Provides Mechanistic Insight into Drosophila Germline Development.

The copackaging of mRNAs into biomolecular condensates called germ granules is a conserved strategy to posttranscriptionally regulate germline mRNAs. In Drosophila melanogaster, mRNAs accumulate in germ granules by forming homotypic clusters, aggregates containing multiple transcripts from the same gene. Nucleated by Oskar (Osk), homotypic clusters are generated through a stochastic seeding and self-recruitment process that requires the 3' untranslated region (UTR) of germ granule mRNAs. Interestingly, the 3' UTR belonging to germ granule mRNAs, such as nanos (nos), have considerable sequence variations among Drosophila species and we hypothesized that this diversity influences homotypic clustering. To test our hypothesis, we investigated the homotypic clustering of nos and polar granule component (pgc) in four Drosophila species and concluded that clustering is a conserved process used to enrich germ granule mRNAs. However, we discovered germ granule phenotypes that included significant changes in the abundance of transcripts present in species' homotypic clusters, which also reflected diversity in the number of coalesced primordial germ cells within their embryonic gonads. By integrating biological data with computational modeling, we found that multiple mechanisms underlie naturally occurring germ granule diversity, including changes in nos, pgc, osk levels and/or homotypic clustering efficacy. Furthermore, we demonstrated how the nos 3' UTR from different species influences nos clustering, causing granules to have ∼70% less nos and increasing the presence of defective primordial germ cells. Our results highlight the impact that evolution has on germ granules, which should provide broader insight into processes that modify compositions and activities of other classes of biomolecular condensate.

Sperm mosaicism: implications for genomic diversity and disease.

While sperm mosaicism has few consequences for men, the offspring and future generations are unwitting recipients of gonadal cell mutations, often yielding severe disease. Recent studies, fueled by emergent technologies, show that sperm mosaicism is a common source of de novo mutations (DNMs) that underlie severe pediatric disease as well as human genetic diversity. Sperm mosaicism can be divided into three types: Type I arises during sperm meiosis and is non-age dependent; Type II arises in spermatogonia and increases as men age; and Type III arises during paternal embryogenesis, spreads throughout the body, and contributes stably to sperm throughout life. Where Types I and II confer little risk of recurrence, Type III may confer identifiable risk to future offspring. These mutations are likely to be the single largest contributor to human genetic diversity. New sequencing approaches may leverage this framework to evaluate and reduce disease risk for future generations.

Long-term expansion with germline potential of human primordial germ cell-like cells in vitro.

Human germ cells perpetuate human genetic and epigenetic information. However, the underlying mechanism remains elusive, due to a lack of appropriate experimental systems. Here, we show that human primordial germ cell-like cells (hPGCLCs) derived from human-induced pluripotent stem cells (hiPSCs) can be propagated to at least ~106 -fold over a period of 4 months under a defined condition in vitro. During expansion, hPGCLCs maintain an early hPGC-like transcriptome and preserve their genome-wide DNA methylation profiles, most likely due to retention of maintenance DNA methyltransferase activity. These characteristics contrast starkly with those of mouse PGCLCs, which, under an analogous condition, show a limited propagation (up to ~50-fold) and persist only around 1 week, yet undergo cell-autonomous genome-wide DNA demethylation. Importantly, upon aggregation culture with mouse embryonic ovarian somatic cells in xenogeneic-reconstituted ovaries, expanded hPGCLCs initiate genome-wide DNA demethylation and differentiate into oogonia/gonocyte-like cells, demonstrating their germline potential. By creating a paradigm for hPGCLC expansion, our study uncovers critical divergences in expansion potential and the mechanism for epigenetic reprogramming between the human and mouse germ cell lineage.

Direct repression of Nanog and Oct4 by OTX2 modulates the contribution of epiblast-derived cells to germline and somatic lineage.

In mammals, the pre-gastrula proximal epiblast gives rise to primordial germ cells (PGCs) or somatic precursors in response to BMP4 and WNT signaling. Entry into the germline requires activation of a naïve-like pluripotency gene regulatory network (GRN). Recent work has shown that suppression of OTX2 expression in the epiblast by BMP4 allows cells to develop a PGC fate in a precise temporal window. However, the mechanisms by which OTX2 suppresses PGC fate are unknown. Here, we show that, in mice, OTX2 prevents epiblast cells from activating the pluripotency GRN by direct repression of Oct4 and Nanog. Loss of this control during PGC differentiation in vitro causes widespread activation of the pluripotency GRN and a deregulated response to LIF, BMP4 and WNT signaling. These abnormalities, in specific cell culture conditions, result in massive germline entry at the expense of somatic mesoderm differentiation. Increased generation of PGCs also occurs in mutant embryos. We propose that the OTX2-mediated repressive control of Oct4 and Nanog is the basis of the mechanism that determines epiblast contribution to germline and somatic lineage.

Mammalian primordial germ cell specification.

The peri-implantation window of mammalian development is the crucial window for primordial germ cell (PGC) specification. Whereas pre-implantation dynamics are relatively conserved between species, the implantation window marks a stage of developmental divergence between key model organisms, and thus potential variance in the cell and molecular mechanisms for PGC specification. In humans, PGC specification is very difficult to study in vivo To address this, the combined use of human and nonhuman primate embryos, and stem cell-based embryo models are essential for determining the origin of PGCs, as are comparative analyses to the equivalent stages of mouse development. Understanding the origin of PGCs in the peri-implantation embryo is crucial not only for accurate modeling of this essential process using stem cells, but also in determining the role of global epigenetic reprogramming upon which sex-specific differentiation into gametes relies.

The initial expansion of the C. elegans syncytial germ line is coupled to incomplete primordial germ cell cytokinesis.

The C. elegans germline is organized as a syncytium in which each germ cell possesses an intercellular bridge that is maintained by a stable actomyosin ring and connected to a common pool of cytoplasm, termed the rachis. How germ cells undergo cytokinesis while maintaining this syncytial architecture is not completely understood. Here, we use live imaging to characterize primordial germ cell (PGC) division in C. elegans first-stage larvae. We show that each PGC possesses a stable intercellular bridge that connects it to a common pool of cytoplasm, which we term the proto-rachis. We further show that the first PGC cytokinesis is incomplete and that the stabilized cytokinetic ring progressively moves towards the proto-rachis and eventually integrates with it. Our results support a model in which the initial expansion of the C. elegans syncytial germline occurs by incomplete cytokinesis, where one daughter germ cell inherits the actomyosin ring that was newly formed by stabilization of the cytokinetic ring, while the other inherits the pre-existing stable actomyosin ring. We propose that such a mechanism of iterative cytokinesis incompletion underpins C. elegans germline expansion and maintenance.

lncCPSET1 acts as a scaffold for MLL2/COMPASS to regulate Bmp4 and promote the formation of chicken primordial germ cells.

Primordial germ cells (PGCs) are the ancestors of female and male germ cells. Recent studies have shown that long non-coding RNA (lncRNA) and histone methylation are key epigenetic factors affecting PGC formation; however, their joint regulatory mechanisms have rarely been studied. Here, we explored the mechanism by which lncCPSET1 and H3K4me2 synergistically regulate the formation of chicken PGCs for the first time. Combined with chromatin immunoprecipitation (CHIP) sequencing and RNA-seq of PGCs transfected with the lncCPSET1 overexpression vector, GO annotation and KEGG enrichment analysis revealed that Wnt and TGF-ß signaling pathways were significantly enriched, and Fzd2, Id1, Id4, and Bmp4 were identified as candidate genes. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that ASH2L, DPY30, WDR5, and RBBP5 overexpression significantly increased the expression of Bmp4, which was up-regulated after lncCPSET1 overexpression as well. It indicated that Bmp4 is a target gene co-regulated by lncCPSET1 and MLL2/COMPASS. Interestingly, co-immunoprecipitation results showed that ASH2L, DPY30 and WDR5 combined and RBBP5 weakly combined with DPY30 and WDR5. lncCPSET1 overexpression significantly increased Dpy30 expression and co-immunoprecipitation showed that interference/overexpression of lncCPSET1 did not affect the binding between the proteins in the complexes, but interference with lncCPSET1 inhibited DPY30 expression, which was confirmed by RNA immunoprecipitation that lncCPSET1 binds to DPY30. Additionally, CHIP-qPCR results showed that DPY30 enriched in the Bmp4 promoter region promoted its transcription, thus promoting the formation of PGCs. This study demonstrated that lncCPSET1 and H3K4me2 synergistically promote PGC formation, providing a reference for the study of the regulatory mechanisms between lncRNA and histone methylation, as well as a molecular basis for elucidating the formation mechanism of PGCs in chickens.

Diaph1 knockout inhibits mouse primordial germ cell proliferation and affects gonadal development.

BACKGROUND: Exploring 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. METHODS: Diaphanous 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. RESULTS: Based 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, 3ß-HSD) were decreased in Leydig cells, and the estradiol-associated factor (FSH receptor, AMH) in granulosa cells were also downregulated. CONCLUSIONS: Overall, 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.