Age-Dependent Alterations in Meiotic Recombination Cause Chromosome Segregation Errors in Spermatocytes.

Faithful chromosome segregation in meiosis requires crossover (CO) recombination, which is regulated to ensure at least one CO per homolog pair. We investigate the failure to ensure COs in juvenile male mice. By monitoring recombination genome-wide using cytological assays and at hotspots using molecular assays, we show that juvenile mouse spermatocytes have fewer COs relative to adults. Analysis of recombination in the absence of MLH3 provides evidence for greater utilization in juveniles of pathways involving structure-selective nucleases and alternative complexes, which can act upon precursors to generate noncrossovers (NCOs) at the expense of COs. We propose that some designated CO sites fail to mature efficiently in juveniles owing to inappropriate activity of these alternative repair pathways, leading to chromosome mis-segregation. We also find lower MutLγ focus density in juvenile human spermatocytes, suggesting that weaker CO maturation efficiency may explain why younger men have a higher risk of fathering children with Down syndrome.

Large Drosophila germline piRNA clusters are evolutionarily labile and dispensable for transposon regulation.

PIWI proteins and their guiding Piwi-interacting small RNAs (piRNAs) are crucial for fertility and transposon defense in the animal germline. In most species, the majority of piRNAs are produced from distinct large genomic loci, called piRNA clusters. It is assumed that germline-expressed piRNA clusters, particularly in Drosophila, act as principal regulators to control transposons dispersed across the genome. Here, using synteny analysis, we show that large clusters are evolutionarily labile, arise at loci characterized by recurrent chromosomal rearrangements, and are mostly species-specific across the Drosophila genus. By engineering chromosomal deletions in D. melanogaster, we demonstrate that the three largest germline clusters, which account for the accumulation of >40% of all transposon-targeting piRNAs in ovaries, are neither required for fertility nor for transposon regulation in trans. We provide further evidence that dispersed elements, rather than the regulatory action of large Drosophila germline clusters in trans, may be central for transposon defense.

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.

Histone demethylase KDM2A recruits HCFC1 and E2F1 to orchestrate male germ cell meiotic entry and progression.

In mammals, the transition from mitosis to meiosis facilitates the successful production of gametes. However, the regulatory mechanisms that control meiotic initiation remain unclear, particularly in the context of complex histone modifications. Herein, we show that KDM2A, acting as a lysine demethylase targeting H3K36me3 in male germ cells, plays an essential role in modulating meiotic entry and progression. Conditional deletion of Kdm2a in mouse pre-meiotic germ cells results in complete male sterility, with spermatogenesis ultimately arrested at the zygotene stage of meiosis. KDM2A deficiency disrupts H3K36me2/3 deposition in c-KIT+ germ cells, characterized by a reduction in H3K36me2 but a dramatic increase in H3K36me3. Furthermore, KDM2A recruits the transcription factor E2F1 and its co-factor HCFC1 to the promoters of key genes required for meiosis entry and progression, such as Stra8, Meiosin, Spo11, and Sycp1. Collectively, our study unveils an essential role for KDM2A in mediating H3K36me2/3 deposition and controlling the programmed gene expression necessary for the transition from mitosis to meiosis during spermatogenesis.

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.

Pioneering meiotic recombination.

To induce cell type-specific forms of gene regulation, pioneer factors open tightly packed, inaccessible chromatin sites, enabling the molecular machinery to act on functionally significant information encoded in DNA. While previous studies of pioneer factors have revealed their functions in transcriptional regulation, pioneer factors that open chromatin for other physiological events remain undetermined. In this issue of Genes & Development, Spruce and colleagues (pp. 398-412) report the functional significance of a "pioneer complex" in mouse meiotic recombination. This complex, comprised of the zinc finger DNA-binding protein PRDM9 and the SNF2 family chromatin remodeler HELLS, exposes nucleosomal DNA to designate the sites of DNA double-strand breaks that initiate meiotic recombination. Both HELLS and PRDM9 are required for the determination of these recombination hot spots. Through the identification of a pioneer complex for meiotic recombination, this study broadens the conceptual scope of pioneer factors, indicating their functional significance in biological processes beyond transcriptional regulation.

HELLS and PRDM9 form a pioneer complex to open chromatin at meiotic recombination hot spots.

Chromatin barriers prevent spurious interactions between regulatory elements and DNA-binding proteins. One such barrier, whose mechanism for overcoming is poorly understood, is access to recombination hot spots during meiosis. Here we show that the chromatin remodeler HELLS and DNA-binding protein PRDM9 function together to open chromatin at hot spots and provide access for the DNA double-strand break (DSB) machinery. Recombination hot spots are decorated by a unique combination of histone modifications not found at other regulatory elements. HELLS is recruited to hot spots by PRDM9 and is necessary for both histone modifications and DNA accessibility at hot spots. In male mice lacking HELLS, DSBs are retargeted to other sites of open chromatin, leading to germ cell death and sterility. Together, these data provide a model for hot spot activation in which HELLS and PRDM9 form a pioneer complex to create a unique epigenomic environment of open chromatin, permitting correct placement and repair of DSBs.

Genome-wide DNA methylation changes in human spermatogenesis.

Sperm production and function require the correct establishment of DNA methylation patterns in the germline. Here, we examined the genome-wide DNA methylation changes during human spermatogenesis and its alterations in disturbed spermatogenesis. We found that spermatogenesis is associated with remodeling of the methylome, comprising a global decline in DNA methylation in primary spermatocytes followed by selective remethylation, resulting in a spermatids/sperm-specific methylome. Hypomethylated regions in spermatids/sperm were enriched in specific transcription factor binding sites for DMRT and SOX family members and spermatid-specific genes. Intriguingly, while SINEs displayed differential methylation throughout spermatogenesis, LINEs appeared to be protected from changes in DNA methylation. In disturbed spermatogenesis, germ cells exhibited considerable DNA methylation changes, which were significantly enriched at transposable elements and genes involved in spermatogenesis. We detected hypomethylation in SVA and L1HS in disturbed spermatogenesis, suggesting an association between the abnormal programming of these regions and failure of germ cells progressing beyond meiosis.

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.

Destabilization of mRNAs enhances competence to initiate meiosis in mouse spermatogenic cells.

The specialized cell cycle of meiosis transforms diploid germ cells into haploid gametes. In mammals, diploid spermatogenic cells acquire the competence to initiate meiosis in response to retinoic acid. Previous mouse studies revealed that MEIOC interacts with RNA-binding proteins YTHDC2 and RBM46 to repress mitotic genes and to promote robust meiotic gene expression in spermatogenic cells that have initiated meiosis. Here, we have used the enhanced resolution of scRNA-seq and bulk RNA-seq of developmentally synchronized spermatogenesis to define how MEIOC molecularly supports early meiosis in spermatogenic cells. We demonstrate that MEIOC mediates transcriptomic changes before meiotic initiation, earlier than previously appreciated. MEIOC, acting with YTHDC2 and RBM46, destabilizes its mRNA targets, including the transcriptional repressors E2f6 and Mga, in mitotic spermatogonia. MEIOC thereby derepresses E2F6- and MGA-repressed genes, including Meiosin and other meiosis-associated genes. This confers on spermatogenic cells the molecular competence to, in response to retinoic acid, fully activate the transcriptional regulator STRA8-MEIOSIN, which is required for the meiotic G1/S phase transition and for meiotic gene expression. We conclude that, in mice, mRNA decay mediated by MEIOC-YTHDC2-RBM46 enhances the competence of spermatogenic cells to initiate meiosis.

Organogenetic transcriptomes of the Drosophila embryo at single cell resolution.

To gain insight into the transcription programs activated during the formation of Drosophila larval structures, we carried out single cell RNA sequencing during two periods of Drosophila embryogenesis: stages 10-12, when most organs are first specified and initiate morphological and physiological specialization; and stages 13-16, when organs achieve their final mature architectures and begin to function. Our data confirm previous findings with regards to functional specialization of some organs - the salivary gland and trachea - and clarify the embryonic functions of another - the plasmatocytes. We also identify two early developmental trajectories in germ cells and uncover a potential role for proteolysis during germline stem cell specialization. We identify the likely cell type of origin for key components of the Drosophila matrisome and several commonly used Drosophila embryonic cell culture lines. Finally, we compare our findings with other recent related studies and with other modalities for identifying tissue-specific gene expression patterns. These data provide a useful community resource for identifying many new players in tissue-specific morphogenesis and functional specialization of developing organs.

YTHDC2 serves a distinct late role in spermatocytes during germ cell differentiation.

Posttranscriptional regulation of gene expression by RNA-binding proteins can enhance the speed and robustness of cell state transitions by controlling RNA stability, localization, or if, when, or where mRNAs are translated. The RNA helicase YTHDC2 is required to shut down components of the mitotic program to facilitate a proper switch from mitosis to meiosis in mouse germ cells. Here, we show that YTHDC2 has a second essential role in promoting meiotic progression in late spermatocytes. Inducing conditional knockout of Ythdc2 during the first wave of spermatogenesis, after initiation of meiotic prophase, allowed YTHDC2-deficient germ cells to advance to the pachytene stage and properly express many meiotic markers. However, the YTHDC2-deficient spermatocytes mis-expressed a number of genes, some up-regulated and some down-regulated, failed to transition to the diplotene stage, and then quickly died. Coimmunoprecipitation experiments revealed that YTHDC2 interacts with several RNA-binding proteins in early or late spermatocytes, with many of the interacting proteins, including MEIOC, localizing to granules, similar to YTHDC2. Our findings suggest that YTHDC2 collaborates with other RNA granule components to facilitate proper progression of germ cells through multiple steps of meiosis via mechanisms influencing posttranscriptional regulation of RNAs.

Control of homologous recombination by the HROB-MCM8-MCM9 pathway.

DNA repair by homologous recombination (HR) is essential for genomic integrity, tumor suppression, and the formation of gametes. HR uses DNA synthesis to repair lesions such as DNA double-strand breaks and stalled DNA replication forks, but despite having a good understanding of the steps leading to homology search and strand invasion, we know much less of the mechanisms that establish recombination-associated DNA polymerization. Here, we report that C17orf53/HROB is an OB-fold-containing factor involved in HR that acts by recruiting the MCM8-MCM9 helicase to sites of DNA damage to promote DNA synthesis. Mice with targeted mutations in Hrob are infertile due to depletion of germ cells and display phenotypes consistent with a prophase I meiotic arrest. The HROB-MCM8-MCM9 pathway acts redundantly with the HELQ helicase, and cells lacking both HROB and HELQ have severely impaired HR, suggesting that they underpin two major routes for the completion of HR downstream from RAD51. The function of HROB in HR is reminiscent of that of gp59, which acts as the replicative helicase loader during bacteriophage T4 recombination-dependent DNA replication. We therefore propose that the loading of MCM8-MCM9 by HROB may similarly be a key step in the establishment of mammalian recombination-associated DNA synthesis.

Ways to unwind with HROB, a new player in homologous recombination.

Homologous recombination (HR) is an important route for repairing DNA double-strand breaks (DSBs). The early stages of HR are well understood, but later stages remain mysterious. In this issue of Genes & Development, Hustedt and colleagues (pp. 1397-1415) reveal HROB as a new player in HR required for recruitment of the MCM8-9 complex, which is paralogous to the MCM2-7 replicative helicase. HROB functions closely with MCM8-9 to promote postsynaptic DNA repair synthesis. This study sheds valuable light on late events in HR and suggests that HROB may load MCM8-9 onto HR intermediates to facilitate the DNA unwinding required for DNA repair synthesis.

P-body-like condensates in the germline.

P-bodies are cytoplasmic condensates that accumulate low-translation mRNAs for temporary storage before translation or degradation. P-bodies have been best characterized in yeast and mammalian tissue culture cells. We describe here related condensates in the germline of animal models. Germline P-bodies have been reported at all stages of germline development from primordial germ cells to gametes. The activity of the universal germ cell fate regulator, Nanos, is linked to the mRNA decay function of P-bodies, and spatially-regulated condensation of P-body like condensates in embryos is required to localize mRNA regulators to primordial germ cells. In most cases, however, it is not known whether P-bodies represent functional compartments or non-functional condensation by-products that arise when ribonucleoprotein complexes saturate the cytoplasm. We speculate that the ubiquity of P-body-like condensates in germ cells reflects the strong reliance of the germline on cytoplasmic, rather than nuclear, mechanisms of gene regulation.

The potential role of the mobile and non-coding genomes in adaptive response.

The tightly regulated feedback loops linking small RNAs (sRNAs) and transposable elements (TEs) offer the opportunity for an adaptive response to changing environments at the molecular level. Environmentally induced changes in TE and sRNA profiles may affect expression of coding genes and trigger an organismic and transgenerational response. Understanding this link may provide a mechanistic explanation for how species can adapt to changing climates and may offer novel molecular targets for biomedical and agricultural applications.

Nucleome programming is required for the foundation of totipotency in mammalian germline development.

Germ cells are unique in engendering totipotency, yet the mechanisms underlying this capacity remain elusive. Here, we perform comprehensive and in-depth nucleome analysis of mouse germ-cell development in vitro, encompassing pluripotent precursors, primordial germ cells (PGCs) before and after epigenetic reprogramming, and spermatogonia/spermatogonial stem cells (SSCs). Although epigenetic reprogramming, including genome-wide DNA de-methylation, creates broadly open chromatin with abundant enhancer-like signatures, the augmented chromatin insulation safeguards transcriptional fidelity. These insulatory constraints are then erased en masse for spermatogonial development. Notably, despite distinguishing epigenetic programming, including global DNA re-methylation, the PGCs-to-spermatogonia/SSCs development entails further euchromatization. This accompanies substantial erasure of lamina-associated domains, generating spermatogonia/SSCs with a minimal peripheral attachment of chromatin except for pericentromeres-an architecture conserved in primates. Accordingly, faulty nucleome maturation, including persistent insulation and improper euchromatization, leads to impaired spermatogenic potential. Given that PGCs after epigenetic reprogramming serve as oogenic progenitors as well, our findings elucidate a principle for the nucleome programming that creates gametogenic progenitors in both sexes, defining a basis for nuclear totipotency.

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.

Transgenerational Epigenetic Inheritance.

Inheritance of genomic DNA underlies the vast majority of biological inheritance, yet it has been clear for decades that additional epigenetic information can be passed on to future generations. Here, we review major model systems for transgenerational epigenetic inheritance via the germline in multicellular organisms. In addition to surveying examples of epivariation that may arise stochastically or in response to unknown stimuli, we also discuss the induction of heritable epigenetic changes by genetic or environmental perturbations. Mechanistically, we discuss the increasingly well-understood molecular pathways responsible for epigenetic inheritance, with a focus on the unusual features of the germline epigenome.