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Our learning about human reproductive development is greatly hampered due to the absence of an adequate model. Animal studies cannot truthfully recapitulate human developmental processes, and studies of human fetal tissues are limited by their availability and ethical restrictions. Innovative three-dimensional (3D) organoid technology utilizing human pluripotent stem cells (hPSCs) offered a new approach to study tissue and organ development in vitro. However, a system for modeling human gonad development has not been established, thus, limiting our ability to study causes of infertility.In our study we utilized the 3D hPSC organoid culture in mini-spin bioreactors. Relying on intrinsic self-organizing and differentiation capabilities of stem cells, we explored whether organoids could mimic the development of human embryonic and fetal gonad.We have developed a simple, bioreactor-based organoid system for modeling early human gonad development. Male hPSC-derived organoids follow the embryonic gonad developmental trajectory and differentiate into multipotent progenitors, which further specialize into testicular supporting and interstitial cells. We demonstrated functional activity of the generated cell types by analyzing the expression of cell type-specific markers. Furthermore, the specification of gonadal progenitors in organoid culture was accompanied by the characteristic architectural tissue organization.This organoid system opens the opportunity for detailed studies of human gonad and germ cell development that can advance our understanding of sex development disorders. Implementation of human gonad organoid technology could be extended to modeling causes of infertility and regenerative medicine applications.

Centrosomes are microtubule-organizing centers comprised of a pair of centrioles and the surrounding pericentriolar material. Abnormalities in centriole number are associated with cell division errors and can contribute to diseases such as cancer. Centriole duplication is limited to once per cell cycle and is controlled by the dosage-sensitive Polo-like kinase 4 (PLK4). Here, we show that PLK4 abundance is translationally controlled through conserved upstream open reading frames (uORFs) in the 5′ UTR of the mRNA. Plk4 uORFs suppress Plk4 translation and prevent excess protein synthesis. Mice with homozygous knockout of Plk4 uORFs (Plk4Δu/Δu) are viable but display dramatically reduced fertility because of a significant depletion of primordial germ cells (PGCs). The remaining PGCs in Plk4Δu/Δu mice contain extra centrioles and display evidence of increased mitotic errors. PGCs undergo hypertranscription and have substantially more Plk4 mRNA than somatic cells. Reducing Plk4 mRNA levels in mice lacking Plk4 uORFs restored PGC numbers and fully rescued fertility. Together, our data uncover a specific requirement for uORF-dependent control of PLK4 translation in counterbalancing the increased Plk4 transcription in PGCs. Thus, uORF-mediated translational suppression of PLK4 has a critical role in preventing centriole amplification and preserving the genomic integrity of future gametes.

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.

Chromosome movements and licensing of synapsis must be tightly regulated during early meiosis to ensure accurate chromosome segregation and avoid aneuploidy, although how these steps are coordinated is not fully understood. Here we show that GRAS-1, the worm homolog of mammalian GRASP/Tamalin and CYTIP, coordinates early meiotic events with cytoskeletal forces outside the nucleus. GRAS-1 localizes close to the nuclear envelope (NE) in early prophase I and interacts with NE and cytoskeleton proteins. Delayed homologous chromosome pairing, synaptonemal complex (SC) assembly, and DNA double-strand break repair progression are partially rescued by the expression of human CYTIP in gras-1 mutants, supporting functional conservation. However, Tamalin, Cytip double knockout mice do not exhibit obvious fertility or meiotic defects, suggesting evolutionary differences between mammals. gras-1 mutants show accelerated chromosome movement during early prophase I, implicating GRAS-1 in regulating chromosome dynamics. GRAS-1-mediated regulation of chromosome movement is DHC-1-dependent, placing it acting within the LINC-controlled pathway, and depends on GRAS-1 phosphorylation at a C-terminal S/T cluster. We propose that GRAS-1 serves as a scaffold for a multi-protein complex coordinating the early steps of homology search and licensing of SC assembly by regulating the pace of chromosome movement in early prophase I.

Homologous recombination (HR) is an essential meiotic process that contributes to the genetic variation of offspring and ensures accurate chromosome segregation. Recombination is facilitated by the formation and repair of programmed DNA double-strand breaks. These DNA breaks are repaired via recombination between maternal and paternal homologous chromosomes and a subset result in the formation of crossovers. HR and crossover formation is facilitated by synapsis of homologous chromosomes by a proteinaceous scaffold structure known as the synaptonemal complex (SC). Recent studies in yeast and worms have indicated that polo-like kinases (PLKs) regulate several events during meiosis, including DNA recombination and SC dynamics. Mammals express four active PLKs (PLK1-4), and our previous work assessing localization and kinase function in mouse spermatocytes suggested that PLK1 coordinates nuclear events during meiotic prophase. Therefore, we conditionally mutated

Background The SMC5/6 complex, cohesin and condensin are the three mammalian members of the structural maintenance of chromosomes (SMC) family, large ring-like protein complexes that are essential for genome maintenance. The SMC5/6 complex is the least characterized complex in mammals; however, it is known to be involved in homologous recombination repair (HRR) and chromosome segregation. Results In this study, a yeast two-hybrid screen was used to help elucidate novel interactions of the kleisin subunit of the SMC5/6 complex, NSMCE4A. This approach discovered an interaction between NSMCE4A and GPS1, a COP9 signalosome (CSN) component, and this interaction was further confirmed by co-immunoprecipitation. Additionally, GPS1 and components of SMC5/6 complex colocalize during interphase and mitosis. CSN is a cullin deNEDDylase and is an important factor for HRR. Depletion of GPS1, which has been shown to negatively impact DNA end resection during HRR, caused an increase in SMC5/6 levels at sites of laser-induced DNA damage. Furthermore, inhibition of the dennedylation function of CSN increased SMC5/6 levels at sites of laser-induced DNA damage. Conclusion Taken together, these data demonstrate for the first time that the SMC5/6 and CSN complexes interact and provides evidence that the CSN complex influences SMC5/6 functions during cell cycle progression and response to DNA damage.

The production of noncanonical mRNA transcripts is associated with cell transformation. Driven by our previous findings on the sensitivity of T cell acute lymphoblastic leukemia (T-ALL) cells to SF3B1 inhibitors, we identified that SF3B1 inhibition blocks T-ALL growth in vivo with no notable associated toxicity. We also revealed protein stabilization of the U2 complex component SF3B1 via deubiquitination. Our studies showed that SF3B1 inhibition perturbs exon skipping, leading to nonsense-mediated decay and diminished levels of DNA damage response–related transcripts, such as the serine/threonine kinase CHEK2, and impaired DNA damage response. We also identified that SF3B1 inhibition leads to a general decrease in R-loop formation. We further demonstrate that clinically used SF3B1 inhibitors synergize with CHEK2 inhibitors and chemotherapeutic drugs to block leukemia growth. Our study provides the proof of principle for posttranslational regulation of splicing components and associated roles and therapeutic implications for the U2 complex in T cell leukemia., Description, The splicing factor SF3B1 is regulated at the protein level and is a therapeutic target in T cell leukemia.

The establishment of bipolar spindles during meiotic divisions ensures faithful chromosome segregation to prevent gamete aneuploidy. We analyzed centriole duplication, as well as centrosome maturation and separation during meiosis I and II using mouse spermatocytes. The first round of centriole duplication occurs during early prophase I, and then, centrosomes mature and begin to separate by the end of prophase I to prime formation of bipolar metaphase I spindles. The second round of centriole duplication occurs at late anaphase I, and subsequently, centrosome separation coordinates bipolar segregation of sister chromatids during meiosis II. Using a germ cell‐specific conditional knockout strategy, we show that Polo‐like kinase 1 and Aurora A kinase are required for centrosome maturation and separation prior to metaphase I, leading to the formation of bipolar metaphase I spindles. Furthermore, we show that PLK1 is required to block the second round of centriole duplication and maturation until anaphase I. Our findings emphasize the importance of maintaining strict spatiotemporal control of cell cycle kinases during meiosis to ensure proficient centrosome biogenesis and, thus, accurate chromosome segregation during spermatogenesis.

Errors during meiotic resumption in oocytes can result in chromosome missegregation and infertility. Several cell cycle kinases have been linked with roles in coordinating events during meiotic resumption, including polo-like kinases (PLKs). Mammals express four kinase-proficient PLKs (PLK1–4). Previous studies assessing the role of PLK1 have relied on RNA knockdown and kinase inhibition approaches, as Plk1 null mutations are embryonically lethal. To further assess the roles of PLK1 during meiotic resumption, we developed a Plk1 conditional knockout (cKO) mouse to specifically mutate Plk1 in oocytes. Despite normal oocyte numbers and follicle maturation, Plk1 cKO mice were infertile. From analysis of meiotic resumption, Plk1 cKO oocytes underwent nuclear envelope breakdown with the same timing as control oocytes. However, Plk1 cKO oocytes failed to form compact bivalent chromosomes, and localization of cohesin and condensin were defective. Furthermore, Plk1 cKO oocytes either failed to organize α-tubulin or developed an abnormally small bipolar spindle. These abnormalities were attributed to aberrant release of the microtubule organizing center (MTOC) linker protein, C-NAP1, and the failure to recruit MTOC components and liquid-like spindle domain (LISD) factors. Ultimately, these defects result in meiosis I arrest before homologous chromosome segregation.

Precise control of chromosome dynamics during meiosis is critical for fertility. A gametocyte undergoing meiosis coordinates formation of the synaptonemal complex (SC) to promote efficient homologous chromosome recombination. Subsequent disassembly of the SC occurs prior to segregation of homologous chromosomes during meiosis I. We examined the requirements of the mammalian Aurora kinases (AURKA, AURKB and AURKC) during SC disassembly and chromosome segregation using a combination of chemical inhibition and gene deletion approaches. We find that both mouse and human spermatocytes fail to disassemble SC lateral elements when the kinase activity of AURKB and AURKC are chemically inhibited. Interestingly, both Aurkb conditional knockout and Aurkc knockout mouse spermatocytes successfully progress through meiosis, and the mice are fertile. In contrast, Aurkb, Aurkc double knockout spermatocytes fail to coordinate disassembly of SC lateral elements with chromosome condensation and segregation, resulting in delayed meiotic progression. In addition, deletion of Aurkb and Aurkc leads to an accumulation of metaphase spermatocytes, chromosome missegregation and aberrant cytokinesis. Collectively, our data demonstrate that AURKB and AURKC functionally compensate for one another ensuring successful mammalian spermatogenesis. This article has an associated First Person interview with the first author of the paper.

Mutations of SMC5/6 components cause developmental defects, including primary microcephaly. To model neurodevelopmental defects, we engineered a mouse wherein Smc5 is conditionally knocked out (cKO) in the developing neocortex. Smc5 cKO mice exhibited neurodevelopmental defects due to neural progenitor cell (NPC) apoptosis, which led to reduction in cortical layer neurons. Smc5 cKO NPCs formed DNA bridges during mitosis and underwent chromosome missegregation. SMC5/6 depletion triggers a CHEK2-p53 DNA damage response, as concomitant deletion of the Trp53 tumor suppressor or Chek2 DNA damage checkpoint kinase rescued Smc5 cKO neurodevelopmental defects. Further assessment using Smc5 cKO and auxin-inducible degron systems demonstrated that absence of SMC5/6 leads to DNA replication stress at late-replicating regions such as pericentromeric heterochromatin. In summary, SMC5/6 is important for completion of DNA replication prior to entering mitosis, which ensures accurate chromosome segregation. Thus, SMC5/6 functions are critical in highly proliferative stem cells during organism development.

Genetic recombination generates novel trait combinations, and understanding how recombination is distributed across the genome is key to modern genetics. The PRDM9 protein defines recombination hotspots; however, megabase-scale recombination patterning is independent of PRDM9. The single round of DNA replication, which precedes recombination in meiosis, may establish these patterns; therefore, we devised an approach to study meiotic replication that includes robust and sensitive mapping of replication origins. We find that meiotic DNA replication is distinct; reduced origin firing slows replication in meiosis, and a distinctive replication pattern in human males underlies the subtelomeric increase in recombination. We detected a robust correlation between replication and both contemporary and historical recombination and found that replication origin density coupled with chromosome size determines the recombination potential of individual chromosomes. Our findings and methods have implications for understanding the mechanisms underlying DNA replication, genetic recombination, and the landscape of mammalian germline variation.

The auxin-inducible degron (AID) system is becoming a widely used method for rapid and reversible degradation of target proteins. This system has been successfully used to study gene and protein functions in eukaryotic cells and common model organisms, such as nematode and fruit fly. To date, applications of the AID system in mammalian stem cell research are limited. Furthermore, standard mouse models harboring the AID system have not been established. Here we have explored the utility of the H11 safe-harbor locus for integration of the TIR1 transgene, an essential component of auxin-based protein degradation system. We have shown that the H11 locus can support constitutive and conditional TIR1 expression in mouse and human embryonic stem cells, as well as in mice. We demonstrate that the AID system can be successfully employed for rapid degradation of stable proteins in embryonic stem cells, which is crucial for investigation of protein functions in quickly changing environments, such as stem cell proliferation and differentiation. As embryonic stem cells possess unlimited proliferative capacity, differentiation potential, and can mimic organ development, we believe that these research tools will be an applicable resource to a broad scientific audience.

Summary:Genetic recombination generates novel trait combinations and understanding how recombination is distributed across the genome is key to modern genetics. The PRDM9 protein defines recombination hotspots, however megabase-scale recombination patterning is independent of PRDM9. The single round of DNA replication, which precedes recombination in meiosis, may establish these patterns, therefore we devised a novel approach to study meiotic replication that includes robust and sensitive mapping of replication origins. We find that meiotic DNA replication is distinct; reduced origin firing slows replication in meiosis and a distinctive replication pattern in human males underlies the sub-telomeric increase in recombination. We detected a robust correlation between replication and both contemporary and ancestral recombination and found that replication origin density coupled with chromosome size determines the recombination potential of individual chromosomes. Our findings and methods have far-reaching implications for understanding the mechanisms underlying DNA replication, genetic recombination, and the landscape of mammalian germline variation.

Precise control of chromosome dynamics during meiosis is critical for fertility. A gametocyte undergoing meiosis coordinates formation of the synaptonemal complex (SC) to promote efficient homologous chromosome recombination. Subsequent disassembly of the SC is required prior to meiotic divisions to ensure accurate segregation of chromosomes. We examined the requirements of the mammalian Aurora kinases (AURKA, B, and C) during SC disassembly and chromosome segregation using a combination of chemical inhibition and gene deletion approaches. We find that both mouse and human spermatocytes fail to disassemble SC lateral elements when AURKB and AURKC are inhibited. Interestingly, both Aurkb conditional knockout and Aurkc knockout spermatocytes successfully progress through meiosis and mice are fertile. In contrast, Aurkb, Aurkc double knockout spermatocytes failed to coordinate disassembly of SC lateral elements with chromosome segregation, resulting in delayed meiotic progression, spindle assembly checkpoint failure, chromosome missegregation, and abnormal spermatids. Collectively, our data demonstrates that AURKB and AURKC functionally compensate for one another ensuring successful mammalian spermatogenesis.SUMMARYChemical inhibition and gene deletion approaches show that Aurora B and Aurora C have overlapping functions that ensure timely disassembly of lateral element components of the synaptonemal complex in mouse and human spermatocytes and ensure accurate chromosome segregation during meiosis.

BACKGROUND: Human testicular cells are greatly valuable to the research community as tools for studying testicular physiology and the effects of environmental pollutants. Because adult testicular cells have a limited self-organization capacity and life span, we investigated whether human pluripotent stem cells (hPSCs) can be used together with testicular cells to move a step closer toward making an optimal model of the human testis. METHODS: We used in vitro culture of donor testicular cells under serum-containing and chemically defined conditions. CRISPR-Cas9 technology was applied to introduce fluorescent transgenes (mCherry2 and EGFP) into hPSCs and testicular cells. hPSC-derived spheroids were co-cultured with human testicular cells in mini-spin bioreactors. RESULTS: Traditional cell culture conditions used for maintenance of testicular somatic cells generally contain serum and pose limitations on evaluating the role of active molecules on cell functions. We established that chemically defined culture conditions can be used to maintain testicular cells without the loss of proliferative activity. These cultures demonstrate marker expression which is characteristic of common testicular cell types: Sertoli, Leydig, endothelial, myoid cells, and macrophages. In order to model testicular physiology, it is important to be able to perform live cell microscopy. Thus, we generated fluorescent protein-expressing human testicular cells and hPSCs and demonstrated that these cell types can be successfully co-cultured for prolonged periods of time in a three-dimensional microenvironment. CONCLUSION: Our research extends the possible applications of human testis-derived somatic cells and shows that they can be used together with hPSCs for further studies of human male reproductive biology. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13770-020-00240-0) contains supplementary material, which is available to authorized users.

During meiotic prophase, cohesin complexes mediate cohesion between sister chromatids and promote pairing and synapsis of homologous chromosomes. Precisely how the activity of cohesin is controlled to promote these events is not fully understood. In metazoans, cohesion establishment between sister chromatids during mitotic divisions is accompanied by recruitment of the cohesion-stabilizing protein Sororin. During somatic cell division cycles, Sororin is recruited in response to DNA replication-dependent modification of the cohesin complex by ESCO acetyltransferases. How Sororin is recruited and acts in meiosis is less clear. Here, we have surveyed the chromosomal localization of Sororin and its relationship to the meiotic cohesins and other chromatin modifiers with the objective of determining how Sororin contributes to meiotic chromosome dynamics. We show that Sororin localizes to the cores of meiotic chromosomes in a manner that is dependent on synapsis and the synaptonemal complex protein SYCP1. In contrast, cohesin, with which Sororin interacts in mitotic cells, shows axial enrichment on meiotic chromosomes even in the absence of synapsis between homologs. Using high-resolution microscopy, we show that Sororin is localized to the central region of the synaptonemal complex. These results indicate that Sororin regulation during meiosis is distinct from its regulation in mitotic cells and may suggest that it interacts with a distinctly different partner to ensure proper chromosome dynamics in meiosis.

Cohesin is an essential structural component of chromosomes that ensures accurate chromosome segregation during mitosis and meiosis. Previous studies have shown that there are cohesin complexes specific to meiosis, required to mediate homologous chromosome pairing, synapsis, recombination, and segregation. Meiosis-specific cohesin complexes consist of two structural maintenance of chromosomes proteins (SMC1α/SMC1β and SMC3), an α-kleisin protein (RAD21, RAD21L, or REC8), and a stromal antigen protein (STAG1, 2, or 3). STAG3 is exclusively expressed during meiosis, and is the predominant STAG protein component of cohesin complexes in primary spermatocytes from mouse, interacting directly with each α-kleisin subunit. REC8 and RAD21L are also meiosis-specific cohesin components. Stag3 mutant spermatocytes arrest in early prophase (“zygotene-like” stage), displaying failed homolog synapsis and persistent DNA damage, as a result of unstable loading of cohesin onto the chromosome axes. Interestingly, Rec8, Rad21L double mutants resulted in an earlier “leptotene-like” arrest, accompanied by complete absence of STAG3 loading. To assess genetic interactions between STAG3 and α-kleisin subunits RAD21L and REC8, our lab generated Stag3, Rad21L, and Stag3, Rec8 double knockout mice, and compared them to the Rec8, Rad21L double mutant. These double mutants are phenotypically distinct from one another, and more severe than each single knockout mutant with regards to chromosome axis formation, cohesin loading, and sister chromatid cohesion. The Stag3, Rad21L, and Stag3, Rec8 double mutants both progress further into prophase I than the Rec8, Rad21L double mutant. Our genetic analysis demonstrates that cohesins containing STAG3 and REC8 are the main complex required for centromeric cohesion, and RAD21L cohesins are required for normal clustering of pericentromeric heterochromatin. Furthermore, the STAG3/REC8 and STAG3/RAD21L cohesins are the primary cohesins required for axis formation.

The structural maintenance of chromosomes (SMC) complex, SMC5/6, is important for genome maintenance in all model eukaryotes. To date, the most extensive studies have focused on the roles of Smc5/6 in lower eukaryotes, such as yeast and fly. In the handful of studies that have used mammalian cells, siRNA was used by most to knockdown SMC5/6 components. RNAi methods have been very important for scientific progression, but they are hindered by incomplete silencing of protein expression and off-target effects. This chapter outlines the use of a conditional knockout approach in mouse embryonic fibroblasts to study the function of the SMC5/6 complex. These cell lines provide an alternative method to study the function and properties of the SMC5/6 complex in mammals.

Male factor infertility is a complex issue presenting many diagnostic and management challenges. It is responsible for about 50% of all causes of infertility and thus carries significant medical, financial, and psychological implications for the couples struggling with conception. Klinefelter syndrome is the most common chromosomal male anomaly associated with male infertility. This review focuses specifically on non-obstructive azoospermia secondary to Klinefelter syndrome and discusses controversies surrounding fertility management in patients with this genetic disorder.

Lifelong mammalian male fertility is maintained through an intricate balance between spermatogonial proliferation and differentiation. DNA damage in spermatogonia, for instance caused by chemo- or radiotherapy, can induce cell cycle arrest or germ cell apoptosis, possibly resulting in male infertility. Spermatogonia are generally more radiosensitive and prone to undergo apoptosis than somatic cells. Among spermatogonial sub-types the response to DNA damage is differentially modulated; undifferentiated spermatogonia, including the spermatogonial stem cells (SSCs), are relatively radio-resistant, whereas differentiating spermatogonia are very radiosensitive. To investigate the molecular mechanisms underlying this difference, we used an in vitro system consisting of mouse male germline stem (GS) cells that can be induced to differentiate. Using RNA-sequencing analysis, we analyzed the response of undifferentiated and differentiating GS cells to ionizing radiation (IR). At the RNA expression level, both undifferentiated and differentiating GS cells showed a very similar response to IR. Protein localization of several genes found to be involved in either spermatogonial differentiation or radiation response was investigated using mouse testis sections. For instance, we found that the transcription factor PDX1 was specifically expressed in undifferentiated spermatogonia and thus may be a novel marker for these cells. Interestingly, also at the protein level, undifferentiated GS cells showed a more pronounced upregulation of p53 in response to IR than differentiating GS cells. The higher p53 protein level in undifferentiated spermatogonia may preferentially induce cell cycle arrest, thereby giving these cells more time to repair inflicted DNA damage and increase their radio-resistance.

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during meiosis can lead to infertility, pregnancy loss or genetic defects. Commencing at the onset of puberty and continuing throughout adulthood, continuous semi-synchronous waves of spermatocytes undergo spermatogenesis and ultimately form haploid sperm. The first wave of mouse spermatocytes undergoing meiotic initiation appear at day 10 post-partum (10 dpp) and are released into the lumen of seminiferous tubules as mature sperm at 35 dpp. Therefore, it is advantageous to utilize mice within this developmental time-window in order to obtain highly enriched populations of interest. Analysis of rare cell stages is more difficult in older mice due to the contribution of successive spermatogenic waves, which increase the diversity of the cellular populations within the tubules. The method described here is an easily implemented technique for the cytological evaluation of the cells found within the seminiferous tubules of mice, including spermatogonia, spermatocytes, and spermatids. The tubule squash technique maintains the integrity of isolated male germ cells and allows examination of cellular structures that are not easily visualized with other techniques. To demonstrate the possible applications of this tubule squash technique, spindle assembly was monitored in spermatocytes progressing through the prophase to metaphase I transition (G2/MI transition). In addition, centrosome duplication, meiotic sex chromosome inactivation (MSCI), and chromosome bouquet formation were assessed as examples of the cytological structures that can be observed using this tubule squash method. This technique can be used to pinpoint specific defects during spermatogenesis that are caused by mutation or exogenous perturbation, and thus, contributes to our molecular understanding of spermatogenesis.

The Smc5/6 complex, along with cohesin and condensin, is a member of the structural maintenance of chromosome (SMC) family, large ring-like protein complexes that are essential for chromatin structure and function. Thanks to numerous studies of the mitotic cell cycle, Smc5/6 has been implicated to have roles in homologous recombination, restart of stalled replication forks, maintenance of ribosomal DNA (rDNA) and heterochromatin, telomerase-independent telomere elongation, and regulation of chromosome topology. The nature of these functions implies that the Smc5/6 complex also contributes to the profound chromatin changes, including meiotic recombination, that characterize meiosis. Only recently, studies in diverse model organisms have focused on the potential meiotic roles of the Smc5/6 complex. Indeed, Smc5/6 appears to be essential for meiotic recombination. However, due to both the complexity of the process of meiosis and the versatility of the Smc5/6 complex, many additional meiotic functions have been described. In this review, we provide a clear overview of the multiple functions found so far for the Smc5/6 complex in meiosis. Additionally, we compare these meiotic functions with the known mitotic functions in an attempt to find a common denominator and thereby create clarity in the field of Smc5/6 research.

The structural maintenance of chromosomes complex SMC5/6 is thought to be essential for DNA repair and chromosome segregation during mitosis and meiosis. To determine the requirements of the SMC5/6 complex during mouse spermatogenesis we combined a conditional knockout allele for Smc5, with four germ cell–specific Cre-recombinase transgenes, Ddx4-Cre, Stra8-Cre, Spo11-Cre, and Hspa2-Cre, to mutate Smc5 in spermatogonia, in spermatocytes before meiotic entry, during early meiotic stages, and during midmeiotic stages, respectively. Conditional mutation of Smc5 resulted in destabilization of the SMC5/6 complex. Despite this, we observed only mild defects in spermatogenesis. Mutation of Smc5 mediated by Ddx4-Cre and Stra8-Cre resulted in partial loss of preleptotene spermatocytes; however, spermatogenesis progresses and mice are fertile. Mutation of Smc5 via Spo11-Cre or Hspa2-Cre did not result in detectable defects of spermatogenesis. Upon exposure to gamma irradiation or etoposide treatment, each conditional Smc5 mutant demonstrated an increase in the number of enlarged round spermatids with multiple acrosomes and supernumerary chromosome content. We propose that the SMC5/6 complex is not acutely required for premeiotic DNA replication and meiotic progression during mouse spermatogenesis; however, when germ cells are challenged by exogenous DNA damage, the SMC5/6 complex ensures genome integrity, and thus, fertility.

SMC complexes include three major classes: cohesin, condensin, and SMC5/6. However, the localization pattern and genetic requirements for the SMC5/6 complex during mammalian oogenesis had not previously been examined. In mouse oocytes, the SMC5/6 complex is enriched at the pericentromeric heterochromatin, and also localizes along chromosome arms during meiosis. The infertility phenotypes of females with a Zp3-Cre-driven conditional knockout (cKO) of Smc5 demonstrated that maternally expressed SMC5 protein is essential for early embryogenesis. Interestingly, protein levels of SMC5/6 complex components in oocytes decline as wild-type females age. When SMC5/6 complexes were completely absent in oocytes during meiotic resumption, homologous chromosomes failed to segregate accurately during meiosis I. Despite what appears to be an inability to resolve concatenation between chromosomes during meiosis, localization of topoisomerase II alpha to bivalents was not affected; however, localization of condensin along the chromosome axes was perturbed. Taken together, these data demonstrate that the SMC5/6 complex is essential for the formation of segregation-competent bivalents during meiosis I, and findings suggest that age-dependent depletion of the SMC5/6 complex in oocytes could contribute to increased incidence of oocyte aneuploidy and spontaneous abortion in aging females.

Background There are several differences associated with the behaviour of the four main experimental Neisseria gonorrhoeae strains, FA1090, FA19, MS11, and F62. Although there is data concerning the gene complements of these strains, the reasons for the behavioural differences are currently unknown. Phase variation is a mechanism that occurs commonly within the Neisseria spp. and leads to switching of genes ON and OFF. This mechanism may provide a means for strains to express different combinations of genes, and differences in the strain-specific repertoire of phase variable genes may underlie the strain differences. Results By genome comparison of the four publicly available neisserial genomes a revised list of 64 genes was created that have the potential to be phase variable in N. gonorrhoeae, excluding the opa and pilC genes. Amplification and sequencing of the repeat-containing regions of these genes allowed determination of the presence of the potentially unstable repeats and the ON/OFF expression state of these genes. 35 of the 64 genes show differences in the composition or length of the repeats, of which 28 are likely to be associated with phase variation. Two genes were expressed differentially between strains causing disseminated infection and uncomplicated gonorrhoea. Further study of one of these in a range of clinical isolates showed this association to be due to sample size and is not maintained in a larger sample. Conclusion The results provide us with more evidence as to which genes identified through comparative genomics are indeed phase variable. The study indicates that there are large differences between these four N. gonorrhoeae strains in terms of gene expression during in vitro growth. It does not, however, identify any clear patterns by which previously reported behavioural differences can be correlated with the phase variable gene repertoire.

Neisseria meningitidis, a causative agent of bacterial meningitis, has a relatively small repertoire of transcription factors, including NMB0573 (annotated AsnC), a member of the Lrp-AsnC family of regulators that are widely expressed in both Bacteria and Archaea. In the present study we show that NMB0573 binds to l-leucine and l-methionine and have solved the structure of the protein with and without bound amino acids. This has shown, for the first time that amino acid binding does not induce significant conformational changes in the structure of an AsnC/Lrp regulator although it does appear to stabilize the octameric assembly of the protein. Transcriptional profiling of wild-type and NMB0573 knock-out strains of N. meningitidis has shown that NMB0573 is associated with an adaptive response to nutrient poor conditions reflected in a reduction in major surface protein expression. On the basis of its structure and the transcriptional response, we propose that NMB0573 is a global regulator in Neisseria controlling responses to nutrient availability through indicators of general amino acid abundance: leucine and methionine.

Correct duplication of stem cell genetic material and its appropriate segregation into daughter cells are requisites for tissue, organ and organism homeostasis. Disruption of stem cell genomic integrity can lead to developmental abnormalities and cancer. Roles of the Smc5/6 structural maintenance of chromosomes complex in pluripotent stem cell genome maintenance have not been investigated, despite its important roles in DNA synthesis, DNA repair and chromosome segregation evaluated in other model systems. Using mouse embryonic stem cells with a conditional knockout allele of Smc5, we showed that Smc5 protein depletion resulted in destabilization of the Smc5/6 complex, accumulation of cells in G2 phase of the cell cycle and apoptosis. Detailed assessment of mitotic mESCs revealed abnormal condensin distribution and perturbed chromosome segregation, accompanied by irregular spindle morphology, lagging chromosomes and DNA bridges. Mutation of Smc5 resulted in retention of Aurora B kinase and enrichment of condensin on chromosome arms. Furthermore, we observed reduced levels of Polo-like kinase 1 at kinetochores during mitosis. Our study reveals crucial requirements of the Smc5/6 complex during cell cycle progression and for stem cell genome maintenance.

Crossing over establishes connections between homologous chromosomes that promote their proper segregation at the first meiotic division. However, there exists a backup system to ensure the correct segregation of those chromosome pairs that fail to cross over. We have found that, in budding yeast, a mutation eliminating the synaptonemal complex protein, Zip1, increases the meiosis I nondisjunction rate of nonexchange chromosomes (NECs). The centromeres of NECs become tethered during meiotic prophase, and this tethering is disrupted by the zip1 mutation. Furthermore, the Zip1 protein often colocalizes to the centromeres of the tethered chromosomes, suggesting that Zip1 plays a direct role in holding NECs together. Zip3, a protein involved in the initiation of synaptonemal complex formation, is also important for NEC segregation. In the absence of Zip3, both the tethering of NECs and the localization of Zip1 to centromeres are impaired. A mutation in the MAD3 gene, which encodes a component of the spindle checkpoint, also increases the nondisjunction of NECs. Together, the zip1 and mad3 mutations have an additive effect, suggesting that these proteins act in parallel pathways to promote NEC segregation. We propose that Mad3 promotes the segregation of NECs that are not tethered by Zip1 at their centromeres.

Microsatellites are powerful molecular genetic markers for many evolutionary and biotechnological investigations, however, development of sufficient microsatellite markers is time-consuming and expensive especially considering the vast numbers of species for which they could be used. In light of the conservative nature of microsatellite loci between related species we describe an alternative approach to microsatellite development. A single round of microsatellite isolation enabled the characterization of sufficient loci for a large number of related python species. From 21 loci isolated in the focal species, an average of 86.2% were conserved within the other species while an average of 60.5% were polymorphic in all 13 python species analysed. Our approach will decrease significantly the expense and time required for microsatellite development for large numbers of related species.

Genome integrity is crucial for safe reproduction. Therefore, chromatin structure and dynamics should be tightly regulated during germ cell development. Chromatin structure and function are in large part determined by the structural maintenance of chromosomes (SMC) protein complexes, of which SMC5/6 recently has been shown to be involved in both spermatogonial differentiation and meiosis during mouse spermatogenesis. We therefore investigated the role of this complex in human spermatogenesis. We found SMC6 to be expressed in the human testis and present in a subset of type Adark and type Apale spermatogonia, all spermatocytes, and round spermatids. During human meiosis, SMC5/6 is located at the synaptonemal complex (SC), the XY body, and at the centromeres during meiotic metaphases. However, in contrast to mouse spermatogenesis, SMC6 is not located at pericentromeric heterochromatin in human spermatogenic cells, indicating subtle but perhaps important differences in not only SMC5/6 function but maybe also in maintenance of genomic integrity at the repetitive pericentromeric regions. Nonetheless, our data clearly indicate that the SMC5/6 complex, as shown in mice, is involved in numerous crucial processes during human spermatogenesis, such as in spermatogonial development, on the SC between synapsed chromosomes, and in DNA double-strand break repair on unsynapsed chromosomes during pachynema.

During meiosis, Structural Maintenance of Chromosome (SMC) complexes underpin two fundamental features of meiosis: homologous recombination and chromosome segregation. While meiotic functions of the cohesin and condensin complexes have been delineated, the role of the third SMC complex, Smc5/6, remains enigmatic. Here we identify specific, essential meiotic functions for the Smc5/6 complex in homologous recombination and the regulation of cohesin. We show that Smc5/6 is enriched at centromeres and cohesin-association sites where it regulates sister-chromatid cohesion and the timely removal of cohesin from chromosomal arms, respectively. Smc5/6 also localizes to recombination hotspots, where it promotes normal formation and resolution of a subset of joint-molecule intermediates. In this regard, Smc5/6 functions independently of the major crossover pathway defined by the MutLγ complex. Furthermore, we show that Smc5/6 is required for stable chromosomal localization of the XPF-family endonuclease, Mus81-Mms4Eme1. Our data suggest that the Smc5/6 complex is required for specific recombination and chromosomal processes throughout meiosis and that in its absence, attempts at cell division with unresolved joint molecules and residual cohesin lead to severe recombination-induced meiotic catastrophe., Author Summary Meiosis is a specialized cell division that exactly halves the number of chromosomes transmitted from each parent to their offspring via gamete cells (such as sperm and eggs). This requires that matching (‘homologous’) chromosomes associate and then separate into different cells such that each gamete contains exactly one complete set of chromosomes. In many organisms, this sequence of events is facilitated by the induction and repair of chromosome breaks via a process called homologous recombination. As homologous chromosomes engage in recombination, matching DNA strands between broken and intact template chromosomes become intertwined in repair intermediates called Joint Molecules. In this study, we show that a highly conserved protein complex called the Structural Maintenance of Chromosomes 5/6 (Smc5/6) complex is important for regulating the choice of recombination template as well as for the resolution of Joint Molecules that is required for chromosomes to separate. Even though Joint Molecules remain unresolved in mutants that lack normal Smc5/6 function, cells still attempt to separate chromosomes and meiosis becomes catastrophic. Thus, Smc5/6 mutants have a two-fold defect: accumulation of unresolved Joint Molecules and a failure to stall meiosis in order to remove these structures.

Cells coordinate spindle formation with DNA repair and morphological modifications to chromosomes prior to their segregation to prevent cell division with damaged chromosomes. Here we uncover a novel and unexpected role for Aurora kinase in preventing the formation of spindles by Clb5-CDK (S-CDK) during meiotic prophase I and when the DDR is active in budding yeast. This is critical since S-CDK is essential for replication during premeiotic S-phase as well as double-strand break induction that facilitates meiotic recombination and, ultimately, chromosome segregation. Furthermore, we find that depletion of Cdc5 polo kinase activity delays spindle formation in DDR-arrested cells and that ectopic expression of Cdc5 in prophase I enhances spindle formation, when Ipl1 is depleted. Our findings establish a new paradigm for Aurora kinase function in both negative and positive regulation of spindle dynamics.

et al., Four members of the structural maintenance of chromosome (SMC) protein family have essential functions in chromosome condensation (SMC2/4) and sister-chromatid cohesion (SMC1/3). The SMC5/6 complex has been implicated in chromosome replication, DNA repair and chromosome segregation in somatic cells, but its possible functions during mammalian meiosis are unknown. Here, we show in mouse spermatocytes that SMC5 and SMC6 are located at the central region of the synaptonemal complex from zygotene until diplotene. During late diplotene both proteins load to the chromocenters, where they colocalize with DNA Topoisomerase IIα, and then accumulate at the inner domain of the centromeres during the first and second meiotic divisions. Interestingly, SMC6 and DNA Topoisomerase IIα colocalize at stretched strands that join kinetochores during the metaphase II to anaphase II transition, and both are observed on stretched lagging chromosomes at anaphase II following treatment with Etoposide. During mitosis, SMC6 and DNA Topoisomerase IIα colocalize at the centromeres and chromatid axes. Our results are consistent with the participation of SMC5 and SMC6 in homologous chromosome synapsis during prophase I, chromosome and centromere structure during meiosis I and mitosis and, with DNA Topoisomerase IIα, in regulating centromere cohesion during meiosis II., This work was supported by Ministerio de Economía y Competitividad (Spain) [grant number SAF2011-28842-C02-01 to J.A.S. and SAF2011-25252 to A.M.P.]; a UK-US Fulbright Distinguished Scholar Award; the US National Institutes of Health [grant number HD069458 to P.W.J.; HD33816 to M.A.H.; HD42137 to John Eppig, M.A.H. and J.C.S.]; the Priority Program SPP 1384 ‘Mechanisms of genome haploidization’ (to M.A. and R.J.) from the German Science Foundation.

During meiosis, accurate coordination of the completion of homologous recombination and synaptonemal complex (SC) disassembly during the prophase to metaphase I (G2/MI) transition is essential to avoid aneuploid gametes and infertility. Previous studies have shown that kinase activity is required to promote meiotic prophase exit. The first step of the G2/MI transition is the disassembly of the central element components of the SC, however the kinase(s) required to trigger this process remains unknown. Here we assess roles of polo-like kinases (PLKs) in mouse spermatocytes, both in vivo and during prophase exit induced ex vivo by the phosphate inhibitor okadaic acid (OA). All four PLKs are expressed during the first wave of spermatogenesis. Only PLK1 (not PLK2-4) localizes to the SC during the G2/MI transition. The SC central element proteins SYCP1, TEX12 and SYCE1 are phosphorylated during the G2/MI transition. However, treatment of pachytene spermatocytes with the PLK inhibitor BI 2536 prevented the OA-induced meiotic prophase exit and inhibited phosphorylation of the central element proteins as well as their removal from the SC. Phosphorylation assays in vitro demonstrated that PLK1, but not PLK2-4, phosphorylates central element proteins SYCP1 and TEX12. These findings provide mechanistic details of the first stage of SC disassembly in mammalian spermatocytes, and reveal that PLK-mediated phosphorylation of central element proteins is required for meiotic prophase exit.

Several protein kinases collaborate to orchestrate and integrate cellular and chromosomal events at the G2/M transition in both mitotic and meiotic cells. During the G2/M transition in meiosis, this includes the completion of crossover recombination, spindle formation, and synaptonemal complex (SC) breakdown. We identified Ipl1/Aurora B kinase as the main regulator of SC disassembly. Mutants lacking Ipl1 or its kinase activity assemble SCs with normal timing, but fail to dissociate the central element component Zip1, as well as its binding partner, Smt3/SUMO, from chromosomes in a timely fashion. Moreover, lack of Ipl1 activity causes delayed SC disassembly in a cdc5 as well as a CDC5-inducible ndt80 mutant. Crossover levels in the ipl1 mutant are similar to those observed in wild type, indicating that full SC disassembly is not a prerequisite for joint molecule resolution and subsequent crossover formation. Moreover, expression of meiosis I and meiosis II-specific B-type cyclins occur normally in ipl1 mutants, despite delayed formation of anaphase I spindles. These observations suggest that Ipl1 coordinates changes to meiotic chromosome structure with resolution of crossovers and cell cycle progression at the end of meiotic prophase.

Following pre-meiotic DNA replication, homologous chromosomes must be paired and become tightly linked to ensure reductional segregation during meiosis I. Therefore initiation of homologous chromosome pairing is vital for meiosis to proceed correctly. A number of factors contribute to the initiation of homologous chromosome pairing including telomere and centromere dynamics, pairing centres, checkpoint proteins and components of the axial element. The present review briefly summarizes recent progress in our understanding of initiation of homologous chromosome pairing during meiosis and discusses the differences that are observed between research organisms.

Background: Tandem repeats contained within coding regions can mediate phase variation when the repeated units change the reading frame of the coding sequence in a copy number dependent manner. Coding tandem repeats are those which do not alter the reading frame with copy number, and the changes in copy number of these repeats may then potentially alter the function or antigenicity of the protein encoded. Three complete neisserial genomes were analyzed and compared to identify coding tandem repeats where the number of copies of the repeat will have some structural consequence for the protein. This is the first study to address coding tandem repeats that may affect protein structures using comparative genomics, combined with a population survey to investigate which show interstrain variability. Results: A total of 28 genes were identified. Of these, 22 contain coding tandem repeats that vary in copy number between the three sequenced strains, three strain specific genes were included for investigation on the basis of having >90% identity between repeated units, and three genes with repeated elements of >250 bp were included although no length variations were seen in the genomes. Amplification, and sequencing of repeats showing altered copy number, of these 28 coding tandem repeat containing regions, from a set of largely unrelated strains, revealed further repeat length variation in several cases. Conclusion: Eighteen genes were identified which have variation in repeat copy number between strains of the same species, twelve of which show greater diversity in repeat copy number than is present in the sequenced genomes. In some cases, this may reflect a mechanism for the generation of antigenic variation, as previously described in other species. However, some of the genes identified encode proteins with cytoplasmic functions, including sugar metabolism, DNA repair, and protein production, in which repeat length variation may have other functions. Coding tandem repeats appear to represent a largely unexplored mechanism of generating diversity in the Neisseria spp.