Component Of Synaptonemal Complex Research Articles

Proximity labeling reveals new functional relationships between meiotic recombination proteins in S. cerevisiae.

Several protein ensembles facilitate crossover recombination and the associated assembly of synaptonemal complex (SC) during meiosis. In yeast, meiosis-specific factors including the DNA helicase Mer3, the "ZZS" complex consisting of Zip4, Zip2, and Spo16, the RING-domain protein Zip3, and the MutSγ heterodimer collaborate with crossover-promoting activity of the SC component, Zip1, to generate crossover-designated recombination intermediates. These ensembles also promote SC formation - the organized assembly of Zip1 with other structural proteins between aligned chromosome axes. We used proximity labeling to investigate spatial relationships between meiotic recombination and SC proteins in S. cerevisiae. We find that recombination initiation and SC factors are dispensable for proximity labeling of Zip3 by ZZS components, but proteins associated with early steps in recombination are required for Zip3 proximity labeling by MutSγ, suggesting that MutSγ joins Zip3 only after a recombination intermediate has been generated. We also find that zip1 separation-of-function mutants that are crossover deficient but still assemble SC fail to generate protein ensembles where Zip3 can engage ZZS and/or MutSγ. The SC structural protein Ecm11 is proximity labeled by ZZS proteins in a Zip4-dependent and Zip1-independent manner, but labeling of Ecm11 by Zip3 and MutSγ requires, at least in part, Zip1. Finally, mass spectrometry analysis of biotinylated proteins in eleven proximity labeling strains uncovered shared proximity targets of SC and crossover-associated proteins, some of which have not previously been implicated in meiotic recombination or SC formation, highlighting the potential of proximity labeling as a discovery tool.

Synaptonemal complex protects double-Holliday junctions during meiosis.

Chromosomal linkages formed through crossover recombination are essential for accurate segregation of homologous chromosomes during meiosis1. DNA events of recombination are spatially and functionally linked to structural components of meiotic chromosomes2. Imperatively, biased resolution of double-Holliday junction (dHJ) intermediates into crossovers3,4 occurs within the synaptonemal complex (SC), the meiosis-specific structure that mediates homolog synapsis during the pachytene stage5,6. However, the SC's role in crossing over remains unclear. Here we show that SC promotes crossover-specific resolution by protecting dHJs from unscheduled and aberrant resolution. When key SC components are conditionally inactivated during pachytene, dHJs are resolved into noncrossover products by Sgs1-Top3-Rmi1 (STR), the yeast ortholog of the human BLM complex7. Cohesin, the core component of SC lateral elements, plays a primary role in chromatin organization and is required to maintain both SCs and crossover recombination complexes (CRCs) during pachytene. SC central region component Zip1 is required to maintain CRCs even when dHJs are stabilized by inactivating STR. Reciprocally, SC stability requires continuous presence of CRCs, an unanticipated interdependence with important implications for SC dynamics. In conclusion, through hierarchical and interdependent functions of its key components, the SC enables crossover-specific dHJ resolution and thereby ensures the linkage and segregation of homologous chromosomes.

P-045 Deciphering meiotic chromatin organization by SCP3

Abstract Study question What’s the pattern of genome-wide chromatin occupancy of SCP3 during meiotic prophase, and how its interaction with chromatin is regulated? Summary answer We document SCP3 enriched at active chromatin regions with largely inherited from leptotene to pachytene stage, which facilitated by transcription and condensate assembly in spermatocytes. What is known already Meiosis is a critical step during the process of gametogenesis to form haploid cells which finally develop into functional gametes. The synaptonemal complex (SC) is a meiosis-specific protein assembly which bridges homologous chromosomes to establish the architecture of meiotic chromosome. Genetic ablation of genes encoding SC components impedes meiosis and gametogenesis in mice and mutation of these genes is frequently found in infertile patients. SCP3 localization changes with chromosome morphology in autosomes and is involved in maintaining the association of sex chromosomes. Male mice with null mutation of SCP3 failed to form SCs, resulting in developmental arrest during meiotic prophase. Study design, size, duration This study included male mice at post-natal day 10 (P10), 4 weeks or adult (8 weeks or older) were maintained on C57BL/6J background. Spermatocytes were performed modified CUT&Tag using antibodies. ChIP-seq data was analyzed together with WGBS and ATAC-seq data. Participants/materials, setting, methods Spermatocytes were obtained by a discontinuous BSA density gradient. Cell pellet was resuspended in culture medium (MEM with 10% FBS) at a concentration of 105 cells/ml and cultured at 37 °C in a 5% CO2 incubator. Spermatocytes were cultured with or without 1,6-hexanediol or α-amanitin. Spermatocytes with or without drug treatment were collected at indicated time points for further examination and then modified CUT&Tag and co-IP using anti-SCP3 and anti-POL II antibodies. Main results and the role of chance Chromatin occupancy of SCP3 in leptotene and pachytene spermatocytes, SCP3-Core sites retained upon treatment of 1,6-hexanediol which dissolves SCP3 condensate, and SCP1 occupancy at meiotic chromatin in pachytene spermatocytes. When we determined inter-peak distances and width of peaks, we observe that: (1) SCP3 occupancy in leptotene stage had greater inter-peak distances and narrower peak width than SCP3 occupancy in pachytene stage, demonstrating gradual accumulated SCP3 at chromatin during meiotic progression; (2) SCP3-Core sites have broader peak width than SCP3-NonCore sites, in agreement with stability of SCP3 occupancy at SCP3-Core sites; (3) SCP1 occupancy had greater inter-peak distances and narrower peak width than SCP3 occupancy in pachytene stage, supporting that SCP1 peaks are a subset of SCP3 peaks and SCP1’s major function in connecting LE and CE respectively. Generally, at pachytene-stage meiosis, SCP3 mainly occupies chromatin regions including transcriptionally active sites, Deu and Alu elements, while SCP1 mainly co-localizes with SCP3 at a subset of regions with open chromatin and high cohesin enrichment to connect meiotic chromosome axes with CE. In this way, a local active chromatin environment is created at meiotic chromosome axes, facilitating homologous chromosome recombination. Limitations, reasons for caution Our test was not successful to examine SCP3 occupancy with as few as less than 1000 spermatocytes using our protocol. It is improvement of efficiency of tagmentation or development of more efficient antibodies against SCP3 will be helpful for clinical sample detection. Wider implications of the findings SCP3 occupancy was largely inherited from leptotene to pachytene stage, facilitated by transcription and condensate assembly, and enriched at specific SINE repeats. Further exploration using knockout mouse models of meiotic axial proteins and epigenetic modifiers will provide valuable information on meiotic chromatin architecture and crosstalk among meiotic chromatin events. Trial registration number N/A

Induction of Meiosis from Human Pluripotent Stem Cells.

An in vitro model of human meiosis would accelerate research into this important reproductive process and development of therapies for infertility. We have developed a method to induce meiosis starting from male or female human pluripotent stem cells. We demonstrate that DNMT1 inhibition, retinoid signaling activation, and overexpression of regulatory factors (anti-apoptotic BCL2, and pro-meiotic HOXB5, BOLL, or MEIOC) rapidly activates meiosis, with leptonema beginning at 6 days, zygonema at 9 days, and pachynema at 12 days. Immunofluorescence microscopy shows key aspects of meiosis, including chromosome synapsis and sex body formation. The meiotic cells express genes similar to meiotic oogonia in vivo, including all synaptonemal complex components and machinery for meiotic recombination. These findings establish an accessible system for inducing human meiosis in vitro.

Multiple reorganizations of the lateral elements of the synaptonemal complex facilitate homolog segregation in Bombyx mori oocytes.

Although Lepidopteran females build a synaptonemal complex (SC) in pachytene, homologs do not crossover, necessitating an alternative method of homolog conjunction. In Bombyx mori oocytes, the SC breaks down at the end of pachytene, and homolog associations are maintained by a large oocyte-specific structure, which we call the bivalent bridge (BB), connecting paired homologs. The BB is derived from at least some components of the SC lateral elements (LEs). It contains the HORMAD protein HOP1 and the LE protein SYCP2 and is formed by the fusion of the two LE derivatives. As diplotene progresses, the BB increases in width and acquires a layered structure with a thick band of HOP1 separating two layers of SYCP2. The HOP1 interacting protein, PCH2, joins the BB in mid-diplotene, and by late-diplotene, it lies in the middle of the HOP1 filament. This structure is maintained through metaphase I. SYCP2 and PCH2 are lost at anaphase I, and the BB no longer connects the separating homologs. However, a key component of the BB, HOP1, remains at the metaphase I plate. These changes in organization of the BB occur simultaneously with the movement of the kinetochore protein, DSN1, from within the BB at mid-diplotene to the edge of the homologs facing the poles by metaphase I. We view these data in context of models in which SC components and regulators can be repurposed to achieve different functions, a fascinating example of evolution achieving homolog conjunction in an alternative way with recycling of SC proteins.

Sexual dimorphic regulation of recombination by the synaptonemal complex in C. elegans.

In sexually reproducing organisms, germ cells faithfully transmit the genome to the next generation by forming haploid gametes, such as eggs and sperm. Although most meiotic proteins are conserved between eggs and sperm, many aspects of meiosis are sexually dimorphic, including the regulation of recombination. The synaptonemal complex (SC), a large ladder-like structure that forms between homologous chromosomes, is essential for regulating meiotic chromosome organization and promoting recombination. To assess whether sex-specific differences in the SC underpin sexually dimorphic aspects of meiosis, we examined Caenorhabditis elegans SC central region proteins (known as SYP proteins) in oogenesis and spermatogenesis and uncovered sex-specific roles for the SYPs in regulating meiotic recombination. We find that SC composition, specifically SYP-2, SYP-3, SYP-5, and SYP-6, is regulated by sex-specific mechanisms throughout meiotic prophase I. During pachytene, both oocytes and spermatocytes differentially regulate the stability of SYP-2 and SYP-3 within an assembled SC. Further, we uncover that the relative amount of SYP-2 and SYP-3 within the SC is independently regulated in both a sex-specific and a recombination-dependent manner. Specifically, we find that SYP-2 regulates the early steps of recombination in both sexes, while SYP-3 controls the timing and positioning of crossover recombination events across the genomic landscape in only oocytes. Finally, we find that SYP-2 and SYP-3 dosage can influence the composition of the other SYPs in the SC via sex-specific mechanisms during pachytene. Taken together, we demonstrate dosage-dependent regulation of individual SC components with sex-specific functions in recombination. These sexual dimorphic features of the SC provide insights into how spermatogenesis and oogenesis adapted similar chromosome structures to differentially regulate and execute recombination.

The RNA-binding protein FUS/TLS interacts with SPO11 and PRDM9 and localize at meiotic recombination hotspots

In mammals, meiotic recombination is initiated by the introduction of DNA double strand breaks (DSBs) into narrow segments of the genome, defined as hotspots, which is carried out by the SPO11/TOPOVIBL complex. A major player in the specification of hotspots is PRDM9, a histone methyltransferase that, following sequence-specific DNA binding, generates trimethylation on lysine 4 (H3K4me3) and lysine 36 (H3K36me3) of histone H3, thus defining the hotspots. PRDM9 activity is key to successful meiosis, since in its absence DSBs are redirected to functional sites and synapsis between homologous chromosomes fails. One protein factor recently implicated in guiding PRDM9 activity at hotspots is EWS, a member of the FET family of proteins that also includes TAF15 and FUS/TLS. Here, we demonstrate that FUS/TLS partially colocalizes with PRDM9 on the meiotic chromosome axes, marked by the synaptonemal complex component SYCP3, and physically interacts with PRDM9. Furthermore, we show that FUS/TLS also interacts with REC114, one of the axis-bound SPO11-auxiliary factors essential for DSB formation. This finding suggests that FUS/TLS is a component of the protein complex that promotes the initiation of meiotic recombination. Accordingly, we document that FUS/TLS coimmunoprecipitates with SPO11 in vitro and in vivo. The interaction occurs with both SPO11β and SPO11α splice isoforms, which are believed to play distinct functions in the formation of DSBs in autosomes and male sex chromosomes, respectively. Finally, using chromatin immunoprecipitation experiments, we show that FUS/TLS is localized at H3K4me3-marked hotspots in autosomes and in the pseudo-autosomal region, the site of genetic exchange between the XY chromosomes.

Polycomb group (PcG) proteins prevent the assembly of abnormal synaptonemal complex structures during meiosis

The synaptonemal complex (SC) is a proteinaceous scaffold that is assembled between paired homologous chromosomes during the onset of meiosis. Timely expression of SC coding genes is essential for SC assembly and successful meiosis. However, SC components have an intrinsic tendency to self-organize into abnormal repetitive structures, which are not assembled between the paired homologs and whose formation is potentially deleterious for meiosis and gametogenesis. This creates an interesting conundrum, where SC genes need to be robustly expressed during meiosis, but their expression must be carefully regulated to prevent the formation of anomalous SC structures. In this manuscript, we show that the Polycomb group protein Sfmbt, the Drosophila ortholog of human MBTD1 and L3MBTL2, is required to avoid excessive expression of SC genes during prophase I. Although SC assembly is normal after Sfmbt depletion, SC disassembly is abnormal with the formation of multiple synaptonemal complexes (polycomplexes) within the oocyte. Overexpression of the SC gene corona and depletion of other Polycomb group proteins are similarly associated with polycomplex formation during SC disassembly. These polycomplexes are highly dynamic and have a well-defined periodic structure. Further confirming the importance of Sfmbt, germ line depletion of this protein is associated with significant metaphase I defects and a reduction in female fertility. Since transcription of SC genes mostly occurs during early prophase I, our results suggest a role of Sfmbt and other Polycomb group proteins in downregulating the expression of these and other early prophase I genes during later stages of meiosis.

Alterations in synaptonemal complex coding genes and human infertility.

About 10% of reproductive-aged couples suffer from infertility. However, the genetic causes of human infertility cases are largely unknown. Meiosis produces haploid gametes for fertilization and errors in meiosis are associated with human infertility in both males and females. Successful meiosis relies on the assembly of the synaptonemal complex (SC) between paired homologous chromosomes during the meiotic prophase. The SC is ultrastructurally and functionally conserved, promoting inter-homologous recombination and crossover formation, thus critical for accurate meiotic chromosome segregation. With whole-genome/exome sequencing and mouse models, a list of mutations in SC coding genes has been linked to human infertility. Here we summarize those findings. We also analyzed SC gene variants present in the general population and presented complex interaction networks associated with SC components. Whether a combination of genetic variations and environmental factors causes human infertility demands further investigations.

The synaptonemal complex central region modulates crossover pathways and feedback control of meiotic double-strand break formation.

The synaptonemal complex (SC) is a proteinaceous structure that mediates homolog engagement and genetic recombination during meiosis. In budding yeast, Zip-Mer-Msh (ZMM) proteins promote crossover (CO) formation and initiate SC formation. During SC elongation, the SUMOylated SC component Ecm11 and the Ecm11-interacting protein Gmc2 facilitate the polymerization of Zip1, an SC central region component. Through physical recombination, cytological, and genetic analyses, we found that ecm11 and gmc2 mutants exhibit chromosome-specific defects in meiotic recombination. CO frequencies on a short chromosome (chromosome III) were reduced, whereas CO and non-crossover frequencies on a long chromosome (chromosome VII) were elevated. Further, in ecm11 and gmc2 mutants, more double-strand breaks (DSBs) were formed on a long chromosome during late prophase I, implying that the Ecm11–Gmc2 (EG) complex is involved in the homeostatic regulation of DSB formation. The EG complex may participate in joint molecule (JM) processing and/or double-Holliday junction resolution for ZMM-dependent CO-designated recombination. Absence of the EG complex ameliorated the JM-processing defect in zmm mutants, suggesting a role for the EG complex in suppressing ZMM-independent recombination. Our results suggest that the SC central region functions as a compartment for sequestering recombination-associated proteins to regulate meiosis specificity during recombination.

SYP-5 regulates meiotic thermotolerance in Caenorhabditis elegans.

Meiosis produces the haploid gametes required by all sexually reproducing organisms, occurring in specific temperature ranges in different organisms. However, how meiotic thermotolerance is regulated remains largely unknown. Using the model organism Caenorhabditis elegans, here, we identified the synaptonemal complex (SC) protein SYP-5 as a critical regulator of meiotic thermotolerance. syp-5-null mutants maintained a high percentage of viable progeny at 20°C but produced significantly fewer viable progeny at 25°C, a permissive temperature in wild-type worms. Cytological analysis of meiotic events in the mutants revealed that while SC assembly and disassembly, as well as DNA double-strand break repair kinetics, were not affected by the elevated temperature, crossover designation, and bivalent formation were significantly affected. More severe homolog segregation errors were also observed at elevated temperature. A temperature switching assay revealed that late meiotic prophase events were not temperature-sensitive and that meiotic defects during pachytene stage were responsible for the reduced viability of syp-5 mutants at the elevated temperature. Moreover, SC polycomplex formation and hexanediol sensitivity analysis suggested that SYP-5 was required for the normal properties of the SC, and charge-interacting elements in SC components were involved in regulating meiotic thermotolerance. Together, these findings provide a novel molecular mechanism for meiotic thermotolerance regulation.

Meiotic chromosome synapsis depends on multivalent SYCE1-SIX6OS1 interactions that are disrupted in cases of human infertility.

Meiotic reductional division depends on the synaptonemal complex (SC), a supramolecular protein assembly that mediates homologous chromosomes synapsis and promotes crossover formation. The mammalian SC has eight structural components, including SYCE1, the only central element protein with known causative mutations in human infertility. We combine mouse genetics, cellular, and biochemical studies to reveal that SYCE1 undergoes multivalent interactions with SC component SIX6OS1. The N terminus of SIX6OS1 binds and disrupts SYCE1's core dimeric structure to form a 1:1 complex, while their downstream sequences provide a distinct second interface. These interfaces are separately disrupted by SYCE1 mutations associated with nonobstructive azoospermia and premature ovarian failure (POF), respectively. Mice harboring SYCE1's POF mutation and a targeted deletion within SIX6OS1's N terminus are infertile with failure of chromosome synapsis. We conclude that both SYCE1-SIX6OS1 binding interfaces are essential for SC assembly, thus explaining how SYCE1's reported clinical mutations give rise to human infertility.

Active transcription and Orc1 drive chromatin association of the AAA+ ATPase Pch2 during meiotic G2/prophase.

Pch2 is an AAA+ protein that controls DNA break formation, recombination and checkpoint signaling during meiotic G2/prophase. Chromosomal association of Pch2 is linked to these processes, and several factors influence the association of Pch2 to euchromatin and the specialized chromatin of the ribosomal (r)DNA array of budding yeast. Here, we describe a comprehensive mapping of Pch2 localization across the budding yeast genome during meiotic G2/prophase. Within non-rDNA chromatin, Pch2 associates with a subset of actively RNA Polymerase II (RNAPII)-dependent transcribed genes. Chromatin immunoprecipitation (ChIP)- and microscopy-based analysis reveals that active transcription is required for chromosomal recruitment of Pch2. Similar to what was previously established for association of Pch2 with rDNA chromatin, we find that Orc1, a component of the Origin Recognition Complex (ORC), is required for the association of Pch2 to these euchromatic, transcribed regions, revealing a broad connection between chromosomal association of Pch2 and Orc1/ORC function. Ectopic mitotic expression is insufficient to drive recruitment of Pch2, despite the presence of active transcription and Orc1/ORC in mitotic cells. This suggests meiosis-specific 'licensing' of Pch2 recruitment to sites of transcription, and accordingly, we find that the synaptonemal complex (SC) component Zip1 is required for the recruitment of Pch2 to transcription-associated binding regions. Interestingly, Pch2 binding patterns are distinct from meiotic axis enrichment sites (as defined by Red1, Hop1, and Rec8). Inactivating RNAPII-dependent transcription/Orc1 does not lead to effects on the chromosomal abundance of Hop1, a known chromosomal client of Pch2, suggesting a complex relationship between SC formation, Pch2 recruitment and Hop1 chromosomal association. We thus report characteristics and dependencies for Pch2 recruitment to meiotic chromosomes, and reveal an unexpected link between Pch2, SC formation, chromatin and active transcription.

Familial primary ovarian insufficiency associated with an SYCE1 point mutation: defective meiosis elucidated in humanized mice.

More than 50% of cases of primary ovarian insufficiency (POI) and nonobstructive azoospermia in humans are classified as idiopathic infertility. Meiotic defects may relate to at least some of these cases. Mutations in genes coding for synaptonemal complex (SC) components have been identified in humans, and hypothesized to be causative for the observed infertile phenotype. Mutation SYCE1 c.721C>T (former c.613C>T)-a familial mutation reported in two sisters with primary amenorrhea-was the first such mutation found in an SC central element component-coding gene. Most fundamental mammalian oogenesis events occur during the embryonic phase, and eventual defects are identified many years later, thus leaving few possibilities to study the condition's etiology and pathogenesis. Aiming to validate an approach to circumvent this difficulty, we have used the CRISPR/Cas9 technology to generate a mouse model with an SYCE1 c.721C>T equivalent genome alteration. We hereby present the characterization of the homozygous mutant mice phenotype, compared to their wild type and heterozygous littermates. Our results strongly support a causative role of this mutation for the POI phenotype in human patients, and the mechanisms involved would relate to defects in homologous chromosome synapsis. No SYCE1 protein was detected in homozygous mutants and Syce1 transcript level was highly diminished, suggesting transcript degradation as the basis of the infertility mechanism. This is the first report on the generation of a humanized mouse model line for the study of an infertility-related human mutation in an SC component-coding gene, thus representing a proof of principle.

Identification of novel synaptonemal complex components in C. elegans.

The synaptonemal complex (SC) is a tripartite protein scaffold that forms between homologous chromosomes during meiosis. Although the SC is essential for stable homologue pairing and crossover recombination in diverse eukaryotes, it is unknown how individual components assemble into the highly conserved SC structure. Here we report the biochemical identification of two new SC components, SYP-5 and SYP-6, in Caenorhabditis elegans. SYP-5 and SYP-6 are paralogous to each other and play redundant roles in synapsis, providing an explanation for why these genes have evaded previous genetic screens. Superresolution microscopy reveals that they localize between the chromosome axes and span the width of the SC in a head-to-head manner, similar to the orientation of other known transverse filament proteins. Using genetic redundancy and structure-function analyses to truncate C-terminal tails of SYP-5/6, we provide evidence supporting the role of SC in both limiting and promoting crossover formation.

Multivalent weak interactions between assembly units drive synaptonemal complex formation.

The synaptonemal complex (SC) is an ordered but highly dynamic structure assembled between homologous chromosomes to control interhomologous crossover formation, ensuring accurate meiotic chromosome segregation. However, the mechanisms regulating SC assembly and dynamics remain unclear. Here, we identified two new SC components, SYP-5 and SYP-6, in Caenorhabditis elegans that have distinct expression patterns and form distinct SC assembly units with other SYPs through stable interactions. SYP-5 and SYP-6 exhibit diverse in vivo SC regulatory functions and distinct phase separation properties in cells. Charge-interacting elements (CIEs) are enriched in SC intrinsically disordered regions (IDRs), and IDR deletion or CIE removal confirmed a requirement for these elements in SC regulation. Our data support the theory that multivalent weak interactions between the SC units drive SC formation and that CIEs confer multivalency to the assembly units.

Slx5p-Slx8p Promotes Accurate Chromosome Segregation by Mediating the Degradation of Synaptonemal Complex Components during Meiosis.

Meiosis increases genetic diversity, yet the genome complement needs to be stable to ensure offspring viability. Both small ubiquitin‐like modifier (SUMO) and ubiquitin have been reported to participate in meiotic regulation, yet functions of the SUMO‐ubiquitination crosstalk in meiosis remain unclear. Here, it is reported that a SUMO‐targeted ubiquitin ligase, Slx8p, promotes accurate chromosome segregation during meiosis, since the deletion of SLX8 leads to increased aneuploidy due to a defect in synaptonemal complex (SC) component degradation. Both the RING domain and SUMO interacting motifs of Slx8p are essential for meiotic progression and maintaining spore viability, and the expression of tetraubiquitin fused with SUMO partially rescues meiotic defects in the SLX8‐deletion strain. Furthermore, Slx5p‐Slx8p can directly add ubiquitin to SUMOylated Zip1p and Ecm11p, and forced degradation of Ecm11p partially rescues the sporulation defects of the SLX8 deletion strain. These findings provide a mechanism for SC disassembly and reveal that the crosstalk between SUMOylation and ubiquitination facilitates accurate chromosome segregation by promoting SC component degradation during meiosis.

X chromosome and autosomal recombination are differentially sensitive to disruptions in SC maintenance

The synaptonemal complex (SC) is a conserved meiotic structure that regulates the repair of double-strand breaks (DSBs) into crossovers or gene conversions. The removal of any central-region SC component, such as the Drosophila melanogaster transverse filament protein C(3)G, causes a complete loss of SC structure and crossovers. To better understand the role of the SC in meiosis, we used CRISPR/Cas9 to construct 3 in-frame deletions within the predicted coiled-coil region of the C(3)G protein. Since these 3 deletion mutations disrupt SC maintenance at different times during pachytene and exhibit distinct defects in key meiotic processes, they allow us to define the stages of pachytene when the SC is necessary for homolog pairing and recombination during pachytene. Our studies demonstrate that the X chromosome and the autosomes display substantially different defects in pairing and recombination when SC structure is disrupted, suggesting that the X chromosome is potentially regulated differently from the autosomes.

Fam9B was associated with human spermatogenesis and male-specific sterility

Spermatogenesis is the process of gamete formation, which includes mitosis in spermatogonia, meiosis in spermatocytes, and spermiogenesis. A key event is the formation of the synaptonemal complex (SC). Mutations of genes encoding SC components (such as SYCP3，synaptonemal complex protein 3) lead to infertility or subfertility due to germ cell death. Fam9B mapped on the human chromosome X (Xp22.3) was more similar to SYCP3 in their amino acid sequences and expressed in human testis. However, the expression and precise underlying mechanisms of Fam9B have not been clearly in testes during the spermatogenesis and infertility. And Fam9B mutation associated with sterility? This study was an analysis of azoospermia, sertoli cell only syndrome (SCOS) and proven fertile patients, including 162 patients diagnosed with azoospermia, 65 patients diagnosed with SCOS and 10 proven fertile patients at the Reproductive Medical Center of Peking University Third Hospital between January 2015 and January 2019. Fam9B was cloned, expressed and identified. Genome DNA sequencing analysis, RT-PCR, Western-blot, Immunohistochemistry and immunocytochemistry were performed. Fam9B was analyzed with blood and testicular biopsy samples from azoospermia patients by genome DNA sequencing analysis. These likely mutations were further screened in SCOS patients and in men proven to be fertile. And Fam9B was cloned, expressed and identified with testicular samples using RT-PCR, western blot, Immunohistochemistry and immunocytochemistry. The concentration of testosterone in azoospermia and SCOS patients were determined, and their relativity was studied by statistic methods. Fam9B mRNAs and protein were detected in human testis, and Fam9B was expressed at different level in patients of azoospermia, SCOS and normal groups. Immunohistochemical staining showed that Fam9B was expressed in the nucleus of primary spermatocyte in fertile persons. Moreover, Fam9B was more similar to SYCP3 expression pattern and located on the SC during the leptotene and diplotene spermatocytes. Furthermore, genome DNA sequencing analysis revealed three fam9b deletion patients and five patients of point mutation in fam9b gene. Spermatogenesis of these three patients was arrested at spermatocytes and accompanied by germ cells lost. Interesting, testosterone concentrations levels of these three patients were decreased compared with controls (P < 0.01) . Fam9B may be a SC related protein participated in human normal spermatogenesis. And mutations of fam9b may be risk of spermatocytes arrest and male-specific sterility.

Crossover recombination and synapsis are linked by adjacent regions within the N terminus of the Zip1 synaptonemal complex protein.

Accurate chromosome segregation during meiosis relies on the prior establishment of at least one crossover recombination event between homologous chromosomes. Most meiotic recombination intermediates that give rise to interhomolog crossovers are embedded within a hallmark chromosomal structure called the synaptonemal complex (SC), but the mechanisms that coordinate the processes of SC assembly (synapsis) and crossover recombination remain poorly understood. Among known structural components of the budding yeast SC, the Zip1 protein is unique for its independent role in promoting crossover recombination; Zip1 is specifically required for the large subset of crossovers that also rely on the meiosis-specific MutSγ complex. Here we report that adjacent regions within Zip1’s N terminus encompass its crossover and synapsis functions. We previously showed that deletion of Zip1 residues 21–163 abolishes tripartite SC assembly and prevents robust SUMOylation of the SC central element component, Ecm11, but allows excess MutSγ crossover recombination. We find the reciprocal phenotype when Zip1 residues 2–9 or 10–14 are deleted; in these mutants SC assembles and Ecm11 is hyperSUMOylated, but MutSγ crossovers are strongly diminished. Interestingly, Zip1 residues 2–9 or 2–14 are required for the normal localization of Zip3, a putative E3 SUMO ligase and pro-MutSγ crossover factor, to Zip1 polycomplex structures and to recombination initiation sites. By contrast, deletion of Zip1 residues 15–20 does not detectably prevent Zip3’s localization at Zip1 polycomplex and supports some MutSγ crossing over but prevents normal SC assembly and Ecm11 SUMOylation. Our results highlight distinct N terminal regions that are differentially critical for Zip1’s roles in crossing over and SC assembly; we speculate that the adjacency of these regions enables Zip1 to serve as a liaison, facilitating crosstalk between the two processes by bringing crossover recombination and synapsis factors within close proximity of one another.