Synaptonemal Complex Research Articles

ZmASY1 interacts with ZmPRD3 and is crucial for meiotic double-strand break formation in maize.

During meiosis, recombination-mediated pairing and synapsis of homologous chromosomes begin with programmed DNA double-strand breaks (DSBs). In yeast and mice, DSBs form in a tethered loop-axis complex, in which DSB sites are located within chromatin loops and tethered to the proteinaceous axial element (AE) by DSB-forming factors. In plants, the molecular connection between DSB sites and chromosome axes is poorly understood. By integrating genetic analysis, immunostaining technology, and protein-protein interaction studies, the putative factors linking DSB formation to chromosome axis were explored in maize meiosis. Here, we report that the AE protein ZmASY1 directly interacts with the DSB-forming protein ZmPRD3 in maize (Zea mays) and mediates DSB formation, synaptonemal complex assembly, and homologous recombination. ZmPRD3 also interacts with ZmPRD1, which plays a central role in organizing the DSB-forming complex. These results suggest that ZmASY1 and ZmPRD3 may work as a key module linking DSB sites to chromosome axes during DSB formation in maize. This mechanism is similar to that described in yeast and recently Arabidopsis involving the homologs Mer2/ZmPRD3 and HOP1/ZmASY1, thus indicating that the process of tethering DSBs in chromatin loops to the chromosome axes may be evolutionarily conserved in diverse taxa.

Image analysis workflows to reveal the spatial organization of cell nuclei and chromosomes

ABSTRACT Nucleus, chromatin, and chromosome organization studies heavily rely on fluorescence microscopy imaging to elucidate the distribution and abundance of structural and regulatory components. Three-dimensional (3D) image stacks are a source of quantitative data on signal intensity level and distribution and on the type and shape of distribution patterns in space. Their analysis can lead to novel insights that are otherwise missed in qualitative-only analyses. Quantitative image analysis requires specific software and workflows for image rendering, processing, segmentation, setting measurement points and reference frames and exporting target data before further numerical processing and plotting. These tasks often call for the development of customized computational scripts and require an expertise that is not broadly available to the community of experimental biologists. Yet, the increasing accessibility of high- and super-resolution imaging methods fuels the demand for user-friendly image analysis workflows. Here, we provide a compendium of strategies developed by participants of a training school from the COST action INDEPTH to analyze the spatial distribution of nuclear and chromosomal signals from 3D image stacks, acquired by diffraction-limited confocal microscopy and super-resolution microscopy methods (SIM and STED). While the examples make use of one specific commercial software package, the workflows can easily be adapted to concurrent commercial and open-source software. The aim is to encourage biologists lacking custom-script-based expertise to venture into quantitative image analysis and to better exploit the discovery potential of their images. Abbreviations: 3D FISH: three-dimensional fluorescence in situ hybridization; 3D: three-dimensional; ASY1: ASYNAPTIC 1; CC: chromocenters; CO: Crossover; DAPI: 4',6-diamidino-2-phenylindole; DMC1: DNA MEIOTIC RECOMBINASE 1; DSB: Double-Strand Break; FISH: fluorescence in situ hybridization; GFP: GREEN FLUORESCENT PROTEIN; HEI10: HUMAN ENHANCER OF INVASION 10; NCO: Non-Crossover; NE: Nuclear Envelope; Oligo-FISH: oligonucleotide fluorescence in situ hybridization; RNPII: RNA Polymerase II; SC: Synaptonemal Complex; SIM: structured illumination microscopy; ZMM (ZIP: MSH4: MSH5 and MER3 proteins); ZYP1: ZIPPER-LIKE PROTEIN 1.

Nonhomologous Chromosome Interactions in Prophase I: Dynamics of Bizarre Meiotic Contacts in the Alay Mole Vole Ellobius alaicus (Mammalia, Rodentia).

Nonhomologous chromosome interactions take place in both somatic and meiotic cells. Prior to this study, we had discovered special contacts through the SYCP3 (synaptonemal complex protein 3) filament between the short arms of nonhomologous acrocentrics at the pachytene stage in the Alay mole vole, and these contacts demonstrate several patterns from proximity to the complete fusion stage. Here, we investigated the nonhomologous chromosome contacts in meiotic prophase I. It turned out that such contacts do not introduce changes into the classic distribution of DNA double-strand breaks. It is noteworthy that not all meiotic contacts were localized in the H3k9me3-positive heterochromatic environment. Both in the mid zygotene and in the early-mid diplotene, three types of contacts (proximity, touching, and anchoring/tethering) were observed, whereas fusion seems to be characteristic only for pachytene. The number of contacts in the mid pachytene is significantly higher than that in the zygotene, and the distance between centromeres in nonhomologous contacts is also the smallest in mid pachytene for all types of contacts. Thus, this work provides a new insight into the behavior of meiotic contacts during prophase I and points to avenues of further research.

Premeiotic pairing of homologous chromosomes during Drosophila male meiosis

In the early stages of meiosis, maternal and paternal chromosomes pair with their homologous partner and recombine to ensure exchange of genetic information and proper segregation. These events can vary drastically between species and between males and females of the same species. In Drosophila, in contrast to females, males do not form synaptonemal complexes (SCs), do not recombine, and have no crossing over; yet, males are able to segregate their chromosomes properly. Here, we investigated the early steps of homolog pairing in Drosophila males. We found that homolog centromeres are not paired in germline stem cells (GSCs) and become paired in the mitotic region before meiotic entry, similarly to females. Surprisingly, male germline cells express SC proteins, which localize to centromeres and promote pairing. We further found that the SUN/KASH (LINC) complex and microtubules are required for homolog pairing as in females. Chromosome movements in males, however, are much slower than in females and we demonstrate that this slow dynamic is compensated in males by having longer cell cycles. In agreement, slowing down cell cycles was sufficient to rescue pairing-defective mutants in female meiosis. Our results demonstrate that although meiosis differs significantly between males and females, sex-specific cell cycle kinetics integrate similar molecular mechanisms to achieve proper centromere pairing.

Meiosis: Deciphering the dialog between recombination and the synaptonemal complex

A meiotic-specific structure, the synaptonemal complex, regulates meiotic recombination, but the 'language' used for this regulation has yet to be fully decoded. A new study identifies a novel phosphorylation site onthesynaptonemal complex that regulates DNA repair pathway choice, revealing a new corner of the 'Rosetta Stone' for meiosis.

Teflon promotes chromosomal recruitment of homolog conjunction proteins during Drosophila male meiosis.

Meiosis in males of higher dipterans is achiasmate. In their spermatocytes, pairing of homologs into bivalent chromosomes does not include synaptonemal complex and crossover formation. While crossovers preserve homolog conjunction until anaphase I during canonical meiosis, an alternative system is used in dipteran males. Mutant screening in Drosophila melanogaster has identified teflon (tef) as being required specifically for alternative homolog conjunction (AHC) of autosomal bivalents. The additional known AHC genes, snm, uno and mnm, are needed for the conjunction of autosomal homologs and of sex chromosomes. Here, we have analyzed the pattern of TEF protein expression. TEF is present in early spermatocytes but cannot be detected on bivalents at the onset of the first meiotic division, in contrast to SNM, UNO and MNM (SUM). TEF binds to polytene chromosomes in larval salivary glands, recruits MNM by direct interaction and thereby, indirectly, also SNM and UNO. However, chromosomal SUM association is not entirely dependent on TEF, and residual autosome conjunction occurs in tef null mutant spermatocytes. The higher tef requirement for autosomal conjunction is likely linked to the quantitative difference in the amount of SUM protein that provides conjunction of autosomes and sex chromosomes, respectively. During normal meiosis, SUM proteins are far more abundant on sex chromosomes compared to autosomes. Beyond promoting SUM recruitment, TEF has a stabilizing effect on SUM proteins. Increased SUM causes excess conjunction and consequential chromosome missegregation during meiosis I after co-overexpression. Similarly, expression of SUM without TEF, and even more potently with TEF, interferes with chromosome segregation during anaphase of mitotic divisions in somatic cells, suggesting that the known AHC proteins are sufficient for establishment of ectopic chromosome conjunction. Overall, our findings suggest that TEF promotes alternative homolog conjunction during male meiosis without being part of the final physical linkage between chromosomes.

Joint control of meiotic crossover patterning by the synaptonemal complex and HEI10 dosage

Meiotic crossovers are limited in number and are prevented from occurring close to each other by crossover interference. In many species, crossover number is subject to sexual dimorphism, and a lower crossover number is associated with shorter chromosome axes lengths. How this patterning is imposed remains poorly understood. Here, we show that overexpression of the Arabidopsis pro-crossover protein HEI10 increases crossovers but maintains some interference and sexual dimorphism. Disrupting the synaptonemal complex by mutating ZYP1 also leads to an increase in crossovers but, in contrast, abolishes interference and disrupts the link between chromosome axis length and crossovers. Crucially, combining HEI10 overexpression and zyp1 mutation leads to a massive and unprecedented increase in crossovers. These observations support and can be predicted by, a recently proposed model in which HEI10 diffusion along the synaptonemal complex drives a coarsening process leading to well-spaced crossover-promoting foci, providing a mechanism for crossover patterning.

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.

Decabromodiphenyl ethane induces male reproductive toxicity by glycolipid metabolism imbalance and meiotic failure

Decabromodiphenyl ethane (DBDPE) is a typical flame retardant found in various electrical and textile items. DBDPE is abundantly available in the surrounding environment and wild animals based on its persistence and bioaccumulation. DBDPE has been shown to cause apoptosis in rat spermatogenic cells, resulting in reproductive toxicity. However, the toxicity of DBDPE on the male reproductive system and the potential mechanisms are still unclear. This study evaluated the effect of DBDPE on the reproductive system in male SD rats and demonstrated the potential mechanisms of reproductive toxicity. DBDPE (0, 5, 50, and 500 mg/kg/day) was administered via gavage to male SD rats for 28 days. DBDPE caused histopathological changes in the testis, reduced sperm quantity and motility, and raised the malformation rate in rats, according to the findings. Furthermore, it caused DNA damage to rat testicular cells. It inhibited the expressions of spermatogenesis‐and oogenesis‐specific helix‐loop‐helix transcription factor 1 (Sohlh1), piwi-like RNA-mediated gene silencing 2 (MILI), cyclin-dependent kinase 2 (CDK2), and CyclinA, resulting in meiotic failure, as well as the expressions of synaptonemal complex proteins 1 and 3 (SYCP1 and SYCP3), leading to chromosomal association disorder in meiosis and spermatocyte cycle arrest. Moreover, DBDPE induced glycolipid metabolism disorder and activated mitochondria-mediated apoptosis pathways in the testes of SD rats. The quantity and quality of sperm might be declining due to these factors. Our findings offer further evidence of the harmful impact of DBDPE on the male reproductive system.

Sex chromosome differentiation via changes in the Y chromosome repeat landscape in African annual killifishes Nothobranchius furzeri and N. kadleci.

Homomorphic sex chromosomes and their turnover are common in teleosts. We investigated the evolution of nascent sex chromosomes in several populations of two sister species of African annual killifishes, Nothobranchius furzeri and N. kadleci, focusing on their under-studied repetitive landscape. We combined bioinformatic analyses of the repeatome with molecular cytogenetic techniques, including comparative genomic hybridization, fluorescence in situ hybridization with satellite sequences, ribosomal RNA genes (rDNA) and bacterial artificial chromosomes (BACs), and immunostaining of SYCP3 and MLH1 proteins to mark lateral elements of synaptonemal complexes and recombination sites, respectively. Both species share the same heteromorphic XY sex chromosome system, which thus evolved prior to their divergence. This was corroborated by sequence analysis of a putative master sex determining (MSD) gene gdf6Y in both species. Based on their divergence, differentiation of the XY sex chromosome pair started approximately 2 million years ago. In all populations, the gdf6Y gene mapped within a region rich in satellite DNA on the Y chromosome long arms. Despite their heteromorphism, X and Y chromosomes mostly pair regularly in meiosis, implying synaptic adjustment. In N. kadleci, Y-linked paracentric inversions like those previously reported in N. furzeri were detected. An inversion involving the MSD gene may suppress occasional recombination in the region, which we otherwise evidenced in the N. furzeri population MZCS-121 of the Limpopo clade lacking this inversion. Y chromosome centromeric repeats were reduced compared with the X chromosome and autosomes, which points to a role of relaxed meiotic drive in shaping the Y chromosome repeat landscape. We speculate that the recombination rate between sex chromosomes was reduced due to heterochiasmy. The observed differences between the repeat accumulations on the X and Y chromosomes probably result from high repeat turnover and may not relate closely to the divergence inferred from earlier SNP analyses.

The piRNA-pathway factor FKBP6 is essential for spermatogenesis but dispensable for control of meiotic LINE-1 expression in humans

Infertility affects around 7% of the male population and can be due to severe spermatogenic failure (SPGF), resulting in no or very few sperm in the ejaculate. We initially identified a homozygous frameshift variant in FKBP6 in a man with extreme oligozoospermia. Subsequently, we screened a total of 2,699 men with SPGF and detected rare bi-allelic loss-of-function variants in FKBP6 in five additional persons. All six individuals had no or extremely few sperm in the ejaculate, which were not suitable for medically assisted reproduction. Evaluation of testicular tissue revealed an arrest at the stage of round spermatids. Lack of FKBP6 expression in the testis was confirmed by RT-qPCR and immunofluorescence staining. In mice, Fkbp6 is essential for spermatogenesis and has been described as being involved in piRNA biogenesis and formation of the synaptonemal complex (SC). We did not detect FKBP6 as part of the SC in normal human spermatocytes, but small RNA sequencing revealed that loss of FKBP6 severely impacted piRNA levels, supporting a role for FKBP6 in piRNA biogenesis in humans. In contrast to findings in piRNA-pathway mouse models, we did not detect an increase in LINE-1 expression in men with pathogenic FKBP6 variants. Based on our findings, FKBP6 reaches a "strong" level of evidence for being associated with male infertility according to the ClinGen criteria, making it directly applicable for clinical diagnostics. This will improve patient care by providing a causal diagnosis and will help to predict chances for successful surgical sperm retrieval.

ATM/ATR kinases link the synaptonemal complex and DNA double-strand break repair pathway choice

DNA double-strand breaks (DSBs) are deleterious lesions, which must be repaired precisely to maintain genomic stability. During meiosis, programmed DSBs are repaired via homologous recombination (HR) while repair using the nonhomologous end joining (NHEJ) pathway is inhibited, thereby ensuring crossover formation and accurate chromosome segregation.1,2 How DSB repair pathway choice is implemented during meiosis is unknown. In C.elegans, meiotic DSB repair takes place in the context of the fully formed, highly dynamic zipper-like structure present between homologous chromosomes called the synaptonemal complex (SC).3,4,5,6,7,8,9 The SC consists of a pair of lateral elements bridged by a central region composed of the SYP proteins in C.elegans. How the structural components of the SC are regulated to maintain the architectural integrity of the assembled SC around DSB repair sites remained unclear. Here, we show that SYP-4, a central region component of the SC, is phosphorylated at Serine 447 in a manner dependent on DSBs and the ATM/ATR DNA damage response kinases. We show that this SYP-4 phosphorylation is critical for preserving the SC structure following exogenous (γ-IR-induced) DSB formation and for promoting normal DSB repair progression and crossover patterning following SPO-11-dependent and exogenous DSBs. We propose a model in which ATM/ATR-dependent phosphorylation of SYP-4 at the S447 site plays important roles both in maintaining the architectural integrity of the SC following DSB formation and in warding off repair via the NHEJ repair pathway, thereby preventing aneuploidy.

RBM46 is essential for gametogenesis and functions in post-transcriptional roles affecting meiotic cohesin subunits.

RBM46 is a germ cell-specific RNA-binding protein required for gametogenesis, but the targets and molecular functions of RBM46 remain unknown. Here, we demonstrate that RBM46 binds at specific motifs in the 3'UTRs of mRNAs encoding multiple meiotic cohesin subunits and show that RBM46 is required for normal synaptonemal complex formation during meiosis initiation. Using a recently reported, high-resolution technique known as LACE-seq and working with low-input cells, we profiled the targets of RBM46 at single-nucleotide resolution in leptotene and zygotene stage gametes. We found that RBM46 preferentially binds target mRNAs containing GCCUAU/GUUCGA motifs in their 3'UTRs regions. In Rbm46 knockout mice, the RBM46-target cohesin subunits displayed unaltered mRNA levels but had reduced translation, resulting in the failed assembly of axial elements, synapsis disruption, and meiotic arrest. Our study thus provides mechanistic insights into the molecular functions of RBM46 in gametogenesis and illustrates the power of LACE-seq for investigations of RNA-binding protein functions when working with low-abundance input materials.

Super-Resolution Microscopy of the Synaptonemal Complex within the Caenorhabditis elegans Germline.

During meiosis, homologous chromosomes must recognize and adhere to one another to allow for their correct segregation. One of the key events that secures the interaction of homologous chromosomes is the assembly of the synaptonemal complex (SC) in meiotic prophase I. Even though there is little sequence homology between protein components within the SC among different species, the general structure of the SC has been highly conserved during evolution. In electron micrographs, the SC appears as a tripartite, ladder-like structure composed of lateral elements or axes, transverse filaments, and a central element. However, precisely identifying the localization of individual components within the complex by electron microscopy to determine the molecular structure of the SC remains challenging. By contrast, fluorescence microscopy allows for the identification of individual protein components within the complex. However, since the SC is only ~100 nm wide, its substructure cannot be resolved by diffraction-limited conventional fluorescence microscopy. Thus, determining the molecular architecture of the SC requires super-resolution light microscopy techniques such as structured illumination microscopy (SIM), stimulated-emission depletion (STED) microscopy, or single-molecule localization microscopy (SMLM). To maintain the structure and interactions of individual components within the SC, it is important to observe the complex in an environment that is close to its native environment in the germ cells. Therefore, we demonstrate an immunohistochemistry and imaging protocol that enables the study of the substructure of the SC in intact, extruded Caenorhabditis elegans germline tissue with SMLM and STED microscopy. Directly fixing the tissue to the coverslip reduces the movement of the samples during imaging and minimizes aberrations in the sample to achieve the high resolution necessary to visualize the substructure of the SC in its biological context.

Coiled-coil structure of meiosis protein TEX12 and conformational regulation by its C-terminal tip

Meiosis protein TEX12 is an essential component of the synaptonemal complex (SC), which mediates homologous chromosome synapsis. It is also recruited to centrosomes in meiosis, and aberrantly in certain cancers, leading to centrosome dysfunction. Within the SC, TEX12 forms an intertwined complex with SYCE2 that undergoes fibrous assembly, driven by TEX12’s C-terminal tip. However, we hitherto lack structural information regarding SYCE2-independent functions of TEX12. Here, we report X-ray crystal structures of TEX12 mutants in three distinct conformations, and utilise solution light and X-ray scattering to determine its wild-type dimeric four-helical coiled-coil structure. TEX12 undergoes conformational change upon C-terminal tip mutations, indicating that the sequence responsible for driving SYCE2-TEX12 assembly within the SC also controls the oligomeric state and conformation of isolated TEX12. Our findings provide the structural basis for SYCE2-independent roles of TEX12, including the possible regulation of SC assembly, and its known functions in meiotic centrosomes and cancer.

Increased expression of SYCP2 predicts poor prognosis in patients suffering from breast carcinoma.

Overexpression of synaptonemal complex protein-2 (SYCP2) has been identified in various human papillomavirus (HPV)–related carcinomas, whereas its significant role in breast carcinoma remains unclear. The aim of this study was to elucidate the prognostic value and potential function of SYCP2 in breast carcinoma. Herein, data for breast carcinoma patients from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas database (TCGA) were analyzed. The enrichment analysis of SYCP2 including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Friends, and GSEA was performed. Kaplan–Meier analysis, Cox regression, and receiver operating characteristic (ROC) curves were employed for determining the predictive value of SYCP2 on clinical outcomes in patients suffering from breast carcinoma. A nomogram was generated to predict the effect arising from SYCP2 on prognosis. The association analysis of SYCP2 gene expression and diverse immune infiltration levels was conducted through ssGSEA and ESTIMATE analysis, which consisted of dendritic cell (DC), neutrophil, eosinophil, macrophage, mast cell, NK cell, and other 18 cell subtypes. The results showed that SYCP2 expression was significantly elevated in breast carcinoma tissues as compared with that of normal tissues (p < 0.001). SYCP2 plays a certain role in pathways related to DNA methylation, keratinocyte differentiation, steroid hormone biosynthesis, and immune infiltration. The high expression of SYCP2 had a significant relationship to age, pathological type, ER expression, and PR expression (p < 0.001). Kaplan–Meier survival analysis showed that patients suffering from breast carcinoma characterized by high-SYCP2 expression had a poorer prognosis than patients with low-SYCP2 expression (p = 0.005). Univariate and multivariate Cox regression analyses revealed that SYCP2 had an independent relationship to overall survival (p = 0.049). Moreover, ROC curves suggested the significant diagnostic ability of SYCP2 for breast carcinoma, and as time went on, SYCP2 had more accurate prognostic efficacy. Furthermore, a high level of SYCP2 expression was found to have a relationship to poor prognosis of breast carcinoma in the subgroups of T3, N0, and M0, and infiltrating ductal carcinoma (HR > 1, p < 0.05). The calibration plot of the nomogram indicated that the SYCP2 model has an effective predictive performance for breast carcinoma patients. Conclusively, SYCP2 plays a vital role in the pathogenesis and progression of human breast carcinoma, so it may serve as a promising prognostic molecular marker of poor survival.

Changes in Ploidy Drive Reproduction Transition and Genomic Diversity in a Polyploid Fish Complex.

Unisexual animals are commonly found in some polyploid species complexes, and most of these species have had a long evolutionary history. However, their method for avoiding genomic decay remains unclear. The polyploid Carassius complex naturally comprises the sexual amphidiploid C. auratus (crucian carp or goldfish) (AABB) and the gynogenetic amphitriploid C. gibelio (gibel carp) (AAABBB). Recently, we developed a fertile synthetic amphitetraploid (AAAABBBB) male from C. gibelio by incorporating a C. auratus genome. In this study, we generated novel amphitriploids (AAABBB) by backcrossing the amphitetraploid male with the amphidiploid C. auratus. Whole-genome resequencing revealed the genomic changes, including recombination and independent assortment between homologs of C. gibelio and C. auratus. The fertility, sex determination system, oocyte development, and fertilization behaviors of the novel amphitriploids were investigated. Approximately 80% of the novel amphitriploid females recovered the unisexual gynogenesis ability. Intriguingly, two types of primary oocyte (with and without homolog synapsis) were discovered, and their distinct development fates were observed. Type I oocytes entered apoptosis due to improper synaptonemal complex assembly and incomplete double-strand break repair, whereas subsequent type II oocytes bypassed meiosis through an alternative ameiotic pathway to develop into mature eggs. Moreover, gynogenesis was stabilized in their offspring, and a new array of diverse gynogenetic amphitriploid clones was produced. These revealed genomic changes and detailed cytological data provide comprehensive evidence that changes in ploidy drive unisexual and sexual reproduction transition, thereby resulting in genomic diversity and allowing C. gibelio avoid genomic decay.

OsRAD51 Plays a Vital Role in Promoting Homologous Recombination in Rice Meiosis.

Meiotic recombination plays a pivotal role in achieving accurate chromosomal segregation and increasing genetic diversity. In the homologous recombination pathway, the detailed mechanisms of how OsRAD51 and OsDMC1 work in rice meiosis remain to be explored. Here, we obtained different types of mutants for Osrad51a1, Osrad51a2, Osdmc1a, and Osdmc1b through CRISPR/Cas9. Both Osrad51a1 and Osrad51a2 exhibited normal vegetative growth and fertility. Osrad51 (Osrad51a1 Osrad51a2) mutant plants show normal vegetative growth but exhibit complete sterility, indicating that OsRAD51A1 and OsRAD51A2 are functionally redundant in rice fertility. In contrast to the wild type, Osrad51 chromosomes are not paired perfectly at pachytene and synaptonemal complex (SC) formation is deficient. Moreover, univalents and multivalent associations were observed at metaphase I, chromosome fragments presented at anaphase I, and crossover formation is basically suppressed in Osrad51 pollen mother cells (PMCs). OsRAD51 foci emerge at leptotene and disappear from late pachytene and chromosome localization of OsRAD51 depends on the formation of double-strand breaks (DSBs). Most OsRAD51 foci can co-localize with OsDMC1 signals. OsRAD51 is essential for the loading of OsDMC1 onto chromosomes, and vice versa. In addition, both OsRAD51 and OsDMC1 can interact with OsFIGL1 and OsBRCA2, two important components in rice meiosis. Moreover, the Osrad51 Osdmc1 (Osrad51a1 Osrad51a2 Osdmc1a Osdmc1b) quadruple mutant PMCs exhibited similar defective phenotypes as Osrad51 in homologous pairing, synapsis, and DSB repair. Taken together, our results suggest that the recombinases DMC1 and RAD51 may functionally depend on each other and play important roles in meiotic recombination during meiosis in rice.

Location and Identification on Chromosome 3B of Bread Wheat of Genes Affecting Chiasma Number.

Understanding meiotic crossover (CO) variation in crops like bread wheat (Triticum aestivum L.) is necessary as COs are essential to create new, original and powerful combinations of genes for traits of agronomical interest. We cytogenetically characterized a set of wheat aneuploid lines missing part or all of chromosome 3B to identify the most influential regions for chiasma formation located on this chromosome. We showed that deletion of the short arm did not change the total number of chiasmata genome-wide, whereas this latter was reduced by ~35% while deleting the long arm. Contrary to what was hypothesized in a previous study, deletion of the long arm does not disturb the initiation of the synaptonemal complex (SC) in early meiotic stages. However, progression of the SC is abnormal, and we never observed its completion when the long arm is deleted. By studying six different deletion lines (missing different parts of the long arm), we revealed that at least two genes located in both the proximal (C-3BL2-0.22) and distal (3BL7-0.63-1.00) deletion bins are involved in the control of chiasmata, each deletion reducing the number of chiasmata by ~15%. We combined sequence analyses of deletion bins with RNA-Seq data derived from meiotic tissues and identified a set of genes for which at least the homoeologous copy on chromosome 3B is expressed and which are involved in DNA processing. Among these genes, eight (CAP-E1/E2, DUO1, MLH1, MPK4, MUS81, RTEL1, SYN4, ZIP4) are known to be involved in the recombination pathway.

Uroplakin 1a Knockout Mice Display Marginal Reduction in Fecundity, Decreased Bacterial Clearance Capacity, and Drastic Changes in the Testicular Transcriptome.

Uroplakins (UPKs) form physical and chemical barriers in the bladder and other urinary tract tissues. We previously reported the identification and localization of UPKs in the male reproductive tract of rat. In this study, we characterized Upk1a knockout mice and report a marginal reduction in fecundity associated with significant decrease in sperm count. Upk1a mice had lower bacterial clearance capacity when challenged with uropathogenic Escherichia coli for 1 to 5days. High-throughput analyses of testicular transcriptome indicated that 1128 genes that are expressed in testis of wild-type mice were completely absent in the knockout, while 2330 genes were found to be expressed only in the testis of knockout mice. Furthermore, differential regulation of 148 (67 upregulated and 81 downregulated) was observed. Gene ontology analyses indicated that processes related to integral components of membrane (plasma membrane), G-protein receptor activity and signaling, olfactory receptor activity and perception of smell, organization of extracellular space/region, immune and inflammatory responses to pathogens, spermatid development, meiotic cell cycle, and formation of synaptonemal complex were affected. Results of this study provide evidence on the possible multi-functional role of Upk1a in male reproductive tract and in other tissues as well.