Centrosome dysfunction associated with somatic expression of the synaptonemal complex protein TEX12

The synaptonemal complex (SC) is a supramolecular protein scaffold that mediates chromosome synapsis and facilitates crossing over during meiosis. In mammals, SC proteins are generally assumed to have no other function. Here, we show that SC protein TEX12 also localises to centrosomes during meiosis independently of chromosome synapsis. In somatic cells, ectopically expressed TEX12 similarly localises to centrosomes, where it is associated with centrosome amplification, a pathology correlated with cancer development. Indeed, TEX12 is identified as a cancer-testis antigen and proliferation of some cancer cells is TEX12-dependent. Moreover, somatic expression of TEX12 is aberrantly activated via retinoic acid signalling, which is commonly disregulated in cancer. Structure-function analysis reveals that phosphorylation of TEX12 on tyrosine 48 is important for centrosome amplification but not for recruitment of TEX12 to centrosomes. We conclude that TEX12 normally localises to meiotic centrosomes, but its misexpression in somatic cells can contribute to pathological amplification and dysfunction of centrosomes in cancers.

FBXO47 is essential for preventing the synaptonemal complex from premature disassembly in mouse male meiosis

Meiotic prophase is a prolonged G2 phase that ensures the completion of numerous meiosis-specific chromosome events. During meiotic prophase, homologous chromosomes undergo synapsis to facilitate meiotic recombination yielding crossovers. It remains largely elusive how homolog synapsis is temporally maintained and destabilized during meiotic prophase. Here we show that FBXO47 is the stabilizer of synaptonemal complex during male meiotic prophase. Disruption of FBXO47 shows severe impact on homologous chromosome synapsis and DSB repair processes, leading to male infertility. Notably, in the absence of FBXO47, although once homologous chromosomes are synapsed, the synaptonemal complex is precociously disassembled before progressing beyond pachytene. Remarkably, Fbxo47 KO spermatocytes remain in earlier stage of meiotic prophase and lack crossovers, despite apparently exhibiting diplotene-like chromosome morphology. We propose that FBXO47 functions independently of SCF E3 ligase, and plays a crucial role in preventing synaptonemal complex from premature disassembly during cell cycle progression of meiotic prophase.

Meiotic Chromosome Synapsis and XY-Body Formation In Vitro

To achieve spermatogenesis in vitro, one of the most challenging processes to mimic is meiosis. Meiotic problems, like incomplete synapsis of the homologous chromosomes, or impaired homologous recombination, can cause failure of crossover formation and subsequent chromosome nondisjunction, eventually leading to aneuploid sperm. These meiotic events are therefore strictly monitored by meiotic checkpoints that initiate apoptosis of aberrant spermatocytes and lead to spermatogenic arrest. However, we recently found that, in vitro derived meiotic cells proceeded to the first meiotic division (MI) stage, despite displaying incomplete chromosome synapsis, no discernible XY-body and lack of crossover formation. We therefore optimized our in vitro culture system of meiosis from male germline stem cells (mGSCs) in order to achieve full chromosome synapsis, XY-body formation and meiotic crossovers. In comparison to previous culture system, the in vitro-generated spermatocytes were transferred after meiotic initiation to a second culture dish. This dish already contained a freshly plated monolayer of proliferatively inactivated immortalized Sertoli cells supporting undifferentiated mGSCs. In this way we aimed to simulate the multiple layers of germ cell types that support spermatogenesis in vivo in the testis. We found that in this optimized culture system, although independent of the undifferentiated mGSCs, meiotic chromosome synapsis was complete and XY body appeared normal. However, meiotic recombination still occurred insufficiently and only few meiotic crossovers were formed, leading to MI-spermatocytes displaying univalent chromosomes (paired sister chromatids). Therefore, considering that meiotic checkpoints are not necessarily fully functional in vitro, meiotic crossover formation should be closely monitored when mimicking gametogenesis in vitro to prevent generation of aneuploid gametes.

Altering the Binding Properties of PRDM9 Partially Restores Fertility across the Species Boundary

Sterility or subfertility of male hybrid offspring is commonly observed. This phenomenon contributes to reproductive barriers between the parental populations, an early step in the process of speciation. One frequent cause of such infertility is a failure of proper chromosome pairing during male meiosis. In subspecies of the house mouse, the likelihood of successful chromosome synapsis is improved by the binding of the histone methyltransferase PRDM9 to both chromosome homologs at matching positions. Using genetic manipulation, we altered PRDM9 binding to occur more often at matched sites, and find that chromosome pairing defects can be rescued, not only in an intersubspecific cross, but also between distinct species. Using different engineered variants, we demonstrate a quantitative link between the degree of matched homolog binding, chromosome synapsis, and rescue of fertility in hybrids between Mus musculus and Mus spretus. The resulting partial restoration of fertility reveals additional mechanisms at play that act to lock-in the reproductive isolation between these two species.

SYMPK Is Required for Meiosis and Involved in Alternative Splicing in Male Germ Cells

SYMPK is a scaffold protein that supports polyadenylation machinery assembly on nascent transcripts and is also involved in alternative splicing in some mammalian somatic cells. However, the role of SYMPK in germ cells remains unknown. Here, we report that SYMPK is highly expressed in male germ cells, and germ cell-specific knockout (cKO) of Sympk in mouse leads to male infertility. Sympk cKODdx4–cre mice showed reduced spermatogonia at P4 and almost no germ cells at P18. Sympk cKOStra8–Cre spermatocytes exhibit defects in homologous chromosome synapsis, DNA double-strand break (DSB) repair, and meiotic recombination. RNA-Seq analyses reveal that SYMPK is associated with alternative splicing, besides regulating the expressions of many genes in spermatogenic cells. Importantly, Sympk deletion results in abnormal alternative splicing and a decreased expression of Sun1. Taken together, our results demonstrate that SYMPK is pivotal for meiotic progression by regulating pre-mRNA alternative splicing in male germ cells.

The Arabidopsis HOP2 Gene Has a Role in Preventing illegitimate Exchanges between Nonhomologous Chromosomes

Meiotic homologous chromosomes pair up and undergo crossing over. In many eukaryotes both intimate pairing and crossing over require the induction of double stranded breaks (DSBs) and subsequent repair via Homologous Recombination (HR). In these organisms, two key proteins are the recombinases RAD51 and DMC1. Recombinase-modulators HOP2 and MND1 have been identified as proteins that assist RAD51 and DMC1 and are needed to promote stabilized pairing. We have probed the nature of the genetic lesions seen in hop2 mutants and looked at the role of HOP2 in the fidelity of genetic exchanges. Using γH2AX as a marker for unrepaired DSBs we found that hop2-1 and mnd1 mutants have different appearance/disappearance for DSBs than wild type, but all DSBs are repaired by mid-late pachytene. Therefore, the bridges and fragments seen from metaphase I onward are due to mis-repaired DSBs, not unrepaired ones. Studying Arabidopsis haploid meiocytes we found that wild type haploids produced the expected five univalents, but hop2-1 haploids suffered many illegitimate exchanges that were stable enough to produce bridged chromosomes during segregation. Our results suggest that HOP2 has a significant active role in preventing nonhomologous associations. We also found evidence that HOP2 plays a role in preventing illegitimate exchanges during repair of radiation-induced DSBs in rapidly dividing petal cells. Thus, HOP2 plays both a positive role in promoting homologous chromosome synapsis and a separable role in preventing nonhomologous chromosome exchanges. Possible mechanisms for this second important role are discussed.

The impact of chromosomal fusions on 3D genome folding and recombination in the germ line

The spatial folding of chromosomes inside the nucleus has regulatory effects on gene expression, yet the impact of genome reshuffling on this organization remains unclear. Here, we take advantage of chromosome conformation capture in combination with single-nucleotide polymorphism (SNP) genotyping and analysis of crossover events to study how the higher-order chromatin organization and recombination landscapes are affected by chromosomal fusions in the mammalian germ line. We demonstrate that chromosomal fusions alter the nuclear architecture during meiosis, including an increased rate of heterologous interactions in primary spermatocytes, and alterations in both chromosome synapsis and axis length. These disturbances in topology were associated with changes in genomic landscapes of recombination, resulting in detectable genomic footprints. Overall, we show that chromosomal fusions impact the dynamic genome topology of germ cells in two ways: (i) altering chromosomal nuclear occupancy and synapsis, and (ii) reshaping landscapes of recombination.

Reproductive Isolation Between Taxonomically Controversial Forms of the Gray Voles (Microtus, Rodentia; Arvicolinae): Cytological Mechanisms and Taxonomical Implications

The formation of hybrid sterility is an important stage of speciation. The voles of the genus Microtus, which is the most speciose genus of rodents, provide a good model for studying the cytological mechanisms of hybrid sterility. The voles of the “mystacinus” group of the subgenus Microtus (2n = 54) comprising several recently diverged forms with unclear taxonomic status are especially interesting. To resolve the taxonomic status of Microtus mystacinus and Microtus kermanensis, we crossed both with Microtus rossiaemeridionalis, and M. kermanensis alone with Microtus arvalis “obscurus” and M. transcaspicus and examined the reproductive performance of their F1 hybrids. All interspecies male hybrids were sterile. Female M. kermanensis × M. arvalis and M. kermanensis × M. transcaspicus hybrids were sterile as well. Therefore, M. mystacinus, M. kermanensis, and M. rossiaemeridionalis could be considered valid species. To gain an insight into the cytological mechanisms of male hybrid sterility, we carried out a histological analysis of spermatogenesis and a cytological analysis of chromosome synapsis, recombination, and epigenetic chromatin modifications in the germ cells of the hybrids using immunolocalization of key meiotic proteins. The hybrids showed wide variation in the onset of spermatogenesis arrest stage, from mature (although abnormal) spermatozoa to spermatogonia only. Chromosome asynapsis was apparently the main cause of meiotic arrest. The degree of asynapsis varied widely across cells, individuals, and the crosses—from partial asynapsis of several small bivalents to complete asynapsis of all chromosomes. The asynapsis was accompanied by a delayed repair of DNA double-strand breaks marked by RAD51 antibodies and silencing of unpaired chromatin marked by γH2A.X antibodies. Overall, the severity of disturbances in spermatogenesis in general and in chromosome synapsis in particular increased in the hybrids with an increase in the phylogenetic distance between their parental species.

Repurposing of synaptonemal complex proteins for kinetochores in Kinetoplastida

Chromosome segregation in eukaryotes is driven by the kinetochore, a macromolecular complex that connects centromeric DNA to microtubules of the spindle apparatus. Kinetochores in well-studied model eukaryotes consist of a core set of proteins that are broadly conserved among distant eukaryotic phyla. By contrast, unicellular flagellates of the class Kinetoplastida have a unique set of 36 kinetochore components. The evolutionary origin and history of these kinetochores remain unknown. Here, we report evidence of homology between axial element components of the synaptonemal complex and three kinetoplastid kinetochore proteins KKT16-18. The synaptonemal complex is a zipper-like structure that assembles between homologous chromosomes during meiosis to promote recombination. By using sensitive homology detection protocols, we identify divergent orthologues of KKT16-18 in most eukaryotic supergroups, including experimentally established chromosomal axis components, such as Red1 and Rec10 in budding and fission yeast, ASY3-4 in plants and SYCP2-3 in vertebrates. Furthermore, we found 12 recurrent duplications within this ancient eukaryotic SYCP 2–3 gene family, providing opportunities for new functional complexes to arise, including KKT16-18 in the kinetoplastid parasite Trypanosoma brucei . We propose the kinetoplastid kinetochore system evolved by repurposing meiotic components of the chromosome synapsis and homologous recombination machinery that were already present in early eukaryotes.