The crucial role of HFM1 in regulating FUS ubiquitination and localization for oocyte meiosis prophase I progression in mice.

BACKGROUND: Helicase for meiosis 1 (HFM1), a putative DNA helicase expressed in germ-line cells, has been reported to be closely associated with premature ovarian insufficiency (POI). However, the underlying molecular mechanism has not been clearly elucidated. The aim of this study was to investigate the function of HFM1 in the first meiotic prophase of mouse oocytes. RESULTS: The results suggested that the deficiency of HFM1 resulting in increased apoptosis and depletion of oocytes in mice, while the oocytes were arrested in the pachytene stage of the first meiotic prophase. In addition, impaired DNA double-strand break repair and disrupted synapsis were observed in the absence of HFM1. Further investigation revealed that knockout of HFM1 promoted ubiquitination and degradation of FUS protein mediated by FBXW11. Additionally, the depletion of HFM1 altered the intranuclear localization of FUS and regulated meiotic- and oocyte development-related genes in oocytes by modulating the expression of BRCA1. CONCLUSIONS: These findings elaborated that the critical role of HFM1 in orchestrating the regulation of DNA double-strand break repair and synapsis to ensure meiosis procession and primordial follicle formation. This study provided insights into the pathogenesis of POI and highlighted the importance of HFM1 in maintaining proper meiotic function in mouse oocytes.

Sororin is an evolutionary conserved antagonist of WAPL.

Cohesin mediates sister chromatid cohesion to enable chromosome segregation and DNA damage repair. To perform these functions, cohesin needs to be protected from WAPL, which otherwise releases cohesin from DNA. It has been proposed that cohesin is protected from WAPL by SORORIN. However, in vivo evidence for this antagonism is missing and SORORIN is only known to exist in vertebrates and insects. It is therefore unknown how important and widespread SORORIN's functions are. Here we report the identification of SORORIN orthologs in Schizosaccharomyces pombe (Sor1) and Arabidopsis thaliana (AtSORORIN). sor1Δ mutants display cohesion defects, which are partially alleviated by wpl1Δ. Atsororin mutant plants display dwarfism, tissue specific cohesion defects and chromosome mis-segregation. Furthermore, Atsororin mutant plants are sterile and separate sister chromatids prematurely at anaphase I. The somatic, but not the meiotic deficiencies can be alleviated by loss of WAPL. These results provide in vivo evidence for SORORIN antagonizing WAPL, reveal that SORORIN is present in organisms beyond the animal kingdom and indicate that it has acquired tissue specific functions in plants.

Post-meiotic mechanism of facultative parthenogenesis in gonochoristic whiptail lizard species.

Facultative parthenogenesis (FP) has historically been regarded as rare in vertebrates, but in recent years incidences have been reported in a growing list of fish, reptile, and bird species. Despite the increasing interest in the phenomenon, the underlying mechanism and evolutionary implications have remained unclear. A common finding across many incidences of FP is either a high degree of homozygosity at microsatellite loci or low levels of heterozygosity detected in next-generation sequencing data. This has led to the proposal that second polar body fusion following the meiotic divisions restores diploidy and thereby mimics fertilization. Here, we show that FP occurring in the gonochoristic Aspidoscelis species A. marmoratus and A. arizonae results in genome-wide homozygosity, an observation inconsistent with polar body fusion as the underlying mechanism of restoration. Instead, a high-quality reference genome for A. marmoratus and analysis of whole-genome sequencing from multiple FP and control animals reveals that a post-meiotic mechanism gives rise to homozygous animals from haploid, unfertilized oocytes. Contrary to the widely held belief that females need to be isolated from males to undergo FP, females housed with conspecific and heterospecific males produced unfertilized eggs that underwent spontaneous development. In addition, offspring arising from both fertilized eggs and parthenogenetic development were observed to arise from a single clutch. Strikingly, our data support a mechanism for facultative parthenogenesis that removes all heterozygosity in a single generation. Complete homozygosity exposes the genetic load and explains the high rate of congenital malformations and embryonic mortality associated with FP in many species. Conversely, for animals that develop normally, FP could potentially exert strong purifying selection as all lethal recessive alleles are purged in a single generation.

Retinoic acid mitigates the NSC319726-induced spermatogenesis dysfunction through cuproptosis-independent mechanisms.

Copper ionophore NSC319726 has attracted researchers' attention in treating diseases, particularly cancers. However, its potential effects on male reproduction during medication are unclear. This study aimed to determine whether NSC319726 exposure affected the male reproductive system. The reproductive toxicity of NSC319726 was evaluated in male mice following a continuous exposure period of 5 weeks. The result showed that NSC319726 exposure caused testis index reduction, spermatogenesis dysfunction, and architectural damage in the testis and epididymis. The exposure interfered with spermatogonia proliferation, meiosis initiation, sperm count, and sperm morphology. The exposure also disturbed androgen synthesis and blood testis barrier integrity. NSC319726 treatment could elevate the copper ions in the testis to induce cuproptosis in the testis. Copper chelator rescued the elevated copper ions in the testis and partly restored the spermatogenesis dysfunction caused by NSC319726. NSC319726 treatment also decreased the level of retinol dehydrogenase 10 (RDH10), thereby inhibiting the conversion of retinol to retinoic acid, causing the inability to initiate meiosis. Retinoic acid treatment could rescue the meiotic initiation and spermatogenesis while not affecting the intracellular copper ion levels. The study provided an insight into the bio-safety of NSC319726. Retinoic acid could be a potential therapy for spermatogenesis impairment in patients undergoing treatment with NSC319726.

Primordial germ cell DNA demethylation and development require DNA translesion synthesis.

Mutations in DNA damage response (DDR) factors are associated with human infertility, which affects up to 15% of the population. The DDR is required during germ cell development and meiosis. One pathway implicated in human fertility is DNA translesion synthesis (TLS), which allows replication impediments to be bypassed. We find that TLS is essential for pre-meiotic germ cell development in the embryo. Loss of the central TLS component, REV1, significantly inhibits the induction of human PGC-like cells (hPGCLCs). This is recapitulated in mice, where deficiencies in TLS initiation (Rev1-/- or PcnaK164R/K164R) or extension (Rev7 -/-) result in a > 150-fold reduction in the number of primordial germ cells (PGCs) and complete sterility. In contrast, the absence of TLS does not impact the growth, function, or homeostasis of somatic tissues. Surprisingly, we find a complete failure in both activation of the germ cell transcriptional program and in DNA demethylation, a critical step in germline epigenetic reprogramming. Our findings show that for normal fertility, DNA repair is required not only for meiotic recombination but for progression through the earliest stages of germ cell development in mammals.

Meiotic drive against chromosome fusions in butterfly hybrids.

Species frequently differ in the number and structure of chromosomes they harbor, but individuals that are heterozygous for chromosomal rearrangements may suffer from reduced fitness. Chromosomal rearrangements like fissions and fusions can hence serve as a mechanism for speciation between incipient lineages, but their evolution poses a paradox. How can rearrangements get fixed between populations if heterozygotes have reduced fitness? One solution is that this process predominantly occurs in small and isolated populations, where genetic drift can override natural selection. However, fixation is also more likely if a novel rearrangement is favored by a transmission bias, such as meiotic drive. Here, we investigate chromosomal transmission distortion in hybrids between two wood white (Leptidea sinapis) butterfly populations with extensive karyotype differences. Using data from two different crossing experiments, we uncover that there is a transmission bias favoring the ancestral chromosomal state for derived fusions, a result that shows that chromosome fusions actually can fix in populations despite being counteracted by meiotic drive. This means that meiotic drive not only can promote runaway chromosome number evolution and speciation, but also that it can be a conservative force acting against karyotypic change and the evolution of reproductive isolation. Based on our results, we suggest a mechanistic model for why chromosome fusion mutations may be opposed by meiotic drive and discuss factors contributing to karyotype evolution in Lepidoptera.

FMNL2 regulates actin for endoplasmic reticulum and mitochondria distribution in oocyte meiosis.

During mammalian oocyte meiosis, spindle migration and asymmetric cytokinesis are unique steps for the successful polar body extrusion. The asymmetry defects of oocytes will lead to the failure of fertilization and embryo implantation. In present study, we reported that an actin nucleating factor Formin-like 2 (FMNL2) played critical roles in the regulation of spindle migration and organelle distribution in mouse and porcine oocytes. Our results showed that FMNL2 mainly localized at the oocyte cortex and periphery of spindle. Depletion of FMNL2 led to the failure of polar body extrusion and large polar bodies in oocytes. Live-cell imaging revealed that the spindle failed to migrate to the oocyte cortex, which caused polar body formation defects, and this might be due to the decreased polymerization of cytoplasmic actin by FMNL2 depletion in the oocytes of both mice and pigs. Furthermore, mass spectrometry analysis indicated that FMNL2 was associated with mitochondria and endoplasmic reticulum (ER)-related proteins, and FMNL2 depletion disrupted the function and distribution of mitochondria and ER, showing with decreased mitochondrial membrane potential and the occurrence of ER stress. Microinjecting Fmnl2-EGFP mRNA into FMNL2-depleted oocytes significantly rescued these defects. Thus, our results indicate that FMNL2 is essential for the actin assembly, which further involves into meiotic spindle migration and ER/mitochondria functions in mammalian oocytes.

Meiotic transcriptional reprogramming mediated by cell-cell communications in humans and mice revealed by scATAC-seq and scRNA-seq.

Meiosis is a highly complex process significantly influenced by transcriptional regulation. However, studies on the mechanisms that govern transcriptomic changes during meiosis, especially in prophase I, are limited. Here, we performed single-cell ATAC-seq of human testis tissues and observed reprogramming during the transition from zygotene to pachytene spermatocytes. This event, conserved in mice, involved the deactivation of genes associated with meiosis after reprogramming and the activation of those related to spermatogenesis before their functional onset. Furthermore, we identified 282 transcriptional regulators (TRs) that underwent activation or deactivation subsequent to this process. Evidence suggested that physical contact signals from Sertoli cells may regulate these TRs in spermatocytes, while secreted ENHO signals may alter metabolic patterns in these cells. Our results further indicated that defective transcriptional reprogramming may be associated with non-obstructive azoospermia (NOA). This study revealed the importance of both physical contact and secreted signals between Sertoli cells and germ cells in meiotic progression.

CPEB3 Maintains Developmental Competence of the Oocyte.

Mammalian oocyte development depends on the temporally controlled translation of maternal transcripts, particularly in the coordination of meiotic and early embryonic development when transcription has ceased. The translation of mRNA is regulated by various RNA-binding proteins. We show that the absence of cytoplasmic polyadenylation element-binding protein 3 (CPEB3) negatively affects female reproductive fitness. CPEB3-depleted oocytes undergo meiosis normally but experience early embryonic arrest due to a disrupted transcriptome, leading to aberrant protein expression and the subsequent failure of embryonic transcription initiation. We found that CPEB3 stabilizes a subset of mRNAs with a significantly longer 3'UTR that is enriched in its distal region with cytoplasmic polyadenylation elements. Overall, our results suggest that CPEB3 is an important maternal factor that regulates the stability and translation of a subclass of mRNAs that are essential for the initiation of embryonic transcription and thus for embryonic development.

Chromosome stability of synthetic Triticum turgidum-Aegilops umbellulata hybrids.

BACKGROUND: Unreduced gamete formation during meiosis plays a critical role in natural polyploidization. However, the unreduced gamete formation mechanisms in Triticum turgidum-Aegilops umbellulata triploid F1 hybrid crosses and the chromsome numbers and compostions in T. turgidum-Ae. umbellulata F2 still not known. RESULTS: In this study, 11 T.turgidum-Ae. umbellulata triploid F1 hybrid crosses were produced by distant hybridization. All of the triploid F1 hybrids had 21 chromosomes and two basic pathways of meiotic restitution, namely first-division restitution (FDR) and single-division meiosis (SDM). Only FDR was found in six of the 11 crosses, while both FDR and SDM occurred in the remaining five crosses. The chromosome numbers in the 127 selfed F2 seeds from the triploid F1 hybrid plants of 10 crosses (no F2 seeds for STU 16) varied from 35 to 43, and the proportions of euploid and aneuploid F2 plants were 49.61% and 50.39%, respectively. In the aneuploid F2 plants, the frequency of chromosome loss/gain varied among genomes. The chromosome loss of the U genome was the highest (26.77%) among the three genomes, followed by that of the B (22.83%) and A (11.81%) genomes, and the chromosome gain for the A, B, and U genomes was 3.94%, 3.94%, and 1.57%, respectively. Of the 21 chromosomes, 7U (16.54%), 5 A (3.94%), and 1B (9.45%) had the highest loss frequency among the U, A, and B genomes. In addition to chromosome loss, seven chromosomes, namely 1 A, 3 A, 5 A, 6 A, 1B, 1U, and 6U, were gained in the aneuploids. CONCLUSION: In the aneuploid F2 plants, the frequency of chromosome loss/gain varied among genomes, chromsomes, and crosses. In addition to variations in chromosome numbers, three types of chromosome translocations including 3UL·2AS, 6UL·1AL, and 4US·6AL were identified in the F2 plants. Furthermore, polymorphic fluorescence in situ hybridization karyotypes for all the U chromosomes were also identified in the F2 plants when compared with the Ae. umbellulata parents. These results provide useful information for our understanding the naturally occurred T. turgidum-Ae. umbellulata amphidiploids.

NLRP4E regulates actin cap formation through SRC and CDC42 during oocyte meiosis.

BACKGROUND: Members of the nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing (NLRP) family regulate various physiological and pathological processes. However, none have been shown to regulate actin cap formation or spindle translocation during the asymmetric division of oocyte meiosis I. NLRP4E has been reported as a candidate protein in female fertility, but its function is unknown. METHODS: Immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR), and western blotting were employed to examine the localization and expression levels of NLRP4E and related proteins in mouse oocytes. small interfering RNA (siRNA) and antibody transfection were used to knock down NLRP4E and other proteins. Immunoprecipitation (IP)-mass spectrometry was used to identify the potential proteins interacting with NLRP4E. Coimmunoprecipitation (Co-IP) was used to verify the protein interactions. Wild type (WT) or mutant NLRP4E messenger RNA (mRNA) was injected into oocytes for rescue experiments. In vitro phosphorylation was employed to examine the activation of steroid receptor coactivator (SRC) by NLRP4E. RESULTS: NLRP4E was more predominant within oocytes compared with other NLRP4 members. NLRP4E knockdown significantly inhibited actin cap formation and spindle translocation toward the cap region, resulting in the failure of polar body extrusion at the end of meiosis I. Mechanistically, GRIN1, and GANO1 activated NLRP4E by phosphorylation at Ser429 and Thr430; p-NLRP4E is translocated and is accumulated in the actin cap region during spindle translocation. Next, we found that p-NLRP4E directly phosphorylated SRC at Tyr418, while p-SRC negatively regulated p-CDC42-S71, an inactive form of CDC42 that promotes actin cap formation and spindle translocation in the GTP-bound form. CONCLUSIONS: NLRP4E activated by GRIN1 and GANO1 regulates actin cap formation and spindle translocation toward the cap region through upregulation of p-SRC-Tyr418 and downregulation of p-CDC42-S71 during meiosis I.

BRCA1 safeguards genome integrity by activating chromosome asynapsis checkpoint to eliminate recombination-defective oocytes.

In the meiotic prophase, programmed DNA double-strand breaks are repaired by meiotic recombination. Recombination-defective meiocytes are eliminated to preserve genome integrity in gametes. BRCA1 is a critical protein in somatic homologous recombination, but studies have suggested that BRCA1 is dispensable for meiotic recombination. Here we show that BRCA1 is essential for meiotic recombination. Interestingly, BRCA1 also has a function in eliminating recombination-defective oocytes. Brca1 knockout (KO) rescues the survival of Dmc1 KO oocytes far more efficiently than removing CHK2, a vital component of the DNA damage checkpoint in oocytes. Mechanistically, BRCA1 activates chromosome asynapsis checkpoint by promoting ATR activity at unsynapsed chromosome axes in Dmc1 KO oocytes. Moreover, Brca1 KO also rescues the survival of asynaptic Spo11 KO oocytes. Collectively, our study not only unveils an unappreciated role of chromosome asynapsis in eliminating recombination-defective oocytes but also reveals the dual functions of BRCA1 in safeguarding oocyte genome integrity.

Premeiotic 24-nt phasiRNAs are present in the Zea genus and unique in biogenesis mechanism and molecular function.

Reproductive phasiRNAs (phased, small interfering RNAs) are broadly present in angiosperms and play crucial roles in sustaining male fertility. While the premeiotic 21-nt (nucleotides) phasiRNAs and meiotic 24-nt phasiRNA pathways have been extensively studied in maize (Zea mays) and rice (Oryza sativa), a third putative category of reproductive phasiRNAs-named premeiotic 24-nt phasiRNAs-have recently been reported in barley (Hordeum vulgare) and wheat (Triticum aestivum). To determine whether premeiotic 24-nt phasiRNAs are also present in maize and related species and begin to characterize their biogenesis and function, we performed a comparative transcriptome and degradome analysis of premeiotic and meiotic anthers from five maize inbred lines and three teosinte species/subspecies. Our data indicate that a substantial subset of the 24-nt phasiRNA loci in maize and teosinte are already highly expressed at the premeiotic phase. The premeiotic 24-nt phasiRNAs are similar to meiotic 24-nt phasiRNAs in genomic origin and dependence on DCL5 (Dicer-like 5) for biogenesis, however, premeiotic 24-nt phasiRNAs are unique in that they are likely i) not triggered by microRNAs, ii) not loaded by AGO18 proteins, and iii) not capable of mediating PHAS precursor cleavage. In addition, we also observed a group of premeiotic 24-nt phasiRNAs in rice using previously published data. Together, our results indicate that the premeiotic 24-nt phasiRNAs constitute a unique class of reproductive phasiRNAs and are present more broadly in the grass family (Poaceae) than previously known.

Bridging the gap between the evolutionary dynamics and the molecular mechanisms of meiosis: A model based exploration of the PRDM9 intra-genomic Red Queen.

Molecular dissection of meiotic recombination in mammals, combined with population-genetic and comparative studies, have revealed a complex evolutionary dynamic characterized by short-lived recombination hotspots. Hotspots are chromosome positions containing DNA sequences where the protein PRDM9 can bind and cause crossing-over. To explain these fast evolutionary dynamic, a so-called intra-genomic Red Queen model has been proposed, based on the interplay between two antagonistic forces: biased gene conversion, mediated by double-strand breaks, resulting in hotspot extinction (the hotspot conversion paradox), followed by positive selection favoring mutant PRDM9 alleles recognizing new sequence motifs. Although this model predicts many empirical observations, the exact causes of the positive selection acting on new PRDM9 alleles is still not well understood. In this direction, experiment on mouse hybrids have suggested that, in addition to targeting double strand breaks, PRDM9 has another role during meiosis. Specifically, PRDM9 symmetric binding (simultaneous binding at the same site on both homologues) would facilitate homology search and, as a result, the pairing of the homologues. Although discovered in hybrids, this second function of PRDM9 could also be involved in the evolutionary dynamic observed within populations. To address this point, here, we present a theoretical model of the evolutionary dynamic of meiotic recombination integrating current knowledge about the molecular function of PRDM9. Our modeling work gives important insights into the selective forces driving the turnover of recombination hotspots. Specifically, the reduced symmetrical binding of PRDM9 caused by the loss of high affinity binding sites induces a net positive selection eliciting new PRDM9 alleles recognizing new targets. The model also offers new insights about the influence of the gene dosage of PRDM9, which can paradoxically result in negative selection on new PRDM9 alleles entering the population, driving their eviction and thus reducing standing variation at this locus.

Agent-based modeling of nuclear chromosome ensembles identifies determinants of homolog pairing during meiosis.

During meiosis, pairing of homologous chromosomes (homologs) ensures the formation of haploid gametes from diploid precursor cells, a prerequisite for sexual reproduction. Pairing during meiotic prophase I facilitates crossover recombination and homolog segregation during the ensuing reductional cell division. Mechanisms that ensure stable homolog alignment in the presence of an excess of non-homologous chromosomes have remained elusive, but rapid chromosome movements appear to play a role in the process. Apart from homolog attraction, provided by early intermediates of homologous recombination, dissociation of non-homologous associations also appears to contribute to homolog pairing, as suggested by the detection of stable non-homologous chromosome associations in pairing-defective mutants. Here, we have developed an agent-based model for homolog pairing derived from the dynamics of a naturally occurring chromosome ensemble. The model simulates unidirectional chromosome movements, as well as collision dynamics determined by attractive and repulsive forces arising from close-range physical interactions. Chromosome number and size as well as movement velocity and repulsive forces are identified as key factors in the kinetics and efficiency of homologous pairing in addition to homolog attraction. Dissociation of interactions between non-homologous chromosomes may contribute to pairing by crowding homologs into a limited nuclear area thus creating preconditions for close-range homolog attraction. Incorporating natural chromosome lengths, the model accurately recapitulates efficiency and kinetics of homolog pairing observed for wild-type and mutant meiosis in budding yeast, and can be adapted to nuclear dimensions and chromosome sets of other organisms.

Targeted suppression of siRNA biogenesis in Arabidopsis pollen promotes triploid seed viability.

In plants, small-interfering RNAs (siRNAs) mediate epigenetic silencing via the RNA-directed DNA methylation (RdDM) pathway, which is particularly prominent during reproduction and seed development. However, there is limited understanding of the origins and dynamics of reproductive siRNAs acting in different cellular and developmental contexts. Here, we used the RNaseIII-like protein RTL1 to suppress siRNA biogenesis in Arabidopsis pollen, and found distinct siRNA subsets produced during pollen development. We demonstrate that RTL1 expression in the late microspore and vegetative cell strongly impairs epigenetic silencing, and resembles RdDM mutants in their ability to bypass interploidy hybridization barriers in the seed. However, germline-specific RTL1 expression did not impact transgenerational inheritance of triploid seed lethality. These results reveal the existence of multiple siRNA subsets accumulated in mature pollen, and suggest that mobile siRNAs involved in the triploid block are produced in germline precursor cells after meiosis, or in the vegetative cell during pollen mitosis.

CXCR3 participates in asymmetric division of mouse oocytes by modulating actin dynamics.

Extensive research has been conducted on the role of CXCR3 in immune responses and inflammation. However, the role of CXCR3 in the reproductive system, particularly in oocyte development, remains unknown. In this study, we present findings on the involvement of CXCR3 in the meiotic division process of mouse oocytes. We found CXCR3 was expressed consistently throughout the entire maturation process of mouse oocyte. Inhibition of CXCR3 impaired the asymmetric division of oocyte, while the injection of Cxcr3 mRNA was capable of restoring these defects. Further study showed that inhibition of CXCR3 perturbed spindle migration by affecting LIMK/cofilin pathway-mediated actin remodeling. Knockout of CXCR3 led to an upregulation of actin-binding protein and an increased ATP level in GV-stage oocytes, while maintaining normal actin dynamics during the process of meiosis. Additionally, we noticed the expression level of DYNLT1 is markedly elevated in CXCR3-null oocytes. DYNLT1 bound with the Arp2/3 complex, and knockdown of DYNLT1 in CXCR3-null oocytes impaired the organization of cytoplasmic actin, suggesting the regulatory role of DYNLT1 in actin organization, and the compensatory expression of DYNLT1 may contribute to maintain normal actin dynamics in CXCR3-knockout oocytes. In summary, our findings provide insights into the intricate network of actin dynamics associated with CXCR3 during oocyte meiosis.

Multiple intersecting pathways are involved in CPEB1 phosphorylation and regulation of translation during mouse oocyte meiosis.

The RNA-binding protein cytoplasmic polyadenylation element binding 1 (CPEB1) plays a fundamental role in regulating mRNA translation in oocytes. However, the specifics of how and which protein kinase cascades modulate CPEB1 activity are still controversial. Using genetic and pharmacological tools, and detailed time courses, we have re-evaluated the relationship between CPEB1 phosphorylation and translation activation during mouse oocyte maturation. We show that both the CDK1/MAPK and AURKA/PLK1 pathways converge on CPEB1 phosphorylation during prometaphase of meiosis I. Only inactivation of the CDK1/MAPK pathway disrupts translation, whereas inactivation of either pathway alone leads to CPEB1 stabilization. However, CPEB1 stabilization induced by inactivation of the AURKA/PLK1 pathway does not affect translation, indicating that destabilization and/or degradation is not linked to translational activation. The accumulation of endogenous CCNB1 protein closely recapitulates the translation data that use an exogenous template. These findings support the overarching hypothesis that the activation of translation during prometaphase in mouse oocytes relies on a CDK1/MAPK-dependent CPEB1 phosphorylation, and that translational activation precedes CPEB1 destabilization.

DNA-binding site II is required for RAD51 recombinogenic activity in Arabidopsis thaliana.

Homologous recombination is a major pathway for the repair of DNA double strand breaks, essential both to maintain genomic integrity and to generate genetic diversity. Mechanistically, homologous recombination involves the use of a homologous DNA molecule as a template to repair the break. In eukaryotes, the search for and invasion of the homologous DNA molecule is carried out by two recombinases, RAD51 in somatic cells and RAD51 and DMC1 in meiotic cells. During recombination, the recombinases bind overhanging single-stranded DNA ends to form a nucleoprotein filament, which is the active species in promoting DNA invasion and strand exchange. RAD51 and DMC1 carry two major DNA-binding sites-essential for nucleofilament formation and DNA strand exchange, respectively. Here, we show that the function of RAD51 DNA-binding site II is conserved in the plant, Arabidopsis. Mutation of three key amino acids in site II does not affect RAD51 nucleofilament formation but inhibits its recombinogenic activity, analogous to results from studies of the yeast and human proteins. We further confirm that recombinogenic function of RAD51 DNA-binding site II is not required for meiotic double-strand break repair when DMC1 is present. The Arabidopsis AtRAD51-II3A separation of function mutant shows a dominant negative phenotype, pointing to distinct biochemical properties of eukaryotic RAD51 proteins.

Medaka Terb1 Mutant Displays Defects of Synaptonemal Complex Formation and Sexual Difference in Gametogenesis.

Formation of the synaptonemal complex (SC) is a prerequisite for proper recombination and chromosomal segregation during meiotic prophase I. One mechanism that ensures SC formation is chromosomal movement, which is driven by the force derived from cytoskeletal motors. Here, we report the phenotype of medaka mutants lacking the telomere repeat binding bouquet formation protein 1 (TERB1), which, in combination with the SUN/KASH protein, mediates chromosomal movement by connecting telomeres and cytoskeletal motors. Mutations in the terb1 gene exhibit defects in SC formation in medaka. Although SC formation was initiated, as seen by the punctate lateral elements and fragmented transverse filaments, it was not completed in the terb1 mutant meiocytes. The mutant phenotype further revealed that the introduction of double strand breaks was independent of synapsis completion. In association with these phenotypes, meiocytes in both the ovaries and testes exhibited an aberrant arrangement of homologous chromosomes. Interestingly, although oogenesis halted at the zygotene-like stage in terb1 mutant, testes continued to produce sperm-like cells with aberrant DNA content. This indicates that the mechanism of meiotic checkpoint is sexually different in medaka, similar to the mammalian checkpoint in which oogenesis proceeds while spermatogenesis is arrested. Moreover, our results suggest that spermatogenesis is mechanistically dissociable from meiosis.