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PLoS Genetics May 2021Meiosis is a cell division process with complex chromosome events where various molecules must work in tandem. To find meiosis-related genes, we screened evolutionarily...
Meiosis is a cell division process with complex chromosome events where various molecules must work in tandem. To find meiosis-related genes, we screened evolutionarily conserved and reproductive tract-enriched genes using the CRISPR/Cas9 system and identified potassium channel tetramerization domain containing 19 (Kctd19) as an essential factor for meiosis. In prophase I, Kctd19 deficiency did not affect synapsis or the DNA damage response, and chiasma structures were also observed in metaphase I spermatocytes of Kctd19 KO mice. However, spermatocytes underwent apoptotic elimination during the metaphase-anaphase transition. We were able to rescue the Kctd19 KO phenotype with an epitope-tagged Kctd19 transgene. By immunoprecipitation-mass spectrometry, we confirmed the association of KCTD19 with zinc finger protein 541 (ZFP541) and histone deacetylase 1 (HDAC1). Phenotyping of Zfp541 KO spermatocytes demonstrated XY chromosome asynapsis and recurrent DNA damage in the late pachytene stage, leading to apoptosis. In summary, our study reveals that KCTD19 associates with ZFP541 and HDAC1, and that both KCTD19 and ZFP541 are essential for meiosis in male mice.
Topics: Anaphase; Animals; CRISPR-Cas Systems; Cell Cycle Proteins; Cell Nucleus; Chromosomal Proteins, Non-Histone; Chromosome Pairing; Conserved Sequence; DNA Damage; Evolution, Molecular; Fertility; Genes, Essential; Histone Deacetylase 1; Male; Meiosis; Meiotic Prophase I; Metaphase; Mice; Nuclear Proteins; Pachytene Stage; Phenotype; Spermatids; Spermatocytes; Testis; Transcription Factors; Transgenes
PubMed: 33961623
DOI: 10.1371/journal.pgen.1009412 -
Proceedings of the National Academy of... Oct 2022The synaptonemal complex (SC) is a proteinaceous scaffold that is assembled between paired homologous chromosomes during the onset of meiosis. Timely expression of SC...
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 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 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.
Topics: Chromosomal Proteins, Non-Histone; Chromosome Pairing; Female; Humans; Meiosis; Meiotic Prophase I; Polycomb-Group Proteins; Synaptonemal Complex
PubMed: 36215502
DOI: 10.1073/pnas.2204701119 -
PLoS Genetics Feb 2024Meiotic recombination between homologous chromosomes is initiated by the formation of hundreds of programmed double-strand breaks (DSBs). Approximately 10% of these DSBs...
Meiotic recombination between homologous chromosomes is initiated by the formation of hundreds of programmed double-strand breaks (DSBs). Approximately 10% of these DSBs result in crossovers (COs), sites of physical DNA exchange between homologs that are critical to correct chromosome segregation. Virtually all COs are formed by coordinated efforts of the MSH4/MSH5 and MLH1/MLH3 heterodimers, the latter representing the defining marks of CO sites. The regulation of CO number and position is poorly understood, but undoubtedly requires the coordinated action of multiple repair pathways. In a previous report, we found gene-trap disruption of the DNA helicase, FANCJ (BRIP1/BACH1), elicited elevated numbers of MLH1 foci and chiasmata. In somatic cells, FANCJ interacts with numerous DNA repair proteins including MLH1, and we hypothesized that FANCJ functions with MLH1 to regulate the major CO pathway. To further elucidate the meiotic function of FANCJ, we produced three new Fancj mutant mouse lines via CRISPR/Cas9 gene editing: a full-gene deletion, truncation of the N-terminal Helicase domain, and a C-terminal dual-tagged allele. We also generated an antibody against the C-terminus of the mouse FANCJ protein. Surprisingly, none of our Fancj mutants show any change in either MLH1 focus counts during pachynema or total CO number at diakinesis of prophase I. We find evidence that FANCJ and MLH1 do not interact in meiosis; further, FANCJ does not co-localize with MSH4, MLH1, or MLH3 in meiosis. Instead, FANCJ co-localizes with BRCA1 and TOPBP1, forming discrete foci along the chromosome cores beginning in early meiotic prophase I and densely localized to unsynapsed chromosome axes in late zygonema and to the XY chromosomes in early pachynema. Fancj mutants also exhibit a subtle persistence of DSBs in pachynema. Collectively, these data indicate a role for FANCJ in early DSB repair, but they rule out a role for FANCJ in MLH1-mediated CO events.
Topics: Animals; Male; Mice; Alleles; DNA Helicases; DNA Repair; Meiosis; Meiotic Prophase I
PubMed: 38377115
DOI: 10.1371/journal.pgen.1011175 -
Journal of Nematology Mar 1993Hermaphrodites were detected in diploid and polyploid isolates of population 86-Va of Meloidogyne hapla. Young hermaphrodites are indistinguishable from normal females....
Hermaphrodites were detected in diploid and polyploid isolates of population 86-Va of Meloidogyne hapla. Young hermaphrodites are indistinguishable from normal females. Initially, hermaphrodite ovaries are filled with oocytes at various stages of development. Hermaphroditism is expressed later when young oocytes in the early pachytene region of the growth zone suddenly advance to diakinesis and proceed with maturation divisions, resulting in spermatid production. Spermatogenesis may be initiated shortly after the fourth molt, or later, after a female has produced some eggs. Spermatogenesis may occur in one or both gonads, and it may be initiated in one gonad before the other. Once initiated, spermatogenesis continues for the entire reproductive life of the hermaphrodite. Several thousand spermatozoa accumulate in the ovotestis. Because they do not pass through the oviduct into the spermatotheca, they do not take part in reproduction (nonfunctional hermaphroditism). Among the progeny of hermaphrodites, ca. 50% are hermaphroditic, and the remainder are apparently normal females which, however, produce about 50% hermaphroditic progeny. Two temperature regimes (20-23 C and 27-30 C) did not influence the percentage of hermaphrodites among the progeny. Hermaphroditism could not be transmitted to nonhermaphroditic isolates following attempted crosses between males of hermaphroditic and females of nonhermaphroditic isolates. Although this result suggests cytoplasmic rather than nuclear inheritance, this conclusion is not definitive.
PubMed: 19279736
DOI: No ID Found -
Genetics Oct 1973Telocentrics for the beta arm of chromosome 4A and the long arm of 6B were used as cytological markers for the determination of chiasma frequency. In concomitant studies...
Telocentrics for the beta arm of chromosome 4A and the long arm of 6B were used as cytological markers for the determination of chiasma frequency. In concomitant studies of recombination, terminal segments of rye and T. umbellulatum chromatin carrying Hp (Hairy peduncle) and Lr9 (Leaf-rust resistance), respectively, marked 4A and 6B. Two temperatures, 21 degrees and 32 degrees , were used for both the 4A and 6B experiments.-Only one chiasma was observed in each heteromorphic bivalent. Because there was a substantial reduction in pairing between diakinesis and metaphase I, all determinations of chiasma frequency were made at diakinesis. In the 21 degrees experiments, agreement was good between genetic recombination and cytological prediction on the basis of the partial chiasmatypy hypothesis that each chiasma represents a crossover. At 32 degrees both chiasma frequency and crossing over, but particularly the latter, were strongly reduced. The fewer crossovers than expected are explained in part by stickiness of chromosomes at the high temperature, sometimes resulting in adjacent chromosomes being wrongly scored as having a chiasma, and in part by premetaphase disjunction of some recombined bivalents and subsequent independent behavior of the two resulting univalents.-Male transmission of the 4A telocentric from the heteromorphic bivalent was unusually high: 51% at 21 degrees and 31% at 32 degrees .
PubMed: 17248642
DOI: 10.1093/genetics/75.2.231 -
Dual DNA staining enables isolation of multiple sub-types of post-replicative mouse male germ cells.Cytometry. Part a : the Journal of the... Jun 2022During spermatogenesis, mammalian male germ cells undergo multiple developmental processes, including meiosis and post-meiotic differentiation (spermiogenesis). To...
During spermatogenesis, mammalian male germ cells undergo multiple developmental processes, including meiosis and post-meiotic differentiation (spermiogenesis). To understand the transitions between different cellular states it is essential to isolate pure populations of cells at different stages of development. Previous approaches enabled the isolation of cells from different stages of meiotic prophase I, but techniques to sub-fractionate unfixed, post-meiotic spermatids have been lacking. Here we report the development of a protocol enabling simultaneous isolation of cells at different stages of meiotic prophase and post-meiotic differentiation from testes of adult mice. This approach builds on existing fluorescence activated cell sorting protocols designed to purify cells in different stages of meiotic prophase I. By utilizing the specific spectral properties that two different DNA dyes (Hoechst 33342 and SYTO 16) exhibit when bound to chromatin of different stage male germ cells, we obtain highly pure populations of cells in relatively large numbers. This FACS protocol will enable immunocytological and molecular characterization studies of fractionated meiotic and haploid germ cells from both wild type and genetically mutant animals.
Topics: Animals; DNA; Germ Cells; Male; Mammals; Meiosis; Mice; Spermatids; Spermatogenesis; Staining and Labeling; Testis
PubMed: 35128792
DOI: 10.1002/cyto.a.24539 -
Nature Communications Jul 2018Meiotic cells undergo genetic exchange between homologs through programmed DNA double-strand break (DSB) formation, recombination and synapsis. In mice, the DNA...
Meiotic cells undergo genetic exchange between homologs through programmed DNA double-strand break (DSB) formation, recombination and synapsis. In mice, the DNA damage-regulated phosphatidylinositol-3-kinase-like kinase (PIKK) ATM regulates all of these processes. However, the meiotic functions of the PIKK ATR have remained elusive, because germline-specific depletion of this kinase is challenging. Here we uncover roles for ATR in male mouse prophase I progression. ATR deletion causes chromosome axis fragmentation and germ cell elimination at mid pachynema. This elimination cannot be rescued by deletion of ATM and the third DNA damage-regulated PIKK, PRKDC, consistent with the existence of a PIKK-independent surveillance mechanism in the mammalian germline. ATR is required for synapsis, in a manner genetically dissociable from DSB formation. ATR also regulates loading of recombinases RAD51 and DMC1 to DSBs and recombination focus dynamics on synapsed and asynapsed chromosomes. Our studies reveal ATR as a critical regulator of mouse meiosis.
Topics: Animals; Ataxia Telangiectasia Mutated Proteins; Cell Cycle Proteins; Chromosome Pairing; Chromosomes, Mammalian; DNA Breaks, Double-Stranded; In Situ Hybridization, Fluorescence; Male; Meiosis; Meiotic Prophase I; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Nuclear Proteins; Phosphate-Binding Proteins; Rad51 Recombinase; Spermatocytes
PubMed: 29976923
DOI: 10.1038/s41467-018-04850-0 -
Cold Spring Harbor Symposia on... 2010The differentiated state of somatic cells is highly stable, but it can be experimentally reversed. The resulting cells can then be redirected into many different...
The differentiated state of somatic cells is highly stable, but it can be experimentally reversed. The resulting cells can then be redirected into many different pathways. Nuclear reprogramming has been achieved by nuclear transfer to eggs, cell fusion, and overexpression of transcription factors. The mechanisms of nuclear reprogramming are not understood, but some insight into them is provided by comparing the efficiencies of different reprogramming strategies. Here, we compare these efficiencies by describing the frequency and rapidity with which reprogramming is induced and by the proportion of cells and level of expression in which reprogramming is achieved. We comment on the mechanisms that lead to successful somatic-cell reprogramming and on those that resist in helping to maintain the differentiated state of somatic cells.
Topics: Animals; Cell Differentiation; Cell Fusion; Cell Lineage; Cellular Reprogramming; Humans; Induced Pluripotent Stem Cells; Meiotic Prophase I; Nuclear Transfer Techniques; Ovum; Transcription Factors
PubMed: 21047900
DOI: 10.1101/sqb.2010.75.002 -
Nature Structural & Molecular Biology Mar 2019In meiotic prophase, chromosomes are organized into compacted loop arrays to promote homolog pairing and recombination. Here, we probe the architecture of the mouse...
In meiotic prophase, chromosomes are organized into compacted loop arrays to promote homolog pairing and recombination. Here, we probe the architecture of the mouse spermatocyte genome in early and late meiotic prophase using chromosome conformation capture (Hi-C). Our data support the established loop array model of meiotic chromosomes, and infer loops averaging 0.8-1.0 megabase pairs (Mb) in early prophase and extending to 1.5-2.0 Mb in late prophase as chromosomes compact and homologs undergo synapsis. Topologically associating domains (TADs) are lost in meiotic prophase, suggesting that assembly of the meiotic chromosome axis alters the activity of chromosome-associated cohesin complexes. While TADs are lost, physically separated A and B compartments are maintained in meiotic prophase. Moreover, meiotic DNA breaks and interhomolog crossovers preferentially form in the gene-dense A compartment, revealing a role for chromatin organization in meiotic recombination. Finally, direct detection of interhomolog contacts genome-wide reveals the structural basis for homolog alignment and juxtaposition by the synaptonemal complex.
Topics: Animals; Chromatin; Chromosome Pairing; Chromosomes; DNA Breaks; Genome; Homologous Recombination; Male; Meiotic Prophase I; Mice; Mice, Inbred C57BL; Spermatocytes; Spermatogenesis; Synaptonemal Complex
PubMed: 30778236
DOI: 10.1038/s41594-019-0187-0 -
PLoS Genetics Apr 2017The meiosis-specific chromosomal events of homolog pairing, synapsis, and recombination occur over an extended meiotic prophase I that is many times longer than prophase...
The meiosis-specific chromosomal events of homolog pairing, synapsis, and recombination occur over an extended meiotic prophase I that is many times longer than prophase of mitosis. Here we show that, in mice, maintenance of an extended meiotic prophase I requires the gene Meioc, a germ-cell specific factor conserved in most metazoans. In mice, Meioc is expressed in male and female germ cells upon initiation of and throughout meiotic prophase I. Mouse germ cells lacking Meioc initiate meiosis: they undergo pre-meiotic DNA replication, they express proteins involved in synapsis and recombination, and a subset of cells progress as far as the zygotene stage of prophase I. However, cells in early meiotic prophase-as early as the preleptotene stage-proceed to condense their chromosomes and assemble a spindle, as if having progressed to metaphase. Meioc-deficient spermatocytes that have initiated synapsis mis-express CYCLIN A2, which is normally expressed in mitotic spermatogonia, suggesting a failure to properly transition to a meiotic cell cycle program. MEIOC interacts with YTHDC2, and the two proteins pull-down an overlapping set of mitosis-associated transcripts. We conclude that when the meiotic chromosomal program is initiated, Meioc is simultaneously induced so as to extend meiotic prophase. Specifically, MEIOC, together with YTHDC2, promotes a meiotic (as opposed to mitotic) cell cycle program via post-transcriptional control of their target transcripts.
Topics: Animals; Cell Cycle Proteins; Chromosome Pairing; Cyclin A2; Gene Expression Regulation, Developmental; Male; Meiosis; Mice; Mitosis; Prophase; RNA-Binding Proteins; Spermatocytes; Spermatogenesis; Spermatogonia
PubMed: 28380054
DOI: 10.1371/journal.pgen.1006704