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BioRxiv : the Preprint Server For... Oct 2023During meiotic prophase I, recombination between homologous parental chromosomes is initiated by the formation of hundreds of programmed double-strand breaks (DSBs),...
During meiotic prophase I, recombination between homologous parental chromosomes is initiated by the formation of hundreds of programmed double-strand breaks (DSBs), each of which must be repaired with absolute fidelity to ensure genome stability of the germline. One outcome of these DSB events is the formation of Crossovers (COs), the sites of physical DNA exchange between homologs that are critical to ensure the correct segregation of parental chromosomes. However, COs account for only a small (~10%) proportion of all DSB repair events; the remaining 90% are repaired as non-crossovers (NCOs), most by synthesis dependent strand annealing. Virtually all COs are formed by coordinated efforts of the MSH4/MSH5 and MLH1/MLH3 heterodimers. The number and positioning of COs is exquisitely controlled via mechanisms that remain poorly understood, but which undoubtedly require the coordinated action of multiple repair pathways downstream of the initiating DSB. In a previous report we found evidence suggesting that the DNA helicase and Fanconi Anemia repair protein, FANCJ (BRIP1/BACH1), functions to regulate meiotic recombination in mouse. A gene-trap disruption of showed an elevated number of MLH1 foci and COs. FANCJ is known to interact with numerous DNA repair proteins in somatic cell repair contexts, including MLH1, BLM, BRCA1, and TOPBP1, and we hypothesized that FANCJ regulates CO formation through a direct interaction with MLH1 to suppress the major CO pathway. To further elucidate the function of FANCJ in meiosis, we produced three new mutant mouse lines via CRISPR/Cas9 gene editing: a full-gene deletion, a mutant line lacking the MLH1 interaction site and the N-terminal region of the Helicase domain, and a C-terminal 6xHIS-HA dual-tagged allele of . We also generated an antibody against the C-terminus of the mouse FANCJ protein. Surprisingly, while Fanconi-like phenotypes are observed within the somatic cell lineages of the full deletion line, none of the mutants show any change in either MLH1 focus counts during pachynema or total CO number at diakinesis of prophase I of meiosis. We find evidence that FANCJ and MLH1 do not interact in meiosis; further, FANCJ does not co-localize with MSH4, MLH1, or MLH3 during late prophase I. Instead, FANCJ forms discrete foci along the chromosome cores beginning in early meiotic prophase I, occasionally co-localizing with MSH4, and then becomes densely localized on unsynapsed chromosome axes in late zygonema and to the XY chromosomes in early pachynema. Strikingly, this localization strongly overlaps with BRCA1 and TOPBP1. mutants also exhibit a subtle persistence of DSBs in pachynema. Collectively, these data suggest a role for FANCJ in early DSB repair events, and possibly in the formation of NCOs, but they rule out a role for FANCJ in MLH1-mediated CO events. Thus, the role of FANCJ in meiotic cells involves different pathways and different interactors to those described in somatic cell lineages.
PubMed: 37873301
DOI: 10.1101/2023.10.06.561296 -
BioRxiv : the Preprint Server For... Oct 2023H3K9 tri-methylation (H3K9me3) plays emerging roles in gene regulation, beyond its accumulation on pericentric constitutive heterochromatin. It remains a mystery why and...
H3K9 tri-methylation (H3K9me3) plays emerging roles in gene regulation, beyond its accumulation on pericentric constitutive heterochromatin. It remains a mystery why and how H3K9me3 undergoes dynamic regulation in male meiosis. Here, we identify a novel, critical regulator of H3K9 methylation and spermatogenic heterochromatin organization: the germline-specific protein ATF7IP2 (MCAF2). We show that, in male meiosis, ATF7IP2 amasses on autosomal and X pericentric heterochromatin, spreads through the entirety of the sex chromosomes, and accumulates on thousands of autosomal promoters and retrotransposon loci. On the sex chromosomes, which undergo meiotic sex chromosome inactivation (MSCI), the DNA damage response pathway recruits ATF7IP2 to X pericentric heterochromatin, where it facilitates the recruitment of SETDB1, a histone methyltransferase that catalyzes H3K9me3. In the absence of ATF7IP2, male germ cells are arrested in meiotic prophase I. Analyses of ATF7IP2-deficient meiosis reveal the protein's essential roles in the maintenance of MSCI, suppression of retrotransposons, and global upregulation of autosomal genes. We propose that ATF7IP2 is a downstream effector of the DDR pathway in meiosis that coordinates the organization of heterochromatin and gene regulation through the spatial regulation of SETDB1-mediated H3K9me3 deposition.
PubMed: 37873266
DOI: 10.1101/2023.09.30.560314 -
Asian Journal of Andrology Oct 2023Meiosis is the process of producing haploid gametes through a series of complex chromosomal events and the coordinated action of various proteins. The mitochondrial...
Meiosis is the process of producing haploid gametes through a series of complex chromosomal events and the coordinated action of various proteins. The mitochondrial protease complex (ClpXP), which consists of caseinolytic mitochondrial matrix peptidase X (ClpX) and caseinolytic protease P (ClpP) and mediates the degradation of misfolded, damaged, and oxidized proteins, is essential for maintaining mitochondrial homeostasis. ClpXP has been implicated in meiosis regulation, but its precise role is currently unknown. In this study, we engineered an inducible male germ cell-specific knockout caseinolytic mitochondrial matrix peptidase X (ClpxcKO) mouse model to investigate the function of ClpX in meiosis. We found that disrupting Clpx in male mice induced germ cell apoptosis and led to an absence of sperm in the epididymis. Specifically, it caused asynapsis of homologous chromosomes and impaired meiotic recombination, resulting in meiotic arrest in the zygotene-to-pachytene transition phase. The loss of ClpX compromised the double-strand break (DSB) repair machinery by markedly reducing the recruitment of DNA repair protein RAD51 homolog 1 (RAD51) to DSB sites. This dysfunction may be due to an insufficient supply of energy from the aberrant mitochondria in ClpxcKO spermatocytes, as discerned by electron microscopy. Furthermore, ubiquitination signals on chromosomes and the expression of oxidative phosphorylation subunits were both significantly attenuated in ClpxcKO spermatocytes. Taken together, we propose that ClpX is essential for maintaining mitochondrial protein homeostasis and ensuring homologous chromosome pairing, synapsis, and recombination in spermatocytes during meiotic prophase I.
PubMed: 37856231
DOI: 10.4103/aja202343 -
BioRxiv : the Preprint Server For... Dec 2023Meiotic progression requires coordinated assembly and disassembly of protein complexes involved in chromosome synapsis and meiotic recombination. The AAA+ ATPase TRIP13...
Meiotic progression requires coordinated assembly and disassembly of protein complexes involved in chromosome synapsis and meiotic recombination. The AAA+ ATPase TRIP13 and its orthologue Pch2 are instrumental in remodeling HORMA domain proteins. Meiosis-specific HORMAD proteins are associated with unsynapsed chromosome axes but depleted from the synaptonemal complex (SC) of synapsed chromosome homologues. Here we report that TRIP13 localizes to the synapsed SC in early pachytene spermatocytes and to telomeres throughout meiotic prophase I. Loss of TRIP13 leads to meiotic arrest and thus sterility in both sexes. -null meiocytes exhibit abnormal persistence of HORMAD1 and HOMRAD2 on synapsed SC and chromosome asynapsis that preferentially affects XY and centromeric ends. These findings confirm the previously reported phenotypes of the hypomorph alleles. heterozygous () mice also exhibit meiotic defects that are less severe than the -null mice, showing that TRIP13 is a dosage-sensitive regulator of meiosis. Localization of TRIP13 to the synapsed SC is independent of SC axial element proteins such as REC8 and SYCP2/SYCP3. The N- or C-terminal FLAG-tagged TRIP13 proteins are functional and recapitulate the localization of native TRIP13 to SC and telomeres in knockin mice. Therefore, the evolutionarily conserved localization of TRIP13/Pch2 to the synapsed chromosomes provides an explanation for dissociation of HORMA domain proteins upon chromosome synapsis in diverse organisms.
PubMed: 37808842
DOI: 10.1101/2023.09.25.559355 -
ELife Oct 2023In sexually reproducing organisms, germ cells faithfully transmit the genome to the next generation by forming haploid gametes, such as eggs and sperm. Although most...
In sexually reproducing organisms, germ cells faithfully transmit the genome to the next generation by forming haploid gametes, such as eggs and sperm. Although most meiotic proteins are conserved between eggs and sperm, many aspects of meiosis are sexually dimorphic, including the regulation of recombination. The synaptonemal complex (SC), a large ladder-like structure that forms between homologous chromosomes, is essential for regulating meiotic chromosome organization and promoting recombination. To assess whether sex-specific differences in the SC underpin sexually dimorphic aspects of meiosis, we examined SC central region proteins (known as SYP proteins) in oogenesis and spermatogenesis and uncovered sex-specific roles for the SYPs in regulating meiotic recombination. We find that SC composition, specifically SYP-2, SYP-3, SYP-5, and SYP-6, is regulated by sex-specific mechanisms throughout meiotic prophase I. During pachytene, both oocytes and spermatocytes differentially regulate the stability of SYP-2 and SYP-3 within an assembled SC. Further, we uncover that the relative amount of SYP-2 and SYP-3 within the SC is independently regulated in both a sex-specific and a recombination-dependent manner. Specifically, we find that SYP-2 regulates the early steps of recombination in both sexes, while SYP-3 controls the timing and positioning of crossover recombination events across the genomic landscape in only oocytes. Finally, we find that SYP-2 and SYP-3 dosage can influence the composition of the other SYPs in the SC via sex-specific mechanisms during pachytene. Taken together, we demonstrate dosage-dependent regulation of individual SC components with sex-specific functions in recombination. These sexual dimorphic features of the SC provide insights into how spermatogenesis and oogenesis adapted similar chromosome structures to differentially regulate and execute recombination.
Topics: Animals; Female; Male; Caenorhabditis elegans; Synaptonemal Complex; Meiosis; Semen; Caenorhabditis elegans Proteins
PubMed: 37796106
DOI: 10.7554/eLife.84538 -
Reproductive Biology and Endocrinology... Oct 2023In human female primordial germ cells, the transition from mitosis to meiosis begins from the fetal stage. In germ cells, meiosis is arrested at the diplotene stage of... (Review)
Review
In human female primordial germ cells, the transition from mitosis to meiosis begins from the fetal stage. In germ cells, meiosis is arrested at the diplotene stage of prophase in meiosis I (MI) after synapsis and recombination of homologous chromosomes, which cannot be segregated. Within the follicle, the maintenance of oocyte meiotic arrest is primarily attributed to high cytoplasmic concentrations of cyclic adenosine monophosphate (cAMP). Depending on the specific species, oocytes can remain arrested for extended periods of time, ranging from months to even years. During estrus phase in animals or the menstrual cycle in humans, the resumption of meiosis occurs in certain oocytes due to a surge of luteinizing hormone (LH) levels. Any factor interfering with this process may lead to impaired oocyte maturation, which in turn affects female reproductive function. Nevertheless, the precise molecular mechanisms underlying this phenomenon has not been systematically summarized yet. To provide a comprehensive understanding of the recently uncovered regulatory network involved in oocyte development and maturation, the progress of the cellular and molecular mechanisms of oocyte nuclear maturation including meiosis arrest and meiosis resumption is summarized. Additionally, the advancements in understanding the molecular cytoplasmic events occurring in oocytes, such as maternal mRNA degradation, posttranslational regulation, and organelle distribution associated with the quality of oocyte maturation, are reviewed. Therefore, understanding the pathways regulating oocyte meiotic arrest and resumption will provide detailed insight into female reproductive system and provide a theoretical basis for further research and potential approaches for novel disease treatments.
Topics: Animals; Female; Humans; Oogenesis; Oocytes; Meiosis; Meiotic Prophase I; Ovarian Follicle
PubMed: 37784186
DOI: 10.1186/s12958-023-01143-0 -
Nucleic Acids Research Oct 2023Several aspects of telomere biology are regulated by the telomeric repeat-containing RNA TERRA. While TERRA expression is conserved through evolution, species-specific...
Several aspects of telomere biology are regulated by the telomeric repeat-containing RNA TERRA. While TERRA expression is conserved through evolution, species-specific mechanisms regulate its biogenesis and function. Here we report on the expression of TERRA in Caenorhabditis elegans. We show that C. elegans TERRA is regulated by the telomere-binding proteins POT-1 and POT-2 which repress TERRA in a telomere-specific manner. C. elegans TERRA transcripts are heterogeneous in length and form discrete nuclear foci, as detected by RNA FISH, in both postmitotic and germline cells; a fraction of TERRA foci localizes to telomeres. Interestingly, in germ cells, TERRA is expressed in all stages of meiotic prophase I, and it increases during pachytene, a stage in meiosis when homologous recombination is ongoing. We used the MS2-GFP system to study the spatiotemporal dynamics of single-telomere TERRA molecules. Single particle tracking revealed different types of motilities, suggesting complex dynamics of TERRA transcripts. Finally, we unveiled distinctive features of C. elegans TERRA, which is regulated by telomere shortening in a telomere-specific manner, and it is upregulated in the telomerase-deficient trt-1; pot-2 double mutant prior to activation of the alternative lengthening mechanism ALT. Interestingly, in these worms TERRA displays distinct dynamics with a higher fraction of fast-moving particles.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; DNA-Binding Proteins; Meiosis; RNA, Long Noncoding; Telomerase; Telomere; Telomere-Binding Proteins
PubMed: 37713629
DOI: 10.1093/nar/gkad742 -
Current Opinion in Genetics &... Oct 2023During fetal oocyte development in mammals, germ cells progress through meiotic prophase I to form primordial follicles with pregranulosa cells. The primordial follicles... (Review)
Review
During fetal oocyte development in mammals, germ cells progress through meiotic prophase I to form primordial follicles with pregranulosa cells. The primordial follicles remain dormant until oogenesis resumes during puberty. Studies in mice have elucidated mechanisms governing oogenesis, leading to the successful induction of functional oocytes from mouse pluripotent stem cells in vitro. Based on the in vivo/in vitro knowledge in mice and the histological and transcriptomic evidence for fetal oocyte development in humans and primates, human/primate oocyte-like cells corresponding to the early stage of oocytes in vivo have been successfully induced in vitro. Here, we discuss recent advances in our understanding of the mechanisms of fetal oocyte development in mammals, as well as in in vitro oogenesis.
Topics: Mice; Animals; Humans; Meiosis; Oocytes; Oogenesis; Mammals; Pluripotent Stem Cells
PubMed: 37556984
DOI: 10.1016/j.gde.2023.102091 -
BioRxiv : the Preprint Server For... Jul 2023During prophase I of meiosis, DNA double-strand breaks form throughout the genome, with a subset repairing as crossover events, enabling the accurate segregation of...
During prophase I of meiosis, DNA double-strand breaks form throughout the genome, with a subset repairing as crossover events, enabling the accurate segregation of homologous chromosomes during the first meiotic division. The mechanism by which DSBs become selected to repair as crossovers is unknown, although the crossover positioning and levels in each cell indicate it is a highly regulated process. One of the proteins that localises to crossover sites is the serine/threonine cyclin-dependent kinase CDK2. Regulation of CDK2 occurs via phosphorylation at tyrosine 15 (Y15) and threonine 160 (T160) inhibiting and activating the kinase, respectively. In this study we use a combination of immunofluorescence staining on spread spermatocytes and fixed testis sections, and STA-PUT gravitational sedimentation to isolate cells at different developmental stages to further investigate the temporal phospho regulation of CDK2 during prophase I. Western blotting reveals differential levels of the two CDK2 isoforms (CDK2 and CDK2) throughout prophase I, with inhibitory phosphorylation of CDK2 at Y15 occurring early in prophase I, localising to telomeres and diminishing as cells enter pachynema. Conversely, the activatory phosphorylation on T160 occurs later, specifically the CDK2 isoform, and T160 signal is detected in spermatogonia and pachytene spermatocytes, where it co-localises with the Class I crossover protein MLH3. Taken together, our data reveals intricate control of CDK2 both with regards to levels of the two CDK2 isoforms, and differential regulation via inhibitory and activatory phosphorylation.
PubMed: 37546989
DOI: 10.1101/2023.07.24.550435 -
International Journal of Molecular... Jul 2023In the oocyte nucleus, called the germinal vesicle (GV) at the prolonged diplotene stage of the meiotic prophase, chromatin undergoes a global rearrangement, which is... (Review)
Review
In the oocyte nucleus, called the germinal vesicle (GV) at the prolonged diplotene stage of the meiotic prophase, chromatin undergoes a global rearrangement, which is often accompanied by the cessation of its transcriptional activity. In many mammals, including mice and humans, chromatin condenses around a special nuclear organelle called the atypical nucleolus or formerly nucleolus-like body. Chromatin configuration is an important indicator of the quality of GV oocytes and largely predicts their ability to resume meiosis and successful embryonic development. In mice, GV oocytes are traditionally divided into the NSN (non-surrounded nucleolus) and SN (surrounded nucleolus) based on the specific chromatin configuration. The NSN-SN transition is a key event in mouse oogenesis and the main prerequisite for the normal development of the embryo. As for humans, there is no single nomenclature for the chromatin configuration at the GV stage. This often leads to discrepancies and misunderstandings, the overcoming of which should expand the scope of the application of mouse oocytes as a model for developing new methods for assessing and improving the quality of human oocytes. As a first approximation and with a certain proviso, the mouse NSN/SN classification can be used for the primary characterization of human GV oocytes. The task of this review is to analyze and discuss the existing classifications of chromatin configuration in mouse and human GV oocytes with an emphasis on transcriptional activity extinction at the end of oocyte growth.
Topics: Humans; Animals; Mice; Chromatin; Meiosis; Meiotic Prophase I; Oocytes; Cell Nucleus; Mammals
PubMed: 37511273
DOI: 10.3390/ijms241411517