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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 -
ELife Jul 2023Impaired spermatogenesis and male infertility are common manifestations associated with mitochondrial diseases, yet the underlying mechanisms linking these conditions...
Impaired spermatogenesis and male infertility are common manifestations associated with mitochondrial diseases, yet the underlying mechanisms linking these conditions remain elusive. In this study, we demonstrate that mice deficient for the mitochondrial intra-membrane rhomboid protease PARL, a recently reported model of the mitochondrial encephalopathy Leigh syndrome, develop early testicular atrophy caused by a complete arrest of spermatogenesis during meiotic prophase I, followed by degeneration and death of arrested spermatocytes. This process is independent of neurodegeneration. Interestingly, genetic modifications of PINK1, PGAM5, and TTC19 - three major substrates of PARL with important roles in mitochondrial homeostasis - fail to reproduce or modify this severe phenotype, indicating that the spermatogenic arrest arises from distinct molecular pathways. We further observed severe abnormalities in mitochondrial ultrastructure in PARL-deficient spermatocytes, along with prominent electron transfer chain defects, disrupted coenzyme Q (CoQ) biosynthesis, and metabolic rewiring. These mitochondrial defects are associated with a germ cell-specific decrease in GPX4 expression leading arrested spermatocytes to ferroptosis - a regulated cell death modality characterized by uncontrolled lipid peroxidation. Our results suggest that mitochondrial defects induced by PARL depletion act as an initiating trigger for ferroptosis in primary spermatocytes through simultaneous effects on GPX4 and CoQ - two major inhibitors of ferroptosis. These findings shed new light on the potential role of ferroptosis in the pathogenesis of mitochondrial diseases and male infertility warranting further investigation.
Topics: Animals; Humans; Male; Mice; Ferroptosis; Infertility, Male; Meiosis; Metalloproteases; Mitochondrial Proteins; Spermatogenesis
PubMed: 37505079
DOI: 10.7554/eLife.84710 -
Methods in Molecular Biology (Clifton,... 2023The mammalian reproductive cycle, including those of humans and mice, begins very early in development. In utero, the ovaries become populated with primordial germ cells...
The mammalian reproductive cycle, including those of humans and mice, begins very early in development. In utero, the ovaries become populated with primordial germ cells (PGCs) that will generate the oogonia. First, these cells proliferate mitotically, and then they trigger the meiotic program and initiate meiotic prophase I. Since these processes happen during gestation, their study had been very limited and challenging. Recently, we reported that, in the naked mole-rat (Heterocephalus glaber) ovary, there is mitotic expansion of the PGCs, and the initiation of the meiotic program occurs postnatally. In this chapter, we present a comprehensive collection of protocols that permit the analysis of naked mole-rat germ cells, from PGCs to oocytes, in meiotic prophase I, using in vivo and in vitro approaches.
Topics: Humans; Female; Mice; Animals; Ovary; Meiotic Prophase I; Meiosis; Oocytes; Germ Cells; Mammals
PubMed: 37464243
DOI: 10.1007/978-1-0716-3259-8_11 -
BioEssays : News and Reviews in... Oct 2023The ovarian reserve defines female reproductive lifespan, which in humans spans decades. The ovarian reserve consists of oocytes residing in primordial follicles...
The ovarian reserve defines female reproductive lifespan, which in humans spans decades. The ovarian reserve consists of oocytes residing in primordial follicles arrested in meiotic prophase I and is maintained independent of DNA replication and cell proliferation, thereby lacking stem cell-based maintenance. Largely unknown is how cellular states of the ovarian reserve are established and maintained for decades. Our recent study revealed that a distinct chromatin state is established during ovarian reserve formation in mice, uncovering a novel window of epigenetic programming in female germline development. We showed that an epigenetic regulator, Polycomb Repressive Complex 1 (PRC1), establishes a repressive chromatin state in perinatal mouse oocytes that is essential for prophase I-arrested oocytes to form the ovarian reserve. Here we discuss the biological roles and mechanisms underlying epigenetic programming in ovarian reserve formation, highlighting current knowledge gaps and emerging research areas in female reproductive biology.
Topics: Humans; Pregnancy; Female; Mice; Animals; Meiosis; Ovarian Reserve; Oocytes; Chromatin; Epigenesis, Genetic
PubMed: 37417392
DOI: 10.1002/bies.202300069 -
Cell Death & Disease Jul 2023Mammalian oocytes spend most of their life in a unique state of cell cycle arrest at meiotic prophase I, during which time they are exposed to countless DNA-damaging...
Mammalian oocytes spend most of their life in a unique state of cell cycle arrest at meiotic prophase I, during which time they are exposed to countless DNA-damaging events. Recent studies have shown that DNA double-strand break repair occurs predominantly via the homologous recombination (HR) pathway in small non-growing meiotically arrested oocytes (primordial follicle stage). However, the DNA repair mechanisms employed by fully grown meiotically arrested oocytes (GV-stage) have not been studied in detail. Here we established a conditional knockout mouse model to explore the role of Ku80, a critical component of the nonhomologous end joining (NHEJ) pathway, in the repair of DNA damage in GV oocytes. GV oocytes lacking Ku80 failed to repair etoposide-induced DNA damage, even when only low levels of damage were sustained. This indicates Ku80 is needed to resolve DSBs and that HR cannot compensate for a compromised NHEJ pathway in fully-grown oocytes. When higher levels of DNA damage were induced, a severe delay in M-phase entry was observed in oocytes lacking XRCC5 compared to wild-type oocytes, suggesting that Ku80-dependent repair of DNA damage is important for the timely release of oocytes from prophase I and resumption of meiosis. Ku80 was also found to be critical for chromosome integrity during meiotic maturation following etoposide exposure. These data demonstrate that Ku80, and NHEJ, are vital for quality control in mammalian GV stage oocytes and reveal that DNA repair pathway choice differs in meiotically arrested oocytes according to growth status.
Topics: Animals; Mice; DNA Damage; DNA End-Joining Repair; DNA Repair; Etoposide; Mammals; Meiosis; Oocytes
PubMed: 37407587
DOI: 10.1038/s41419-023-05886-x -
BioRxiv : the Preprint Server For... Nov 2023Meiotic sex chromosome inactivation (MSCI) is a critical feature of meiotic prophase I progression in males. While the ATR kinase and its activator TOPBP1 are key...
Meiotic sex chromosome inactivation (MSCI) is a critical feature of meiotic prophase I progression in males. While the ATR kinase and its activator TOPBP1 are key drivers of MSCI within the specialized sex body (SB) domain of the nucleus, how they promote silencing remains unclear given their multifaceted meiotic functions that also include DNA repair, chromosome synapsis and SB formation. Here we report a novel mutant mouse harboring mutations in the TOPBP1-BRCT5 domain. males are infertile, with impaired MSCI despite displaying grossly normal events of early prophase I, including synapsis and SB formation. Specific ATR-dependent events are disrupted including phosphorylation and localization of the RNA:DNA helicase Senataxin. spermatocytes initiate, but cannot maintain ongoing, MSCI. These findings reveal a non-canonical role for the ATR-TOPBP1 signaling axis in MSCI dynamics at advanced stages in pachynema and establish the first mouse mutant that separates ATR signaling and MSCI from SB formation.
PubMed: 37398453
DOI: 10.1101/2023.05.31.543071 -
Nucleic Acids Research Aug 2023DNA-RNA hybrids play various roles in many physiological progresses, but how this chromatin structure is dynamically regulated during spermatogenesis remains largely...
DNA-RNA hybrids play various roles in many physiological progresses, but how this chromatin structure is dynamically regulated during spermatogenesis remains largely unknown. Here, we show that germ cell-specific knockout of Rnaseh1, a specialized enzyme that degrades the RNA within DNA-RNA hybrids, impairs spermatogenesis and causes male infertility. Notably, Rnaseh1 knockout results in incomplete DNA repair and meiotic prophase I arrest. These defects arise from the altered RAD51 and DMC1 recruitment in zygotene spermatocytes. Furthermore, single-molecule experiments show that RNase H1 promotes recombinase recruitment to DNA by degrading RNA within DNA-RNA hybrids and allows nucleoprotein filaments formation. Overall, we uncover a function of RNase H1 in meiotic recombination, during which it processes DNA-RNA hybrids and facilitates recombinase recruitment.
Topics: Humans; Male; Cell Cycle Proteins; DNA; Meiosis; Rad51 Recombinase; Recombinases; Spermatocytes; Ribonuclease H
PubMed: 37378420
DOI: 10.1093/nar/gkad524 -
Nature Communications Jun 2023In mammals, the production of mature oocytes necessitates rigorous regulation of the discontinuous meiotic cell-cycle progression at both the transcriptional and...
In mammals, the production of mature oocytes necessitates rigorous regulation of the discontinuous meiotic cell-cycle progression at both the transcriptional and post-transcriptional levels. However, the factors underlying this sophisticated but explicit process remain largely unclear. Here we characterize the function of N-acetyltransferase 10 (Nat10), a writer for N4-acetylcytidine (ac4C) on RNA molecules, in mouse oocyte development. We provide genetic evidence that Nat10 is essential for oocyte meiotic prophase I progression, oocyte growth and maturation by sculpting the maternal transcriptome through timely degradation of poly(A) tail mRNAs. This is achieved through the ac4C deposition on the key CCR4-NOT complex transcripts. Importantly, we devise a method for examining the poly(A) tail length (PAT), termed Hairpin Adaptor-poly(A) tail length (HA-PAT), which outperforms conventional methods in terms of cost, sensitivity, and efficiency. In summary, these findings provide genetic evidence that unveils the indispensable role of maternal Nat10 in oocyte development.
Topics: Animals; Mice; Mammals; Meiosis; Oocytes; Oogenesis; RNA, Messenger
PubMed: 37349316
DOI: 10.1038/s41467-023-39256-0 -
Frontiers in Plant Science 2023During meiosis, the chromosome axes and synaptonemal complex mediate chromosome pairing and homologous recombination to maintain genomic stability and accurate...
During meiosis, the chromosome axes and synaptonemal complex mediate chromosome pairing and homologous recombination to maintain genomic stability and accurate chromosome segregation. In plants, ASYNAPSIS 1 (ASY1) is a key component of the chromosome axis that promotes inter-homolog recombination, synapsis and crossover formation. Here, the function of ASY1 has been cytologically characterized in a series of hypomorphic wheat mutants. In tetraploid wheat, hypomorphic mutants experience a reduction in chiasmata (crossovers) in a dosage-specific manner, resulting in failure to maintain crossover (CO) assurance. In mutants with only one functional copy of ASY1, distal chiasmata are maintained at the expense of proximal and interstitial chiasmata, indicating that ASY1 is required to promote chiasma formation away from the chromosome ends. Meiotic prophase I progression is delayed in asy1 hypomorphic mutants and is arrested in null mutants. In both tetraploid and hexaploid wheat, single asy1 mutants exhibit a high degree of ectopic recombination between multiple chromosomes at metaphase I. To explore the nature of the ectopic recombination, asy1b-2 was crossed with wheat-wild relative . Homoeologous chiasmata increased 3.75-fold in compared to wild type/, indicating that ASY1 suppresses chiasma formation between divergent, but related chromosomes. These data suggest that ASY1 promotes recombination along the chromosome arms of homologous chromosomes whilst suppressing recombination between non-homologous chromosomes. Therefore, mutants could be utilized to increase recombination between wheat wild relatives and elite varieties for expediting introgression of important agronomic traits.
PubMed: 37284727
DOI: 10.3389/fpls.2023.1188347