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Rec8 cleavage triggers chiasmata resolution and sister centromere disjunction of bivalent chromosomes. (A) Wild-type and Rec8 TEV/TEV oocytes expressing H2B-mCherry, Securin-EGFP, and Flag-Mad2 were cultured for 14-16 h. Metaphase I-arrested oocytes were injected with TEV protease mRNA (time 0), and chromosome movements were visualized by time-lapse confocal microscopy (h:mm). Still images from a representative movie of each genotype are shown. The left panels show the H2B-mCherry channel and the right panels show H2B-mCherry and Securin-EGFP pseudocolored in red and green, respectively. Bar, 10 mm. (B) Wild-type and Rec8 TEV/TEV oocytes expressing TEV protease were matured in vitro for 5 h. Chromosome spreads were prepared and stained with Hoechst to visualize DNA (red) and CREST to mark centromeres (green).
Source publication
During female meiosis, bivalent chromosomes are thought to be held together from birth until ovulation by sister chromatid cohesion mediated by cohesin complexes whose ring structure depends on kleisin subunits, either Rec8 or Scc1. Because cohesion is established at DNA replication in the embryo, its maintenance for such a long time may require co...
Contexts in source publication
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... of IBMX, GV breakdown (GVBD) is followed by alignment of bivalents on the meta- phase I spindle. Their conversion to U-shaped dyad chro- mosomes (chiasmata resolution) occurs as Securin-EGFP fluorescence levels drop due to APC/C activation and is accompanied by chromosome segregation followed by polar body extrusion (PBE) in <1 h (Supplemental Fig. 3A), a course of events identical in Rec8 and Rec8 TEV/TEV ...
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... of Mad2 causes a metaphase I arrest ( Wassmann et al. 2003). After cultur- ing for 14 h post-GVBD, arrested oocytes were reinjected, this time with mRNA for TEV protease. TEV mRNA injection triggered in Rec8 TEV/TEV (n = 5), but not in Rec8 oocytes (n = 7), conversion of bivalents first into dyads and subsequently into individual chromatids (Fig. 3A). TEV- induced Rec8 cleavage did not cause PBE for at least 4 h, presumably because cytokinesis requires cyclin-dependent kinase (CDK) inactivation. Importantly, Securin-EGFP lev- els remained high throughout, demonstrating that APC/C and separase had not been activated (Supplemental Fig. 4A,B). The resolution of chiasmata must ...
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... pattern suggests that TEV had indeed converted bivalents to chromatids. Meiosis I chromosome segregation normally occurs 6-8 h af- ter GVBD. Rec8 TEV-injected and Rec8 TEV/TEV -unin- jected oocytes (n = 26) contained 20 bivalent chromo- somes, while Rec8 TEV/TEV oocytes (n = 18) injected with TEV contained only single chromatids (74-80 per cell) (Fig. 3B). Because similar experiments conducted with Scc1 TEVMyc/TEVMyc oocytes revealed that meiotic chromo- somes were unaffected by TEV-induced Scc1 cleavage (Supplemental Fig. 3B) and little or no Scc1 can be de- tected on bivalent chromosomes (Supplemental Fig. 3C), despite Scc1 being abundant in GVs (see Fig. 6B, below), we conclude that ...
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... -unin- jected oocytes (n = 26) contained 20 bivalent chromo- somes, while Rec8 TEV/TEV oocytes (n = 18) injected with TEV contained only single chromatids (74-80 per cell) (Fig. 3B). Because similar experiments conducted with Scc1 TEVMyc/TEVMyc oocytes revealed that meiotic chromo- somes were unaffected by TEV-induced Scc1 cleavage (Supplemental Fig. 3B) and little or no Scc1 can be de- tected on bivalent chromosomes (Supplemental Fig. 3C), despite Scc1 being abundant in GVs (see Fig. 6B, below), we conclude that bivalent chromosomes are held to- gether by cohesin containing Rec8. If we assume that this is engaged in holding sister chromatids together, then our experiments also ...
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... oocytes (n = 18) injected with TEV contained only single chromatids (74-80 per cell) (Fig. 3B). Because similar experiments conducted with Scc1 TEVMyc/TEVMyc oocytes revealed that meiotic chromo- somes were unaffected by TEV-induced Scc1 cleavage (Supplemental Fig. 3B) and little or no Scc1 can be de- tected on bivalent chromosomes (Supplemental Fig. 3C), despite Scc1 being abundant in GVs (see Fig. 6B, below), we conclude that bivalent chromosomes are held to- gether by cohesin containing Rec8. If we assume that this is engaged in holding sister chromatids together, then our experiments also demonstrate that it is sister chro- matid cohesion and not some ''chiasma binder'' protein ...
Citations
... Unlike mitosis, meiosis comprises only one cycle of DNA replication followed by two rounds of chromosome segregation to produce haploid gametes. Therefore, in mammals the loading of cohesin proteins, essential for the fidelity of chromosome segregation, occurs only once during S phase of fetal life, and these proteins must be retained until meiotic resumption, potentially decades later (Tachibana-Konwalski et al., 2010, Burkhardt et al., 2016. ...
... During ageing, there is a gradual decrease in meiotic cohesin on the chromosomes of mammalian eggs (Lister et al., 2010). Indeed, the cohesin that holds sister chromatids together throughout the prolonged prophase I arrest in primordial follicles is not replenished during the growing phase of oocytes, at least in mice (Tachibana-Konwalski et al., 2010). This means that the cohesin that is critical for proper chromosome segregation is the same cohesin that is laid down in the fetus, perhaps 40 years or earlier in humans. ...
Advanced maternal age is a major cause of infertility, miscarriage, and congenital abnormalities. This is principally caused by a decrease in oocyte quality and developmental competence with age. Oocyte ageing is characterised by an increase in chromosome missegregation and aneuploidy. However, the underlying mechanisms of age-related aneuploidy have not been fully elucidated and are still under active investigation. In addition to chromosome missegregation, oocyte ageing is also accompanied by metabolic dysfunction. In this review, we integrate old and new perspectives on oocyte ageing, chromosome segregation and metabolism in mammalian oocytes and make direct links between these processes. We consider age-related alterations to chromosome segregation machinery, including the loss of cohesion, microtubule stability and the integrity of the spindle assembly checkpoint. We focus on how metabolic dysfunction in the ageing oocyte disrupts chromosome segregation machinery to contribute to and exacerbate age-related aneuploidy. More specifically, we discuss how mitochondrial function, ATP production and the generation of free radicals are altered during ageing. We also explore recent developments in oocyte metabolic ageing, including altered redox reactions (NAD ⁺ metabolism) and the interactions between oocytes and their somatic nurse cells. Throughout the review we integrate the mechanisms by which changes in oocyte metabolism influence age-related chromosome missegregation.
... Zygote arrest 1 (ZAR1) is the first oocyte-specific maternal-effect gene known to function in the oocyte-to-embryo transition [43]. Rec8-containing cohesin, bound to Smc3/Smc1a or Smc3/Smc1b, maintains the bivalent cohesion in mammalian meiosis [44]. Sister chromatid cohesion mediated by the cohesin complex is essential for chromosome segregation in mitosis and meiosis [45]. ...
The development of the ovarian antral follicle is a complex, highly regulated process. Oocytes orchestrate and coordinate the development of mammalian ovarian follicles, and the rate of follicular development is governed by a developmental program intrinsic to the oocyte. Characterizing oocyte signatures during this dynamic process is critical for understanding oocyte maturation and follicular development. Although the transcriptional signature of sheep oocytes matured in vitro and preovulatory oocytes have been previously described, the transcriptional changes of oocytes in antral follicles have not. Here, we used single-cell transcriptomics (SmartSeq2) to characterize sheep oocytes from small, medium, and large antral follicles. We characterized the transcriptomic landscape of sheep oocytes during antral follicle development, identifying unique features in the transcriptional atlas, stage-specific molecular signatures, oocyte-secreted factors, and transcription factor networks. Notably, we identified the specific expression of 222 genes in the LO, 8 and 6 genes that were stage-specific in the MO and SO, respectively. We also elucidated signaling pathways in each antral follicle size that may reflect oocyte quality and in vitro maturation competency. Additionally, we discovered key biological processes that drive the transition from small to large antral follicles, revealing hub genes involved in follicle recruitment and selection. Thus, our work provides a comprehensive characterization of the single-oocyte transcriptome, filling a gap in the mapping of the molecular landscape of sheep oogenesis. We also provide key insights into the transcriptional regulation of the critical sizes of antral follicular development, which is essential for understanding how the oocyte orchestrates follicular development.
... In mammalian oocytes, cohesin is established in utero after which oocytes enter a long arrest in meiotic prophase I. Meiosis is not resumed until the oocyte is ovulated, potentially several decades later. Evidence from mice indicates that there is no cohesin turnover during this period, 4,5 suggesting that cohesin laid down in the fetus must last throughout the female reproductive lifespan, up to age $50 years in humans. However, female fertility declines before this, largely due to the loss of oocyte euploidy. ...
... Zygote arrest 1 (ZAR1) is the first oocyte-specific maternal-effect gene known to function in the oocyte-to-embryo transition [42]. Rec8containing cohesin, bound to Smc3/Smc1a or Smc3/Smc1b, maintains bivalent cohesion in mammalian meiosis [43]. Sister chromatid cohesion mediated by the cohesin complex is essential for chromosome segregation in mitosis and meiosis [44]. ...
The development of the ovarian antral follicle is a complex, highly regulated process. Oocytes orchestrate and coordinate the development of mammalian ovarian follicles, and the rate of follicular development is governed by a developmental program intrinsic to the oocyte. Characterizing oocyte signatures during this dynamic process is critical for understanding oocyte maturation and follicular development. Although the transcriptional signature of sheep oocytes matured in vitro and preovulatory oocytes have been previously described, the transcriptional changes of oocytes in antral follicles have not. Here, we used single-cell transcriptomics (SmartSeq2) to characterize sheep oocytes from small, medium, and large antral follicles. We characterized the transcriptomic landscape of sheep oocytes during antral follicle development, identifying unique features in the transcriptional atlas, stage-specific molecular signatures, oocyte-secreted factors, and transcription factor networks. Notably, we identified specific gene and signaling pathways in each antral follicle stage that may reflect oocyte quality and in vitro maturation competency. Additionally, we discovered key biological processes that drive the transition from small to large antral follicles, revealing hub genes involved in follicle recruitment and selection. Thus, our work provides a comprehensive characterization of the single-oocyte transcriptome, filling a gap in the mapping of the molecular landscape of sheep oogenesis. We also provide key insights into the transcriptional regulation of the critical stages of antral follicular development, which is essential for understanding how the oocyte orchestrates follicular development.
... In old mouse, the reason why cohesion between sister chromatids diminishes, demonstrating by increased distance between sister kinetochores, can be partly explained by a loss of chromosome-related meiotic recombination protein REC8 homolog (REC8) whose responsibility is to protect chromatins and sister chromatids from segregation [158]. And since the total REC8 level has been estimated to be of no difference between young and old oocyte, it is possible that loss of cohesion with advanced age is not able to be effectively replaced, which has been proven in other study [158,159]. Nevertheless, similar alteration has illustrated to occur in meiotic cohesion protein, structural maintenance of chromosomes protein 1B (SMC1B) as well as cohesion subunit SA-3 (STAG3) that is located between sister chromatids [160]. ...
Ovarian aging is a natural and physiological aging process characterized by loss of quantity and quality of oocyte or follicular pool. As it is generally accepted that women are born with a finite follicle pool that will go through constant decline without renewing, which, together with decreased oocyte quality, makes a severe situation for women who is of advanced age but desperate for a healthy baby. The aim of our review was to investigate mechanisms leading to ovarian aging by discussing both extra- and intra- ovarian factors and to identify genetic characteristics of ovarian aging. The mechanisms were identified as both extra-ovarian alternation of hypothalamic–pituitary-ovarian axis and intra-ovarian alternation of ovary itself, including telomere, mitochondria, oxidative stress, DNA damage, protein homeostasis, aneuploidy, apoptosis and autophagy. Moreover, here we reviewed related Genome-wide association studies (GWAS studies) from 2009 to 2021 and next generation sequencing (NGS) studies of primary ovarian insufficiency (POI) in order to describe genetic characteristics of ovarian aging. It is reasonable to wish more reliable anti-aging interventions for ovarian aging as the exploration of mechanisms and genetics being progressing.
... This association is predominantly achieved by cohesin, a multisubunit protein complex that links chromosome arms and the centromeres of sister chromatids (2)(3)(4). During oocyte meiosis I, cohesin is removed from chromosome arms by Separase-mediated cleavage of its meiosis-specific component Rec8 (2,(5)(6)(7). Simultaneously, cohesin between sister chromatid centromeres is protected by Shugoshin2 shielding Rec8 from Separase activity (8)(9)(10). When coupled with microtubule-based pulling forces that are exerted on the chromosomes' kinetochores (1,11,12), this localized cohesion protection provides oocytes with a mechanism to selectively separate homologs at anaphase I without disrupting sister chromatid linkage. ...
Aging-related centromeric cohesion loss underlies premature separation of sister chromatids and egg aneuploidy in reproductively older females. Here, we show that F-actin maintains chromatid association after cohesion deterioration in aged eggs. F-actin disruption in aged mouse eggs exacerbated untimely dissociation of sister chromatids, while its removal in young eggs induced extensive chromatid separation events generally only seen in advanced reproductive ages. In young eggs containing experimentally reduced cohesion, F-actin removal accelerated premature splitting and scattering of sister chromatids in a microtubule dynamics–dependent manner, suggesting that actin counteracts chromatid-pulling spindle forces. Consistently, F-actin stabilization restricted scattering of unpaired chromatids generated by complete degradation of centromeric cohesion proteins. We conclude that actin mitigates egg aneuploidies arising from age-related cohesion depletion by limiting microtubule-driven separation and dispersion of sister chromatids. This is supported by our finding that spindle-associated F-actin structures are disrupted in eggs of reproductively older females.
... Notably, aneuploidy occurs in more than 50% of eggs from women aged 35 years and above [11,63]. Data from mice suggest that cohesion levels on chromosomes could determine the U-shaped curve [64][65][66][67][68]. Sister chromatids are bound together by many ring-like protein structures called cohesion complexes that are installed during DNA replication [66]. ...
Ovarian reserve is essential for fertility and influences healthy aging in women. Advanced maternal age correlates with the progressive loss of both the quantity and quality of oocytes. The molecular mechanisms and various contributing factors underlying ovarian aging have been uncovered. In this review, we highlight some of critical factors that impact oocyte quantity and quality during aging. Germ cell and follicle reserve at birth determines reproductive lifespan and timing the menopause in female mammals. Accelerated diminishing ovarian reserve leads to premature ovarian aging or insufficiency. Poor oocyte quality with increasing age could result from chromosomal cohesion deterioration and misaligned chromosomes, telomere shortening, DNA damage and associated genetic mutations, oxidative stress, mitochondrial dysfunction and epigenetic alteration. We also discuss the intervention strategies to delay ovarian aging. Both the efficacy of senotherapies by antioxidants against reproductive aging and mitochondrial therapy are discussed. Functional oocytes and ovarioids could be rejuvenated from pluripotent stem cells or somatic cells. We propose directions for future interventions. As couples increasingly begin delaying parenthood in life worldwide, understanding the molecular mechanisms during female reproductive aging and potential intervention strategies could benefit women in making earlier choices about their reproductive health.
... In the case of other factors implicated in TAD and loop establishment and maintenance, namely cohesin or WAPL, a key regulator of cohesion turnover (Wutz et al. 2017), the analysis is rather complicated. Although their deletion has been shown to affect the higher-order genome organization in the zygote , their involvement in faithful chromosome segregation in oocyte meiosis (Silva et al. 2020) or embryonic mitosis (Tachibana-Konwalski et al. 2010) is limiting the use of the genetic knockout models. Nevertheless, the weak chromatin compartmentalization seems to be important for a successful development and the depletion of cohesin was shown to be beneficial for the reprogramming by promoting the early expressed zygotic genes (Zhang et al. 2020). ...
In brief
Understanding the establishment of post-fertilization totipotency has broad implications for modern biotechnologies. This review summarizes the current knowledge of putative egg components governing this process following natural fertilization and after somatic cell nuclear transfer.
Abstract
The mammalian oocyte is a unique cell, and comprehending its physiology and biology is essential for understanding fertilization, totipotency and early events of embryogenesis. Consequently, research in these areas influences the outcomes of various technologies, for example, the production and conservation of laboratory and large animals with rare and valuable genotypes, the rescue of the species near extinction, as well as success in human assisted reproduction. Nevertheless, even the most advanced and sophisticated reproductive technologies of today do not always guarantee a favorable outcome. Elucidating the interactions of oocyte components with its natural partner cell – the sperm or an ‘unnatural’ somatic nucleus, when the somatic cell nucleus transfer is used is essential for understanding how totipotency is established and thus defining the requirements for normal development. One of the crucial aspects is the stoichiometry of different reprogramming and remodeling factors present in the oocyte and their balance. Here, we discuss how these factors, in combination, may lead to the formation of a new organism. We focus on the laboratory mouse and its genetic models, as this species has been instrumental in shaping our understanding of early post-fertilization events.
... A direct assessment of the contribution of REC-8 and COH-3/4 cohesin to SCC under normal conditions requires the ability to specifically remove these complexes in a temporally-resolved manner after normal chromosome morphogenesis. For example, in mouse oocytes arrested at metaphase I, TEV-mediated removal of REC8 revealed that SCC is exclusively provided by REC8 cohesin, despite the presence of SCC1 cohesin (Tachibana-Konwalski et al., 2010). ...
... These results are consistent with REC-8 complexes providing SCC in metaphase I oocytes, and suggest that the weak chromatid attachments that remain in diakinesis bivalents following REC-8 removal are resolved in metaphase I oocytes. This may be due to the assembly of the first meiotic spindle, which may be sufficient to pull apart weakly attached chromatids, or to the resolution of a crossover-associated chromosome structure between diakinesis and metaphase I. Regardless, the clear conclusion from our temporally-resolved kleisin removal experiments is that, similar to mouse oocytes (Tachibana-Konwalski et al., 2010), SCC is exclusively provided by REC-8 cohesin in metaphase I oocytes of C. elegans. ...
... We reveal a clear distribution of functions between stably-bound and low abundance REC-8 complexes, which provide SCC and DSB repair, and high-abundance COH-3/4 complexes that ensure the integrity of axial elements and associate dynamically with pachytene chromosomes in a process controlled by WAPL-1 and SCC-2. Our studies support functional conservation between worm and mouse REC8 cohesin, with REC8 complexes providing SCC and being largely refractory to WAPLmediated removal in both organisms (Crawley et al., 2016;Silva et al., 2020;Tachibana-Konwalski et al., 2010), and suggest that functional conservation also likely extends to COH-3/4-RAD21L complexes. ...
The cohesin complex plays essential roles in chromosome segregation, 3D genome organisation, and DNA damage repair through its ability to modify DNA topology. In higher eukaryotes, meiotic chromosome function, and therefore fertility, requires cohesin complexes containing meiosis-specific kleisin subunits: REC8 and RAD21L in mammals and REC-8 and COH-3/4 in C. elegans . How these complexes perform the multiple functions of cohesin during meiosis and whether this involves different modes of DNA binding or dynamic association with chromosomes is poorly understood. Combining time-resolved methods of protein removal with live imaging and exploiting the temporospatial organisation of the C. elegans germline, we show that REC-8 complexes provide sister chromatid cohesion (SCC) and DNA repair, while COH-3/4 complexes control higher-order chromosome structure. High-abundance COH-3/4 complexes associate dynamically with individual chromatids in a manner dependent on cohesin loading (SCC-2) and removal (WAPL-1) factors. In contrast, low-abundance REC-8 complexes associate stably with chromosomes, tethering sister chromatids from S-phase until the meiotic divisions. Our results reveal that kleisin identity determines the function of meiotic cohesin by controlling the mode and regulation of cohesin-DNA association, and are consistent with a model in which SCC and DNA looping are performed by variant cohesin complexes that coexist on chromosomes.
... Previous studies have shown that the cohesin complex forms a ring-like structure that can entrap two sister chromatids [5][6][7] . During the metaphase-to-anaphase transition, separase cleaves RAD21, which opens the cohesin ring and releases sister chromatids for segregation to daughter cells [8][9][10][11] . Besides its role in sister-chromatid cohesion, cohesin is an ATP-dependent motor protein that extrudes chromatin loops during interphase 12-14 . ...
... The unstructured region in which the TEV sites were inserted contains one of two naturally occurring separase cleavage sites 8 . Previous work has shown that cleavage of RAD21 (or Scc1) by separase, or TEV, in this region suffices to disrupt sister-chromatid cohesion [8][9][10][11] . A comparison of HAP1 cells to HAP1-RAD21 TEV nuclei showed that the RAD21 protein levels, RAD21 and CTCF chromatin binding, and loop formation were unaffected by the insertion of the TEV cleavage sites ( Fig. 2b and Supplementary Fig. 6d-i). ...
... Some CTCF-CTCF interactions can be contacts between sister chromatids that are mediated by cohesive cohesin complexes 36 . Previous studies had shown that [8][9][10][11] . To rule out that any of our results are due to a loss of inter-sister interactions, we sorted G1 nuclei after TEV treatment and formaldehyde fixation of HAP1-RAD21 TEV nuclei. ...
The ring-like cohesin complex mediates sister-chromatid cohesion by encircling pairs of sister chromatids. Cohesin also extrudes loops along chromatids. Whether the two activities involve similar mechanisms of DNA engagement is not known. We implemented an experimental approach based on isolated nuclei carrying engineered cleavable RAD21 proteins to precisely control cohesin ring integrity so that its role in chromatin looping could be studied under defined experimental conditions. This approach allowed us to identify cohesin complexes with distinct biochemical, and possibly structural, properties that mediate different sets of chromatin loops. When RAD21 is cleaved and the cohesin ring is opened, cohesin complexes at CTCF sites are released from DNA and loops at these elements are lost. In contrast, cohesin-dependent loops within chromatin domains that are not anchored at pairs of CTCF sites are more resistant to RAD21 cleavage. The results show that the cohesin complex mediates loops in different ways depending on the genomic context and suggests that it undergoes structural changes as it dynamically extrudes and encounters CTCF sites. Liu and Dekker test the importance of cohesin ring integrity for genome architecture: cohesin ring opening via Rad21 cleavage causes loss of CTCF–CTCF loops but maintains dynamic intra-domain loops, suggesting distinct cohesin engagement modes.
