ORCID Profile
0000-0002-8155-0981
Current Organisation
University of Southampton
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Publisher: Bioscientifica
Date: 10-2010
DOI: 10.1530/REP-10-0223
Abstract: Previous studies have established that when maturing mouse oocytes are continuously incubated with the Aurora inhibitor ZM447439, meiotic maturation is blocked. In this study, we observe that by altering the time of addition of the inhibitor, oocyte maturation can actually be accelerated by 1 h as measured by the timing of polar body extrusion. ZM447439 also had the ability to overcome a spindle assembly checkpoint (SAC) arrest caused by nocodazole and so rescue polar body extrusion. Consistent with the ability of the SAC to inhibit cyclin B1 degradation by blocking activation of the anaphase-promoting complex, we could also observe a rescue in cyclin B1 degradation when ZM447439 was added to nocodazole-treated oocytes. The acceleration of the first meiotic ision by ZM447439, which has not been achieved previously, and its effects on the SAC are all consistent with the proposed mitotic role of Aurora B in activating the SAC. We hypothesize that Aurora kinase activity controls the SAC in meiosis I, despite differences to the mitotic cell cycle ision in spindle architecture brought about by the meiotic mono-orientation of sister kinetochores.
Publisher: Springer Science and Business Media LLC
Date: 27-07-2018
DOI: 10.1038/S41467-018-05338-7
Abstract: In the first meiotic ision (MI) of oocytes, the cortically positioned spindle causes bivalent segregation in which only the centre-facing homologue pairs are retained. ‘Selfish’ chromosomes are known to exist, which bias their spindle orientation and hence retention in the egg, a process known as ‘meiotic drive’. Here we report on this phenomenon in oocytes from F 1 hybrid mice, where parental strain differences in centromere size allows distinction of the two homologue pairs of a bivalent. Bivalents with centromere and kinetochore asymmetry show meiotic drive by rotating during prometaphase, in a process dependent on aurora kinase activity. Cortically positioned homologue pairs appear to be under greater stretch than their centre-facing partners. Additionally the cortex spindle-half contain a greater density of tubulin and microtubule organising centres. A model is presented in which meiotic drive is explained by the impact of microtubule force asymmetry on chromosomes with different sized centromeres and kinetochores.
Publisher: Rockefeller University Press
Date: 06-08-2018
Abstract: Mouse female meiotic spindles assemble from acentriolar microtubule-organizing centers (aMTOCs) that fragment into discrete foci. These are further sorted and clustered to form spindle poles, thus providing balanced forces for faithful chromosome segregation. To assess the impact of aMTOC biogenesis on spindle assembly, we genetically induced their precocious fragmentation in mouse oocytes using conditional overexpression of Plk4, a master microtubule-organizing center regulator. Excessive microtubule nucleation from these fragmented aMTOCs accelerated spindle assembly dynamics. Prematurely formed spindles promoted the breakage of three different fragilized bivalents, generated by the presence of recombined Lox P sites. Reducing the density of microtubules significantly diminished the extent of chromosome breakage. Thus, improper spindle forces can lead to widely described yet unexplained chromosomal structural anomalies with disruptive consequences on the ability of the gamete to transmit an uncorrupted genome.
Publisher: Informa UK Limited
Date: 23-04-2014
DOI: 10.4161/CC.28897
Publisher: Cold Spring Harbor Laboratory
Date: 09-04-2021
DOI: 10.1101/2021.04.08.439026
Abstract: The future of single cell ersity screens involves ever-larger s le sizes, dictating the need for higher throughput methods with low analytical noise to accurately describe the nature of the cellular system. Current approaches are limited by the Poisson statistic, requiring dilute cell suspensions and associated losses in throughput. In this contribution, we apply Dean entrainment to both cell and bead inputs, defining different volume packets to effect efficient co-encapsulation. Volume ratio scaling was explored to identify optimal conditions. This enabled the co-encapsulation of single cells with reporter beads at rates of ~1 million cells/hour, while increasing assay signal-to-noise with cell multiplet rates of ~2.5% and capturing ~70% of cells. The method, called Pirouette-seq, extends our capacity to investigate biological systems. Pirouette-seq involves cell and reporter bead inertial ordering for efficient co-encapsulation, achieving a throughput of 1 million cells/hour, a 2.5% multiplet rate and a 70% cell capture efficiency.
Publisher: The Company of Biologists
Date: 2014
DOI: 10.1242/DEV.100206
Abstract: As women get older their oocytes become susceptible to chromosome mis-segregation. This generates aneuploid embryos, leading to increased infertility and birth defects. Here we examined the provenance of aneuploidy by tracking chromosomes and their kinetochores in oocytes from young and aged mice. Changes consistent with chromosome cohesion deterioration were found with age, including increased interkinetochore distance and loss of the centromeric protector of cohesion SGO2 in metaphase II arrested (metII) eggs, as well as a rise in the number of weakly attached bivalents in meiosis I (MI) and lagging chromosomes at anaphase I. However, there were no MI errors in congression or biorientation. Instead, premature separation of dyads in meiosis II was the major segregation defect in aged eggs and these were associated with very low levels of SGO2. These data show that although considerable cohesion loss occurs during MI, its consequences are observed during meiosis II, when centromeric cohesion is needed to maintain dyad integrity.
Publisher: Springer Science and Business Media LLC
Date: 18-03-2014
DOI: 10.1038/NCOMMS4444
Abstract: The spindle assembly checkpoint (SAC) prevents aneuploidy by coupling anaphase onset, through anaphase-promoting complex (APC) activation, with chromosome attachment to spindle microtubules. Here, we examine APC activity in oocytes, noted for their susceptibility to chromosome mis-segregation during the first meiotic ision (MI). We find that MI oocytes only contain sub-maximal APC activity, measured through cyclin B1-GFP degradation, because inhibition of SAC proteins when the APC is normally fully active increases cyclin B1 degradation twofold and reduces the length of this ision by 2 h. In addition, inhibiting the SAC component Mps1 only when the APC is already active increases aneuploidy rates in the resulting egg by up to 30%. We therefore establish that the activities of SAC proteins and the APC co-exist in oocytes, and such concurrence has a vital role in reducing aneuploidy rates by extending MI, probably by allowing time for numerous erroneous microtubule attachments to be corrected.
Publisher: The Company of Biologists
Date: 06-2012
DOI: 10.1242/DEV.077040
Abstract: Homologous chromosome segregation errors during meiosis I are common and generate aneuploid embryos. Here, we provide a reason for this susceptibility to mis-segregation by live cell imaging of mouse oocytes. Our results show that stable kinetochore-microtubule attachments form in mid-prometaphase, 3-4 hours before anaphase. This coincided with the loss of Mad2 from kinetochores and with the start of anaphase-promoting complex/cyclosome (APC/C)-mediated cyclin B1 destruction. Therefore, the spindle assembly checkpoint (SAC) ceased to inhibit the APC/C from mid-prometaphase. This timing did not coincide with bivalent congression in one-third of all oocytes examined. Non-aligned bivalents were weakly positive for Mad2, under less tension than congressed bivalents and, by live-cell imaging, appeared to be in the process of establishing correct bi-orientation. The time from when the APC/C became active until anaphase onset was affected by the rate of loss of CDK1 activity, rather than by these non-aligned bivalents, which occasionally persisted until anaphase, resulting in homolog non-disjunction. We conclude that, in oocytes, a few erroneous attachments of bivalent kinetochores to microtubules do not generate a sufficient SAC ‘wait anaphase’ signal to inhibit the APC/C.
Publisher: American Society for Cell Biology (ASCB)
Date: 15-10-2012
Abstract: FZR1 is an anaphase-promoting complex (APC) activator best known for its role in the mitotic cell cycle at M-phase exit, in G1, and in maintaining genome integrity. Previous studies also established that it prevents meiotic resumption, equivalent to the G2/M transition. Here we report that mouse oocytes lacking FZR1 undergo passage through meiosis I that is accelerated by ∼1 h, and this is due to an earlier onset of spindle assembly checkpoint (SAC) satisfaction and APC CDC20 activity. However, loss of FZR1 did not compromise SAC functionality instead, earlier SAC satisfaction was achieved because the bipolar meiotic spindle was assembled more quickly in the absence of FZR1. This novel regulation of spindle assembly by FZR1 led to premature bivalent attachment to microtubules and loss of kinetochore-bound MAD2. Bivalents, however, were observed to congress poorly, leading to nondisjunction rates of 25%. We conclude that in mouse oocytes FZR1 controls the timing of assembly of the bipolar spindle and in so doing the timing of SAC satisfaction and APC CDC20 activity. This study implicates FZR1 as a major regulator of prometaphase whose activity helps to prevent chromosome nondisjunction.
Publisher: Springer New York
Date: 2017
DOI: 10.1007/978-1-4939-6340-9_13
Abstract: Accurate chromosome segregation is necessary so that genetic material is equally shared among daughter cells. However, maturing mammalian oocytes are particularly prone to chromosome segregation errors, making them a valuable tool for identifying the causes of mis-segregation. Factors such as aging, cohesion loss, DNA damage, and the roles of a plethora of kinetochore and cell cycle-related proteins are involved. To study chromosome segregation in oocytes in a live setting is an imaging challenge that requires advanced techniques. Here we describe a method for examining chromosomes in live oocytes in detail as they undergo maturation. Our method is based on tracking the "center of brightness" of fluorescently labeled chromosomes. Here we describe how to set up our software and run experiments on a Leica TCS SP8 confocal microscope, but the method would be transferable to other microscopes with computer-aided microscopy.
Publisher: Elsevier BV
Date: 07-2012
DOI: 10.1016/J.YEXCR.2012.02.012
Abstract: Aneuploidy is a leading cause of early embryo loss, miscarriage and birth defects in humans. It is predominantly brought about by the mis-segregation of homologous chromosomes (bivalents) in the first meiotic ision (MI) of the oocyte, with advanced maternal age being a risk factor. Although its etiology is likely to be multifactorial the predominating factors remain amenable for study in models such as mice. Homologous chromosome separation in MI is achieved by the mono-orientation of functionally paired sister kinetochores but despite this unique ision the Spindle Assembly Checkpoint (SAC), which prevents sister chromatid mis-segregation in mitosis, is functional in mouse oocytes. However, it remains to be fully established what types of error the SAC respond to, for ex le the presence of univalents, and how sensitive it is to attachment or tension defects in bivalent alignment. Such errors may increase with advanced maternal age as chromosomes lose their cohesive ties and the oocyte has less capacity to service the metabolic needs associated with meiotic ision. Environmental insults and hormonal changes could also affect the fidelity of this process. Here we review how all these factors converge on the meiotic spindle during MI to cause mis-segregation errors.
Publisher: Elsevier
Date: 2013
Publisher: Springer Science and Business Media LLC
Date: 15-06-2016
DOI: 10.1038/SREP27991
Abstract: Whether the adult mammalian ovary contains oogonial stem cells (OSCs) is controversial. They have been isolated by a live-cell sorting method using the germ cell marker DDX4, which has previously been assumed to be cytoplasmic, not surface-bound. Furthermore their stem cell and germ cell characteristics remain disputed. Here we show that although OSC-like cells can be isolated from the ovary using an antibody to DDX4, there is no good in silico modelling to support the existence of a surface-bound DDX4. Furthermore these cells when isolated were not expressing DDX4 and did not initially possess germline identity. Despite these unremarkable beginnings, they acquired some pre-meiotic markers in culture, including DDX4, but critically never expressed oocyte-specific markers and furthermore were not immortal but died after a few months. Our results suggest that freshly isolated OSCs are not germ stem cells and are not being isolated by their DDX4 expression. However it may be that culture induces some pre-meiotic markers. In summary the present study offers weight to the dogma that the adult ovary is populated by a fixed number of oocytes and that adult de novo production is a rare or insignificant event.
Publisher: Springer Science and Business Media LLC
Date: 14-11-2016
DOI: 10.1038/SREP36994
Abstract: Mouse oocytes respond to DNA damage by arresting in meiosis I through activity of the Spindle Assembly Checkpoint (SAC) and DNA Damage Response (DDR) pathways. It is currently not known if DNA damage is the primary trigger for arrest, or if the pathway is sensitive to levels of DNA damage experienced physiologically. Here, using follicular fluid from patients with the disease endometriosis, which affects 10% of women and is associated with reduced fertility, we find raised levels of Reactive Oxygen Species (ROS), which generate DNA damage and turn on the DDR-SAC pathway. Only follicular fluid from patients with endometriosis, and not controls, produced ROS and damaged DNA in the oocyte. This activated ATM kinase, leading to SAC mediated metaphase I arrest. Completion of meiosis I could be restored by ROS scavengers, showing this is the primary trigger for arrest and offering a novel clinical therapeutic treatment. This study establishes a clinical relevance to the DDR induced SAC in oocytes. It helps explain how oocytes respond to a highly prevalent human disease and the reduced fertility associated with endometriosis.
Publisher: Springer Science and Business Media LLC
Date: 02-11-2015
DOI: 10.1038/NCOMMS9553
Abstract: Extensive damage to maternal DNA during meiosis causes infertility, birth defects and abortions. However, it is unknown if fully grown oocytes have a mechanism to prevent the creation of DNA-damaged embryos. Here we show that DNA damage activates a pathway involving the spindle assembly checkpoint (SAC) in response to chemically induced double strand breaks, UVB and ionizing radiation. DNA damage can occur either before or after nuclear envelope breakdown, and provides an effective block to anaphase-promoting complex activity, and consequently the formation of mature eggs. This contrasts with somatic cells, where DNA damage fails to affect mitotic progression. However, it uncovers a second function for the meiotic SAC, which in the context of detecting microtubule–kinetochore errors has hitherto been labelled as weak or ineffectual in mammalian oocytes. We propose that its essential role in the detection of DNA damage sheds new light on its biological purpose in mammalian female meiosis.
Publisher: Rockefeller University Press
Date: 04-10-2017
Abstract: The spindle assembly checkpoint (SAC) prevents chromosome missegregation by coupling anaphase onset with correct chromosome attachment and tension to microtubules. It does this by generating a diffusible signal from free kinetochores into the cytoplasm, inhibiting the anaphase-promoting complex (APC). The volume in which this signal remains effective is unknown. This raises the possibility that cell volume may be the reason the SAC is weak, and chromosome segregation error-prone, in mammalian oocytes. Here, by a process of serial bisection, we analyzed the influence of oocyte volume on the ability of the SAC to inhibit bivalent segregation in meiosis I. We were able to generate oocytes with cytoplasmic volumes reduced by 86% and observed changes in APC activity consistent with increased SAC control. However, bivalent biorientation remained uncoupled from APC activity, leading to error-prone chromosome segregation. We conclude that volume is one factor contributing to SAC weakness in oocytes. However, additional factors likely uncouple chromosome biorientation with APC activity.
Publisher: Oxford University Press (OUP)
Date: 02-2013
DOI: 10.1095/BIOLREPROD.112.104786
Abstract: It is becoming clear that reduced chromosome cohesion is an important factor in the rise of maternal age-related aneuploidy. This reduction in cohesion has been observed both in human and mouse oocytes, and it can be measured directly by an increase with respect to maternal age in interkinetochore (iKT) distance between a sister chromatid pair. We have observed variations in iKT distance even in oocytes from young mice and wondered if such differences may predispose those oocytes displaying the greatest iKT distances to be becoming aneuploid. Therefore, we used two methods, one pharmacological (Aurora kinase inhibitor) and one genetic (Fzr1 knockout), to raise aneuploidy rates in oocytes from young mice (age, 1-3 mo) and to examine if those oocytes that were aneuploid had greater iKT distances. We observed that for both Aurora kinase inhibition and Fzr1 knockout, iKT distances were significantly greater in those oocytes that became aneuploid compared to those that remained euploid. Based on these results, we propose that in idual oocytes undergo loss in chromosomal cohesion at different rates and that the greater this loss, the greater the risk for becoming aneuploid.
Publisher: Rockefeller University Press
Date: 11-08-2021
Abstract: The repetitive sequences of DNA centromeric regions form the structural basis for kinetochore assembly. Recently they were found to be transcriptionally active in mitosis, with their RNAs providing noncoding functions. Here we explore the role, in mouse oocytes, of transcripts generated from within the minor satellite repeats. Depletion of minor satellite transcripts delayed progression through meiosis I by activation of the spindle assembly checkpoint. Arrested oocytes had poorly congressed chromosomes, and centromeres were frequently split by microtubules. Thus, we have demonstrated that the centromeric RNA plays a specific role in female meiosis I compared with mitosis and is required for maintaining the structural integrity of centromeres. This may contribute to the high aneuploidy rates observed in female meiosis.
Publisher: The Company of Biologists
Date: 15-09-2013
DOI: 10.1242/DEV.090589
Abstract: Mammalian oocytes are particularly error prone in segregating their chromosomes during their two meiotic isions. This results in the creation of an embryo that has inherited the wrong number of chromosomes: it is aneuploid. The incidence of aneuploidy rises significantly with maternal age and so there is much interest in understanding this association and the underlying causes of aneuploidy. The spindle assembly checkpoint, a surveillance mechanism that operates in all cells to prevent chromosome mis-segregation, and the cohesive ties that hold those chromosomes together, have thus both been the subject of intensive investigation in oocytes. It is possible that a lowered sensitivity of the spindle assembly checkpoint to certain types of chromosome attachment error may endow oocytes with an innate susceptibility to aneuploidy, which is made worse by an age-related loss in the factors that hold the chromosomes together.
Publisher: Humana Press
Date: 05-10-2012
DOI: 10.1007/978-1-62703-191-2_6
Abstract: The first meiotic ision of mammalian oocytes physiologically occurs in the ovary in the hours preceding ovulation. Fortunately, oocytes removed from their follicular environment will readily undergo this process in culture. Their large size, optical transparency, and efficiency in translating exogenous cRNA make mouse oocytes very amenable to study this process in detail using fluorescence imaging-based techniques. Here we describe the process of microinjecting cRNA to proteins of interest that have been coupled to a fluorescent protein using cyclin B1 as an ex le.
Publisher: Springer London
Date: 12-10-2012
Publisher: Cold Spring Harbor Laboratory
Date: 25-06-2018
DOI: 10.1101/350272
Abstract: Mouse female meiotic spindles assemble from acentriolar MTOCs (aMTOCs) that fragment into discrete foci. These are further sorted and clustered to form spindle poles, thus providing balanced forces for faithful chromosome segregation. To assess the impact of aMTOCs biogenesis on spindle assembly, we genetically induced their precocious fragmentation in mouse oocytes using conditional overexpression of Plk4, a master MTOC regulator. Excessive microtubule nucleation from these fragmented aMTOCs accelerated spindle assembly dynamics. Prematurely formed spindles promoted the breakage of three different fragilized bivalents, generated by the presence of recombined Lox P sites. Reducing the density of microtubules diminished the extent of chromosome breakage. Thus, improper spindle forces can lead to widely described yet unexplained chromosomal structural anomalies with disruptive consequences on the ability of the gamete to transmit an uncorrupted genome.
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Simon Lane.