Oocyte and embryo culture under oil profoundly alters effective concentrations of small molecule inhibitors.

Culture of oocytes and embryos in media under oil is a cornerstone of fertility treatment, and extensively employed in experimental investigation of early mammalian development. It has been noted anecdotally by some that certain small molecule inhibitors might lose activity in oil-covered culture systems, presumably by drug partitioning into the oil. Here we took a pseudo-pharmacological approach to appraise this formally using mouse oocytes and embryos. Using different culture dish designs with defined media:oil volume ratios, we show that the EC50 of the widely employed microtubule poison nocodazole shifts as a function of the media:oil ratio, such that nocodazole concentrations that prevent cell division in oil-free culture fail to in oil-covered media drops. Relatively subtle changes in culture dish design lead to measurable changes in EC50. This effect is not specific to one type of culture oil, and can be readily observed both in oocyte and embryo culture experiments. We subsequently applied a similar approach to a small panel of widely employed cell cycle-related inhibitors, finding that most lose activity in standard oil-covered oocyte/embryo culture systems. Our data suggest that loss of small molecule activity in oil-covered oocyte and embryo culture is a widespread phenomenon with potentially far-reaching implications for data reproducibility, and we recommend avoiding oil-covered culture for experiments employing inhibitors/drugs wherever possible.

Spindle assembly checkpoint insensitivity allows meiosis-II despite chromosomal defects in aged eggs.

Chromosome segregation errors in mammalian oocyte meiosis lead to developmentally compromised aneuploid embryos and become more common with advancing maternal age. Known contributors include age-related chromosome cohesion loss and spindle assembly checkpoint (SAC) fallibility in meiosis-I. But how effective the SAC is in meiosis-II and how this might contribute to age-related aneuploidy is unknown. Here, we developed genetic and pharmacological approaches to directly address the function of the SAC in meiosis-II. We show that the SAC is insensitive in meiosis-II oocytes and that as a result misaligned chromosomes are randomly segregated. Whilst SAC ineffectiveness in meiosis-II is not age-related, it becomes most prejudicial in oocytes from older females because chromosomes that prematurely separate by age-related cohesion loss become misaligned in meiosis-II. We show that in the absence of a robust SAC in meiosis-II these age-related misaligned chromatids are missegregated and lead to aneuploidy. Our data demonstrate that the SAC fails to prevent cell division in the presence of misaligned chromosomes in oocyte meiosis-II, which explains how age-related cohesion loss can give rise to aneuploid embryos.

Imaging the subcellular viscoelastic properties of mouse oocytes.

In recent years, cellular biomechanical properties have been investigated as an alternative to morphological assessments for oocyte selection in reproductive science. Despite the high relevance of cell viscoelasticity characterization, the reconstruction of spatially distributed viscoelastic parameter images in such materials remains a major challenge. Here, a framework for mapping viscoelasticity at the subcellular scale is proposed and applied to live mouse oocytes. The strategy relies on the principles of optical microelastography for imaging in combination with the overlapping subzone nonlinear inversion technique for complex-valued shear modulus reconstruction. The three-dimensional nature of the viscoelasticity equations was accommodated by applying an oocyte geometry-based 3D mechanical motion model to the measured wave field. Five domains-nucleolus, nucleus, cytoplasm, perivitelline space, and zona pellucida-could be visually differentiated in both oocyte storage and loss modulus maps, and statistically significant differences were observed between most of these domains in either property reconstruction. The method proposed herein presents excellent potential for biomechanical-based monitoring of oocyte health and complex transformations across lifespan. It also shows appreciable latitude for generalization to cells of arbitrary shape using conventional microscopy equipment.

Kinetochore deterioration concommitant with centromere weakening during aging in mouse oocyte meiosis-I.

Age-related oocyte aneuploidy occurs as a result of chromosome segregation errors in female meiosis-I and meiosis-II, and is caused by a progressive age-related deterioration of the chromosome segregation machinery. Here, we assess the impact of age upon the kinetochore, the multi-protein structure that forms the link between the chromosome and spindle microtubules. We find that in meiosis-I the outer kinetochore assembles at germinal vesicle breakdown, but that a substantially smaller outer kinetochore is assembled in oocytes from aged mice. We show this correlates with a weaker centromere in aged oocytes and, using nuclear transfer approaches to generate young-aged hybrid oocytes, we show that outer kinetochore assembly always mirrors the status of the centromere, regardless of cytoplasmic age. Finally, we show that weaker kinetochores in aged oocytes are associated with thinner microtubule bundles, that are more likely to be mis-attached. We conclude that progressive loss of the centromere with advancing maternal age underpins a loss of the outer kinetochore in meiosis-I, which likely contributes to chromosome segregation fallibility in oocytes from older females.

Absence of a robust mitotic timer mechanism in early preimplantation mouse embryos leads to chromosome instability.

Preimplantation embryos often consist of a combination of euploid and aneuploid cells, suggesting that safeguards preventing the generation and propagation of aneuploid cells in somatic cells might be deficient in embryos. In somatic cells, a mitotic timer mechanism has been described, in which even a small increase in the duration of M phase can cause a cell cycle arrest in the subsequent interphase, preventing further propagation of cells that have undergone a potentially hazardously long M phase. Here, we report that cell divisions in the mouse embryo and embryonic development continue even after a mitotic prolongation of several hours. However, similar M-phase extensions caused cohesion fatigue, resulting in prematurely separated sister chromatids and the production of micronuclei. Only extreme prolongation of M phase caused a subsequent interphase arrest, through a mechanism involving DNA damage. Our data suggest that the simultaneous absence of a robust mitotic timer and susceptibility of the embryo to cohesion fatigue could contribute to chromosome instability in mammalian embryos. This article has an associated 'The people behind the papers' interview.

Cell size and polarization determine cytokinesis furrow ingression dynamics in mouse embryos.

Cytokinesis is the final step of cell division during which a contractile ring forms a furrow that partitions the cytoplasm in two. How furrow ingression is spatiotemporally regulated and how it is adapted to complex cellular environments and developmental transitions remain poorly understood. Here, we examine furrow ingression dynamics in the context of the early mouse embryo and find that cell size is a powerful determinant of furrow ingression speed during reductive cell divisions. In addition, the emergence of cell polarity and the assembly of the apical domain in outer cells locally inhibits the recruitment of cytokinesis components and thereby negatively regulates furrow ingression specifically on one side of the furrow. We show that this biasing of cytokinesis is not dependent upon cell­cell adhesion or shape but rather is cell intrinsic and is caused by a paucity of cytokinetic machinery in the apical domain. The results thus reveal that in the mouse embryo cell polarity directly regulates the recruitment of cytokinetic machinery in a cell-autonomous manner and that subcellular organization can instigate differential force generation and constriction speed in different zones of the cytokinetic furrow.

Distinct classes of lagging chromosome underpin age-related oocyte aneuploidy in mouse.

Chromosome segregation errors that cause oocyte aneuploidy increase in frequency with maternal age and are considered a major contributing factor of age-related fertility decline in females. Lagging anaphase chromosomes are a common age-associated phenomenon in oocytes, but whether anaphase laggards actually missegregate and cause aneuploidy is unclear. Here, we show that lagging chromosomes in mouse oocytes comprise two mechanistically distinct classes of chromosome motion that we refer to as "class-I" and "class-II" laggards. We use imaging approaches and mechanistic interventions to dissociate the two classes and find that whereas class-II laggards are largely benign, class-I laggards frequently directly lead to aneuploidy. Most notably, a controlled prolongation of meiosis I specifically lessens class-I lagging to prevent aneuploidy. Our data thus reveal lagging chromosomes to be a cause of age-related aneuploidy in mouse oocytes and suggest that manipulating the cell cycle could increase the yield of useful oocytes in some contexts.

The impact of embryo binucleation depends upon its origin.

Summary: Preimplantation embryos frequently contain binucleated cells, but reports differ as to whether binucleation affects development and whether such embryos should be used clinically. In this Point Of View article, we propose a possible explanation for this disparity: binucleation can arise by distinct routes, one that produces healthy blastomeres and one that directly threatens embryo viability.

Human oocytes harboring damaged DNA can complete meiosis I.

OBJECTIVE: To determine whether human oocytes possess a checkpoint to prevent completion of meiosis I when DNA is damaged. DESIGN: DNA damage is considered a major threat to the establishment of healthy eggs and embryos. Recent studies found that mouse oocytes with damaged DNA can resume meiosis and undergo germinal vesicle breakdown (GVBD), but then arrest in metaphase of meiosis I in a process involving spindle assembly checkpoint (SAC) signaling. Such a mechanism could help prevent the generation of metaphase II (MII) eggs with damaged DNA. Here, we compared the impact of DNA-damaging agents with nondamaged control samples in mouse and human oocytes. SETTING: University-affiliated clinic and research center. PATIENT(S): Patients undergoing ICSI cycles donated GV-stage oocytes after informed consent; 149 human oocytes were collected over 2 years (from 50 patients aged 27-44 years). INTERVENTIONS(S): Mice and human oocytes were treated with DNA-damaging drugs. MAIN OUTCOME MEASURE(S): Oocytes were monitored to evaluate GVBD and polar body extrusion (PBE), in addition to DNA damage assessment with the use of γH2AX antibodies and confocal microscopy. RESULT(S): Whereas DNA damage in mouse oocytes delays or prevents oocyte maturation, most human oocytes harboring experimentally induced DNA damage progress through meiosis I and subsequently form an MII egg, revealing the absence of a DNA damage-induced SAC response. Analysis of the resulting MII eggs revealed damaged DNA and chaotic spindle apparatus, despite the oocyte appearing morphologically normal. CONCLUSION(S): Our data indicate that experimentally induced DNA damage does not prevent PBE in human oocytes and can persist in morphologically normal looking MII eggs.

Chromosome dynamics and spindle microtubule establishment in mouse embryos.

Chromosome segregation errors in mammalian embryos are common and jeopardize embryo health. Here, we perform for the first time 4-Dimensional imaging and tracking of chromosomes and centromeres through each preimplantation mitotic cell division in mouse embryos to define the normal dynamics of chromosome segregation. We show that a microtubule (MT)-dependent inward movement of chromosomes occurs at the time of nuclear envelope breakdown (NEBD), particularly in the earliest cell divisions, to position chromosomes prior to spindle assembly. Establishment of a rudimentary metaphase plate occurs immediately after NEBD, and is followed by a progressive alignment and biorientation of mitotic chromosomes. Stable end-on kinetochore-MT attachments form rapidly and attachment errors are uncommon. Altogether our data describe a rapid and efficient spindle assembly pathway that apparently minimizes the need for canonical MT attachment error correction in normally dividing embryos.

Tetraploidy causes chromosomal instability in acentriolar mouse embryos.

Tetraploidisation is considered a common event in the evolution of chromosomal instability (CIN) in cancer cells. The current model for how tetraploidy drives CIN in mammalian cells is that a doubling of the number of centrioles that accompany the genome doubling event leads to multipolar spindle formation and chromosome segregation errors. By exploiting the unusual scenario of mouse blastomeres, which lack centrioles until the ~64-cell stage, we show that tetraploidy can drive CIN by an entirely distinct mechanism. Tetraploid blastomeres assemble bipolar spindles dictated by microtubule organising centres, and multipolar spindles are rare. Rather, kinetochore-microtubule turnover is altered, leading to microtubule attachment defects and anaphase chromosome segregation errors. The resulting blastomeres become chromosomally unstable and exhibit a dramatic increase in whole chromosome aneuploidies. Our results thus reveal an unexpected mechanism by which tetraploidy drives CIN, in which the acquisition of chromosomally-unstable microtubule dynamics contributes to chromosome segregation errors following tetraploidisation.

Cell-Size-Independent Spindle Checkpoint Failure Underlies Chromosome Segregation Error in Mouse Embryos.

Chromosome segregation errors during mammalian preimplantation development cause "mosaic" embryos comprising a mixture of euploid and aneuploid cells, which reduce the potential for a successful pregnancy [1-5], but why these errors are common is unknown. In most cells, chromosome segregation error is averted by the spindle assembly checkpoint (SAC), which prevents anaphase-promoting complex (APC/C) activation and anaphase onset until chromosomes are aligned with kinetochores attached to spindle microtubules [6, 7], but little is known about the SAC's role in the early mammalian embryo. In C. elegans, the SAC is weak in early embryos, and it strengthens during early embryogenesis as a result of progressively lessening cell size [8, 9]. Here, using live imaging, micromanipulation, gene knockdown, and pharmacological approaches, we show that this is not the case in mammalian embryos. Misaligned chromosomes in the early mouse embryo can recruit SAC components to mount a checkpoint signal, but this signal fails to prevent anaphase onset, leading to high levels of chromosome segregation error. We find that failure of the SAC to prolong mitosis is not attributable to cell size. We show that mild chemical inhibition of APC/C can extend mitosis, thereby allowing more time for correct chromosome alignment and reducing segregation errors. SAC-APC/C disconnect thus presents a mechanistic explanation for frequent chromosome segregation errors in early mammalian embryos. Moreover, our data provide proof of principle that modulation of the SAC-APC/C axis can increase the likelihood of error-free chromosome segregation in cultured mammalian embryos.

Segregating Chromosomes in the Mammalian Oocyte.

Chromosome segregation errors in human oocytes lead to aneuploid embryos that cause infertility and birth defects. Here we provide an overview of the chromosome-segregation process in the mammalian oocyte, highlighting mechanistic differences between oocytes and somatic cells that render oocytes so prone to segregation error. These differences include the extremely large size of the oocyte cytoplasm, the unique geometry of meiosis-I chromosomes, idiosyncratic function of the spindle assembly checkpoint, and dramatically altered oocyte cell-cycle control and spindle assembly, as compared to typical somatic cells. We summarise recent work suggesting that aging leads to a further deterioration in fidelity of chromosome segregation by impacting multiple components of the chromosome-segregation machinery. In addition, we compare and contrast recent results from mouse and human oocytes, which exhibit overlapping defects to differing extents. We conclude that the striking propensity of the oocyte to mis-segregate chromosomes reflects the unique challenges faced by the spindle in a highly unusual cellular environment.

Monitoring Microtubule Dynamics in the Mouse Egg Using Photoactivatable-GFP-Tubulin.

Fluorescence photoactivation provides a strategy for monitoring protein kinetics within living cells. In particular, fluorescence photoactivation of a subpopulation of microtubule subunits within the spindle using photoactivatable fluorescent tubulin constructs has proven useful for assessing a variety of features of spindle microtubule dynamics, including poleward microtubule movement, microtubule depolymerization, and microtubule turnover, in various cellular settings. The current chapter describes a method for monitoring microtubule dynamics within the mouse egg spindle by photoactivation of photoactivatable-GFP-tubulin, followed by time-lapse confocal imaging.

Meiosis: SUMO Keeps a Tight Grip on Sister Chromatids.

Maintaining cohesion between sister chromatids during the first meiotic cell division is crucial for preventing oocyte aneuploidy. In a new paper in Current Biology, Yi and colleagues present evidence that the Small Ubiquitin-related Modifier (SUMO) pathway protects centromeric sister cohesion during the meiosis I-II transition in mouse oocytes.

Electrical-assisted microinjection for analysis of fertilization and cell division in mammalian oocytes and early embryos.

Microinjection is an essential approach in the study of mammalian oocytes and early embryos, and is useful for the introduction of many molecules and reagents. Whereas microinjection into germinal vesicle stage oocytes is relatively simple using various microinjection setups, metaphase-II mouse eggs are notoriously fragile, and nondamaging microinjection can be difficult to achieve. Here we describe a microinjection method that is based on electrophysiology, which vastly reduces microinjection damage, especially in metaphase-II eggs. When optimized, this approach allows for over 90% oocyte survival, increasing confidence in experimental results.

Correlative Live Imaging and Immunofluorescence for Analysis of Chromosome Segregation in Mouse Preimplantation Embryos.

Chromothripsis is a phenomenon observed in cancer cells, wherein a single or few chromosome(s) exhibit vast genomic rearrangements. Recent studies elucidated a striking series of events in which defective segregation of chromosomes causes their incorporation into micronuclei, where they are subject to extensive DNA damage prior to re-joining the main mass of chromosomes in a subsequent cell cycle, which provide an appealing mechanism for the etiology of chromothripsis. Micronuclei are well known to be common in human preimplantation embryos. We recently showed that, unlike in cancer cells, in mouse preimplantation embryos the micronuclei are maintained during multiple cell generations and apparently fail to re-join the main set of chromosomes. This unexpected finding could safeguard the early embryonic genome from chromothripsis. Here, we describe an approach that combines live and immunofluorescence imaging methods that was pivotal in that study to reveal the lack of a functional kinetochore in chromosomes from mouse embryo micronuclei.

Mammalian Oocytes Locally Remodel Follicular Architecture to Provide the Foundation for Germline-Soma Communication.

Germ cells develop in a microenvironment created by the somatic cells of the gonad [1-3]. Although in males, the germ and somatic support cells lie in direct contact, in females, a thick extracellular coat surrounds the oocyte, physically separating it from the somatic follicle cells [4]. To bypass this barrier to communication, narrow cytoplasmic extensions of the follicle cells traverse the extracellular coat to reach the oocyte plasma membrane [5-9]. These delicate structures provide the sole platform for the contact-mediated communication between the oocyte and its follicular environment that is indispensable for production of a fertilizable egg [8, 10-15]. Identifying the mechanisms underlying their formation should uncover conserved regulators of fertility. We show here in mice that these structures, termed transzonal projections (TZPs), are specialized filopodia whose number amplifies enormously as oocytes grow, enabling increased germ-soma communication. By creating chimeric complexes of genetically tagged oocytes and follicle cells, we demonstrate that follicle cells elaborate new TZPs that push through the extracellular coat to reach the oocyte surface. We further show that growth-differentiation factor 9, produced by the oocyte, drives the formation of new TZPs, uncovering a key yet unanticipated role for the germ cell in building these essential bridges of communication. Moreover, TZP number and germline-soma communication are strikingly reduced in reproductively aged females. Thus, the growing oocyte locally remodels follicular architecture to ensure that its developmental needs are met, and an inability of somatic follicle cells to respond appropriately to oocyte-derived cues may contribute to human infertility.

Ultrafast imaging of cell elasticity with optical microelastography.

Elasticity is a fundamental cellular property that is related to the anatomy, functionality, and pathological state of cells and tissues. However, current techniques based on cell deformation, atomic force microscopy, or Brillouin scattering are rather slow and do not always accurately represent cell elasticity. Here, we have developed an alternative technique by applying shear wave elastography to the micrometer scale. Elastic waves were mechanically induced in live mammalian oocytes using a vibrating micropipette. These audible frequency waves were observed optically at 200,000 frames per second and tracked with an optical flow algorithm. Whole-cell elasticity was then mapped using an elastography method inspired by the seismology field. Using this approach we show that the elasticity of mouse oocytes is decreased when the oocyte cytoskeleton is disrupted with cytochalasin B. The technique is fast (less than 1 ms for data acquisition), precise (spatial resolution of a few micrometers), able to map internal cell structures, and robust and thus represents a tractable option for interrogating biomechanical properties of diverse cell types.