Mechanical Characterization of Murine Oocytes by Atomic Force Microscopy.

The quality of murine and human oocytes correlates to their mechanical properties, which are tightly regulated to reach the blastocyst stage after fertilization. Oocytes are nonadherent spherical cells with a diameter over 80 µm. Their mechanical properties have been studied in our lab and others using the micropipette aspiration technique, particularly to obtain the oocyte cortical tension. Micropipette aspiration is affordable but has a low throughput and induces cell-scale deformation. Here we present a step-by-step protocol to characterize the mechanical properties of oocytes using atomic force microscopy (AFM), which is minimally invasive and has a much higher throughput. We used electron microscopy grids to immobilize oocytes. This allowed us to obtain local and reproducible measurements of the cortical tension of murine oocytes during their meiotic divisions. Cortical tension values obtained by AFM are in agreement with the ones previously obtained by micropipette aspiration. Our protocol could help characterize the biophysical properties of oocytes or other types of large nonadherent samples in fundamental and medical research.

Aberrant cortex contractions impact mammalian oocyte quality.

The cortex controls cell shape. In mouse oocytes, the cortex thickens in an Arp2/3-complex-dependent manner, ensuring chromosome positioning and segregation. Surprisingly, we identify that mouse oocytes lacking the Arp2/3 complex undergo cortical actin remodeling upon division, followed by cortical contractions that are unprecedented in mammalian oocytes. Using genetics, imaging, and machine learning, we show that these contractions stir the cytoplasm, resulting in impaired organelle organization and activity. Oocyte capacity to avoid polyspermy is impacted, leading to a reduced female fertility. We could diminish contractions and rescue cytoplasmic anomalies. Similar contractions were observed in human oocytes collected as byproducts during IVF (in vitro fertilization) procedures. These contractions correlate with increased cytoplasmic motion, but not with defects in spindle assembly or aneuploidy in mice or humans. Our study highlights a multiscale effect connecting cortical F-actin, contractions, and cytoplasmic organization and affecting oocyte quality, with implications for female fertility.

Meiosis: Actin and microtubule networks drive chromosome clustering in oocytes.

Actin and microtubule networks in human and porcine oocytes sequentially gather chromosomes in a cluster shortly after nuclear envelope breakdown to ensure their complete capture by the meiotic spindle.

Filopodia-like protrusions of adjacent somatic cells shape the developmental potential of oocytes.

The oocyte must grow and mature before fertilization, thanks to a close dialogue with the somatic cells that surround it. Part of this communication is through filopodia-like protrusions, called transzonal projections (TZPs), sent by the somatic cells to the oocyte membrane. To investigate the contribution of TZPs to oocyte quality, we impaired their structure by generating a full knockout mouse of the TZP structural component myosin-X (MYO10). Using spinning disk and super-resolution microscopy combined with a machine-learning approach to phenotype oocyte morphology, we show that the lack of Myo10 decreases TZP density during oocyte growth. Reduction in TZPs does not prevent oocyte growth but impairs oocyte-matrix integrity. Importantly, we reveal by transcriptomic analysis that gene expression is altered in TZP-deprived oocytes and that oocyte maturation and subsequent early embryonic development are partially affected, effectively reducing mouse fertility. We propose that TZPs play a role in the structural integrity of the germline-somatic complex, which is essential for regulating gene expression in the oocyte and thus its developmental potential.

Methods for Assessing Oocyte Quality: A Review of Literature.

The rate of infertility continues to rise in the world for several reasons, including the age of conception and current lifestyle. We list in this paper potential non-invasive and invasive techniques to assess oocyte quality. We searched the database PubMed using the terms "oocytes AND quality AND evaluation". In the first part, we study the morphological criteria, compartment by compartment, to then focus in a second part on more objective techniques such as genetics, molecular, apoptosis, or human follicular fluid that contain biologically active molecules. The main criteria used to assess oocyte quality are morphological; however, several other techniques have been studied in women to improve oocyte quality assessment, but most of them are invasive and not usable in routine.

Cytoplasmic forces functionally reorganize nuclear condensates in oocytes.

Cells remodel their cytoplasm with force-generating cytoskeletal motors. Their activity generates random forces that stir the cytoplasm, agitating and displacing membrane-bound organelles like the nucleus in somatic and germ cells. These forces are transmitted inside the nucleus, yet their consequences on liquid-like biomolecular condensates residing in the nucleus remain unexplored. Here, we probe experimentally and computationally diverse nuclear condensates, that include nuclear speckles, Cajal bodies, and nucleoli, during cytoplasmic remodeling of female germ cells named oocytes. We discover that growing mammalian oocytes deploy cytoplasmic forces to timely impose multiscale reorganization of nuclear condensates for the success of meiotic divisions. These cytoplasmic forces accelerate nuclear condensate collision-coalescence and molecular kinetics within condensates. Disrupting the forces decelerates nuclear condensate reorganization on both scales, which correlates with compromised condensate-associated mRNA processing and hindered oocyte divisions that drive female fertility. We establish that cytoplasmic forces can reorganize nuclear condensates in an evolutionary conserved fashion in insects. Our work implies that cells evolved a mechanism, based on cytoplasmic force tuning, to functionally regulate a broad range of nuclear condensates across scales. This finding opens new perspectives when studying condensate-associated pathologies like cancer, neurodegeneration and viral infections.

An interpretable and versatile machine learning approach for oocyte phenotyping.

Meiotic maturation is a crucial step of oocyte formation, allowing its potential fertilization and embryo development. Elucidating this process is important for both fundamental research and assisted reproductive technology. However, few computational tools based on non-invasive measurements are available to characterize oocyte meiotic maturation. Here, we develop a computational framework to phenotype oocytes based on images acquired in transmitted light. We trained neural networks to segment the contour of oocytes and their zona pellucida using oocytes from diverse species. We defined a comprehensive set of morphological features to describe an oocyte. These steps were implemented in an open-source Fiji plugin. We present a feature-based machine learning pipeline to recognize oocyte populations and determine morphological differences between them. We first demonstrate its potential to screen oocytes from different strains and automatically identify their morphological characteristics. Its second application is to predict and characterize the maturation potential of oocytes. We identify the texture of the zona pellucida and cytoplasmic particle size as features to assess mouse oocyte maturation potential and tested whether these features were applicable to the developmental potential of human oocytes. This article has an associated First Person interview with the first author of the paper.

MYO10 promotes transzonal projection-dependent germ line-somatic contact during mammalian folliculogenesis†.

Granulosa cells of growing ovarian follicles elaborate filopodia-like structures termed transzonal projections (TZPs) that supply the enclosed oocyte with factors essential for its development. Little is known, however, of the mechanisms underlying the generation of TZPs. We show in mouse and human that filopodia, defined by an actin backbone, emerge from granulosa cells in early stage primary follicles and that actin-rich TZPs become detectable as soon as a space corresponding to the zona pellucida appears. mRNA encoding Myosin10 (MYO10), a motor protein that accumulates at the base and tips of filopodia and has been implicated in their initiation and elongation, is present in granulosa cells and oocytes of growing follicles. MYO10 protein accumulates in foci located mainly between the oocyte and innermost layer of granulosa cells, where it colocalizes with actin. In both mouse and human, the number of MYO10 foci increases as oocytes grow, corresponding to the increase in the number of actin-TZPs. RNAi-mediated depletion of MYO10 in cultured mouse granulosa cell-oocyte complexes is associated with a 52% reduction in the number of MYO10 foci and a 28% reduction in the number of actin-TZPs. Moreover, incubation of cumulus-oocyte complexes in the presence of epidermal growth factor, which triggers a 93% reduction in the number of actin-TZPs, is associated with a 55% reduction in the number of MYO10 foci. These results suggest that granulosa cells possess an ability to elaborate filopodia, which when directed toward the oocyte become actin-TZPs, and that MYO10 increases the efficiency of formation or maintenance of actin-TZPs.

Myosin-X is dispensable for spindle morphogenesis and positioning in the mouse oocyte.

Off-center spindle positioning in mammalian oocytes enables asymmetric divisions in size, which are important for subsequent embryogenesis. The migration of the meiosis I spindle from the oocyte center to its cortex is mediated by F-actin. Specifically, an F-actin cage surrounds the microtubule spindle and applies forces to it. To better understand how F-actin transmits forces to the spindle, we studied a potential direct link between F-actin and microtubules. For this, we tested the implication of myosin-X, a known F-actin and microtubule binder involved in spindle morphogenesis and/or positioning in somatic cells, amphibian oocytes and embryos. Using a mouse strain conditionally invalidated for myosin-X in oocytes and by live-cell imaging, we show that myosin-X is not localized on the spindle, and is dispensable for spindle and F-actin assembly. It is not required for force transmission as spindle migration and chromosome alignment occur normally. More broadly, myosin-X is dispensable for oocyte developmental potential and female fertility. We therefore exclude a role for myosin-X in transmitting F-actin-mediated forces to the spindle, opening new perspectives regarding this mechanism in mouse oocytes, which differ from most mitotic cells.

Artificially decreasing cortical tension generates aneuploidy in mouse oocytes.

Human and mouse oocytes' developmental potential can be predicted by their mechanical properties. Their development into blastocysts requires a specific stiffness window. In this study, we combine live-cell and computational imaging, laser ablation, and biophysical measurements to investigate how deregulation of cortex tension in the oocyte contributes to early developmental failure. We focus on extra-soft cells, the most common defect in a natural population. Using two independent tools to artificially decrease cortical tension, we show that chromosome alignment is impaired in extra-soft mouse oocytes, despite normal spindle morphogenesis and dynamics, inducing aneuploidy. The main cause is a cytoplasmic increase in myosin-II activity that could sterically hinder chromosome capture. We describe here an original mode of generation of aneuploidies that could be very common in oocytes and could contribute to the high aneuploidy rate observed during female meiosis, a leading cause of infertility and congenital disorders.

Active diffusion in oocytes nonspecifically centers large objects during prophase I and meiosis I.

Nucleus centering in mouse oocytes results from a gradient of actin-positive vesicle activity and is essential for developmental success. Here, we analyze 3D model simulations to demonstrate how a gradient in the persistence of actin-positive vesicles can center objects of different sizes. We test model predictions by tracking the transport of exogenous passive tracers. The gradient of activity induces a centering force, akin to an effective pressure gradient, leading to the centering of oil droplets with velocities comparable to nuclear ones. Simulations and experimental measurements show that passive particles subjected to the gradient exhibit biased diffusion toward the center. Strikingly, we observe that the centering mechanism is maintained in meiosis I despite chromosome movement in the opposite direction; thus, it can counteract a process that specifically off-centers the spindle. In conclusion, our findings reconcile how common molecular players can participate in the two opposing functions of chromosome centering versus off-centering.

A computational model of the early stages of acentriolar meiotic spindle assembly.

The mitotic spindle is an ensemble of microtubules responsible for the repartition of the chromosomal content between the two daughter cells during division. In metazoans, spindle assembly is a gradual process involving dynamic microtubules and recruitment of numerous associated proteins and motors. During mitosis, centrosomes organize and nucleate the majority of spindle microtubules. In contrast, oocytes lack canonical centrosomes but are still able to form bipolar spindles, starting from an initial ball that self-organizes in several hours. Interfering with early steps of meiotic spindle assembly can lead to erroneous chromosome segregation. Although not fully elucidated, this process is known to rely on antagonistic activities of plus end- and minus end-directed motors. We developed a model of early meiotic spindle assembly in mouse oocytes, including key factors such as microtubule dynamics and chromosome movement. We explored how the balance between plus end- and minus end-directed motors, as well as the influence of microtubule nucleation, impacts spindle morphology. In a refined model, we added spatial regulation of microtubule stability and minus-end clustering. We could reproduce the features of early stages of spindle assembly from 12 different experimental perturbations and predict eight additional perturbations. With its ability to characterize and predict chromosome individualization, this model can help deepen our understanding of spindle assembly.

Nuclear positioning as an integrator of cell fate.

Cells are the building units of living organisms and consequently adapt to their environment by modulating their intracellular architecture, in particular the position of their nucleus. Important efforts have been made to decipher the molecular mechanisms involved in nuclear positioning. The LINC complex at the nuclear envelope is a very important part of the molecular connectivity between the cell outside and the intranuclear compartment, and thus emerged as a central player in nuclear mechanotransduction. More recent concepts in nuclear mechanotransduction came from studies involving nuclear confined migration, compression or swelling. Also, the effect of nuclear mechanosensitive properties in driving cell differentiation raises the question of nuclear mechanotransduction and gene expression and recent efforts have been done to tackle it, even though it remains difficult to address in a direct manner. Eventually, an original mechanism of nucleus positioning, mechanotransduction and regulation of gene expression in the non-adherent, non-polarized mouse oocyte, highlights the fact that nuclear positioning is an important developmental issue.

Chromosome structural anomalies due to aberrant spindle forces exerted at gene editing sites in meiosis.

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.

Shifting meiotic to mitotic spindle assembly in oocytes disrupts chromosome alignment.

Mitotic spindles assemble from two centrosomes, which are major microtubule-organizing centers (MTOCs) that contain centrioles. Meiotic spindles in oocytes, however, lack centrioles. In mouse oocytes, spindle microtubules are nucleated from multiple acentriolar MTOCs that are sorted and clustered prior to completion of spindle assembly in an "inside-out" mechanism, ending with establishment of the poles. We used HSET (kinesin-14) as a tool to shift meiotic spindle assembly toward a mitotic "outside-in" mode and analyzed the consequences on the fidelity of the division. We show that HSET levels must be tightly gated in meiosis I and that even slight overexpression of HSET forces spindle morphogenesis to become more mitotic-like: rapid spindle bipolarization and pole assembly coupled with focused poles. The unusual length of meiosis I is not sufficient to correct these early spindle morphogenesis defects, resulting in severe chromosome alignment abnormalities. Thus, the unique "inside-out" mechanism of meiotic spindle assembly is essential to prevent chromosomal misalignment and production of aneuploidy gametes.

Control of nucleus positioning in mouse oocytes.

The position of the nucleus in a cell can instruct morphogenesis in some cases, conveying spatial and temporal information and abnormal nuclear positioning can lead to disease. In oocytes from worm, sea urchin, frog and some fish, nucleus position regulates embryo development, it marks the animal pole and in Drosophila it defines the future dorso-ventral axis of the embryo and of the adult body plan. However, in mammals, the oocyte nucleus is centrally located and does not instruct any future embryo axis. Yet an off-center nucleus correlates with poor outcome for mouse and human oocyte development. This is surprising since oocytes further undergo two extremely asymmetric divisions in terms of the size of the daughter cells (enabling polar body extrusion), requiring an off-centering of their chromosomes. In this review we address not only the bio-physical mechanism controlling nucleus positioning via an actin-mediated pressure gradient, but we also speculate on potential biological relevance of nuclear positioning in mammalian oocytes and early embryos.

Asymmetries and Symmetries in the Mouse Oocyte and Zygote.

Mammalian oocytes grow periodically after puberty thanks to the dialogue with their niche in the follicle. This communication between somatic and germ cells promotes the accumulation, inside the oocyte, of maternal RNAs, proteins and other molecules that will sustain the two gamete divisions and early embryo development up to its implantation. In order to preserve their stock of maternal products, oocytes from all species divide twice minimizing the volume of their daughter cells to their own benefit. For this, they undergo asymmetric divisions in size where one main objective is to locate the division spindle with its chromosomes off-centred. In this chapter, we will review how this main objective is reached with an emphasis on the role of actin microfilaments in this process in mouse oocytes, the most studied example in mammals. This chapter is subdivided into three parts: I-General features of asymmetric divisions in mouse oocytes, II-Mechanism of chromosome positioning by actin in mouse oocytes and III-Switch from asymmetric to symmetric division at the oocyte-to-embryo transition.

Meiotic spindle assembly and chromosome segregation in oocytes.

Oocytes accumulate maternal stores (proteins, mRNAs, metabolites, etc.) during their growth in the ovary to support development after fertilization. To preserve this cytoplasmic maternal inheritance, they accomplish the difficult task of partitioning their cytoplasm unequally while dividing their chromosomes equally. Added to this complexity, most oocytes, for reasons still speculative, lack the major microtubule organizing centers that most cells use to assemble and position their spindles, namely canonical centrosomes. In this review, we will address recent work on the mechanisms of meiotic spindle assembly and chromosome alignment/segregation in female gametes to try to understand the origin of errors of oocyte meiotic divisions. The challenge of oocyte divisions appears indeed not trivial because in both mice and humans oocyte meiotic divisions are prone to chromosome segregation errors, a leading cause of frequent miscarriages and congenital defects.

Oocyte Maturation and Development.

Sexual reproduction is essential for many organisms to propagate themselves. It requires the formation of haploid female and male gametes: oocytes and sperms. These specialized cells are generated through meiosis, a particular type of cell division that produces cells with recombined genomes that differ from their parental origin. In this review, we highlight the end process of female meiosis, the divisions per se, and how they can give rise to a functional female gamete preparing itself for the ensuing zygotic development. In particular, we discuss why such an essential process in the propagation of species is so poorly controlled, producing a strong percentage of abnormal female gametes in the end. Eventually, we examine aspects related to the lack of centrosomes in female oocytes, the asymmetry in size of the mammalian oocyte upon division, and in mammals the direct consequences of these long-lived cells in the ovary.