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.

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.

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.

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.

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.

The SIOD disorder protein SMARCAL1 is an RPA-interacting protein involved in replication fork restart.

The integrity of genomic DNA is continuously challenged by the presence of DNA base lesions or DNA strand breaks. Here we report the identification of a new DNA damage response protein, SMARCAL1 (SWI/SNF-related, matrix associated, actin-dependent regulator of chromatin, subfamily a-like 1), which is a member of the SNF2 family and is mutated in Schimke immunoosseous dysplasia (SIOD). We demonstrate that SMARCAL1 directly interacts with Replication protein A (RPA) and is recruited to sites of DNA damage in an RPA-dependent manner. SMARCAL1-depleted cells display sensitivity to DNA-damaging agents that induce replication fork collapse, and exhibit slower fork recovery and delayed entry into mitosis following S-phase arrest. Furthermore, SIOD patient fibroblasts reconstituted with SMARCAL1 exhibit faster cell cycle progression after S-phase arrest. Thus, the symptoms of SIOD may be caused, at least in part, by defects in the cellular response to DNA replication stress.

Actin-based spindle positioning: new insights from female gametes.

Asymmetric divisions are essential in metazoan development, where they promote the emergence of cell lineages. The mitotic spindle has astral microtubules that contact the cortex, which act as a sensor of cell geometry and as an integrator to orient cell division. Recent advances in live imaging revealed novel pools and roles of F-actin in somatic cells and in oocytes. In somatic cells, cytoplasmic F-actin is involved in spindle architecture and positioning. In starfish and mouse oocytes, newly discovered meshes of F-actin control chromosome gathering and spindle positioning. Because oocytes lack centrosomes and astral microtubules, F-actin networks are key players in the positioning of spindles by transmitting forces over long distances. Oocytes also achieve highly asymmetric divisions, and thus are excellent models to study the roles of these newly discovered F-actin networks in spindle positioning. Moreover, recent studies in mammalian oocytes provide a further understanding of the organisation of F-actin networks and their biophysical properties. In this Commentary, we present examples of the role of F-actin in spindle positioning and asymmetric divisions, with an emphasis on the most up-to-date studies from mammalian oocytes. We also address specific technical issues in the field, namely live imaging of F-actin networks and stress the need for interdisciplinary approaches.

Cohesin acetylation speeds the replication fork.

Cohesin not only links sister chromatids but also inhibits the transcriptional machinery's interaction with and movement along chromatin. In contrast, replication forks must traverse such cohesin-associated obstructions to duplicate the entire genome in S phase. How this occurs is unknown. Through single-molecule analysis, we demonstrate that the replication factor C (RFC)-CTF18 clamp loader (RFC(CTF18)) controls the velocity, spacing and restart activity of replication forks in human cells and is required for robust acetylation of cohesin's SMC3 subunit and sister chromatid cohesion. Unexpectedly, we discovered that cohesin acetylation itself is a central determinant of fork processivity, as slow-moving replication forks were found in cells lacking the Eco1-related acetyltransferases ESCO1 or ESCO2 (refs 8-10) (including those derived from Roberts' syndrome patients, in whom ESCO2 is biallelically mutated) and in cells expressing a form of SMC3 that cannot be acetylated. This defect was a consequence of cohesin's hyperstable interaction with two regulatory cofactors, WAPL and PDS5A (refs 12, 13); removal of either cofactor allowed forks to progress rapidly without ESCO1, ESCO2, or RFC(CTF18). Our results show a novel mechanism for clamp-loader-dependent fork progression, mediated by the post-translational modification and structural remodelling of the cohesin ring. Loss of this regulatory mechanism leads to the spontaneous accrual of DNA damage and may contribute to the abnormalities of the Roberts' syndrome cohesinopathy.

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.

Spindle positioning in mammalian oocytes.

To preserve the maternal stores accumulated during oogenesis for further embryo development, oocytes divide asymmetrically which minimizes the volume of cytoplasm lost with each set of haploid genome. To ensure asymmetric division to occur, oocytes have to position their division spindle asymmetrically as well as tailor the size of daughter cells to the chromatin mass. In this review, we will discuss the recent advances in the field, with emphasis on the control mechanisms involved in meiotic spindle positioning in mammalian oocytes.

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.

A centriole- and RanGTP-independent spindle assembly pathway in meiosis I of vertebrate oocytes.

Spindle formation is essential for stable inheritance of genetic material. Experiments in various systems indicate that Ran GTPase is crucial for meiotic and mitotic spindle assembly. Such an important role for Ran in chromatin-induced spindle assembly was initially demonstrated in Xenopus laevis egg extracts. However, the requirement of RanGTP in living meiotic cells has not been shown. In this study, we used a fluorescence resonance energy transfer probe to measure RanGTP-regulated release of importin beta. A RanGTP-regulated gradient was established during meiosis I and was centered on chromosomes throughout mouse meiotic maturation. Manipulating levels of RanGTP in mice and X. laevis oocytes did not inhibit assembly of functional meiosis I spindles. However, meiosis II spindle assembly did not tolerate changes in the level of RanGTP in both species. These findings suggest that a mechanism common to vertebrates promotes meiosis I spindle formation in the absence of chromatin-induced microtubule production and centriole-based microtubule organizing centers.

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.

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.

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.

Functional dissection of mitotic regulators through gene targeting in human somatic cells.

With the human genome fully sequenced (1, 2), biologists continue to face the challenging task of evaluating the function of each of the approximately 25,000 genes contained within it. Gene targeting in human cells provides a powerful and unique experimental tool in this regard (3-8). Although somewhat more involved than RNAi or pharmacological approaches, somatic cell gene targeting is a precise technique that avoids both incomplete knockdown and off-target effects, but is still much quicker than analogous manipulations in the mouse. Moreover, immortal knockout cell lines provide excellent platforms for both complementation analysis and biochemical purification of multiprotein complexes in native form. Here we present a detailed gene-targeting protocol that was recently applied to the mitotic regulator Polo-like kinase 1 (Plk1) (9).