Oocyte Mechanics and Morphogenesis

I. Massaging the oocyte nucleus for a good transcriptome

I. 1. A non-specific pressure gradient that positions large objects in the centre of the oocyte

The position of the nucleus in a cell can instruct morphogenesisandabnormal nuclear positioning can lead to disease. In many species,oocyte nucleus position regulates embryo axis development. However, in fully grown mouse and human oocytes, the nucleus is centrally located and does not instruct any future embryo axis. Yet an off-centre nucleus correlates with a poor outcome for mouse and human oocyte development, arguing thatcentral positioning is important. This is surprising since oocytes further undergo two extremely asymmetric divisions in terms of the size of the daughter cells, requiring an off-centring of their chromosomes (Fig 1). Our group has discovered that in the mouse, the nucleus is positioned at the centre of the oocyte via an acto-myosin gradient of pressure (Almonacid 2015).

We demonstrated, using bio-physical and 3D-agent based simulations that this gradient is non-specific and propels large objects towards the oocyte centre both during Prophase I and meiosis I(Fig 2), allowing the oocyte to preserve its large organelle content, despite undergoing asymmetric divisions (Colin 2019).

I. 2. A mechano-transduction cascade to control gene expression in the oocyte

We discovered the biological significance of nucleus centring in mouse oocytes. The nucleus of mouse oocytes is centered at the very end of their growth in the follicle. In all species, the end of oocyte growth is associated with a massive and unique reduction in global transcription. The regulation of oocyte transcription and the abundance of key maternal transcripts is of major importance for future embryo development. We showed the existence of a mechano-transduction cascade (Fig 3A), dependent on the presence of the actin nucleator Formin 2, whereby the cytoskeleton-induced nuclear envelope fluctuations have an important impact on the oocyte transcriptome (Almonacid 2019).

In addition, these fluctuations reorganize nuclear condensates such as splicing speckles, modulating mRNA processing (Fig 3B). Hence, we think that the actin meshwork, promoting nuclear centring, also allows to modulate the amount and quality of maternal mRNAs (Al Jord BioRxiv). Importantly our work has also revisited the dogma claiming that oocyte transcription is totally shut-down at that developmental stage. It suggests that few loci are still transcriptionally active and respond to F-actin dependant mechano-transduction.

II. Cortical tension controls the geometry of division as well as oocyte ploidy

II. 1. A switch in cortex tension is essential for the return to symmetric division after fertilization

In mitotic cells, spindle positioning relies on interactions between astral microtubules and the cortex. In order for astral microtubule/cortex interactions to efficiently transmit forces, the cortex has to be stiff. The increase in cortical tension in mitosis ensures correct spindle positioning. Since mouse oocytes lack true centrosomes and their associated astral microtubules, their spindle positioning depends only on microfilaments whose nucleation is regulated by the spatial location of F-actin nucleators as well as force generating elements (Myosin-II).

We discovered thatin oocytes, the nucleation of a cortical F-actin thickening by the Arp2/3 complex excludes Myosin-II from the cortex, decreasing cortex tension (Chaigne 2013, 2015). Importantly, cortex softening favours asymmetry of meiotic spindle positioning, whereas we could demonstrate that cortex stiffening in early zygotes after fertilization (Fig 4), promotes symmetry of the division with spindle centring (Chaigne 2016).The abrupt change in cortex properties between oocyte and early zygote is essential for the meiotic to mitotic transition and appears to be conserved in humans.

II. 2. A too low cortex tension impacts chromosome segregation

It has been shown that human and mouse oocytes developmental potential is accurately predicted by mechanical properties within hours after fertilization. Their stiffness has to be tightly gated in order for them to develop into blastocysts. If they are too stiff or too soft, a rather frequent defect, embryos will cease development.

By artificially manipulating cortex tension (Fig 5), we have demonstrated that a too soft cortex induces defects in chromosome alignment and aneuploidy (Bennabi 2020). Importantly and quite unexpected from previous work, we could exclude the contribution of Myosin-X, a microtubule/actin crosslinker, in mediating the link between the cortex and the spindle (Crozet 2021). We have thus described a new 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.

III. Perturbing a centrosomal spindle assembly promotes chromosome anomalies

III. 1. Shifting meiotic to mitotic spindle assembly in oocytes disrupts chromosome alignment

We showed that in mouse oocytes, spindle microtubules are nucleated from multiple aMTOCs (acentriolar MTOCs) that are sorted and clustered prior to completion of spindle assembly in an ‘inside-out’ mechanism, ending with the establishment of spindle poles. We could simulate bipolar assembly of acentrosomal meiotic spindle (Letort 2019) and experimentally shift this assembly towards a mitotic ‘outside-in’ mode and analyze the consequences on the fidelity of the division (Bennabi 2018). We could show that if the unique ‘inside-out’ mechanism of meiotic spindle assembly is certainly fragile, it is essential to prevent chromosomal misalignment and the production of aneuploid gametes.

III. 2. Premature increase in microtubule assembly triggers fragile bivalent breakage

We perturbed the process of aMTOCs fragmentation, leading to improper spindle forces. We demonstrated that this leads to widely described, but never explained, chromosomal structural anomalies having severe disruptive consequences on the ability of the gamete to transmit an uncorrupted genome (Manil-Ségalen 2018).

Altogether, our work has highlighted the impact of actin meshes in the control of chromosome positioning and on the developmental potential of the oocyte. Interestingly, nucleus position as well as cortex properties constitute non-invasive predictors of early embryo development quality that could be used easily in Assisted Reproduction Technics where up to now embryo selection consists of a highly subjective morphological assessment. In particular, we developed an AI-based approach to automatically discover novel features able to predict before-hand female gamete developmental potential (Letort 2022).