RhoA- and Cdc42-induced antagonistic forces underlie symmetry breaking and spindle rotation in mouse oocytes

Mammalian oocyte meiotic divisions are highly asymmetric and produce a large haploid gamete and 2 small polar bodies. This relies on the ability of the cell to break symmetry and position its spindle close to the cortex before anaphase occurs. In metaphase II–arrested mouse oocytes, the spindle is actively maintained close and parallel to the cortex, until fertilization triggers sister chromatid segregation and the rotation of the spindle. The latter must indeed reorient perpendicular to the cortex to enable cytokinesis ring closure at the base of the polar body. However, the mechanisms underlying symmetry breaking and spindle rotation have remained elusive. In this study, we show that spindle rotation results from 2 antagonistic forces. First, an inward contraction of the cytokinesis furrow dependent on RhoA signaling, and second, an outward attraction exerted on both sets of chromatids by a Ran/Cdc42-dependent polarization of the actomyosin cortex. By combining live segmentation and tracking with numerical modeling, we demonstrate that this configuration becomes unstable as the ingression progresses. This leads to spontaneous symmetry breaking, which implies that neither the rotation direction nor the set of chromatids that eventually gets discarded are biologically predetermined.


Reviewer #2
The authors have done an excellent job in improving the manuscript based on previous reviewer comments. The low nocodazole experiment demonstrating that a cortical attractive force on chromosome clusters still exists during anaphase II is a nice addition. The work is a significant advance over previous work. However, there are two related issues that frustrate this reviewer and would frustrate any reader. First, the mechanism of the attractive force has not been determined. It is implied that cytoplasmic flow toward the cortex is the attractive force. The authors call this We are grateful to Reviewer 2 for these positive remarks and encouragements. The low nocodazole experiments have indeed brought new evidence concerning the maintenance of cortical attraction forces during anaphase II. In this condition, we observed that DNA clusters, who eventually detached from the meiotic spindle, rapidly relocated to the overlying cortex. This was accompanied by a local flow of cytoplasmic particles, and we therefore considered that outward oriented flows, hence referred to as metaphase-like flows, could play a role in attracting DNA clusters to the cortex after the anaphase onset. However, monitoring metaphase-like flows proved challenging since the cytoplasmic streaming pattern in activated oocytes was largely dominated by inward oriented flows resulting from the cytokinetic furrow ingression. To overcome this difficulty, we prevented the cytokinetic contraction using inhibitors such as the Bi-2536 (PLK1 inhibitor) or the Y-27632 (ROCK1 inhibitor) and clearly demonstrated that metaphase-like flows still occur during anaphase II (Fig 2H and Fig 6F). Because similarly oriented flows were previously described to promote chromosomes/spindle attraction in late metaphase I and metaphase II (Yi et al, 2013;Yi et al, 2011) and because inhibiting these flows using Cdc42T17N resulted in spindle relocation to the center of the oocyte (Fig 6C and Fig 6F), we considered metaphase-like flows as a probable explanation for the maintenance of cortical attractive forces during anaphase II. Note that we acknowledge the hypothetical nature of our assertion in the manuscript by writing that cortical attraction forces "most likely" result from metaphase-like flows (see Results line 243 and line 293).
To go further, we would like to stress that both metaphase-like and cytokinetic flows can influence the movement of cytoplasmic particles. Indeed, the isolated effect of the metaphase-like flow can be observed in Bi-2536 and Y-27632treated oocytes (Fig 2H and Fig 6F) while the isolated effect of cytokinetic flows can be observed in Cdc42T17Ninjected oocytes (Fig 6F). However, these two types of flows have different origins with, on the one hand, cytokinetic flows resulting from the pressure exerted by the invaginating cortex on the cytoplasm while, on the other hand, metaphase-like flows result from the polarized cortical flow of actin filaments (Yi et al, 2011). Consequently, the cytoplasmic streaming pattern we monitored using PIV is a composite of these two antagonistic flows (oriented in opposite direction) that necessarily interfere with each other. In our view, the metaphase-like flows are transiently masked by the stronger cytokinetic flows that arise during the peak of cytokinetic contraction. The fact that cytokinetic flows were found to be reinforced upon Cdc42 inhibition, a condition in which metaphase-like flows were prevented (see Fig 5F, S5C and S5D Fig), support this idea. In any case, more investigations will be needed to fully understand how cytoplasmic flows influence DNA cluster positioning during anaphase II. Improving the spatiotemporal resolution of confocal acquisition, as well as using more discrete tracking algorithms could probably help resolving the short-ranged metaphase-like flows throughout the second meiotic division. However, our aim here was primarily to enquire whether cortical attractive forces persisted during anaphase II, and we are pleased that this reviewer was satisfied by our new sets of data.
To answer this comment, we further elaborated on these considerations in the manuscript (see Discussion line 388-396).

Reviewer #3
The authors have done a tremendous amount of work in response to all reviewer comments and returned with a much more robust manuscript with a stronger body of evidence to support their overall conclusions. I probably would have not attempted imaging spindle rotation in IVF eggs in reponse to reviewer 2's comment 6 as this was not going to be easy from the outset, as the authors found out. Perhaps a SrCl2 activation would have done the job. Their effort is admirable nonetheless and now we at least have it on record someone tested this approach. I am overall satisfied with the revised manuscript and fully support its publication.
We are very pleased to hear these kind remarks from a leader in the field. Thank you for your encouragements and wise advice for improvements.