Epithelial Cell Transforming Protein 2 (ECT2) Depletion Blocks Polar Body Extrusion and Generates Mouse Oocytes Containing Two Metaphase II Spindles

Abstract

Completion of the first meiosis in oocytes is achieved by the extrusion of the first polar body (PBI), a particular example of cell division. In mitosis, the small GTPase RhoA, which is activated by epithelial cell transforming protein 2 (ECT2), orchestrates contractile ring constriction, thus enabling cytokinesis. However, the involvement of this pathway in mammalian oocytes has not been established. To characterize the role of ECT2 in PBI emission in mouse oocytes, the small interfering RNA approach was employed. We found that ECT2 depletion significantly reduces PBI emission, induces first metaphase arrest, and generates oocytes containing two properly formed spindles of the second metaphase. Moreover, we describe, for the first time, that before PBI emission, RhoA forms a ring that is preceded by a dome-like accumulation at the oocyte cortex, next to the spindle. This unique mode of RhoA translocation failed to occur in the absence of ECT2. We further found that the Rho-dependent kinase, a main RhoA effector, is essential for PBI emission. In addition, we demonstrate herein that ECT2 is subjected to phosphorylation/dephosphorylation throughout meiosis in oocytes and further reveal that PBI emission is temporally associated with ECT2 dephosphorylation. Our data provide the first demonstration that an active cyclin-dependent kinase 1, the catalytic subunit of the maturation-promoting factor, phosphorylates ECT2 during the first meiotic metaphase and that cyclin-dependent kinase 1 inactivation at anaphase allows ECT2 dephosphorylation. In conclusion, our study demonstrates the indispensable role of the maturation-promoting factor/ECT2/RhoA pathway in PBI extrusion in mouse oocytes.

Actin cytoskeleton in cell polarity and asymmetric division during mouse oocyte maturation

“…However, unlike in mitotic cells, actomyosin ring assembly and contraction appear to be regulated distinctly in mouse oocytes, as these two events are temporally separated [Deng et al, 2007;Elbaz et al, 2010]. For example, the actomyosin ring forms upon stimulation from chromatin signal during the MII arrest, whereas ring contraction does not occur until anaphase onset after sperm entry.…”

“…Moreover, the actomyosin ring assembly is dependent on the Ran GTPase mediated chromatin signaling, but it is unclear whether RhoA signaling is also involved in this process. Actomyosin contraction, on the other hand, requires the Ect2/RhoA pathway at least during first polar body extrusion [Elbaz et al, 2010]. Ect2 might not be involved in actomyosin assembly during metaphase, as it has been found that its activation, through dephosphorylation, requires CDK1 inactivation at anaphase [Elbaz et al, 2010].…”

Polar body emission

“…It seems reasonable to assume that it occurs similarly to that in frog oocytes [Azoury et al, 2008;Wang et al, 2008]. However, at least one study has reported that in fact furrowing and polar body protrusion begins when meiosis I spindle is parallel to the cortex and only become perpendicular after furrow constricts [Elbaz et al, 2010], as depicted in Fig. 3C.…”

“…Over-expression of a dominant negative Ect2 mutant abolishes RhoA activation and prevents the contractile ring formation in frog oocytes . Similarly depletion of Ect2 by RNA interference in mouse oocytes abrogates formation of the RhoA contractile ring and inhibits first polar body emission [Elbaz et al, 2010], as does direct inhibition of RhoA by the C3 toxin [Zhong et al, 2005].…”

“…The functional requirement of both a RhoGEF (Ect2) and a RhoGAP (MacRacGAP) for cytokinesis means that RhoA must transit between GTP-bound state and GDP-bound state during cytokinesis . Ect2 is similarly localized to the central spindle in anaphase and the mid body at the end of cytokinesis Elbaz et al, 2010] during polar body emission. Over-expression of a dominant negative Ect2 mutant abolishes RhoA activation and prevents the contractile ring formation in frog oocytes .…”