Reproduction Abstracts (2014) 1 P355 | DOI: 10.1530/repabs.1.P355

Cdh1- and Cdc20-independent APC activity in mouse oocytes

Ibtissem Nabti1 & John Carroll2

1University College London, London, UK; 2Monash University, Clayton, Victoria, Australia.

Introduction: The anaphase-promoting complex (APC) is an E3-ubiquitin ligase responsible for regulated destruction of substrates at specific stages of the cell cycle. Two APC co-activators, Cdc20 and Cdh1, mediate the timing and selectivity of substrates recognition. Progression through meiosis in oocytes utilises the same molecular players, although the start–stop nature of the female meiosis invokes additional levels of regulation. Recently, we reported that Cdk1 and MAPK play compensatory roles in regulating the APC by supressing its activity early in prometapahse I, thereby allowing accumulation of the APC substrates essential for meiosis I. Here, we investigate which co-activator is responsible for prematurely activating the APC when Cdk1 and MAPK are inhibited during prometaphase I.

Materials and methods: To deplete Cdc20 and Cdh1, we used antisense morpholino oligonucleotides, which were microinjected in prophase-arrested mouse oocytes 24 h before use. Cdk1 and MAPK were inhibited using Roscovitine and UO126 respectively. Western blotting and time-lapse imaging of oocytes expressing the APC substrates tagged to GFP were used to examine their stability.

Results and discussion: To date, the accumulated evidence suggests that the APC is only active in the presence of its known co-activators. Here, we report that depletion of Cdh1 and Cdc20 fails to inhibit the APC-mediated destruction of substrates that happens when Cdk1 and MAPK are inhibited during prometaphase I. Furthermore, a slow component of the APC-mediated destruction persists after depletion of Cdh1 and Cdc20 in prophase-arrested oocytes. These data suggest that alternative mechanisms for regulating APC activity may exist in mouse oocytes.

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