133. SPATIAL REGULATION OF APC^CDH1 INDUCED CYCLIN B1 DEGRADATION MAINTAINS GV ARREST IN MOUSE OOCYTES

Abstract

Within the mammalian ovary, oocytes remain prophase I arrested until a hormonal cue triggers meiotic resumption. The E3 ubiquitin ligase, Anaphase-Promoting Complex with its co-activator Cdh1 (APC^Cdh1) is known to be essential for this process by promoting cyclin B1 degradation via the 26S proteasome. Cyclin B1 is the regulatory subunit of Maturation-Promoting Factor, forming a heterodimer with CDK1, which is essential for nuclear envelope breakdown (NEB). Here we describe the intracellular partitioning that would explain how Cdh1 activity is fine-tuned, such that cyclin B1 is maintained at sufficient levels to allow oocytes to resume meiosis but not so high as to cause premature meiosis re-entry. Using RT-PCR we detected only one splice variant in mouse oocytes, Cdh1α, which possessed a nuclear localisation signal. By immunofluorescence we confirmed the nuclear location of Cdh1 and degradation machinery components including essential subunits of the APC and 26S proteasome. In all systems studied, cyclin B1 shuttles between the cytoplasm and nucleus, with nuclear localisation occurring just before NEB. We reasoned therefore that the nuclear localisation of the APC^Cdh1 and 26S proteasome would aid in maintaining low nuclear levels of cyclin B1. Using two GFP-coupled cyclin B1 mutants, which differed in their intracellular location we found that nuclear accumulation of cyclin B1 was necessary in order for it to promote meiotic resumption because over-expression of nuclear-cyclin B1 accelerated entry into meiosis, whereas cytoplasmic-cyclin B1 did not. However, in milrinone-arrested GV oocytes rates of nuclear-cyclin B1 degradation were 5 fold higher than cytoplasmic-cyclin B1. Therefore we conclude that in oocytes, an increase in the nuclear-cytoplasmic ratio of cyclin B1 is an essential step in meiotic resumption, and that nuclear APC^Cdh1 activity guards against early meiotic resumption, until the degradation machinery is overwhelmed by cyclin B1 translocation.