SummaryFully-grown mammalian oocytes maintain a prophase I, germinal-vesicle stage arrest in the ovary for extended periods before a mid-cycle luteinizing surge induces entry into the first meiotic division. Cdh1 is an activator of the Anaphase-Promoting Complex (APC), and APC cdh1 is normally restricted to late M -early G1 of the cell cycle. Here we find that APC cdh1 is active in mouse oocytes and is necessary to maintain prophase arrest.Fully-grown mammalian oocytes remain arrested at prophase I within antral follicles until stimulated to enter the first meiotic division by a mid-cycle surge in luteinising hormone. An oolemma receptor maintains this arrest by raising protein kinase A activity1 which inhibits Maturation-Promoting Factor (CDK1-cyclin B1) by affecting the phosphorylation status of CDK12. Oocytes can resume meiosis spontaneously, manifest by germinal vesicle breakdown (GVB), when released into culture media, but remain arrested if agents such as the phosphodiesterase inhibitor milrinone3, are added to maintain protein kinase A.Raising cyclin B1 levels in milrinone-arrested oocytes by microinjection of its cRNA coupled to GFP induced GVB. Spatially the cyclin B1-GFP expressed in oocytes mirrored the distribution reported in adult cells 4 ( Supplementary Information, Fig S1a). Cytoplasmic cyclin B1 entered the nucleus before GVB and became associated with chromatin afterwards. However, the GVB rate in these oocytes was <15% by 5 h (Fig 1a), and never exceeded 20%, even after 24 h. The proteasomal inhibitor MG132 had a mild stimulatory effect on GVB over 5 h, and when combined with cyclin B1 the rate GVB increased 2-3 fold compared to cyclin B1 alone (Fig 1a and see Supplementary Information, Table S1). The increased rate of GVB was likely caused by increased cyclin B1-GFP since levels doubled with MG132 (Fig 1b).Cyclin B1 degradation requires polyubiquitination by the Anaphase-Promoting Complex (APC) followed by proteasomal degradation5. In mitosis, the APC needs one of two essential co-activators, cdc20 and cdh1, which are both present in mouse eggs6. APC cdc20 and APC cdh1 both degrade substrates such as cyclin B1 that contain a Destruction-(D)-box. Therefore we repeated the above cyclin B1 experiment using Δ90-cyclin B1, an N-terminal truncation which removes the D-box 7. Δ90-cyclin B1 cRNA induced 70% GVB rates by 5 h (Fig 1a), and 80% by 24 h; rates that are 4-5 fold higher than cyclin B1-GFP and with the MG132 data are consistent with cyclin B1 being degraded in GV oocytes.Cyclin B1 degradation in oocytes, where MPF is low, is likely due to APC cdh1 because APC cdc20 requires high MPF levels for activity8. Oocytes do contain cdh1 (see Supplementary Information, Fig S1b) therefore to examine if APC cdh1 was active at this time, in addition to cyclin B1, we coupled two further APC cdh1 substrates to GFP, injected their cRNA and measured their stability following protein synthesis inhibition. We used cdc20 itself and a mutant form of securin (securin dm ) in which its D-Box has been mutated. Bo...

SummaryThe first female meiotic division (MI) is uniquely prone to chromosome segregation errors through non-disjunction, resulting in trisomies and early pregnancy loss1. Here, we show a fundamental difference in the control of mammalian meiosis which may underlie such susceptibility. It involved a reversal in the well-established timing of activation of the AnaphasePromoting Complex (APC)2, 3 by its co-activators cdc20 and cdh1. APC cdh1 was active first, during prometaphase I, and was needed in order to allow homologue congression, since loss of cdh1 speeded up MI, leading to premature chromosome segregation and a non-disjunction phenotype. APC cdh1 targeted cdc20 for degradation but not securin and cyclin B1. These were degraded later in MI through APC cdc20 , making cdc20 re-synthesis essential for successful meiotic progression. The switch from APC cdh1 to APC cdc20 activity was controlled by increasing CDK1 and cdh1 loss. These findings demonstrate a fundamentally different mechanism of control for the first meiotic division in mammalian oocytes not observed in meioses of other species.The E3 ligase activity of the Anaphase-Promoting Factor (APC) bound to its co-activator cdh1 (APC cdh1 ) is commonly associated with late M-and early G1-phases of the cell cycle, where it contributes to M-phase exit by degradation of mitotic proteins, while simultaneously preventing precocious DNA replication3-6. APC cdh1 activity is also observed in germinal vesicle stage (GV) mouse oocytes, equivalent to late G2, where it contributes to cyclin B1 degradation and as such is required for maintenance of GV arrest7, 8.We wanted to establish if cdh1 had any role in meiosis I (MI) after GV breakdown (GVB), independent of its role in maintaining GV arrest. Therefore, we examined the ability of oocytes to progress through MI following microinjection with a cdh1 antisense morpholino (cdh1 MO ). Culture in milrinone-containing medium for 24 h following cdh1 MO microinjection is sufficient to reduce cdh1 levels by >90% (hereafter 'cdh1 knockdown oocytes') but maintain GV arrest in the majority of oocytes, with longer term culture (48h) needed to promote GVB7. In cdh1 knockdown oocytes, which maintained arrest over 24 h, we found that progression through MI was accelerated following milrinone wash-out. Oocytes extruded a polar body (PB), which forms on completion of MI, 1.5 h earlier than non-injected oocytes (Fig 1a). This effect was attributed specifically to loss of cdh1, since it Correspondence should be addressed to KTJ. (email: k.t.jones@ncl.ac.uk). AUTHOR CONTRIBUTIONS K.T.J. directed the work. A.R. and S.M. performed most the experiments; with HY.C making the initial observations on the effects of the cdh1 MO , I.N. performing some of the Westerns, and M.L. making some of the constructs. KTJ wrote the paper in consultation with A.R. and S.M. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests. was not observed in mock cdh1 depleted oocytes through addition of a 5-base-mismatch cdh1 morpholino (5...

Newcastle-upon-Tyne, UK Cdc20 and cdh1 are coactivators of the anaphase-promoting complex (APC). APC cdc20 is necessary for the metaphaseanaphase transition and, at the end of mitosis, vertebrate cdc20 itself becomes a target for degradation through KEN-boxdependent APC cdh1 activity. By studying the degradation of fluorescent protein chimaeras in mammalian oocytes and early embryos, we found that cdc20 was degraded through two independent degradation signals (degrons), the KEN box and a newly described CRY box. In both oocytes and G1-stage embryos, the rate of degradation through the CRY box was greater than through the KEN box, although both were mediated by APC cdh1 . Thus, mammalian oocytes and embryos have the capacity to recognize two degrons in cdc20.

Separase not only triggers anaphase of meiosis I by proteolytic cleavage of cohesin on chromosome arms; in vitro vertebrate separase also acts as a direct inhibitor of cyclin-dependent kinase 1 (Cdk1) upon liberation from inhibitory securin. Blocking separase-Cdk1 complex formation by microinjection of anti-separase antibodies prevents polar body extrusion in vertebrate oocytes. Importantly, proper meiotic maturation is rescued by chemical inhibition of Cdk1 or expression of Cdk1-binding separase fragments lacking cohesin-cleaving activity.The cohesin complex holds sister chromatids together and at anaphase is cleaved by the protease separase1,2. Until then separase is kept inactive by mutually exclusive association with cylin-dependent kinase 1 (Cdk1) or securin3,4,5. At metaphase the anaphase-promoting complex or cyclosome (APC/C) mediates degradation of securin and cyclin B1, the regulatory subunit of Cdk1, freeing separase to cleave cohesin6. Biochemically, Cdk1 activity is itself switched off by separase-Cdk1 complex formation4. However it is unclear whether separase acts as a Cdk1 inhibitor in vivo. During meiosis I of vertebrate eggs, fall in cdk1 activity is transient and not associated with complete loss in cyclin B7,8, raising the possibility that separase-dependent inactivation of Cdk1 might be necessary for meiotic maturation.Cdk1 binding has only been demonstrated for human separase4. In addition to phosphorylation of Ser1126 it requires a short sequence showing weak homology to S. cerevisiae Cdc6 and probably binding cyclin B1. To investigate the role of separasedependent Cdk1 inactivation in meiosis we raised antibodies against the Xenopus sequences corresponding to the two known Cdk1-binding determinants (amino acids 1123-54 plus 1381-1422). Both antibodies recognized recombinant Xenopus separase (Fig. 1A, lanes 1 and 8). The anti-aa 1381-1422 also detected and immuno-precipitated proteins of 240 and 180 kDa from meiotic Xenopus egg extract (lanes 2 to 5). Consistent with these representing full-length and self-cleaved endogenous separase, respectively, the same bands were recognized by anti-aa 1123-54 (lane 6). We incubated recombinant Xenopus separasesecurin complexes in anaphase-arrested extracts to degrade securin and then re-isolated separase via N-terminal HA-tags3. Cdk1 co-purified with Xenopus separase demonstrating that human and frog separase share Cdk1-binding despite low conservation of CBDs at 4 Correspondence should be addressed to KTJ and OS

Mammalian eggs remain arrested at metaphase of the second meiotic division (metII) for an indeterminate time before fertilization. During this period, which can last several hours, the continued attachment of sister chromatids is thought to be achieved by inhibition of the protease separase. Separase is known to be inhibited by binding either securin or Maturation (M-Phase)-Promoting Factor, a heterodimer of CDK1/cyclin B1. However, the relative contribution of securin and CDK/cyclin B1 to sister chromatid attachment during metII arrest has not been assessed. Although there are conditions in which either CDK1/cyclinB1 activity or securin can prevent sister chromatid disjunction, principally by overexpression of non-degradable cyclin B1 or securin, we find here that separase activity is primarily regulated by securin and not CDK1/cyclin B1. Thus the CDK1 inhibitor roscovitine and an antibody we designed to block the interaction of CDK1/cyclin B1 with separase, both failed to induce sister disjunction. In contrast, securin morpholino knockdown specifically induced loss of sister attachment, that could be restored by securin cRNA rescue. During metII arrest separase appears primarily regulated by securin binding, not CDK1/cyclin B1.

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