In mitosis, the spindle checkpoint protein Mad2 averts aneuploidy by delaying anaphase onset until chromosomes align. Here we show that depletion of Mad2 in meiosis I mouse oocytes induced an increased incidence of aneuploidy. Proteolysis of cyclin B and securin commenced earlier in Mad2-depleted oocytes, resulting in a shortened duration of meiosis I. Furthermore, overexpression of Mad2 inhibited homolog disjunction. We conclude that Mad2 delays the onset of cyclin B and securin degradation and averts aneuploidy during meiosis I in mammalian oocytes. The data suggest a link between trisomies such as Down syndrome and defective oocyte spindle checkpoint function.Supplemental material is available at http://www.genesdev.org.Received April 16, 2004; revised version accepted October 14, 2004. During meiosis, one round of DNA synthesis is followed by the sequential segregation of homologous chromosomes at meiosis I and sister chromatids at meiosis II. Although female meiosis I errors are the major genetic cause of miscarriage and mental retardation in humans, little is known about their molecular origins (Hassold and Hunt 2001). The spindle checkpoint is the principal mechanism for preventing chromosome missegregation during mitosis by delaying anaphase until completion of chromosome alignment (Mussachio and Hardwick 2002). Checkpoint proteins such as Mad2 prevent activation of a multi-subunit ubiquitin ligase called the anaphase-promoting complex or cyclosome (APC/C) by sequestering the APC/C activator protein, Cdc20 (Peters 2002). Once chromosomes establish a stable bipolar orientation on the mitotic spindle, the APC/C directs proteolysis of securin and cyclin B, thereby inducing anaphase and cytokinesis (Peters 2002). As in mitosis, securin and cyclin B destruction are required for homolog disjunction and exit from meiosis I in mouse oocytes (Herbert et al. 2003).In mammalian somatic cells, the Mad2-dependent spindle checkpoint is required for mitotic arrest in the presence of spindle poisons (Li and Benezra 1996) and to delay anaphase until proper chromosome alignment in the absence of spindle damage (Gorbsky et al. 1998). During meiosis I in budding yeast, Mad2 is also required to arrest cells following spindle disruption and to avert nondisjunction in unperturbed cells (Shonn et al. 2000(Shonn et al. , 2003. In mouse oocytes, a dominant-negative variant of human Mad2 (hMad2) destabilizes cyclin-dependent kinase 1 (Cdk1) activity in the presence of the spindle poison nocodazole, indicating that Mad2 is required for meiosis I arrest in the face of spindle damage (Wassmann et al. 2003). It is not known, however, whether Mad2 regulates progression through meiosis I in unperturbed oocytes.Conventional genetic approaches have not been informative regarding checkpoint function in female meiosis. For example, deletion of Mad2 and Bub3 causes mouse embryonic lethality (Dobles et al. 2000;Kalitsis et al. 2000). Furthermore, using such approaches, it would be difficult to distinguish between errors that occurred duri...

Disjunction of pairs of homologous chromosomes during the first meiotic division (MI) requires anaphase-promoting complex (APC)-mediated activation of separase in budding yeast and Caenorhabditis elegans, but not Xenopus laevis. It is not clear which model best fits the mammalian system. Here we show that homologue disjunction in mouse oocytes is dependent on proteolysis of the separase inhibitor securin and the Cdk1 regulatory sub-unit cyclin B1. Proteolysis of both proteins was entirely dependent on their conserved destruction box (D-box) motifs, through which they are targeted to the APC. These data indicate that the mechanisms regulating homologue disjunction in mammalian oocytes are similar to those of budding yeast and C.elegans.

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...

During interkinesis, a metaphase II (MetII) spindle is built immediately after the completion of meiosis I. Oocytes then remain MetII arrested until fertilization. In mouse, we find that early mitotic inhibitor 2 (Emi2), which is an anaphase-promoting complex inhibitor, is involved in both the establishment and the maintenance of MetII arrest. In MetII oocytes, Emi2 needs to be degraded for oocytes to exit meiosis, and such degradation, as visualized by fluorescent protein tagging, occurred tens of minutes ahead of cyclin B1.Emi2 antisense morpholino knockdown during oocyte maturation did not affect polar body (PB) extrusion. However, in interkinesis the central spindle microtubules from meiosis I persisted for a short time, and a MetII spindle failed to assemble. The chromatin in the oocyte quickly decondensed and a nucleus formed. All of these effects were caused by the essential role of Emi2 in stabilizing cyclin B1 after the first PB extrusion because in Emi2 knockdown oocytes a MetII spindle was recovered by Emi2 rescue or by expression of nondegradable cyclin B1 after meiosis I.

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...

Cyclin B1, the regulatory component of M phase-promoting factor (MPF), is degraded during the metaphase-anaphase transition in an anaphase-promoting complex/cyclosome (APC/C)-dependent process. MPF activity is stable in eggs, and a sperm-triggered Ca(2+) signal is needed to promote cyclin degradation. In frogs, a single Ca(2+) spike promotes cell cycle resumption, but, in mammals, the Ca(2+) signal is more complex, consisting of a series of spikes that stop several hours after sperm fusion. Using dual imaging in mouse eggs, we have examined how the Ca(2+) signal generates cyclin B1 destruction using destructible and nondestructible GFP-tagged constructs. APC/C activity was present in unfertilized eggs, giving cyclin B1 a half-life of 1.15 +/- 0.28 hr. However, APC/C-dependent cyclin degradation was elevated 6-fold when sperm raised cytosolic Ca(2+) levels above 600 nM. This activation was transitory since cyclin B1 levels recovered between Ca(2+) spikes. For continued cyclin degradation at basal Ca(2+) levels, multiple spikes were needed. APC/C-mediated degradation was observed until eggs had completed meiosis with the formation of pronuclei, and, at this time, Ca(2+) spikes stopped. Therefore, the physiological need for a repetitive Ca(2+) signal in mammals is to ensure long-term cyclin destruction during a protracted exit from meiosis.

Mouse eggs arrest at metaphase II following ovulation and are only triggered to complete meiosis when fertilized. Sperm break the cell-cycle arrest by a long-lasting series of Ca2+ spikes that lead to an activation of the anaphase-promoting complex/cyclosome. The signal transduction pathway is not fully resolved but both protein kinase C (PKC) and calmodulin-dependent protein kinase II (CamKII) activities increase at fertilization and previous pharmacological studies have implicated both in cell-cycle resumption. We have used a combination of pharmacological inhibitors and constitutively active cRNA constructs of PKCalpha and CamKIIalpha microinjected into mouse eggs to show that it is CamKII and not PKC that is the sufficient trigger for cell-cycle resumption from metaphase II arrest. Constitutively active PKC constructs had no effect on the resumption of meiosis but caused an immediate and persistent elevation in intracellular Ca2+ when store-operated Ca2+ entry was stimulated. With respect to resumption of meiosis, the effects of constitutively active CamKII on eggs were the same as sperm. Eggs underwent second polar body extrusion and pronucleus formation with normal timings; while both securin and cyclin B1 destruction, visualised by coupling to fluorescent protein tags, were complete by the time of polar body extrusion. Induction of a spindle checkpoint by overexpression of Mad2 or by spindle poisons blocked CamKII-induced resumption of meiosis, but the Ca2+ chelator BAPTA did not. Furthermore direct measurement of Ca2+ levels showed that CamKII did not induce exit from metaphase II arrest by raising Ca2+. Therefore, we conclude that PKCs may play an important role in maintaining Ca2+ spiking at fertilization by promoting store-operated Ca2+ entry, while CamKII transduces cell-cycle resumption, and lies downstream of sperm-induced Ca2+ release but upstream of a spindle checkpoint. These data, combined with the knowledge that CamKII activity increase at fertilization, suggest that mouse eggs undergo cell-cycle resumption through stimulation of CamKII.

Mad2 is a pivotal component of the spindle assembly checkpoint (SAC) which inhibits anaphase promoting complex/cyclosome (APC/C) activity by sequestering Cdc20 thereby regulating the destruction of securin and cyclin B. During mitosis, spindle depolymerisation induces a robust Mad2-dependent arrest due to inhibition of securin and cyclin B destruction. In contrast to mitosis, the molecular details underpinning the meiosis I arrest experienced by mouse oocytes exposed to spindle depolymerisation remain incompletely characterised. Notably, the role of Mad2 and the fate of the anaphase-marker, securin, are unexplored. As shown previously, we find that spindle depolymerisation by nocodazole inhibits first polar body extrusion (PBE) and stabilises cyclin B and cyclin-dependent kinase 1 activity in mouse oocytes. Here we show that stabilisation of cyclin B in nocodazole can be sustained for several hours and is associated with stabilisation of securin. These effects are SAC-mediated as, in oocytes depleted of the majority of Mad2 by morpholino antisense, securin and cyclin B are destabilised and 15% of oocytes undergo PBE. This reflects premature APC/C activation as a mutant form of cyclin B lacking its APC/C degradation signal is stable in Mad2-depleted oocytes. Moreover, homologues do not disjoin during the prolonged meiosis I arrest (>18 h) induced by nocodaozole indicating that a non-cleavage mechanism is insufficient on its own for resolution of arm cohesion in mammalian oocytes. In conclusion, when all kinetochores lack attachment and tension, mouse oocytes mount a robust Mad2-dependent meiosis I arrest which inhibits the destruction of securin and cyclin B.