APCcdh1 activity in mouse oocytes prevents entry into the first meiotic division

Abstract

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...

The APC/C in female mammalian meiosis I

“…The importance of APC/C Cdh1 for restraining Cdk1 through low-level cyclin B1 degradation was initially demonstrated in vitro using morpholino antisensemediated depletion of Cdh1 in fully grown oocytes denuded of their surrounding follicular cells (Reis et al 2006). It was found that such Cdh1-depleted oocytes exhibited an increased susceptibility to undergoing GVBD even under conditions in which cAMP levels were chemically sustained using the phosphodiesterase inhibitor, milrinone.…”

“…APC/C Cdh1 -mediated proteolysis suppresses cyclin B1 levels to sustain G2/prophase arrest (Reis et al 2006). Conversely, Emi1 and securin serve to limit such proteolysis, thereby allowing cyclin B1 accumulation and Cdk1 activation required for GVBD (Marangos et al 2007, Marangos & Carroll 2008.…”

“…A novel aspect of APC/C regulation is the involvement of APC/C Cdh1 in regulating G2/prophase arrest in mammalian oocytes (Reis et al 2006). Important inputs have since been shown to provide opposing influences on APC/C Cdh1 , altogether furnishing highly sophisticated fine-tuning of cyclin levels and hence of Cdk1 activity that maintains GV arrest on the one hand while facilitating re-entry into MI on the other hand.…”

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

“…Microinjection of oocytes is a powerful method to study mechanisms that regulate meiotic maturation 10,11,12,13 . This method provides an economical way to test hypotheses prior to making a large investment in developing transgenic and targeted mouse models.…”

Le point faible méiotique : la première division

“…Les preuves montrant que l'APC/C est requis en méiose I sont indirectes, et aucun activateur de l'APC/C spécifique de la méiose n'a été mis en évidence chez les mammifères, comme cela a été le cas dans d'autres espèces [32]. Il semblerait que l'activateur requis pour l'entrée en méiose I soit Cdh1 [33]. Un rôle difficile à concilier avec le fait que Cdh1 est requis pour la sortie de mitose, et que des substrats de l'APC/ C qui sont dégradés de manière dépendante de Cdh1 peuvent cependant …”