Maintenance of sister chromatid attachment in mouse eggs through maturation-promoting factor activity.
Mammalian eggs naturally arrest at metaphase of the second meiotic division, until sperm triggers a series of Ca(2+) spikes that result in activation of the anaphase-promoting complex/cyclosome (APC/C). APC/C activation at metaphase targets destruction-box containing substrates, such as cyclin B1 and securin, for degradation, and as such eggs complete the second meiotic division. Cyclin B1 degradation reduces maturation (M-phase)-promoting factor (MPF) activity and securin degradation allows sister chromatid separation. Here we examined the second meiotic division in mouse eggs following expression of a cyclin B1 construct with an N-terminal 90 amino acid deletion (Delta 90 cyclin B1) that was visualized by coupling to EGFP. This cyclin construct was not an APC/C substrate, and so following fertilization, sperm were incapable of stimulating Delta 90 cyclin B1 degradation. In these eggs, chromatin remained condensed and no pronuclei formed. As a consequence of the lack of pronucleus formation, sperm-triggered Ca(2+) spiking continued indefinitely, consistent with a current model in which the sperm-activating factor is localized to the nucleus. Because Ca(2+) spiking was not inhibited by Delta 90 cyclin B1, the degradation timing of securin, visualized by coupling it to EGFP, was unaffected. However, despite rapid securin degradation, sister chromatids remained attached. This was a direct consequence of MPF activity because separation was induced following application of the MPF inhibitor roscovitine. Similar observations regarding the ability of MPF to prevent sister chromatid separation have recently been made in Xenopus egg extracts and in HeLa cells. The results presented here show this mechanism can also occur in intact mammalian eggs and further that this mechanism appears conserved among vertebrates. We present a model in which metaphase II arrest is maintained primarily by MPF levels only.
Cell and Developmental Physiology Research Group, Institute of Cell and Molecular Biosciences, The Medical School, Framlington Place, University of Newcastle, NE2 4HH, UK.
Suzanne
S
Madgwick
Victoria L
VL
Nixon
Heng-Yu
HY
Chang
Mary
M
Herbert
Mark
M
Levasseur
Keith T
KT
Jones
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002467:Cell Nucleus / Q000502:physiology; D002842:Chromatids / Q000187:drug effects / Q000502:physiology*; D023902:Chromosome Pairing / Q000502:physiology; D019926:Cyclin B / Q000235:genetics* / Q000378:metabolism; D056744:Cyclin B1; D005260:Female; D005306:Fertilization / Q000502:physiology; D006131:Growth Inhibitors / Q000494:pharmacology; D016200:Maturation-Promoting Factor / Q000502:physiology*; D000090204:Mesothelin; D051379:Mice; D010063:Ovum / Q000502:physiology*; D011687:Purines / Q000494:pharmacology; D000077546:Roscovitine; D013997:Time Factors
C534700:Ccnb1 protein, mouse; D019926:Cyclin B; D056744:Cyclin B1; D006131:Growth Inhibitors; C000715528:Msln protein, mouse; D011687:Purines; D000077546:Roscovitine; D016200:Maturation-Promoting Factor; D000090204:Mesothelin; D002118:Calcium