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New B-type cyclin synthesis is required between meiosis I and II during Xenopus oocyte maturation

Helfrid Hochegger1, Andrea Klotzbücher2, Jane Kirk1, Mike Howell1, Katherine le Guellec3,*, Kate Fletcher1, Tod Duncan1, Muhammad Sohail4 and Tim Hunt1,{ddagger}

1 ICRF Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, UK
2 Institut für Molekulare Medizin, Klinik für Tumorbiologie, Universität Freiburg, Breisacher Strasse 117, 79121 Freiburg, Germany
3 Unité de Biologie et Genetique du Development, CNRS UPR 41, Université Rennes I, Avenue du General Leclerc, 35042 Rennes, France
4 Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
* Deceased June 2001



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  		Fig. 1. Specificity of the antisera against Xenopus B-type cyclins. (A) Five coupled transcription-translation reactions were set up, programmed with no added DNA (-) or with the indicated cyclin plasmids. (B) Aliquots were analyzed by SDS-PAGE in sets of 5, transferred to nitrocellulose and immunoblotted separately with the indicated affinity-purified antisera. The volume of translation mix was identical in all lanes. (C) The sensitivity of the antisera against cyclins B1, B2 and B4 were tested against dilutions of the bacterial antigens, and quantitated by scanning the immunoblots.

 


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  		Fig. 2. Characterisation of X. laevis B-type cyclins. (A) Sequence alignment of cyclins B1, B2, B4 and B5; bars show the conservation, the destruction boxes are marked by a rectangle, and the nuclear export sequences indicated in red. (B) The dendrogram shows the family of mitotic cyclins in X. laevis and X. tropicalis. The scale bar represents 10 amino acid substitutions in the compared length of 180 residues. (C) Egg extracts were labelled with 35S-labelled amino acids and immunoprecipitated with the indicated antisera. These immunoprecipitates were analysed by autoradiography and assayed for binding of Cdc2 and histone H1 kinase activity. (D) Immunoblot showing the destruction of each B-type cyclin after addition of CaCl2 to an egg extract. (E) Cyclin levels during early embryogenesis determined by immunoblotting with the indicated antibodies. MBT occurred at about 10 hours in this experiment.

 


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  		Fig. 3. Oocyte maturation time course. White spot formation started 3.25 hours after addition of progesterone, and half the oocytes had undergone GVBD by 4.5 hours. All the panels are immunoblots using the indicated antisera, except for autoradiograph of the histone H1 kinase assay.

 


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  		Fig. 4. Characterisation of anti-B-type cyclin antisense oligonucleotides. (A) Sequence alignment of oligonucleotide ‘cyc8’ with different B-type cyclins. Mismatches are shown in red. (B) Effects of oligonucleotides ‘cyc8’ and anti B5-2 on cyclin synthesis monitored by the indicated immunoprecipitates of [35S]methionine-labelled egg extracts. (C) Northern blot analysis of mRNA isolated from oocytes which were, uninjected (C), injected with a mixture of 75 ng ‘cyc8’ and 25 ng of anti B5-2 (AS) or injected with the same oligonucleotides in the sense orientation (S).

 


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  		Fig. 5. Cytological characterisation of oocyte maturation (minutes after white spot formation are shown above each panel). (A) Images from a time lapse video of maturing oocytes. An uninjected ‘Control’ oocyte is compared with an ‘Antisense’ injected oocyte containing 75 ng ‘cyc8’ and 25 ng of anti B5-2 antisense oligonucleotides. (B) Spindles and chromosomes during MI. Microtubules are green, phosphorylated histone H3 red. (C) MII meiotic spindles of control, antisense- and sense-injected oocytes. (D) Meiotic spindles in CHX-treated oocytes. Scale bars, 10 µm.

 


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  		Fig. 6. Analysis of the MI/MII transition in progesterone-treated oocytes (Control), oocytes that were injected with 75 ng ‘cyc8’ and 25 ng of anti B5-2 oligonucleotides (Antisense) and oocytes treated with 100 µg/ml CHX 10 minutes after GVBD (CHX). (A) Immunoblots with indicated antibodies and histone H1 kinase assay of oocytes at the indicated times after GVBD; -P denotes stage VI oocytes. (B) Quantitation of the data for cyclins B1 and B2 and histone H1 kinase activity shown in A.

 


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  		Fig. 7. Effects of Arbacia cyclin B {Delta}90 on oocytes injected with 100 ng of anti B1/B4 antisense oligonucleotide. (A) Images of progesterone-treated oocytes 3 hours after GVBD (6 hours after addition of progesterone). Control, uninjected oocytes; Antisense, 100 ng anti B1/B4 antisense oligonucleotide injected 15 minutes after addition of progesterone; Antisense + cyclin B {Delta}90, as before with 100 ng cyclin B {Delta}90 injected 1.5 hours after GVBD. (B) RNase protection assay of mRNA cleavage in these oocytes; C, uninjected controls; S, sense; AS, antisense injected. (C) Histone H1 kinase assays and c-mos immunoblots of the oocytes. Cyclin B {Delta}90 levels were as indicated above each lane. (D) Meiotic spindles; Control, 6 hours after progesterone addition in an uninjected oocyte; Antisense + cyclin B {Delta}90, oocytes injected with 100 ng anti B1/B4 antisense oligonucleotide 15 minutes after progesterone addition and 100 ng cyclin B {Delta}90 1.5 hours after GVBD, sampled 1.5 hours later; Cyclin B {Delta}90, as before without antisense. Scale bar, 10 µm.

 


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  		Fig. 8. Effects of indestructible cyclin B1 mRNA and CHX. (A) Experimental design; dm indicates mRNA encoding indestructible cyclin B1 (RXXL to AXXA mutant). (B) Images of MII-arrested oocytes, CHX-treated oocytes and oocytes that were injected with mRNA encoding indestructible cyclin B1 30 minutes before addition of CHX. (C) Immunoblots and histone H1 kinase assays of oocytes subjected to the indicated treatments.

 


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  		Fig. 9. Cyclin stability and spindle appearance in oocytes treated with CHX 30 minutes after injection of indestructible cyclin B1 mRNA. (A) Destruction assays in oocytes using 35S-labelled cyclin B1, either wild type (WT) or destruction-box mutant (dm) injected into the indicated oocytes 3 hours after GVBD. Control, oocytes with no other treatment; CHX, cycloheximide added 2 hours after GVBD; Cyclin B1 dm + CHX as before, with indestructible cyclin B1 mRNA injected 1.5 hours after GVBD. (B) Control MII spindles in a progesterone-treated oocyte and images of spindles from 3 oocytes treated with CHX after translation of indestructible cyclin B1. Scale bar, 10 µm.

 

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© The Company of Biologists Ltd 2001