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SPECIAL INVITED LECTURESMALE REPRODUCTIVE TRACT/SPERM FUNCTIONWHY CHROMOSOMES SEPARATE AT MEIOTIC ~APHASEBob Moor and Yanfeng DaiLaboratory ofProtein FunctionThe pattern of chromosome segregation at anaphase determines whether the cell division will be equatorial(mitotic) or reductional (meiotic). During mitosis sister chromatids lose cohesion in early anaphase andsegregate to opposite poles ofthe dividing cell. By contrast, in anaphase I of meiosis sister chromatids remaintightly associated and homologous chromosomes move together to the same pole; sister chromatids onlyseparate at anaphase II. A variety of proteins, most notably the cohesions, are required to maintain the bindingbetween sister chromatids throughout the first meiotic division (MI). The sequential proteolysis of selectedclasses of the chromatid binding proteins and cyclins, mediated by the anaphase-promoting complex (APC),controls both the separation of homologous chromosomes and their ordered poleward movement duringanaphase I of meiosis. Before this stage suppression of APe function by proteins of the MAD and cdc20 genefamilies prevents premature chromatid separation and the meiotic errors that otherwise result in irreversible fetaldefects. We shall focus in our communication specifically on the intracellular signals that regulate APC functionand anaphase progression during meiosis in mammals.We have cloned and determined the expression patterns of MAD2 and cdc 20, changes in their spatialdistribution during meiosis and the consequences on meiosis of up- or down-regulating these proteins before orduring anaphase. Interactions between MAD2, cdc20 and upstream regulatory proteins have been analysedusing yeast two-hybrid and co-precipitation approached. Both MAD2 and cdc20 are stored in the oocytecytoplasm as inactive masked proteins. After activation, cdc20 translocates to the kinetochores while MAD2 islocalised most heavily around the spindle pole. Up-regulation ofMAD2 blocks oocytes in metaphase I while upregulationof cdc20 has no discernible effect on meiosis. By contrast the down-regulation of MAD2 inducespecific but dramatic spindle distortions and hastens entry into anaphase I while the down regulation of cdc 20blocks oocytes in metaphase I. These combined results emphasise the key role of MAD2 and cdc20 proteinsduring meiosis.Events in the upstream signalling pathway have been investigated by precipitation and translation studies. Ourresults demonstrate that MAD2 binds strongly to cdc20 which, in tum, activates the APe and inducesproteolysis of the cohesions and cyclins. Moreover, cdc20 binds the ERK proteins and is linked to the meioticcycle regulator mos via the Mos-MAPK pathway. In an extension of our studies of the anaphase signallingnetwork we next examined the regulation of Mos. Transcripts of the c-mos proto-oncogene are stored duringmeiosis as masked messengers and occur in three different forms which differ only in the 3' untranslated region(3 'UTR). Hybrid messages consisting of a luciferase coding region coupled to the mos 3'UTR have beeninjected into oocytes to study tlle mechanisms of mos mRNA translational control. We show that mostranslation is regulated not only by the length ofthe 3'UTR but also by specific V-rich and A-rich motifs which.in tUlR act in different ways on tile long and short form mos transcripts. We propose that transcript length actsas a rheostat to control both the level of translation and the stage during meiosis at which the messages aredegraded while the A-rich and V-rich motifs act as on-off switches. Although we have cloned some of the RNAbinding proteins we have yet to demonstrate how signals from the membrane interact with these bindingproteins to activate the 3' motifs in stored meiosis-related transcripts.IMPACT OF EPIDIDYMAL MATURATION ON THE REDOX REGULATED PATHWAYSCONTROLLING SPERM CAPACITATIONBeverley Lewis and John Aitken* . .*School ofBiological and Chemical Sciences, University ofNewcastle and MRC Reproductive BIOlogyUnit, Edinburgh.IntroductionAs spermatozoa progress through the c.Pididymisthey acquire the competence to capaCItate. Thecellular mechanisms regulating this centralelement of the maturation process are notunderstood. .Tyrosine phosphorylation is widely regarded asone of the keystones of capacitation (1). Incapacitating spermatozoa, tyrosinephosphorylation is controll~d by a re~oxregulated cAMP mediated SIgnal transductioncascade that appears to be unique to this celltype (2-4). Since the function~l comp~t~nce ofthis unusual pathway dunng epIdIdymalmaturation has not been examined, an analysisofthis system was undertaken using the rat as ananimal model.MethodscAMP was monitored using the Biomol, formatA cyclic AMP enzyme immunoassay kit,acetylated version (Biomol" PA, ~SA).Tyrosine phosphorylation was detected WIth thePY20 monoclonal antibody (Affiniti, Exeter,UK) using conventional Western blot protoc~ls.Immunolocalization of the sites of tyrosmephosphorylation was achieved using an alkalinephosphatase labeled second antibody.ResultsThe induction of reactive oxygen speciesgeneration with NADPH resulted in an increasein intracellular cAMP in both caput and caudalspermatozoa, providing a phosphodiesteraseinhibitor was present in the form ofpentoxifylline (Fig la). However only ir: maturecaudal epididymal spermatozoa did thIS redoxinduction of cAMP generation result in anincrease in tyrosine phosphorylation (Fig 1b).The functional development of this cAMPtyrosinephosphorylation path~ay d';1ringepididymal maturation was as.soc~ated WIth ashift in the cellular 10callzatlOn of thephosphorylation sites from the acrosome (Fig.1c) to the sperm tail (Fig. 1d)ConclusionsRedox regulation of cAMP generation is aconsistent feature of spenn biochemistrythroughout all stages of ~pididyrnalmaturation. However only In maturespennatozoa does cAMP induce thephosphorylation of a novel set of proteinsmany of which are located in the sJ?enn ~ail.In this location these phosphoprotelns mIghtplay a key role in controlling the motilitychanges (hyperactivation) asssociated with theattainment ofa capacitated state.These results contribute to our understanding ofthe cellular mechanisms responsible forepididymal sperm maturation. Spe.cifically,they focus attention on the mechamsms bywhich cAMP stimulates tyrosinephosphorylation, as the key event controlli~g thepotential ofmaturing spermatozoa to capaCItate.WGo).,ac..2.0(00x 1.5li')E~ 1.0a.~