DevelopmentalBiology248,331–342(2002) doi:10.1006/dbio.2002.0728 View metadata, citation and similar papers at core.ac.uk brought to you by CORE TargetedDisruptionoftheAkap4GeneCauses provided by Elsevier - Publisher Connector DefectsinSpermFlagellumandMotility KiyoshiMiki,*,1 WilliamD.Willis,*PaulaR.Brown,* EugeniaH.Goulding,*KerryD.Fulcher,†andEdwardM.Eddy*,2 *GameteBiologySection,LaboratoryofReproductiveandDevelopmentalToxicology, NationalInstituteofEnvironmentalHealthSciences,NationalInstitutesofHealth, ResearchTrianglePark,NorthCarolina27709;and†PointLomaNazareneUniversity, SanDiego,California92106 A-kinaseanchoringproteins(AKAPs)tethercyclicAMP-dependentproteinkinasesandtherebylocalizephosphorylationof targetproteinsandinitiationofsignal-transductionprocessestriggeredbycyclicAMP.AKAPscanalsobescaffoldsfor kinasesandphosphatasesandformmacromolecularcomplexeswithotherproteinsinvolvedinsignaltransduction.Akap4 istranscribedonlyinthepostmeioticphaseofspermatogenesisandencodesthemostabundantproteininthefibrous sheath,anovelcytoskeletalstructurepresentintheprincipalpieceofthespermflagellum.Previousstudiesindicatedthat cyclicAMP-dependentsignalingprocessesareimportantintheregulationofspermmotility,andgenetargetingwasused heretotestthehypothesisthatAKAP4isascaffoldforproteincomplexesinvolvedinregulatingflagellarfunction.Sperm numberswerenotreducedinmalemicelackingAKAP4,butspermfailedtoshowprogressivemotilityandmalemicewere infertile.Thefibroussheathanlagenformed,butthedefinitivefibroussheathdidnotdevelop,theflagellumwasshortened, andproteinsusuallyassociatedwiththefibroussheathwereabsentorsubstantiallyreducedinamount.However,theother cytoskeletalcomponentsoftheflagellumwerepresentandappearedfullydeveloped.WeconcludethatAKAP4isascaffold proteinrequiredfortheorganizationandintegrityofthefibroussheathandthateffectivespermmotilityislostinthe absenceofAKAP4becausesignaltransductionandglycolyticenzymesfailtobecomeassociatedwiththefibrous sheath.©2002ElsevierScience(USA) INTRODUCTION asshape,movement,anddivision(DivianiandScott,2001). AKAPsalsocanserveasscaffoldsforsignal-transduction CyclicAMP(cAMP)servesasasecondmessengerfor complexesthatcontainphosphatases,kinases,andother variousbiologicalstimulibytriggeringcAMP-dependent componentsthatarebelievedtocoordinatethephosphory- proteinkinases(PKA)tophosphorylateserineandthreo- lationstatusoftargetproteins(EdwardsandScott,2000; nineresiduesonothersignalcascadeproteinstoactivateor ColledgeandScott,1999).Inaddition,AKAPcomplexes modifytheirfunctions.PKAsoftenaretetheredtoA-kinase maybeassociatedwithupstreamactivatorsordownstream anchoringproteins(AKAPs)associatedwithparticularcel- substratesofPKA-associatedsignalingpathways(Colledge lularcomponents,positioningtheminspecificsubcellular etal.,2000;DodgeandScott,2000). sitesandprovidingamechanismfordefiningwhichpro- Mammalianspermareuniqueandhighlypolarizedcells teinsbecomephosphorylated.Forexample,AKAP-linked withahaploidgenometightlypackagedintheheadanda signalingcomplexesassociatedwiththecytoskeletonare flagellumthatgeneratesthemotileforceneededtodeliver believedtoregulatefundamentalcellularproperties,such thespermtotheegg.Theflagellumisdividedintoamiddle piece,principalpiece,andendpiece.Themiddlepieceis adjacenttotheheadandisdistinguishedbythepresenceof 1 Presentaddress:DepartmentofCellandDevelopmentalBiol- ogy,SchoolofMedicine,UniversityofNorthCarolinaatChapel themitochondrialsheath.Theprincipalpieceoccupies Hill,NC27599-7090. approximatelythree-fourthsofthelengthoftheflagellum, 2 Towhomcorrespondenceshouldbeaddressed.Fax:(919)541- whiletheendpieceisashortsegmentatthetipofthe 3800.E-mail:[email protected]. flagellum.Themajorcytoskeletalcomponentsofthefla- 0012-1606/02$35.00 ©2002ElsevierScience(USA) Allrightsreserved. 331 332 Miki et al. gellum are the axoneme, fibrous sheath, and outer dense latory subunits of PKA or the more widely expressed RI␣ or fibers. The axoneme extends the full length of the flagellum RII␣ subunits. RI␣ and RII␣ are present in the flagellum and is composed of nine microtubule doublets surrounding (Horowitz et al., 1984; Visconti et al., 1997; Johnson et al., a central pair of microtubules. Sperm are the only cell type 1997; Burton et al., 1999). Mice lacking RI␤ or RII␤ showed with a flagellum, but the “nine-plus-two” complex of specific defects in synaptic function in the hippocampus microtubules that forms the axoneme is also present in and cellular metabolism in adipose tissue, respectively, but cilia. The fibrous sheath defines the extent of the principal were fertile (Brandon et al., 1995; Cummings et al., 1996). piece and consists of two longitudinal columns connected The RI␣ mutation was an embryonic lethal (unpublished by closely arrayed circumferential ribs. The outer dense results referred to in Amieux et al., 1997), while RII␣- fibers are associated with the microtubule doublets at the deficient mice had no obvious physiological defects and periphery of the axoneme, beginning at the sperm head and were fertile (Burton et al., 1997). However, compensatory ending in the middle region of the principal piece. They regulation of RI␣ protein levels in the RII␣-deficient mice taper along their length and are enclosed by the mitochon- (Amieux et al., 1997) complicated the interpretation of drial sheath in the middle piece and the fibrous sheath in these results. An alternative approach to examine PKA the principal piece. The classical view has been that, while function was used in this study, generating mice lacking an the axoneme functions as the motor of the sperm, the AKAP that is synthesized only in spermatogenic cells. It fibrous sheath and outer dense fibers serve a mechanical was found that mutation of the Akap4 gene disrupts sperm role by modulating the degree and shape of flagellar bending motility and causes male mice to be infertile. To our (Fawcett, 1975). AKAP4 is the major protein in the fibrous knowledge, this is the first use of gene targeting to explore sheath and is the product of an X-linked gene (Moss et al., the roles of an AKAP and the effects of disruption of PKA 1997) expressed only in spermatids (Carrera et al., 1994; anchoring within a specific cell type. These studies demon- Fulcher et al., 1995). AKAP4 is unusual among AKAPs in strate that AKAP4 is an important component of the fibrous having a RI␣-specific binding domain distinct from the sheath and is required for anchoring the signal transduction prototype RII binding domain and another domain that proteins and glycolytic enzymes necessary for flagellar binds either RI␣ or RII␣ (Visconti et al., 1997; Miki and function. Eddy, 1998, 1999). It is assumed that AKAP4 recruits PKA to the fibrous sheath to facilitate phosphorylation of neigh- boring proteins that regulate flagellar function. MATERIALS AND METHODS Sperm acquire the ability to bind to the zona pellucida, exhibit progressive motility upon release, and respond to Construction of Targeting Plasmid increased levels of intracellular cAMP as they are moved The Akap4 gene was isolated from a P1 phage library of 129Sv through the epididymis (e.g., Lewis and Aitken, 2001). mouse genomic DNA (Incyte Genomics, St. Louis, MO). The DNA Increased production of cAMP and the phosphorylation of restriction fragment containing exons 4 and 5 was digested with proteins occur during passage of sperm through the female HindIII and BglII and cloned into a thymidine kinase (TK) vector, reproductive tract (Morton and Albagli, 1973; Tash and pMC1TKbpA (gift of Dr. Yuji Mishina). The fragment containing Means, 1983; Visconti et al., 1995a). This happens concur- exons 6 and 7 was digested with EcoRV and XbaI and then rently with the development of vigorous flagellar move- subjected to partial PmlI digestion to generate two fragments. The ment with a high-amplitude waveform, referred to as hy- fragment containing exon 6 was subcloned into pPGKneobpAFRT2(B) peractivated motility (Yanagimachi, 1970). Capacitation is (gift of Dr. Yuji Mishina) containing the neo gene surrounded by frt recognition sequences for Drosophila friptase. The fragment con- the process whereby sperm gain the ability to undergo the taining exon 7 was subcloned into the SpeI site outside the loxP acrosome reaction and occurs concurrently with cAMP- cassette of pBS246 (Life Technologies, Grand Island, NY). Exon 6 mediated phosphorylation of flagellar proteins (Visconti et and the neo cassette were excised from pPGKneobpAFRT2(B) and al., 1995b). Furthermore, mouse sperm require glucose to subcloned into the EcoRV site within the loxP cassette of pBS246. become hyperactivated and to fertilize eggs in vitro (Brooks, This plasmid was combined with exons 4 and 5 cloned into 1976; Fraser and Quinn, 1981), suggesting that glycolysis pMC1TKbpA to produce the gene-targeting construct pAKAP4KO generates ATP needed for these processes. Glyceraldehyde (Fig. 1). 3-phosphate dehydrogenase-S (GAPDS) is a germ cell- specific glycolytic enzyme bound to the fibrous sheath Generation of Mutant Mice (Bunch et al., 1998; Westhoff and Kamp, 1997). In earlier studies, glycolytic enzyme activities were found in the The Akap4 gene is located on the X-chromosome and was detergent-insoluble fraction of sperm (Mohri et al., 1965; mutated in vivo by using the CRE/loxP method because of concern that male chimeras produced with XY ES cells would be unable to Harrison, 1971; Storey and Kayne, 1978). These and other transmit a single copy mutant allele to the next generation. proteins associated with the fibrous sheath are prospective Transfection of pAKAP4KO DNA and screening of targeted TC-1 direct