Promoter Mutation in Ccna2 Reveals Novel Functions of the Protein in Spermatogenesis Manuel Torres, Lindsey N. Kent, Maria Cuitino, Yannis Hadjiannis, Jim Dowdle and Gustavo Leone

INTRODUCTIONThecyclinsareasetofproteinsthatplayakeyroleinregula4ngthecellcyclebyac4va4ngtheCDKs,whichinpart

coordinate the events that lead to cellular division (both mito4c and meio4c). Cyclin A2 (CCNA2) is the regulatorysubunitofCDK1and2;together,theyphosphorylatespecificproteinsattheSphaseandduringtheG2/Mtransi4on.1

During spermatogenesis (Fig 3C), germ cells called Spermatogonial Stem Cells (SSCs) undergo subsequentdifferen4a4oneventsun4lspermatozoaareproduced.SSCsare themaincell typethatexpressesCcna2,andare thesourceofcon4nuousproduc4onofsperm.2 Interes4ngly,duringdifferen4a4on,Ccna2expression isdownregulatedinorderforsubsequentmeio4ceventstooccur.3

This temporal specificity of Ccna2 expression in the cell cycle is mediated mostly by the E2Fs, a family oftranscrip4onfactorsthatregulatetheexpressionofmanycellcyclerelatedgenes.4Therelevanceoftheprecise4mingandexpressionlevelofE2F-drivenCcna2expressionhasnotbeenthoroughlystudiedinvivo.

Toevaluate the roll of E2F-mediated regula4onofCcna2 in vivo,wehavegeneratedmicewith amutatedE2FbindingsiteinthepromoterofCcna2(Ccna2m).Althoughmiceareviableandappearhealthy,weobservedinfer4lityandtes4cularatrophyinmaleshomozygousforthepromotermuta4on(Fig2B).

METHODS •  AmouselinewithanullE2Fbindingsiteatthe

Ccna2locuswasgenerated(Fig1)•  Mouseviabilityandfer4litywastracked

throughoutlife.•  Micewerecollectedat2,3,4,6and12weeksof

age.Tes4swereweighedandthenfixed.•  Tes4cularatrophywasassessedviahistological

analysisandcomparingtestes:bodyweightra4osbetweencohorts.

•  Sampleswerefixedwitheitherbouin’sfixa4veforhistologicalanalysisandformalinformarkeranalysisviaimmunohistochemistry(IHC)

RESULTS Analysis of breeding revealed that mutants are completely infer4le, and that one copy of the func4onal

promotersiteissufficientforfer4lity(Fig2A).Histologicalanalysisrevealedadelayed1stwaveofspermatogenesisat 2 weeks followed by progressive degenera4on of the seminiferous tubules; degenera4on peaks at 4 weeks.Surprisingly,theseminiferoustubulesbegintorecovertheircellpopula4onsastheyreachadulthood(Fig3A).IHCconfirmed thepresenceofCCNA2 inmutantSSC,which indicates that thepromotermuta4ondoesnotpreventCCNA2expression(Fig3B).

ACKNOWLEDGEMENTS Funding provided by The Ohio State’s Undergraduate Research Office: Research Scholarship Award and Undergraduate Summer Research Fellowship.

College of Arts and Sciences / Department of Molecular Genetics

Fig1:Diagramofmouselines.ThewildtypelocusofCcna2(Ccna2+)hasanE2F site 11 base pairs from the transcrip4on start site. The mouse linegenerated(Ccna2m)hasfourbasepairsmutatedatthebindingsiteanda5xMYCtaginserteda]erthetransla4onstartsite.

Ccna2+

Ccna2m TAGTCTTAAG ATCAGAATTC

MutatedE2FSite

TAGTCGCGGG ATCAGCGCCC

WildtypeE2FSite

MYC

Bibliographies 1.Wolgemuth,D.J.,Manterola,M.,&Vasileva,A.(2013).Roleofcyclinsincontrollingprogressionofmammalianspermatogenesis.Int.J.Dev.Biol.TheInterna9onalJournal

ofDevelopmentalBiology,57(2-3-4),159-1682.Hermo,L.,Pelle4er,R.,Cyr,D.G.,&Smith,C.E.(2009).Surfingthewave,cycle,lifehistory,andgenes/proteinsexpressedbytes4culargermcells.Part1:Backgroundto

spermatogenesis,spermatogonia,andspermatocytes.MicroscopyResearchandTechniqueMicrosc.Res.Tech.,73(4),241-278.3.Peckham,M.,&Knibbs,A.(2003,January).HistologyGuide|Male.RetrievedFebruary13,2016,fromwww.histology.leeds.ac.uk/male/sertoli_cells4.Henglein,B.,Chenivesse,X.,Wang,J.,Eick,D.,&Brechot,C.(1994).Structureandcellcycle-regulatedtranscrip4onofthehumancyclinAgene.Proceedingsofthe

Na9onalAcademyofSciences,91(12),5490-5494.

CONCLUSIONS/FUTURE PROJECTIONSThedatasuggeststhatE2Fregula4onofCcna2isessen4alforasuccessful1stwaveofspermatogenesis.Since

cellpopula4onsseemtorecover,furtherbreedingstudieswillbeconductedonoldermicetoiden4fyifeventuallythereisacompleterecoveryoffer4lity(upto24weeksofage).Themolecularpathwaycausingthisphenotypeiscurrently being studied through sperma4d squashprepara4ons andmarker analysis via quan4ta4vePCR (qPCR)andIHC.PreliminarydatasuggeststhatthephenotyperesemblesthoseofCdk2mutantmice,butfurthertestsarerequiredinordertoconfirmthis.

Fig2:AdultCcna2m/mmalemiceareinfer7leandhavetes7cularatrophy.(A) Adult male mice of each group were placed with females and theirfer4litywasevaluated.Ccna2m/mmalesmatedsuccessfullyasmeasuredbythepresenceofacopula4onplug,butnolilerswereobserved.Analysisofthemalereproduc4ontractrevealedextremetes4cularatrophyinCcna2m/

madultmiceasmeasuredby thebody : testesweight ra4o.BothCcna2+/+andCcna2+/mmicepresentednormaltes4culardevelopment,butCcna2m/mhada three-fold reduc4onof tes4cularmass. (B)Representa4veexamplesof6week-oldCcna2+/+andCcna2m/mtestesoutlinedinred.

Genotype Fer7lemales(%)

Testes:bodyweightra7o(%)

Ccna2+/+ 100%(n=5) 0.62%(n=5)

Ccna2+/m 100%(n=8) 0.58%(n=7)

Ccna2m/m 0%(n=4) 0.19%(n=4)

A B

Ccna2+/+

Ccna2m/m

Fig3:Ccna2m/mmaleshaveadefec7ve1stwaveofspermatogenesis(A)H&Estainedsec4onsofbouin’s-fixedtestes (10x).At2weeks, thewild-type’sseminiferous tubuleshaveZygotenespermatocytes(blackarrow),whilethemutantiss4llatthepre-Leptotenestage.At3weeks,Ccna2+/+maleshavesecondarymeiocytes(redarrow)whilethetubulesinthemutantbegintodegenerate;thisdegenera4onpeaksat4weeks.However,tubulesbegintorecovertheircellpopula4ons as they reach adulthood. No viable sperm was found in the epididymis (6->12weeks).(B)IHCusinganan4bodyagainstCCNA2todetectexpressionintestesfrom4week-oldmice (4x). CCNA2-posi4ve cells are dividing SSCs. (C) Stages of spermatogenesis andCcna2’stemporalloca4oninthedifferen4a4onprocess.

Ccna2

+/+

Ccna2

m/m

2 wk 3 wk 4 wk 6 wk 12 wkA

B

AdaptedfromWolgemuthet.al.(2013)

C

Ccna2

+/+

Ccna2

m/m

Ccna2