Sperm storage in Hemidactylus mabouia : morphological and ultrastructural aspects of a reproductive strategy.

ContentslistsavailableatScienceDirect

Animal

Reproduction

Science

j o u r n a l ho me p ag e:ww w . e l s e v i e r . c o m / l o c a t e / a n i r e p r o s c i

Sperm

storage

in

Hemidactylus

mabouia:

Morphological

and

ultrastructural

aspects

of

a

reproductive

strategy

Katiane

de

Oliveira

Pinto

Coelho

Nogueira

_,

_Sirlene

_Souza

_Rodrigues

_Sartori

_,

Vinícius

Albano

Araújo

_,

_Clóvis

_Andrade

_Neves

_,

_Benjamin

_Kolisnyk

b_{UniversidadeFederaldeVic¸osa,Brazil}

c_{RobartsResearchInstitute,TheUniversityofWesternOntario,Canada}

a

r

t

i

c

l

e

i

n

f

o

Received10July2014

Receivedinrevisedform19June2015 Accepted25June2015

Availableonline27June2015 Keywords: Spermatozoa Spermstorage Lizard Uterinetube Reproductivestrategy Reptile

a

b

s

t

r

a

c

t

Spermstorageisacommonphenomenoninmostfemalereptiles.Evidenceofspermstorage isbasedontheobservationthatfemalefertilizationoccursevenwhenfemalesare sepa-ratedfrommales,aswellasthepresenceofagglomeratesofspermatozoainspecificregions oftheoviducts.Lizardsarecapableofstoringspermintheuterinetube,vagina,orinboth regions.However,representativesoftheGekkonidaefamilycommonlystore spermato-zoaintheuterinetube,whichisconsideredanancestralcharacterstateforSquamates. Usingcomparativetechniquesoflight,transmissionandscanningelectronmicroscopy,we observedstoredspermorganizedincompactbundleswiththeirheadsfacingthebottomof thecryptsoftheuterinetube,indicatingchemotacticattraction.Thealignmentand pack-ingofspermatozoainHemidactylusmabouiaindicatesthattheprocessofevacuationof thecryptsforfertilizationmayberelatedtothepassageoftheeggthatexerts mechani-calpressureagainstthewallsoftheuterinetube,causingitsdistensionandthereleaseof spermatozoa.Weconcludethatthespermstorageregionandthemorphological organi-zationofthecryptsintheuterinetubeofH.mabouiaissimilartootherpreviouslystudied speciesoflizards,supportingthenotionthatspermstorageisacommonreproductive strategyamongfemalereptiles.

©2015ElsevierB.V.Allrightsreserved.

1. Introduction

Spermstorageintheoviductduringthereproductive cycleisacommonphenomenonamongstfemalereptiles, where inseminationand ovulation/fertilization occurin distinctperiods(Schuett,1992;SeverandHamlett,2002). In 1943, Ludwig and Hahn described sperm storage in

∗ Correspondingauthorat:UniversidadeFederaldeOuroPreto, Cam-pusMorrodoCruzeiro,InstitutodeCiênciasExataseBiológicas(ICEB), DepartamentodeCiênciasBiológicas-DECBI,CEP35400-000/OuroPreto, MG,Brazil.Tel.:+553135591215.

E-mail addresses: katiane@nupeb.ufop.br, kaolli@yahoo.com.br (K.d.O.P.C.Nogueira).

theoviductsofCrotalusviridisviridisfemales.SaintGirons (1962)andFox (1963)reportedthefirststudiesof such spermstorageinlizardsinthe1960s.

Evidence for spermstorageis basedonthe observa-tionthatfemalefertilizationoccursevenwhenthefemales are separated from males and is related to the pres-enceofagglomeratesofspermatozoain specificregions oftheoviducts(AdamsandCooper,1988).However,the existence of these spermatozoa agglomerates does not guaranteespermstorage.Phagocytosissitesareobserved inthereptilianoviductwherealargeamountof spermato-zoa couldbe present. Additionally, phagocytosis occurs withinthestoragesitesthemselvesinlizards(Severand Hopkins, 2004;SiegelandServer, 2007;Nogueira etal., http://dx.doi.org/10.1016/j.anireprosci.2015.06.022

2011a,b).Severalspermatozoahavebeenobservedinthe vaginaofEumecesegregiuslizards(SchaferandRoeding, 1973);however,thesefindingsdonotindicatethisregion asastoragesiteduetotheabsenceofstructuresspecialized forthisfunction,suchascrypts.Similarly,inHemidactylus mabouia,intensephagocytosisofspermatozoabyvaginal epithelialcellshasbeenobserved,despitethefactthatno structuresspecializedinstoragewereobserved;thus,this oviductregionischaracterizedasanexclusivelyphagocytic site(Nogueiraetal.,2011a,b).

Lizardsstorespermatozoaintheuterinetube,vagina orbothoftheseregions(SeverandHopkins,2004;Siegel andServer,2007).Conversely,membersoftheGekkonidae familygenerallystorespermintheuterinetube(Cuellar, 1966; Murphy-Walker and Haley, 1996; Girling et al., 1997),and thisfeatureisconsideredtobetheancestral stateforSquamatesaccordingtothehypothesisof phy-logenybasedonspermatozoastoragetubulesproposedby

SeverandHamlettin2002.

Variousstudieshavedescribedandcomparedthe struc-turalandultrastructuralaspectsofstoragesitesinlizards (Cuellar,1966;ConnerandCrews,1980;Bou-Reslietal., 1981;Girlingetal.,1997,1998;SeverandHamlett,2002; SeverandHopkins,2004).Here,wedescribeforthefirst timetheuterinetubespermstorageprocessinH.mabouia, thusprovidingnewinsightsintotheunderstandingofthis importantreproductivestrategyinamemberofthe Squa-mata.

2. Materialsandmethods

2.1. Animals

TenfemaleH.mabouializardsmeasuringfrom52.9to 59.3mmwerecollectedunderlicensenumber10504-1of InstitutoBrasileirodoMeioAmbienteedosRecursos Nat-uraisRenováveis(IBAMA).Thecarcasseswerefixedina bufferedformalinsolution(Carsonetal.,1973)for24hat roomtemperatureanddepositedattheJoãoMoojen Ani-malScienceMuseumoftheUniversidadeFederaldeVic¸osa (UFV),MinasGerais,Brazil.Theexperimentwasconducted inaccordancewiththeethicalprinciplesfortheutilization oflaboratoryanimalspublishedintheColégioBrasileiro deExperimentac¸ãoAnimal(COBEA).Identificationand col-lectionofthereproductiveorganswasperformedinsitu viaamedianlongitudinalincisionintheventralregionof theanimals.Thecollectedmaterialwasdepositedinvials containingsalinesolution.

2.2. Opticalmicroscopy

Uterinetubefragments werefixedina buffered for-malin solution (Carson et al., 1973) for 24h at room temperature and dehydrated in increasing concentra-tionsofethanol.Thefragmentswereembeddedinglycol methacrylate(Historesin®_{,Leica)and sectionedat2␮m}

in an automatic microtome (Reichert-Jung, Germany). The obtained sections were stained with 1% toluidine blue/sodiumborateandmountedonslideswithEntellan®

(Merck)foranalysiswithanOlympusBX-60microscope.

2.3. Transmissionelectronmicroscopy

Uterinetubefragments (approximately1mm2_)were

fixed in Karnovsky’s solution (Sheehan and Hrapchak, 1980)for4h.Thematerialwasthenwashedwith0.1M sodiumcacodylate buffer(pH 7.2)and post-fixed in 1% osmiumtetroxidefor2h.Thematerialwassubsequently dehydratedin increasingconcentrations of acetoneand embeddedinEponresin.Ultrathinsectionswereobtained inanultramicrotomeandcontraststainedwith2%uranyl acetateand0.2%leadcitrate.Thematerialwasexamined and photographed using an EM109-Zeiss transmission electron microscope in the Nucleus of Microscopy and MicroanalysisatFederalUniversityofVic¸osa,Brazil.

3. Resultsanddiscussion

TheuterinetubeofH.mabouiaiscomposedoffour dis-tinctregionsthatarenamedasfollowsfromtheanterior totheposteriorside:infundibulum,uterinetube,uterus andvagina(Nogueiraetal.,2011a,b).TheuterinetubeofH. mabouiaisinternallycoatedwithasimpleepithelium con-tainingsecretorynon-ciliatedcellsandciliatedcells(Fig.1A andB).Crypts,whicharebranchedepithelialinvaginations formedfromtheepithelialinnerlining,areobservedinside theuterinetube.Thewallsofthecryptscontainboth cili-atedandnon-ciliatedcells;however,veryfewciliatedcells arepresentatthebottom.Thisorganizationissimilarto previousdescriptionsofcryptsinotherlizardspecies( Bou-Reslietal.,1981;Murphy-WalkerandHaley,1996;Girling etal.,1998;SeverandHamlett,2002;YamamotoandOta, 2006;Eckstutetal.,2009).SeverandHopkins(2004)and

Bou-Reslietal.(1981)describednumerousciliatedcells intheneckofthecryptsbutnotatthebottomof Acan-thodactylusscutellatus(Gekkonidae)andScincellalaterale uterinetubes.IntheuterinetubeofAnolissagrei,thedistal portionofthecryptisnotspecialized;however,the prox-imalportionexhibitsasimilarproportionofciliatedand non-ciliatedcells (Severand Hamlett,2002).Indeed,no apparentpatternregardingtheproportionofciliatedand non-ciliatedcellsisnotedamongpreviouslystudiedlizard species.

Thenon-ciliatedcellspresentintheuterinetubeofH. mabouiaproduceametachromaticsecretionwhenstained withtoluidineblue.Thesecretionproducedbynon-ciliated epithelialcellslocatedatthebottomofthecryptsmaybe relatedtoavarietyoffunctions,suchasnutrition(Cuellar, 1966; Bou-Resli et al., 1981), defense (Bou-Resli et al., 1981),chemotaxis(SaintGirons,1973;AdamsandCooper, 1988)andmaintenanceofanaqueousenvironment,which isappropriateforspermatozoasurvival.However,further investigationmustbeperformedtoclarifytheroleofthe non-ciliatedcellsecretions.

Imagesobtainedusingscanning electronmicroscopy (Fig.2)demonstratethatthebottomofthecryptsinthe uterinetubeofH.mabouiaexhibitasmoothsurfacedueto thepresenceofnon-ciliatedcells.Theroughendoplasmic reticulumofthenon-ciliatedcellsishighlydeveloped,and thenucleoliaregenerallyclearlyvisibleandexhibitloose chromatin.Somemitochondriaandnumerousvesicles dis-persedinthecytoplasmcanbeobserved(Figs.3and4).

Fig.1. Lightmicroscopy(A)andtransmissionelectronmicroscopy(B)oftheuterinetube.Notetheepitheliumcomposedofciliatedcells(blackarrows) andnon-ciliatedcells(blackarrowhead).Ciliatedcellsexhibitelongatedbasalcorpuscles(cycles)ontheapicalcytoplasmandseveralmitochondria(white arrowhead).Notethepresenceofclusteredspermatozoa(whitearrows)atthebottomofthecrypts.L,lumen.Bars:AandB,3␮m.

Fig.2. Scanningelectronmicroscopyoftheuterinetube.Notesperm stor-age(blackarrows)andthesmoothsurface(arrowhead)atthebottomof thecrypts.Bar.5␮m.

Fig.3. Transmissionelectronmicroscopyoftheuterinetubecrypt.Note thepredominanceofthenon-ciliatedcells.Roughendoplasmicreticulum (blackarrowhead);mitochondria(whitearrowshead);spermatozoon (blackarrow);L,lumen.Bar:2␮m.

Ciliatedcellsalsocontainanucleuswithloosechromatin, severalmitochondria,elongatedbasalcorpuscles onthe apicalcytoplasmandmoderateamountsofendoplasmic reticulumconnectedtothenuclearregion(Fig.1).

H.mabouiastoresspermatthebottomofthecrypts; thespermareclusteredinbundleswiththeirheads fac-ingtowardstheblindendofthecryptsandtailspositioned towardsthelumen(Figs.1AandBand2).Thissperm orga-nizationmayberelatedtothechemotacticattractionto substancespresentwithinsecretionsinthisregion. More-over,thedensepackingofspermcouldactasamethod todecreasetheiractivityandmaintaintheirviability dur-ingstoragetime.Thisspermarrangementduringstorage waspreviouslyobservedinotherlizardspecies(Fox,1963; ConnerandCrews,1980;SeverandHopkins,2004;Eckstut et al., 2009)and snakes (Hoffman and Wimsatt, 1972; Halpertetal.,1982;PerkinsandPalmer,1996).The oppo-siteorientationofsperm,withtheirheadsfacingthelumen of storage tubules, was observed in Seminatrix pygaea (SiegelandServer,2007).

Fig.4.Transmissionelectronmicroscopyoftheuterinetubecrypt.Note thenucleuswithloosechromatin(blackarrowhead)andnumerous vesi-clesinthecytoplasmofthenon-ciliatedcells(whitearrowshead).Note thepresenceofspermatozoaintransversesections(arrows)immersedin largeamountsofsecretion(sr).Bar:2␮m.

Fig.5. Transmissionelectronmicroscopyoftheuterinetubecrypt.Notethatthespermatozoadonotestablishdirectcontactwithepithelialcells.Bars:A, 2␮m;BandC,1␮m.

The sperm stored in the uterine tube crypts of H. mabouia does not establish direct contact with epithe-lialcells(Fig.5), asobservedinAnolis carolinenses(Fox, 1963). However, in other lizards, such as A. scutellatus and E.egregius, spermwasobservedin theintercellular spacesandthecytoplasmofepithelialcells(Bou-Reslietal., 1981; Schafer and Roeding,1973).AccordingtoCuellar (1966)andAdamsandCooper(1988),spermalsoappeared topenetratetheepithelialcells inHolbrookiapropinqua. Thiscontactbetweenspermandepithelialcellswasalso observedin Thamnophissirtalis,Thamnophissirtalis pari-etalis andAgkistrodonpiscivorus (Hoffman and Wimsatt, 1972; Halpert et al., 1982; Siegel and Server, 2007). In thegartersnake,spermwasobservedbetweenthe stor-age tubules, and this interaction was compared to the interactionbetweenspermandSertolicellsinmammalian seminiferoustubules(HoffmanandWimsatt,1972).

The spermstored intheuterinetube mustreachits lumenforfertilizationtooccur.Thealignmentandpacking ofsperminH.mabouiadonotindicatecryptevacuation asanactiveprocess.Thespermreleasemechanismmay berelatedtothepassageoftheeggthroughtheuterine tube.Eggsareapproximatelysixmillimetersintheirlargest diameterandexertmechanicalpressureagainstthetube wall, causing its distension and possibly sperm release, assuggested forotherspecies (Fox, 1956;Hoffman and Wimsatt,1972;SiegelandServer,2007).Thelackofdirect contactbetweenthestoredspermandepithelialcellsofthe cryptgroundisastrongevidencetosupportthis hypothe-sis.

ThespermstorageperiodinH.mabouiawasnot moni-tored;however,inthetropicalgeckoH.frenatus,whichis phylogeneticallyclosetoH.mabouia,storageoccursfora minimumperiodof36weeks(Murphy-WalkerandHaley, 1996).H. frenatus and H.mabouia present reproductive

cyclesthat extendthroughoutthe year,which suggests similarstorageperiodsforbothspecies.Thepossibilityof placinglittersovertheyearfromeggfertilizationbystored spermdecreasespredationrisksandincreases reproduc-tive competition between species that share the same niche.In Floridaand Texas, H.mabouia, H.frenatusand H.garnottiexhibitreproductiveadvantagesovertheir con-generH.turcicus,whichexhibitsseasonalreproductionand isbeingeliminatedinthoseregions(Eckstutetal.,2009).

Basedonourobservations,weconcludethatthecrypts oftheuterinetubeofH.mabouiaarethespecificsiteof spermstorageinthisspecies.Furthermore,the morpho-logicalorganizationissimilartootherpreviouslystudied speciesoflizards.Thesefindingssupportthenotionthat spermstorageisacommonreproductivestrategyamong femalereptiles.

Conflictofinterest

None.

Acknowledgments

We thankthe Núcleode MicroscopiaeMicroanálise oftheUniversidadeFederaldeVic¸osaandDr.Gwenaelle Pound-LanaforassistancewithEnglishrevisions.

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