Spermvelocityandlongevitytradeof feachother andinuencefertilizationi ntheseaurchin Lytechinusvariegatus Don R.Levitan Department of Biological Science,Florida StateU niversity,Tallahassee,FL323 06-1100,USA ([email protected] ) Thetheoretical prediction that fast sperm shouldbe more e¡ective atfertilizing eggs has never been documentedempirically .Interspeci¢c comparisonssuggest an inverserelationship between sperm velocity andsperm longevity,butthis trade-o¡has never been demonstrated withina species. Here Iinvestigate howsperm velocityand sperm longevityin£ uence the patterns offertilizationin the seaurchin Lytechinus variegatus.Inthe laboratory,Iexamined1 1male ^femalepairs of sea urchins forvariation in sperm velocityand sperm longevity,anddetermined the correlationsof these traits withthe percentageof eggs fertilized withserially diluted sperm. Males withfaster sperm hadhigher rates offertilization than males withslower sperm. Within individualmales, assperm agedthey slowed down and showed a reduced percentageactivity and lower rates offertilization. Across males, the averagevelocity of freshly spawned sperm wasinversely related to sperm longevity.Theseresults establish the possibilitythat sperm traits are adaptedfor varying conditions along a continuumfrom sperm limitationto sperm competition. Keywords: sperm; fertilization;life-history strategies; echinoids
Here Iuse laboratoryexperiments toshow that, in the 1.INTRODUCTION sea urchin Lytechinus variegatus, bothsperm velocityand Afundamentalcharacteristic ofsperm is their abilityto sperm longevityin£ uence their fertilizationrates andthat swim, yet,to the author’sknowledge,no study to date has these traits arenegatively correlated. This result suggests documentedhow sperm velocityin£ uences fertilization that di¡erent combinationsof sperm velocityand sperm rates infree-spawning organisms. F ree-spawninginvert- longevitymay re£ ect selection onsperm traits inorder to ebrates arecharacterized by `primitive’ sperm whichare optimize fertilizationsuccess undervarying conditions of simple instructure andthought to be adapted for sperm limitationand sperm competition. swimmingin search ofeggs in the sea(F ranzen1987) . Theorypredicts that fast sperm shouldcollide with eggs 2. METHODS more frequentlythan slow sperm (Rothschild& Swann 1951; Vogel et al.1982)and variation in sperm velocity (a) Urchin andgamete collection within(Levitan et al.1991;Levitan1 993)and between Lytechinusvariegatus isa common,shallow-water sea urchin (Gray1 955;Levitan 1993) species hasbeen documented, foundin thegrass £ atsof the Caribbean and the Gulf of but notests havedetermined whetherthis variationin Mexico.Experiments were conducted in thesummer and sperm velocityin£ uences their fertilizationrates. autumnof 1996 with seaurchins collected from the Gulf of Anothercharacteristic ofsperm is their relatively Mexicoo¡ Turkey Point of the Florida panhandle (29 850’ N, short lifespan. In many species, sperm losetheir 84830’ W).Oneach experimental day ,onemale sea urchin and viabilitywithin seconds tominutes after dilutionin onefemale sea urchin were used as replicates. Gametes were seawater(Levitan 1 995).Incontrast, eggsare typically obtainedby injection of each sea urchin with 1mlof 0.55M KCl. viablefor several hours after spawning(see, forexample, Spermwere collected with apipetteas theywere extruded from Pennington1 985). thegonopore of the male sea urchin and were placed in aPetri Swimmingfast andswimming for a longperiod both dishon ice. Eggs were collected by inversion of the spawning require energy,soa trade-o¡between the twoseems femaleurchin on a Petridish containing ¢ lteredseawater at likely.Theoptimal combination of speed andendurance ambienttemperature ( ca. 20 8C). maydepend on the chancesof fertilization. I thasbeen suggestedthat fast sperm areadvantageous under (b) Laboratory experiment conditionsof sperm competitionand that long-lived Afterspawning, the egg concentration was adjusted to sperm areadvantageous under conditions of sperm approximately¢ veeggs per microlitre in astockegg suspension. limitation(Levitan 1993, 1 998 b).Evidencefor this trade- Spermwere then diluted to one of a varietyof concentrations o¡has been implied by correlations across closelyrelated (1/25^1/1500dilutionor 1 .25 103^3.78 105 sperm ml71) as a £ £ species (Levitan1993) ,buta negativecorrelation between stocksperm suspension. Because ` dry’(undiluted) sperm can velocityand longevity has never been demonstrated retainviability for many hours whereas diluted sperm age withina species. Intraspeci¢c comparisonsprovide the rapidly(Levitan et al.1991;Levitan1993) ,onlyone sperm- most direct evidenceof a trade-o¡, becausethey are free dilutiontrial was performed at a time.Each sperm dilution was fromother trait di¡erences whichcan confound compari- testedfor its fertilization rate and sperm velocity at a varietyof sons across species (Ro¡1992) . ages.Although both the eggs and sperm were ageing in this
Proc. R.Soc.Lond. B (2000) 267, 531^534 531 © 2000The RoyalSociety Received 2November1 999 Accepted 13December1999 532D .R.Levitan Sperm velocity and sperm longevity in sea urchins experiment,the egg viability in echinoids,as in mostinver- 5 tebrates,is several hours longer than the dilute sperm viability andis less likely to cause di¡ erences in thefertilization rates (reviewedby Levitan 1995) . 4 Onemillilitre of the ¢ rststock sperm suspension was placed intoeach of four to six pairs of scintillationvials containing 8 ml
of¢ lteredseawater .Onevial in thepair was used to assay the 0 5 F fertilizationrate and the other for microscopic examination of the 3 g o spermswimming velocity .Eachof thefour to six pairs was used l fora di¡erent sperm age. Immediately after the addition of the sperm,the `time 0’ vialswere treated. One millilitre of the egg 2 suspensionwas added to the fertilization rate vial, bringing the y = - 13.322x + 6.0379 ¢nalvolume to 10 ml. T enseconds later ,additionalfertilization R2 = 0.6199 wasstopped by the addition of 10ml of0.55 M KCl(Schuel1 984). Istoppedfertilization after only 1 0stoeliminate the e¡ ects of 1 spermageing within eachsperm age treatment. After 1 h, atleast 0.1 0.2 0.3 100eggs were inspected with acompoundmicroscope for the sperm velocity (mm s - 1) presenceof afertilizationmembrane or furtherdevelopment. Simultaneouslyto the addition of the eggs to the fertilization Figure1. In£ uence of spermvelocity on fertilization in ratevial, a sampleof a few dropswas taken from the sperm freshlydiluted sperm. The F50 isthenumber of spermper microlitreneeded to fertilize50% of afemale’seggs; a low velocityvial and placed on a glassslide. A glasscover-slip with valueindicates high-performa ncegametes. aclaysupport on each corner was placed over the sperm specimenand pressed down until the top of the specimen (c) Estimating the fertilization rates touchedthe cover-slip. The slidewas then placed on the stage of Icalculatedthe amount of sperm needed to fertilize 50% of acompoundmicroscope and videotaped at 400magni¢cation £ the eggs (F )by¢ ttingthe fertilization data to V ogel et al.’s for2 min(Levitan et al.1991;Levitan1993) .The planeof focus 50 (1982)fertilization kinetics model. This modelpredicts the wasset midway between the glass slide and cover-slip to minim- proportionof eggs fertilized given the sperm ( S ,in spermper izethe in£ uence of the glass walls on sperm movement (walls 0 microlitre)and egg ( E ,in eggsper microlitre) concentrations, greaterthan several body lengths away have a negligiblein£ u- 0 sperm^eggcontact time ( t,10s) and two rate constants, i.e. the enceon swimming cells) (Winet 1 973).The ¢eldof view was fertilizationrate constant ( b, in mm3 s71)andthe collision rate shiftedat 1 5sintervalsto minimize the risk of sampling a sper- constant (b , in mm3 s71): matozoonmore than once. 0 Ateachof several sperm ages ranging from 5 to1 20min,an S 0 0 E0t aliquotof eggs was placed into one of the next pair of vials. ¿ 1 exp (1 e¡ ) . (1) 1 ˆ ¡ ¡ 0E0 ¡ When timepermitted, another dilution was made and another © ª ageseries begun. The spermdilutions and ages at egg addition The best¢ ttedvalues of and 0 wereestimated for each sperm variedbetween replicates to cover a rangeof overlapping ageand sperm dilution trial within eachreplicate by the values. Marquartmethod of nonlinear regression with theSAS statis- Iestimatedthe egg concentration by counting the number of ticalprogram (see Levitan et al.(1991)andLevitan ( 1993, eggsin replicate0. 1mlaliquotsof thestock egg suspension. The 1996a)forsimilar analyses) .With theserate constants, equation spermconcentration was estimated for each stock sperm suspen- (1)wassolved for the F50 givenan egg concentration of sionby replicate counts using a haemocytometer.Eleven repli- 0.5 eggs ml71 and a sperm^eggcontact time of 10s foreach trial. catesof the entire experiment were conducted and each replicateused an independent male and female urchin. 3. RESULTS (i) Sperm activity Thesperm concentrationdid not in£ uence the sperm Itracedsperm movement onto acetate sheets by marking the velocityover the rangeof concentrations tested ( p 0.79 ˆ sperm’spositionin eachvideo frame while itmoved through the and R2 0.0003),soI pooledthe measurements ofsperm ˆ ¢eldof view.Onlysperm that swam parallel to the glass slide and velocityacross allconcentrations in order to test forvaria- remainedin theplane of focuswere scored. Sperm that were not tionin the sperm velocityacross males andages. The movingor were swimming through the plane of focus were averagevelocities of freshly diluted sperm rangedfrom ignored(Levitan et al.1991;Levitan1993) .Distancewas cali- 0.153to 0.275 mm s 71 across allmales. Linearregression bratedfrom the image of a stagemicrometer videotaped at the revealedthat males withslow sperm neededup to two samemagni¢ cation before the addition of sperm. I measured orders ofmagnitude more sperm tofertilize 50%of a spermdistances with MTV computersoftware by scanning each female’s eggsthan did males withfaster sperm (¢gure 1 ). acetatesheet image and tracing the sperm paths with acomputer Asthe sperm aged,they slowed down (¢ gure 2 a) and mouse.The spermvelocity was estimated from 25 sperm per their abilityto fertilize eggsdecreased (¢gure 2 b). A treatmentas thedistance moved divided by the number of frames multiple regression revealedsigni¢ cant e¡ ects ofboth in which thesperm was scored (at 30 framess 71). sperm velocity( p 5 0.0001)andsperm age( p 5 0.0001) The spermvideotapes were also examined for the percentage onthe amountof sperm neededto fertilize 50%of a ofactive sperm at each dilution and age. The ¢rst100 sperm female’s eggs( R2 0.70).After 1h,two orders of encounteredon the videotape were scored as either moving or magnitudemore spermˆ wereneeded to fertilize 50%of a non-moving. female’s eggsand after 2hfertilizationwas nil.
Proc. R.Soc.Lond. B (2000) Sperm velocity and sperm longevity in sea urchins D.R. Levitan533
0.300 (a) (a)
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Figure2. Sperm age in£ uences sperm velocity and fertilizatione¤ ciency. ( a)Spermvelocity decreases with sperm age. (b)Fertilizatione¤ ciency decreases with sperm 0.1 age.No dataplotted at 120minbecause no fertilizationtook - 1 - 0.6 - 0.2 0 0.2 placeat thissperm age. slope of % activity over age (min)
Thesperm velocitywas negatively correlated with Figure3. Sperm longevity and the trade-o¡ of sperm sperm longevity.Asthe sperm aged,the percentageof velocityand longevity. ( a)The percentageof activesperm activesperm decreased (¢gure 3 a).Theslope of this decreaseswith spermage ( R2 0.24 and p 5 0.0001). ˆ decrease inactivity with age is anindication of that (b)Trade-o¡of theslope of therelationship between sperm male’saveragesperm longevity.Amale’sinitialaverage ageand percentage activity calculated for each male and the averageinitial sperm velocity for each male ( R2 0.38 and sperm velocitywas negatively correlated with his average ˆ sperm longevity(¢ gure 3 b).Fast sperm havelower endur- p 5 0.05).The twomales observed for 120 minare highlighted ancethan do slow sperm. with solidsquares.
neededto achieve 50% fertilization. Because males di¡er 4.DISCUSSION bymore than0.2 mm s 71 intheir averagevelocity of Thepresent studyprovides two novel ¢ ndings.The ¢ rst freshlydiluted sperm andthe velocitydi¡ erence betweena isthe evidencethat sperm velocityin£ uences the fertiliza- male’sfresh andaged sperm wasalso more than tionrate. Thesecond is that sperm velocityand sperm 0.2 mm s71,variationin sperm velocitywithin a species longevityare inversely related within species. Both¢ nd- mayplay an important role in fertilization in nature. ingsincrease ourbasic understanding of the fertilization However,acorrelationbetween the sperm velocityand dynamicsand life-history trade-o¡ s involvedin resource fertilizationrate is notdirect evidencethat ahigher allocationwithin individual sperm. sperm velocityresults inhigher rates offertilization. Thenegative correlation between sperm velocityand Sperm velocitymight, for example, be correlated with the amountof sperm neededto fertilize eggsindicates that some othertrait whichresults inhigher rates offertiliza- a 0.1mm s71 decrease insperm velocityis correlatedwith tion.F ast sperm mightbe overall `better’ sperm. For anorder of magnitude increase inthe number ofsperm example,studies onsperm velocityand fertility in
Proc. R.Soc.Lond. B (2000) 534D .R. Levitan Sperm velocity and sperm longevity in sea urchins humanshave documented that males ininfertile couples IthankA. Jaeger,S. Kellyand K. Silvestrefor technical assis- release fewer,slower and more abnormalsperm thando tance.G. F arley,A.Jaeger,G. LeBuhn, M. McCartney,T. fertile sperm donors(Morales et al.1988;Barratt et al. McGovern,A. Thistle,J .Travis,C. Swansonand A. Winn made helpfulcomments on the manuscript. This workwas funded by 1993).Inthe present case, however,fast sperm agemore theUS National Science F oundation. rapidlythan slow sperm, suggestingthat velocityand endurancetrade o¡ each other ,andthat fast sperm are notnecessarily `better’ inalltraits. REFERENCES Thissecond ¢ nding,that fast sperm havereduced Barratt,C. L.R., T omlinson,M. J.& Cooke,I. D. 1 993 endurance,has important implications for evolutionary Prognosticsigni¢ cance of computerized motility analysis for trade-o¡s ingamete allocation. 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Levitan,D .R.1995The ecologyof fertilization in free-spawning competingfor eggs and virgin eggs are quickly fertilized, invertebrates.In Ecologyof marine invertebrate larvae (ed. L. selection mayfavour velocity over longevity .Conversely, McEdward),pp.1 23^156.Boca Raton, FL: CR CPress. whensperm arelimiting and must drift viawater Levitan,D .R.1996 a E¡ectsof gamete traits on fertilization in currents to¢ ndeggs,selection mayfavour longevity over thesea and the evolution of sexual dimorphism. Nature 382, velocity(Levitan 1 993,1998 a,b). 153^155. Theseexpectations match the interspeci¢c di¡erences Levitan,D. R. 1996 b Predictingoptimal and unique egg sizes in insperm velocityand longevity among congeneric sea free-spawningmarine invertebrates. Am. Nat. 148, 174^188. urchins. Amongthree species of Strongylocentrotus sea Levitan,D. R. 1998 a DoesBateman’ sprincipleapply to broad- urchins, Strongylocentrotus purpuratus hasthe fastest but cast-spawningorganisms ?Eggtraits in£ uence in situ shortest-lived sperm (Levitan1 993).Itlivesat a high fertilizationrates among congeneric sea urchins. Evolution 52, populationdensity where female fertilization rates are 1043^1056. Levitan,D. R. 1998 b Spermlimitation, gamete competition and near10 0%(Levitan 1 998 a,1999)and, presumably , sexualselection in externalfertilizers. In Spermcompetition and sperm oftencompete forfertilizations (Levitan 199 8 b). sexualselection (ed.T .R.Birkhead& A.Moller),pp.1 75^215. Strongylocentrotus droebachiensis hasthe slowest butlongest- SanDiego, CA: AcademicPress. livedsperm (Levitan1993) .Itlivesat the lowestpopula- Levitan,D. R. 1999The fertilizationecology of three tiondensity (Levitan 1 998 a),wherefemale fertilization congenericsea urchins from the northeastern Paci¢ c. 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Strongylocentrotus purpuratus, the 241^247. species withthe highestlevels of female fertilization, has Pennington,J .T.1985The ecologyof fertilization of echinoid small numerouseggs and requires the most sperm to eggs:theconsequence of sperm dilution, adult aggregation, fertilize 50%of a female’seggs,whereas S.droebachiensis andsynchronous spawning. Biol. Bull. 169, 417^430. hasthe largestand fewest eggsand requires the fewest Ro¡, D.A. 1992 Theevolution of life histories: theory and analysis . sperm tofertilize 50%of a female’seggs.Again London:Chapman & Hall. S.fr anciscanus hasintermediate eggtraits andan inter- Rothschild,L. & Swann,M. 1951The fertilizationreaction in mediate fertilizationperformance (Levitan 1 993,1 998 a). thesea urchin. The probabilityof a successfulsperm ^egg Previousstudies haveindicated that eggtraits insea collision. J. Exp. Biol. 28, 403^416. Schuel,H. 1984The preventionof polyspermic fertilization in urchins represent di¡erent pointsalong an adaptive conti- seaurchins. Biol. Bull. 167, 271^309. nuumfrom sperm limitationto sperm competition Styan,C. A.1998Polyspermy ,eggsize, and the fertilization (Levitan1993, 1 996 a,b, 1998a,b;Styan1 998).Thepresent kineticsof free-spawning marine invertebrates. Am. Nat. 152, studyyields the ¢rst direct evidencethat sperm traits also 290^297. in£uence fertilization and trade o¡ against each other . Vogel,H., Czihak, G., Chang, P .&Wolf,W .1982F ertilization Di¡erent combinationsof both sperm andegg traits kineticsof sea urchin eggs. Math. Biosci . 58, 189^216. mighttherefore beselected inorder to optimize fertiliza- Winet,H. 1973W alldrag on free-moving ciliated micro- tionunder varying environmental conditions. organisms. J. Exp. Biol. 59, 753^766.
Proc. R.Soc.Lond. B (2000)