Proc. Natl. Acad. Sci. USAVol. 92, pp. 11039-11043, November 1995Physiology

Sperm capacitation in humans is transient and correlates withchemotactic responsiveness to follicular factors

(chemotaxis/fertilization/mammalian reproduction/acrosome reaction/sperm selection)

ANAT COHEN_DAYAG*, ILAN TUR-KASPAt, JEHOSHUA DORt, SHLOMO MASHIACHt, AND MICHAEL EISENBACH*t*Department of Membrane Research and Biophysics, Weizmann Institute of Science, 76100 Rehovot, Israel; and tDepartment of Obstetrics and Gynecology,Sheba Medical Center, Tel Aviv University Medical School, 52621 Tel Hashomer, Israel

Communicated by Julius Adler, University of Wisconsin, Madison, WI, August 11, 1995

ABSTRACT In humans, only a small fraction (2-12%) ofa sperm population can respond by chemoattraction to fol-licular factors. This recent finding led to the hypothesis thatchemotaxis provides a mechanism for selective recruitment offunctionally mature spermatozoa (i.e., of capacitated sperma-tozoa, which possess the potential to undergo the acrosomereaction and fertilize the egg). This study aimed to examinethis possibility. Capacitated spermatozoa were identified bytheir ability to undergo the acrosome reaction upon stimula-tion with phorbol 12-myristate 13-acetate. Under capacitatingconditions, only a small portion (2-14%) of the spermatozoawere found to be capacitated. The spermatozoa were thenseparated according to their chemotactic activity, which re-sulted in a subpopulation enriched with chemotactically re-sponsive spermatozoa and a subpopulation depleted of suchspermatozoa. The level of capacitated spermatozoa in theformer was -13-fold higher than that in the latter. Thecapacitated state was temporary (50 min < life span < 240min), and it was synchronous with the chemotactic activity. Acontinuous process of replacement of capacitated/chemotac-tic spermatozoa within a sperm population was observed.Spermatozoa that had stopped being capacitated did notbecome capacitated again, which indicates that the capaci-tated state is acquired only once in a sperm's lifetime. A totalsperm population depleted of capacitated spermatozoa stoppedbeing chemotactic. When capacitated spermatozoa reappeared,chemotactic activity was restored. These observations suggestthat spermatozoa acquire their chemotactic responsiveness aspart of the capacitation process and lose this responsivenesswhen the capacitated state is terminated.We suggest that the roleofsperm chemotaxis in sperm-egg interaction in vivo may indeedbe selective recruitment of capacitated spermatozoa for fertiliz-ing the egg.

Human spermatozoa are attracted to follicular factors in vitro,and the attraction is correlated with egg fertilizability (1). Theattraction results from chemotaxis and is accompanied byspeed enhancement (ref. 2; for reviews, see refs. 3 and 4).Unlike the case of species with external fertilization in whichmost, if not all, of the sperm population is chemotacticallyresponsive (for reviews, see refs. 5 and 6), in humans only asmall fraction of the sperm population (2-12%) is chemotac-tically responsive at any given time (7). The identity of theresponsive spermatozoa in humans changes with time byturnover: chemotactic spermatozoa lose their activity whileothers acquire it (7). This raised the possibility that sperma-tozoa are selectively chemotactic only at a certain physiologicalstage. The capacitated stage-i.e., the stage at which sperma-tozoa possess the potential to undergo the acrosome reaction(a release of proteolytic enzymes enabling sperm penetrationthrough the egg coat) and to fertilize the egg (for recent

reviews, see refs. 8-13)-seemed a reasonable possibility (3,7). This study investigates this possibility and finds a linkagebetween sperm capacitation and chemotaxis.

MATERIALS AND METHODS

Spermatozoa. Human ejaculates were collected by mastur-bation from normal healthy donors and washed, as described(2), with Biggers, Whitten, and Whittingham medium (14)supplemented with Hepes (1 mM, pH 7.4) (this solution ishereafter referred to as BWW). To promote capacitation, thespermatozoa were resuspended in BWW to a concentration of108 cells per ml and incubated for 2 h (unless indicatedotherwise) at 35°C or room temperature (there was no statis-tically significant difference in the percentage of capacitatedspermatozoa between the two temperatures).Sperm Separation. Separation of spermatozoa according to

their chemotactic activity was carried out by a separationapparatus as described earlier (7). Briefly, the apparatusconsisted of two chambers connected by a tube: one chambercontained spermatozoa and the other contained an attractant.The active fraction of follicular fluid which remains afterprecipitation by 90% acetone (2), diluted 1:1000, was used asan attractant. The apparatus was incubated for 1 h ("theseparation time") during which net movement of spermatozoato the attractant-containing chamber was observed. As shownearlier (7), the population of spermatozoa that migrated to theattractant-containing chamber was enriched with chemotacticspermatozoa; the population of spermatozoa that remained inthe original chamber and did not follow the attractant wasdepleted of chemotactic spermatozoa. The sperm suspensionsfrom both chambers were collected and washed twice bycentrifugation (120 x g, 15 min). The original population wassimilarly washed as a control.Chemotaxis Assays. The responsiveness of spermatozoa to

the attractant was determined, as described (2), in a sealedchamber (15) in which the spermatozoa choose between a wellcontaining the attractant and a well containing BWW. Thesperm concentration used in the chemotaxis assays was 1-5 x108 cells per ml.Determination of the Fraction of Capacitated Spermatozoa.

Capacitated spermatozoa were identified according to theirability to undergo the acrosome reaction. In practice, wedetermined the number of capacitated spermatozoa from thedifference between the number of acrosome-reacted cellsbefore and after stimulation with phorbol 12-myristate 13-acetate (PMA; Sigma), which is known to induce the acrosomereaction only in capacitated spermatozoa (16, 17). For stim-ulation, spermatozoa (108 cells per ml) were incubated withPMA (5 ,uM from a stock solution in dimethyl sulfoxide; the

Abbreviations: BWW, Biggers, Whitten and Whittingham mediumsupplemented with Hepes; PKC, protein kinase C; PMA, phorbol12-myristate 13-acetate.tTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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final concentration of dimethyl sulfoxide in the sperm samplesdid not exceed 0.5%) for 30 min at 35°C. Acrosome-reactedspermatozoa were identified by the acrosomal marker fluo-rescein isothiocyanate-conjugated Pisum sativum agglutinin(Sigma), using a modification of previously reported stainingmethods (18-20). Briefly, spermatozoa (45 Al of 108 cells perml) were washed (twice at 120 x g for 10 min at roomtemperature) with BWW lacking glucose and bovine serumalbumin, cooled on ice for 10 min, and then permeabilized by95% methanol for 30 s. The acrosomal marker, conjugated P.sativum agglutinin (5 ,A of 1 mg/ml), was added, and themixture was incubated on ice for an additional 30 min, followedby washing with the same medium and fixation with 2%(vol/vol) formaldehyde for 30 min on ice. An aliquot of thesperm suspension was smeared on a microscope slide, airdried, and then covered with a mounting fluid (Mowiol,Hoechst) and a cover glass. The slides were inspected using aZeiss Axiovert 35 microscope equipped with a x 100 oilobjective. Two fluorescence patterns were observed: a uniformfluorescence over the whole acrosomal region and fluores-cence restricted to the equatorial acrosomal region, indicativeof acrosome-intact and acrosome-reacted spermatozoa, re-spectively (19, 20). Cells were inspected by an independentobserver, who categorized 100-200 cells on each slide.

Statistical Analysis. STATVIEW (BrainPower, Calabasas,CA) software was used for statistical calculations. Analyses ofintergroup variance were performed by ANOVA repeatedmeasures. Significance at 99% was calculated according to theFisher protected least significance difference test.

RESULTSTo determine whether or not sperm capacitation and chemo-taxis are correlated, we separated human spermatozoa (whichhad been preincubated under capacitating conditions) accord-ing to their chemotactic activity and measured the percentageof capacitated spermatozoa (i.e., the incremental increase inthe percentage of acrosome-reacted spermatozoa as a result ofPMA stimulation) in each of the separated subpopulations aswell as in the total population. The separation was carried out

by collecting the spermatozoa that migrated to an attractant-containing chamber. As a control for random separation, theseparation procedure was repeated with BWW in place of theattractant. As an additional control, we studied a total spermpopulation that had been incubated with (but not separatedby) the attractant. The level of capacitated spermatozoa in thechemotactically enriched subpopulation was 13 ± 4 (mean +SD of the enriched to depleted ratio of five experiments) timeshigher than that in the remaining, chemotactically depletedsubpopulation (Fig. 1A). In contrast, the percentages ofcapacitated spermatozoa were similar in the subpopulationsand in the total population when the separation had beencarried out substituting BWW for the attractant (Fig. 1B). Theincubation with the attractant did not affect the percentage ofcapacitated spermatozoa (Fig. 1C), indicating that the in-creased percentage of capacitated spermatozoa in the chemo-tactically enriched subpopulation in Fig. 1A ("Passed" bar) isthe result of chemotactic attraction and not the result of theincubation with the attractant during the separation. Theseresults indicate that sperm separation by chemotaxis alsoresults in separation according to capacitation. Similar to thelevel of chemotactic cells in a total sperm population (7), thelevel of capacitated spermatozoa varied from 2% to 14%depending on the sperm sample (the mean ± SD of 24 spermsamples from five donors was 7.7% ± 4.1%; the interassayvariation was <2%). Taken together, the observations imply acorrelation between capacitation and chemotaxis.We have previously shown that, after separation, the che-

motactically enriched sperm subpopulation loses its chemo-tactic responsiveness >50-100 min after the separation, and,at the same time, the chemotactically depleted subpopulationacquires such responsiveness, indicative of turnover of chemo-tactic spermatozoa (7). To further investigate the possiblerelationship between chemotaxis and capacitation, we deter-mined the level of capacitated spermatozoa in the separatedsubpopulations for extended periods of time. As shown in Fig.2, the percentage of capacitated spermatozoa in the chemo-tactically enriched subpopulation remained high for at least 50min after the separation but, within an additional 50 min,decreased by over 6-fold. Thereafter, the level of capacitated

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FIG. 1. Percentage of capacitated cells in sperm populations. Unintentionally, in this particular experiment, the sperm donors had lower levelsof capacitated spermatozoa than the average of all the donors used for the 24 sperm samples mentioned earlier in the text. The percentage ofcapacitated spermatozoa was measured immediately after the washing step that follows the separation and was calculated from the differencebetween the number of acrosome-reacted cells before and after stimulation with PMA (i.e., the level of capacitated spermatozoa is the percentageof spermatozoa that underwent PMA-stimulated acrosome reaction on top of the basal level of acrosome-reacted spermatozoa). The total numberof spermatozoa was considered as 100%. Prior to PMA stimulation, the level of acrosome-reacted spermatozoa varied in the sperm samples from3% to 24% (the mean SD of 24 sperm samples from five donors was 8.7% t 4.7%). (A) Sperm subpopulations obtained by separation with theattractant (diluted 1:1000). Passed, chemotactically enriched; Remained, chemotactically depleted. The results are the mean SD of fiveexperiments (5000 cells in total) performed with three sperm donors. The differences between the subpopulations separated by chemotaxis andbetween them and the total sperm population were significant at 99% according to the Fisher test. The levels of acrosome-reacted spermatozoaprior to PMA stimulation in this specific experiment were 7.0% ± 1.9%, 7.4% 3.8%, and 7.8% 3.0% for the passed, remained, and totalpopulations, respectively. The level of acrosome-reacted spermatozoa did not change significantly as a result of the separation procedure andwashing; immediately after seminal fluid removal, the level was 7.3% 3.1%. (B) Sperm subpopulations obtained, as a control for randomseparation, substituting BWW for the attractant. The results are the mean ± SD of three experiments (1800 cells in total) performed with threesperm donors. (C) The total sperm population after incubation with the attractant (diluted 1:1000) for the indicated periods of time and washingit off (twice for 10 min at 120 x g). Control, fraction of capacitated spermatozoa in the total sperm population after 60 min incubation with BWW.The results are the mean ± SD of five experiments (6000 cells in total) performed with four sperm donors.

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FIG. 2. Time-dependent changes in the percentage of capacitatedspermatozoa in each sperm population. The results are the mean ± SDof three experiments (7200 cells in total) performed with three spermdonors and an attractant from a single follicular fluid. The percentageof capacitated spermatozoa was calculated as described in the legendto Fig. 1. The difference between the results at 50 and 100 min werestatistically significant at 99% (according to the Fisher test) in thechemotactically enriched (Enriched) and chemotactically depleted(Depleted) subpopulations. The levels of acrosome-reacted sperma-tozoa prior to PMA stimulation were, at all time points, 7.5% ± 1.6%,7.8% ± 3.0%, and 7.0% ± 2.5% for the enriched, depleted, and totalsubpopulations, respectively. The total number of spermatozoa wasconsidered as 100%.

spermatozoa remained low for as long as the spermatozoawere motile and alive, indicating that a spermatozoon canbecome capacitated only once during its lifetime. During thesame time window of >50-100 min after the separation, thepercentage of capacitated spermatozoa in the chemotacticallydepleted subpopulation increased, reaching the value of thetotal population, and remained at this value thereafter. Thepercentage of capacitated cells in the total sperm populationremained unchanged throughout the experiment.The above results have demonstrated that a sperm popula-

tion depleted of chemotactic cells is not capacitated. As a finalexamination of this relationship, we carried out the inverseexperiment. We depleted, by PMA stimulation, a total spermpopulation of capacitated cells and measured its chemotacticactivity. A control portion of the same sperm population wassimilarly treated without PMA. An additional control samplewas incubated with PMA for 3.75 h. As shown in Fig. 3, thePMA-stimulated sperm population had no chemotactic activ-ity for at least 40 min after washing off the PMA, unlike the"No PMA" control population, which retained the activity.This indicates that, as expected, a sperm population depletedof capacitated cells is unable to respond chemotactically.When we remeasured the chemotactic activity 2-3 h afterPMA removal, the activity was fully restored (Fig. 3), in linewith the notion of turnover. The control sample, which wasincubated with PMA for 3.75 h, had no chemotactic activity(data not shown). These results, taken together with the otherobservations made in this study, strongly suggest that capaci-tated spermatozoa, and only capacitated spermatozoa, arechemotactically responsive to follicular factors.

In view of the transient nature of the capacitated state, wewished to estimate its life span. The lower limit of the span is>50 min, as spermatozoa remain capacitated for at least 50min after the separation (Fig. 2). To estimate the upper limit,we first determined more accurately the time period duringwhich spermatozoa become capacitated. For this purpose, westudied the kinetics of sperm capacitation in a total spermpopulation after washing off its seminal fluid [seminal fluidremoval initiates capacitation (10)]. As shown in Fig. 4, thetiming of capacitation and the percentage of capacitatedspermatozoa varied extensively between sperm samples, evenbetween samples of a single donor. Spermatozoa becamecapacitated between 40 and 130 min, depending on the sample.

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FIG. 3. Time-dependent chemotactic activity of a sperm popula-tion depleted of capacitated spermatozoa. A sperm population wasdepleted of capacitated spermatozoa by stimulating the acrosomereaction with 5 ,uM PMA for 30 min at 35°C. The spermatozoa werethen washed twice with BWW (10 min at 120 x g, room temperature)and tested for chemotactic activity. The end of the last centrifugationwas considered as the time of PMA removal (i.e., as zero time). As acontrol, spermatozoa were similarly incubated with BWW instead ofPMA and examined 5-10 min after the washings ("No PMA"). Therelative chemotactic activity was calculated as the ratio of the maximalsperm densities near the attractant-containing well and BWW-containing well, integrated over the whole observation period. Thedifference between the 20-40 min column and each of the other twocolumns was significant at 99% according to the Fisher test. The 20-40min column and the control column each represent the mean ± SD ofseven different experiments (three sperm donors and an attractantfrom two follicular fluids were used). In five experiments, the che-motactic activity was measured both at 20-40 min and 2-3 h afterPMA removal.

This suggests that the upper limit of the capacitated life spanis <240 min: 120-min incubation for capacitation, minus 40min for the minimal time required for capacitation (accordingto Fig. 4), plus 60 min separation time, plus <100 min duringwhich the cells may remain capacitated (according to Fig. 2).It should be noted that, in most cases, once established, asteady state of the percentage of capacitated spermatozoa wasmaintained for as long as we measured (30 h; data not shown).

DISCUSSIONAlthough the phenomenon of capacitation has been known formore than 40 years (21, 22), its molecular mechanism is stillobscure. This study indicates that the capacitated state inhuman spermatozoa is transient, it demonstrates the occur-

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FIG. 4. The kinetics of capacitation in a total sperm population.After seminal fluid removal, a total sperm population was incubatedat 35°C, and, at the indicated time points, samples were taken and thepercentage of capacitated spermatozoa was determined as describedin the legend to Fig. 1. The total number of spermatozoa wasconsidered as 100%. The different symbols represent three differentsperm donors. The differently formatted lines represent differentejaculates from the same donor.

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rence of a continuous process of replacement of capacitatedspermatozoa within a sperm population, and it points to a

correlation between sperm capacitation and chemotaxis to-ward follicular factors. These findings are discussed below.We identified capacitated spermatozoa by their ability to

undergo the acrosome reaction. Bielfeld et al. (23) recentlyquestioned this criterion on the basis of their finding thatspermatozoa that had been incubated in an albumin-freemedium and considered noncapacitated were able to undergothe acrosome reaction. However, the presumption that sper-

matozoa suspended in an albumin-free medium were nonca-pacitated did not take into account earlier findings (10) thatthere is no particular component in sperm-capacitating me-

dium that is absolutely necessary for capacitation. Indeed, veryrecently, human spermatozoa incubated in an albumin-freemedium under the conditions of Bielfeld et al. were demon-strated to be capacitated (A.C.-D., I.T.-K., and M.E., unpub-lished results), indicating that, after all, spermatozoa must becapacitated for undergoing the acrosome reaction. We usedPMA, a known activator of protein kinase C (PKC), to inducethe acrosome reaction. The use of PMA as an activator of theacrosome reaction is well established (16, 17, 24, 25). PKC wasidentified and localized in human sperm (26), and its involve-ment in the acrosome reaction has been demonstrated by a

number of groups (e.g., refs. 17, 24, 27, and 28). Recently PKCwas even demonstrated to be involved in an acrosome reactioninduced by the zona pellucida-the physiological activator ofthe acrosome reaction (25). Other functional assays for ca-

pacitation that measure the sperm-fertilizing potential (e.g.,zona pellucida binding, egg penetration, and oocyte fusionassays) were not adequate for investigating the turnover pro-cesses of this study, because these assays require prolongedincubation periods (several hours) with the spermatozoa (29)[even though the fertilization itself requires <1 h (30)].

This study suggests that spermatozoa acquire their chemo-tactic responsiveness as part of the capacitation process andlose this responsiveness when the capacitated state is termi-nated. This conclusion is based on the following observations:(i) The percentage of capacitated cells in the total spermpopulation (2-14%) is similar to the percentage of chemotacticspermatozoa (2-12%). (ii) A subpopulation enriched withchemotactic spermatozoa is also enriched with capacitatedspermatozoa, and vice versa; a subpopulation depleted ofchemotactic spermatozoa is depleted of capacitated sperma-tozoa (Fig. 1). (iii) Chemotactic responsiveness of a totalsperm population is lost upon intentional depletion of capac-itated spermatozoa; such responsiveness is regained afterfurther incubation under capacitating conditions (Fig. 3). (iv)Both the capacitated state and the chemotactic responsivenessare temporary and appear only once in the sperm's lifetime,and there is synchrony between them (Fig. 2 and ref. 7). Eventhough each of these observations alone is not a direct proof forthe linkage between capacitation and chemotaxis (a directproof can only be obtained from studying capacitation andchemotaxis of individual spermatozoa throughout their lifespan- an apparently impossible mission by current tech-niques), all the observations taken together strongly suggestthat the linkage does exist. In other words, the linkage betweencapacitation and chemotaxis relies on the similar percentages,kinetics, and turnover of capacitated and chemotactic sper-matozoa and on the fact that deliberate depletion of capaci-tated spermatozoa results in total loss of chemotaxis, and, viceversa, depletion of chemotactic spermatozoa results in deple-tion of capacitated spermatozoa.The observation that the capacitated state is temporary

apparently contradicts a common belief that a spermatozoonremains capacitated until stimulated to undergo the acrosomereaction. Our observations suggest that the capacitated stateexists for only >50 min to <4 h. If, during this period, thecapacitated spermatozoon encounters the zona pellucida (or is

stimulated in vitro by other means), it will undergo theacrosome reaction. If not, it will stop being capacitated (i.e., itwill not be able to undergo the acrosome reaction upon

stimulation) and will be "out of the game." The nature of thepostcapacitated state is not known; it is, however, acrosome

intact as evident from the observation that the decrease in thepercentage of capacitated spermatozoa (e.g., between 50 and100 min in Fig. 2) did not result in a parallel increase in thepercentage of acrosome-reacted spermatozoa. Our finding ofturnover of capacitated spermatozoa is in line with the turn-over of hyperactivated§ spermatozoa (33) and with earliersuggestions of asynchrony (34) and turnover (3) of capacita-tion.Our finding that the fraction of capacitated cells in a sperm

population is low is in line with published findings that only a

small proportion of a sperm population is able to fertilize theegg in vivo (35), to undergo the zona pellucida-stimulatedacrosome reaction (36), to bind mannose [a suggested molec-ular marker of human sperm capacitation (9, 37)] undercapacitating conditions (38, 39), or to be hyperactivated (40-43). There are, however, quantitative differences between thefindings. Since capacitation is composed of many processes

(for recent reviews, see refs. 8-10 and 12), these differencesmay be due to different stages of capacitation measured by thevarious techniques. The technique that assays the ability ofcells to undergo the acrosome reaction (as in this study or inref. 36) measures, by definition, one of the final stages ofcapacitation.What is the in vivo physiological significance of the in vitro

observations made in this study? Our observations, whichimply that only capacitated spermatozoa respond chemotac-tically to an attractant secreted from the egg or its surroundingcells, suggest that, in vivo, the role of human sperm chemotaxisis not to direct as many spermatozoa as possible to the egg butrather to recruit a selective population of spermatozoa (i.e.,capacitated spermatozoa) for fertilizing the egg. The role ofthe observed turnover of capacitated spermatozoa is possiblyto ensure availability of capacitated spermatozoa for an ex-tended period of time in spite of the short life span of thecapacitated state. This proposed role, which was suggestedearlier as a hypothesis (3), is in line with published observa-tions reviewed elsewhere (3). To conclude, sperm chemotaxisin humans seems to provide an efficient way for selectingfertilizing spermatozoa and thereby increasing the chances ofan egg to be fertilized.

§Hyperactivation is a motility pattern associated with sperm capaci-tation (31, 32).

We thank Drs. R. Shalgi, L. Yogev, R. Gamzu, and H. Yavetz forhelpful discussions and for helping us to assess the efficacy offunctional fertilization tests for the needs of this study. This study wassupported by grants from the Israel Ministry of Science and the Arts,the Israel Ministry of Health, and from the Mordoch Migan deSalonique Foundation.

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