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

Sperm capacitation in humans is transient and correlates with chemotactic responsiveness to follicular factors (chemotaxis/fertilization/mammalian reproduction//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%) of reviews, see refs. 8-13)-seemed a reasonable possibility (3, a sperm population can respond by chemoattraction to fol- 7). This study investigates this possibility and finds a linkage licular factors. This recent finding led to the hypothesis that between sperm capacitation and chemotaxis. chemotaxis provides a mechanism for selective recruitment of functionally mature spermatozoa (i.e., of capacitated sperma- tozoa, which possess the potential to undergo the acrosome MATERIALS AND METHODS reaction and fertilize the egg). This study aimed to examine Spermatozoa. Human ejaculates were collected by mastur- this possibility. Capacitated spermatozoa were identified by bation from normal healthy donors and washed, as described their ability to undergo the acrosome reaction upon stimula- (2), with Biggers, Whitten, and Whittingham medium (14) tion with phorbol 12-myristate 13-acetate. Under capacitating supplemented with Hepes (1 mM, pH 7.4) (this solution is conditions, only a small portion (2-14%) of the spermatozoa hereafter referred to as BWW). To promote capacitation, the were found to be capacitated. The spermatozoa were then spermatozoa were resuspended in BWW to a concentration of separated according to their chemotactic activity, which re- 108 cells per ml and incubated for 2 h (unless indicated sulted in a subpopulation enriched with chemotactically re- otherwise) at 35°C or room temperature (there was no statis- sponsive spermatozoa and a subpopulation depleted of such tically significant difference in the percentage of capacitated spermatozoa. The level of capacitated spermatozoa in the spermatozoa between the two temperatures). former was -13-fold higher than that in the latter. The Sperm Separation. Separation of spermatozoa according to capacitated state was temporary (50 min < life span < 240 their chemotactic activity was carried out by a separation min), and it was synchronous with the chemotactic activity. A apparatus as described earlier (7). Briefly, the apparatus continuous process of replacement of capacitated/chemotac- consisted of two chambers connected by a tube: one chamber tic spermatozoa within a sperm population was observed. contained spermatozoa and the other contained an attractant. Spermatozoa that had stopped being capacitated did not The active fraction of follicular fluid which remains after become capacitated again, which indicates that the capaci- precipitation by 90% acetone (2), diluted 1:1000, was used as tated state is acquired only once in a sperm's lifetime. A total an attractant. The apparatus was incubated for 1 h ("the sperm population depleted of capacitated spermatozoa stopped separation time") during which net movement of spermatozoa being chemotactic. When capacitated spermatozoa reappeared, to the attractant-containing chamber was observed. As shown chemotactic activity was restored. These observations suggest earlier (7), the population of spermatozoa that migrated to the that spermatozoa acquire their chemotactic responsiveness as attractant-containing chamber was enriched with chemotactic part of the capacitation process and lose this responsiveness spermatozoa; the population of spermatozoa that remained in when the capacitated state is terminated. We suggest that the role the original chamber and did not follow the attractant was ofsperm chemotaxis in sperm-egg interaction in vivo may indeed depleted of chemotactic spermatozoa. The sperm suspensions be selective recruitment of capacitated spermatozoa for fertiliz- from both chambers were collected and washed twice by ing the egg. centrifugation (120 x g, 15 min). The original population was similarly washed as a control. Human spermatozoa are attracted to follicular factors in vitro, Chemotaxis Assays. The responsiveness of spermatozoa to and the attraction is correlated with egg fertilizability (1). The the attractant was determined, as described (2), in a sealed attraction results from chemotaxis and is accompanied by chamber (15) in which the spermatozoa choose between a well speed enhancement (ref. 2; for reviews, see refs. 3 and 4). containing the attractant and a well containing BWW. The Unlike the case of species with external fertilization in which sperm concentration used in the chemotaxis assays was 1-5 x most, if not all, of the sperm population is chemotactically 108 cells per ml. responsive (for reviews, see refs. 5 and 6), in humans only a Determination ofthe Fraction of Capacitated Spermatozoa. small fraction of the sperm population (2-12%) is chemotac- Capacitated spermatozoa were identified according to their tically responsive at any given time (7). The identity of the ability to undergo the acrosome reaction. In practice, we responsive spermatozoa in humans changes with time by determined the number of capacitated spermatozoa from the turnover: chemotactic spermatozoa lose their activity while difference between the number of acrosome-reacted cells others acquire it (7). This raised the possibility that sperma- before and after stimulation with phorbol 12-myristate 13- tozoa are selectively chemotactic only at a certain physiological acetate (PMA; Sigma), which is known to induce the acrosome stage. The capacitated stage-i.e., the stage at which sperma- reaction only in capacitated spermatozoa (16, 17). For stim- tozoa possess the potential to undergo the acrosome reaction ulation, spermatozoa (108 cells per ml) were incubated with (a release of proteolytic enabling sperm penetration PMA (5 ,uM from a stock solution in dimethyl sulfoxide; the through the egg coat) and to fertilize the egg (for recent Abbreviations: BWW, Biggers, Whitten and Whittingham medium The publication costs of this article were defrayed in part by page charge supplemented with Hepes; PKC, protein kinase C; PMA, phorbol payment. This article must therefore be hereby marked "advertisement" in 12-myristate 13-acetate. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 11039 Downloaded by guest on October 1, 2021 11040 Physiology: Cohen-Dayag et al. Proc. Natl. Acad. Sci. USA 92 (1995) final concentration of dimethyl sulfoxide in the sperm samples by collecting the spermatozoa that migrated to an attractant- did not exceed 0.5%) for 30 min at 35°C. Acrosome-reacted containing chamber. As a control for random separation, the spermatozoa were identified by the acrosomal marker fluo- separation procedure was repeated with BWW in place of the rescein isothiocyanate-conjugated Pisum sativum agglutinin attractant. As an additional control, we studied a total sperm (Sigma), using a modification of previously reported staining population that had been incubated with (but not separated methods (18-20). Briefly, spermatozoa (45 Al of 108 cells per by) the attractant. The level of capacitated spermatozoa in the ml) were washed (twice at 120 x g for 10 min at room chemotactically enriched subpopulation was 13 ± 4 (mean + temperature) with BWW lacking glucose and bovine serum SD of the enriched to depleted ratio of five experiments) times , cooled on ice for 10 min, and then permeabilized by higher than that in the remaining, chemotactically depleted 95% methanol for 30 s. The acrosomal marker, conjugated P. subpopulation (Fig. 1A). In contrast, the percentages of sativum agglutinin (5 ,A of 1 mg/ml), was added, and the capacitated spermatozoa were similar in the subpopulations mixture was incubated on ice for an additional 30 min, followed and in the total population when the separation had been by washing with the same medium and fixation with 2% carried out substituting BWW for the attractant (Fig. 1B). The (vol/vol) formaldehyde for 30 min on ice. An aliquot of the incubation with the attractant did not affect the percentage of sperm suspension was smeared on a microscope slide, air capacitated spermatozoa (Fig. 1C), indicating that the in- dried, and then covered with a mounting fluid (Mowiol, creased percentage of capacitated spermatozoa in the chemo- Hoechst) and a cover glass. The slides were inspected using a tactically enriched subpopulation in Fig. 1A ("Passed" bar) is Zeiss Axiovert 35 microscope equipped with a x 100 oil the result of chemotactic attraction and not the result of the objective. Two fluorescence patterns were observed: a uniform incubation with the attractant during the separation. These fluorescence over the whole acrosomal region and fluores- results indicate that sperm separation by chemotaxis also cence restricted to the equatorial acrosomal region, indicative results in separation according to capacitation. Similar to the of acrosome-intact and acrosome-reacted spermatozoa, re- level of chemotactic cells in a total sperm population (7), the spectively (19, 20). Cells were inspected by an independent level of capacitated spermatozoa varied from 2% to 14% observer, who categorized 100-200 cells on each slide. depending on the sperm sample (the mean ± SD of 24 sperm Statistical Analysis. STATVIEW (BrainPower, Calabasas, samples from five donors was 7.7% ± 4.1%; the interassay CA) software was used for statistical calculations. Analyses of variation was <2%). Taken together, the observations imply a intergroup variance were performed by ANOVA repeated correlation between capacitation and chemotaxis. measures. Significance at 99% was calculated according to the We have previously shown that, after separation, the che- Fisher protected least significance difference test. motactically enriched sperm subpopulation loses its chemo- tactic responsiveness >50-100 min after the separation, and, at the same time, the chemotactically depleted subpopulation RESULTS acquires such responsiveness, indicative of turnover of chemo- To determine whether or not sperm capacitation and chemo- tactic spermatozoa (7). To further investigate the possible taxis are correlated, we separated human spermatozoa (which relationship between chemotaxis and capacitation, we deter- had been preincubated under capacitating conditions) accord- mined the level of capacitated spermatozoa in the separated ing to their chemotactic activity and measured the percentage subpopulations for extended periods of time. As shown in Fig. of capacitated spermatozoa (i.e., the incremental increase in 2, the percentage of capacitated spermatozoa in the chemo- the percentage of acrosome-reacted spermatozoa as a result of tactically enriched subpopulation remained high for at least 50 PMA stimulation) in each of the separated subpopulations as min after the separation but, within an additional 50 min, well as in the total population. The separation was carried out decreased by over 6-fold. Thereafter, the level of capacitated

25 25 - B 25 20 < 20 - 20 -

i J _ 15 15 - 15 -

L- 10 10 10 - 5()- -T- T T7- c: 5 C-

0 0-- Passed Remainecd Total Passed Remained Total 0 15 30 45 60 Control Population Population Incubation time, min

FIG. 1. Percentage of capacitated cells in sperm populations. Unintentionally, in this particular experiment, the sperm donors had lower levels of capacitated spermatozoa than the average of all the donors used for the 24 sperm samples mentioned earlier in the text. The percentage of capacitated spermatozoa was measured immediately after the washing step that follows the separation and was calculated from the difference between the number of acrosome-reacted cells before and after stimulation with PMA (i.e., the level of capacitated spermatozoa is the percentage of spermatozoa that underwent PMA-stimulated acrosome reaction on top of the basal level of acrosome-reacted spermatozoa). The total number of spermatozoa was considered as 100%. Prior to PMA stimulation, the level of acrosome-reacted spermatozoa varied in the sperm samples from 3% 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 the attractant (diluted 1:1000). Passed, chemotactically enriched; Remained, chemotactically depleted. The results are the mean SD of five experiments (5000 cells in total) performed with three sperm donors. The differences between the subpopulations separated by chemotaxis and between them and the total sperm population were significant at 99% according to the Fisher test. The levels of acrosome-reacted spermatozoa prior 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 total populations, respectively. The level of acrosome-reacted spermatozoa did not change significantly as a result of the separation procedure and washing; immediately after seminal fluid removal, the level was 7.3% 3.1%. (B) Sperm subpopulations obtained, as a control for random separation, substituting BWW for the attractant. The results are the mean ± SD of three experiments (1800 cells in total) performed with three sperm donors. (C) The total sperm population after incubation with the attractant (diluted 1:1000) for the indicated periods of time and washing it 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. Downloaded by guest on October 1, 2021 Physiology: Cohen-Dayag et al. Proc. Natl. Acad. Sci. USA 92 (1995) 11041

30 1.50 ;8 * O min _ ma ._: 0 F 50 min N u T 0 20 T~~~~~~E 10lO min _ _~~~~~~200 min C) L E 1.25 a CO u U 10

c;5 Icu 0 1.00 -_I E;nriched Depleted Total No PMA 20-40 120-180 Population Time after PMA removal, min FIG. 2. Time-dependent changes in the percentage of capacitated FIG. 3. Time-dependent chemotactic activity of a sperm popula- spermatozoa in each sperm population. The results are the mean ± SD tion depleted of capacitated spermatozoa. A sperm population was of three experiments (7200 cells in total) performed with three sperm depleted of capacitated spermatozoa by stimulating the acrosome donors and an attractant from a single follicular fluid. The percentage reaction with 5 ,uM PMA for 30 min at 35°C. The spermatozoa were of capacitated spermatozoa was calculated as described in the legend then washed twice with BWW (10 min at 120 x g, room temperature) to Fig. 1. The difference between the results at 50 and 100 min were and tested for chemotactic activity. The end of the last centrifugation statistically significant at 99% (according to the Fisher test) in the was considered as the time of PMA removal (i.e., as zero time). As a chemotactically enriched (Enriched) and chemotactically depleted control, spermatozoa were similarly incubated with BWW instead of (Depleted) subpopulations. The levels of acrosome-reacted sperma- PMA and examined 5-10 min after the washings ("No PMA"). The tozoa prior to PMA stimulation were, at all time points, 7.5% ± 1.6%, relative chemotactic activity was calculated as the ratio of the maximal 7.8% ± 3.0%, and 7.0% ± 2.5% for the enriched, depleted, and total sperm densities near the attractant-containing well and BWW- subpopulations, respectively. The total number of spermatozoa was containing well, integrated over the whole observation period. The considered as 100%. difference between the 20-40 min column and each of the other two columns was significant at 99% according to the Fisher test. The 20-40 spermatozoa remained low for as long as the spermatozoa min column and the control column each represent the mean ± SD of were motile and alive, indicating that a can seven different experiments (three sperm donors and an attractant its lifetime. the from two follicular fluids were used). In five experiments, the che- become capacitated only once during During motactic activity was measured both at 20-40 min and 2-3 h after same time window of >50-100 min after the separation, the PMA removal. percentage of capacitated spermatozoa in the chemotactically depleted subpopulation increased, reaching the value of the This suggests that the upper limit of the capacitated life span total population, and remained at this value thereafter. The is <240 min: 120-min incubation for capacitation, minus 40 percentage of capacitated cells in the total sperm population remained unchanged throughout the experiment. min for the minimal time required for capacitation (according The above results have demonstrated that a sperm popula- to Fig. 4), plus 60 min separation time, plus <100 min during tion depleted of chemotactic cells is not capacitated. As a final which the cells may remain capacitated (according to Fig. 2). examination of this relationship, we carried out the inverse It should be noted that, in most cases, once established, a experiment. We depleted, by PMA stimulation, a total sperm steady state of the percentage of capacitated spermatozoa was population of capacitated cells and measured its chemotactic maintained for as long as we measured (30 h; data not shown). activity. A control portion of the same sperm population was similarly treated without PMA. An additional control sample DISCUSSION was incubated with PMA for 3.75 h. As shown in Fig. 3, the PMA-stimulated sperm population had no chemotactic activ- Although the phenomenon of capacitation has been known for ity for at least 40 min after washing off the PMA, unlike the more than 40 years (21, 22), its molecular mechanism is still "No PMA" control population, which retained the activity. obscure. This study indicates that the capacitated state in This indicates that, as expected, a sperm population depleted human spermatozoa is transient, it demonstrates the occur- of capacitated cells is unable to respond chemotactically. When we remeasured the chemotactic activity 2-3 h after 20 PMA removal, the activity was fully restored (Fig. 3), in line with the notion of turnover. The control sample, which was incubated with PMA for 3.75 h, had no chemotactic activity 0 15- (data not shown). These results, taken together with the other observations made in this study, strongly suggest that capaci- tated spermatozoa, and only capacitated spermatozoa, are 00'0 chemotactically responsive to follicular factors. In view of the transient nature of the capacitated state, we .55 wished to estimate its life span. The lower limit of the span is u >50 min, as spermatozoa remain capacitated for at least 50 U0 min after the separation (Fig. 2). To estimate the upper limit, 0 50 100 150 200 250 we first determined more accurately the time period during Time, mi which spermatozoa become capacitated. For this purpose, we studied the kinetics of sperm capacitation in a total sperm FIG. 4. The kinetics of capacitation in a total sperm population. after off its seminal fluid [seminal fluid After seminal fluid removal, a total sperm population was incubated population washing at 35°C, and, at the indicated time points, samples were taken and the removal initiates capacitation (10)]. As shown in Fig. 4, the percentage of capacitated spermatozoa was determined as described timing of capacitation and the percentage of capacitated in the legend to Fig. 1. The total number of spermatozoa was spermatozoa varied extensively between sperm samples, even considered as 100%. The different symbols represent three different between samples of a single donor. Spermatozoa became sperm donors. The differently formatted lines represent different capacitated between 40 and 130 min, depending on the sample. ejaculates from the same donor. Downloaded by guest on October 1, 2021 11042 Physiology: Cohen-Dayag et al. Proc. Natl. Acad. Sci. USA 92 (1995)

rence of a continuous process of replacement of capacitated stimulated in vitro by other means), it will undergo the spermatozoa within a sperm population, and it points to a acrosome reaction. If not, it will stop being capacitated (i.e., it correlation between sperm capacitation and chemotaxis to- will not be able to undergo the acrosome reaction upon ward follicular factors. These findings are discussed below. stimulation) and will be "out of the game." The nature of the We identified capacitated spermatozoa by their ability to postcapacitated state is not known; it is, however, acrosome undergo the acrosome reaction. Bielfeld et al. (23) recently intact as evident from the observation that the decrease in the questioned this criterion on the basis of their finding that percentage of capacitated spermatozoa (e.g., between 50 and spermatozoa that had been incubated in an albumin-free 100 min in Fig. 2) did not result in a parallel increase in the medium and considered noncapacitated were able to undergo percentage of acrosome-reacted spermatozoa. Our finding of the acrosome reaction. However, the presumption that sper- turnover of capacitated spermatozoa is in line with the turn- matozoa suspended in an albumin-free medium were nonca- over of hyperactivated§ spermatozoa (33) and with earlier pacitated did not take into account earlier findings (10) that suggestions of asynchrony (34) and turnover (3) of capacita- there is no particular component in sperm-capacitating me- tion. dium that is absolutely necessary for capacitation. Indeed, very Our finding that the fraction of capacitated cells in a sperm recently, human spermatozoa incubated in an albumin-free population is low is in line with published findings that only a medium under the conditions of Bielfeld et al. were demon- small proportion of a sperm population is able to fertilize the strated to be capacitated (A.C.-D., I.T.-K., and M.E., unpub- egg in vivo (35), to undergo the -stimulated lished results), indicating that, after all, spermatozoa must be acrosome reaction (36), to bind mannose [a suggested molec- capacitated for undergoing the acrosome reaction. We used ular marker of human sperm capacitation (9, 37)] under PMA, a known activator of protein kinase C (PKC), to induce capacitating conditions (38, 39), or to be hyperactivated (40- as an the acrosome reaction. The use of PMA activator of the 43). There are, however, quantitative differences between the acrosome was reaction is well established (16, 17, 24, 25). PKC findings. Since capacitation is composed of many processes and localized in human sperm (26), and its involve- identified (for recent reviews, see refs. 8-10 and 12), these differences ment in the acrosome reaction has been demonstrated by a may be due to different stages of capacitation measured by the number of groups (e.g., refs. 17, 24, 27, and 28). Recently PKC various techniques. The technique that assays the ability of was even demonstrated to be involved in an acrosome reaction cells to undergo the acrosome reaction (as in this study or in induced by the zona pellucida-the physiological activator of ref. 36) measures, by definition, one of the final stages of the acrosome reaction (25). Other functional assays for ca- capacitation. that measure the sperm-fertilizing potential (e.g., pacitation What is the in vivo physiological significance of the in vitro zona binding, egg penetration, and oocyte fusion pellucida observations made in this study? Our observations, which assays) were not adequate for investigating the turnover pro- spermatozoa respond chemotac- cesses of this study, because these assays require prolonged imply that only capacitated incubation periods (several hours) with the spermatozoa (29) tically to an attractant secreted from the egg or its surrounding [even though the fertilization itself requires <1 h (30)]. cells, suggest that, in vivo, the role of human This study suggests that spermatozoa acquire their chemo- is not to direct as many spermatozoa as possible to the egg but tactic responsiveness as part of the capacitation process and rather to recruit a selective population of spermatozoa (i.e., lose this responsiveness when the capacitated state is termi- capacitated spermatozoa) for fertilizing the egg. The role of nated. This conclusion is based on the following observations: the observed turnover of capacitated spermatozoa is possibly (i) The percentage of capacitated cells in the total sperm to ensure availability of capacitated spermatozoa for an ex- population (2-14%) is similar to the percentage ofchemotactic tended period of time in spite of the short life span of the spermatozoa (2-12%). (ii) A subpopulation enriched with capacitated state. This proposed role, which was suggested chemotactic spermatozoa is also enriched with capacitated earlier as a hypothesis (3), is in line with published observa- spermatozoa, and vice versa; a subpopulation depleted of tions reviewed elsewhere (3). To conclude, sperm chemotaxis chemotactic spermatozoa is depleted of capacitated sperma- in humans seems to provide an efficient way for selecting tozoa (Fig. 1). (iii) Chemotactic responsiveness of a total fertilizing spermatozoa and thereby increasing the chances of sperm population is lost upon intentional depletion of capac- an egg to be fertilized. itated spermatozoa; such responsiveness is regained after further incubation under capacitating conditions (Fig. 3). (iv) §Hyperactivation is a motility pattern associated with sperm capaci- Both the capacitated state and the chemotactic responsiveness tation (31, 32). are temporary and appear only once in the sperm's lifetime, and there is synchrony between them (Fig. 2 and ref. 7). Even We thank Drs. R. Shalgi, L. Yogev, R. Gamzu, and H. Yavetz for though each of these observations alone is not a direct proof for helpful discussions and for helping us to assess the efficacy of the linkage between capacitation and chemotaxis (a direct functional fertilization tests for the needs of this study. This study was proof can only be obtained from studying capacitation and supported by grants from the Israel Ministry of Science and the Arts, chemotaxis of individual spermatozoa throughout their life the Israel Ministry of Health, and from the Mordoch Migan de span- an apparently impossible mission by current tech- Salonique Foundation. niques), all the observations taken together strongly suggest that the linkage does exist. In other words, the linkage between 1. Ralt, D., Goldenberg, M., Fetterolf, P., Thompson, D., Dor, J., capacitation and chemotaxis relies on the similar percentages, Mashiach, S., Garbers, D. L. & Eisenbach, M. (1991) Proc. Natl. kinetics, and turnover of capacitated and chemotactic sper- Acad. Sci. USA 88, 2840-2844. matozoa and on the fact that deliberate depletion of capaci- 2. 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