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Human Sperm Chemotaxis: Is Progesterone a Chemoattractant?¹

^a Department of Biological Chemistry, The Weizmann Institute of Science, 76100 Rehovot, Israel ^b IVF Unit, Barzilai Medical Center, Ben-Gurion University of the Negev, 78306 Ashkelon, Israel ^c Department of Obstetrics and Gynecology, Sheba Medical Center, Tel Aviv University Medical School, 52621 Tel Hashomer, Israel

	ABSTRACT

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Follicular fluid (FF) induces sperm chemotaxis in human spermatozoa. Progesterone also causes sperm accumulation. However, sperm accumulation can be caused by chemotaxis, chemokinesis, and trapping of various kinds. It has been suggested that progesterone also induces chemotaxis in human spermatozoa. In view of the physiological significance of sperm chemotaxis in human fertilization and its potential clinical implications, it is important to determine unequivocally whether chemotaxis is induced by progesterone and, if so, whether progesterone in FF is the chemoattractant. To resolve these questions we looked for characteristic changes in the direction of sperm swimming toward pure progesterone as well as toward FF before and after progesterone removal. Progesterone caused sperm accumulation and hyperactivation-like motility, but it caused very few changes in the direction of sperm swimming that are characteristic of chemotaxis. Removal of progesterone (and other steroids) from FF by charcoal treatment abolished the sperm hyperactivation-like motility but not sperm chemotaxis. These results suggest that while progesterone might be a weak chemoattractant, it is not the major chemoattractant in FF. Progesterone probably causes human sperm accumulation mainly by inducing hyperactivation-like motility and, as a consequence, sperm trapping.

	INTRODUCTION

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Human sperm chemotaxis to follicular fluid (FF) in vitro appears to be well established (see [1–3] for reviews), and the presence of attractant in FF correlates well with successful fertilization of the egg [4]. In addition, as spermatozoa swim up the gradient of FF (or an active fraction thereof), their speed is enhanced and they acquire hyperactivation-like motility (i.e., wide amplitude and marked lateral displacement of the head [5, 6]) [7]. In a given sperm population, only 2–12% (depending on the sperm sample) of the spermatozoa are chemotactically responsive [8]. Only capacitated spermatozoa (those possessing the potential to undergo the acrosome reaction and fertilize the egg) are chemotactic. In vivo, human sperm chemotaxis most likely recruits capacitated spermatozoa to fertilize the egg [9]. This suggests that sperm chemotaxis has an essential role in fertilization [2] and that, as such, it may have potential clinical implications for treatment of infertility and for contraception [10].

The identity of the chemoattractant(s) present in human FF is not known (see [3] for a review), but it has been suggested recently that progesterone is the chemoattractant in FF [11, 12]. However, earlier results demonstrated the lack of correlation between sperm accumulation in FF and the level of progesterone in the FF [4], as well as between the HPLC reversed-phase elution profiles of the active fractions of FF and progesterone [13]. In view of the physiological significance of human sperm chemotaxis, it is essential to determine unequivocally whether progesterone is the chemoattractant in FF.

Sperm accumulation can be caused by chemotaxis, chemokinesis, and trapping of various kinds [7], but directed turning behavior is a clear-cut criterion of sperm chemotaxis [3, 7, 14–16]. We use this criterion to examine whether the sperm accumulation in the presence of progesterone [11] is a consequence of chemotaxis, and whether progesterone removal from FF results in loss of directed turning behavior.

	MATERIALS AND METHODS

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Chemicals

Progesterone, dextran (M_r 79 400), polyvinyl pyrrolidone (PVP, M_r 40 000), BSA (fraction V), dimethyl sulfoxide, pyruvic acid, and Hepes were obtained from Sigma Chemical Company (St. Louis, MO). All other chemicals, including acid-activated charcoal, were from Merck (Darmstadt, Germany). For the experiments, progesterone (from a stock solution of 10 mg/ml in ethanol) was diluted in an aqueous solution of PBS containing PVP (35 mg/ml), BSA (3.5%), and glucose (0.5%) [17]. This solution—without progesterone—is referred to hereafter as PVP buffer.

Spermatozoa

Human semen samples were obtained from healthy donors with normal sperm density, motility, and morphology (according to WHO guidelines of 1992) after 3 days of sexual abstinence. Samples were allowed to liquefy for 30–60 min at room temperature and then washed twice (300 x g, 10 min) with Biggers, Whitten, and Whittingham medium—95 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 20 mM sodium lactate, 5 mM glucose, 0.25 mM sodium pyruvate, 25 mM NaHCO₃, pH 7.4 [18], supplemented with Hepes (10 mM, pH 7.4) and 3 mg/ml BSA. (This solution is referred to hereafter as BWW). The supernatants were discarded, the final pellets were resuspended in BWW, and the sperm concentration was adjusted to 1–5 x 10⁸ cells/ml. Every sperm sample was analyzed for percentage motile cells using a Makler counting chamber (Sefi Medical Instruments Ltd., Haifa, Israel) and a computerized sperm analysis software program (ScanArray, Galai, Migdal Haemek, Israel). The sperm suspensions were then incubated under an atmosphere of 5% CO₂ at 37°C for 3 h to induce capacitation [9].

FFs

Clear human FFs were obtained from women undergoing transvaginal oocyte retrieval for in vitro fertilization treatment (approved by the hospital's Helsinki committee). The fluids were acetone precipitated (90% v:v final concentration) [7], and the resulting supernatants were dried, resuspended in H₂O to the original volume, and, unless indicated otherwise, further diluted with BWW for the experiments. Hereafter the term FF will denote the suspension of this supernatant fraction in BWW.

Progesterone Removal from FF

Progesterone as well as other steroids was removed from FF by adding dextran-coated charcoal, essentially as described by Fehl et al. [19] with some minor modifications. Briefly, FF (500 µl, 10 times diluted with PBS) was incubated with 250 µl dextran (0.05%)-coated charcoal (0.5%) for 3 h at 4°C with rotation. The dextran-coated charcoal was then removed by centrifugation at 16 000 x g for 5 min at 4°C, and the supernatant was collected. This procedure was repeated once more, and the final supernatant—termed charcoal-treated FF—was used for the experiments.

Estimation of the Progesterone Level in FF

The progesterone levels in active FF and charcoal-treated FF were determined by Immulite progesterone (Diagnostic Products Corporation, Los Angeles, CA)—an automatic system based on solid-phase chemiluminescent enzyme immunoassay specific for progesterone, with a sensitivity of 0.2–40 ng/ml progesterone.

Chemotaxis and Accumulation Assays

Sperm accumulation and chemotaxis were measured by a microscopic assay in a sealed chamber [20] as described earlier [7]. The spermatozoa chose between a well containing the test substance (FF or progesterone) and a well containing a control medium (BWW or PVP buffer). The behavior of the spermatozoa in the chamber was recorded on video and then analyzed. The sperm concentration used in the assays was 1–5 x 10⁸ cells/ml. For determining the relative sperm accumulation, the area between the wells was divided into serial zones [7]. In each zone the sperm density, integrated over the whole observation period (10 min), was compared between the experimental and the control wells. The ratio between these densities was calculated for each zone. The calculated value for the zone with maximal ratio was termed "relative sperm accumulation."

The trajectories made by the swimming spermatozoa in these microscopic assays were obtained by playing backward video recordings of these assays and tracing the recorded tracks of spermatozoa found near the wells in the sealed chamber [7]. The tracings were made manually by drawing on transparencies. The starting point of each trajectory was its first appearance on the video screen. The trajectory ended either when the spermatozoon entered the well or when it left the video screen. Accordingly, the duration and length of the recorded tracks varied. The recorded field was 1.6 x 1.2 mm.

Sperm accumulation was also measured in a separation apparatus. The measurement was carried out either with an apparatus consisting of two chambers connected by a tube [8], or with an apparatus consisting of three rather than two chambers. Each of the three chambers was 5 mm in diameter and 10.25 mm in depth. The middle chamber was connected to the other two chambers by a tube 17.5 mm long and 2.5 mm in diameter. One chamber contained spermatozoa preincubated under capacitating conditions as described above, and the other(s) contained the test substance(s). When the three-chamber apparatus was used, the middle chamber contained spermatozoa, and each of the side chambers contained a different test substance (e.g., FF and charcoal-treated FF, or FF and progesterone). The apparatus was incubated for 1 h, during which a net movement of spermatozoa was observed. Spermatozoa that had accumulated in the chambers containing the test substances were collected and counted.

Statistical Analysis

InStat 2.01 software package (Graph Pad Software, San Diego, CA) was used for statistical calculations. The significance of the difference between the treatments using the three-chambered apparatus was calculated by Student's t-test. The number of analyzed tracks required for statistical significance was determined by incrementally increasing the number of tracks analyzed until the average of the percentage of spermatozoa showing directional changes remained unchanged [7].

	RESULTS

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Sperm Accumulation in Progesterone

We employed two techniques for investigating whether spermatozoa are accumulated in progesterone: the microscopic assay in a sealed chamber [7, 20] and the separation assay [8]. As shown in Figure 1, 1 µg/ml progesterone caused sperm accumulation in the microscopic assay (panel A), albeit to a somewhat lesser extent than did 10-fold-diluted FF (panel B). At this dilution, measurement showed that the FF contained 0.6 µg/ml progesterone. The accumulation was observed in the concentration range of 1–100 µg/ml progesterone. Similar results were obtained with the separation assay: sperm accumulation in progesterone or in FF was higher than in the control media—PVP buffer and BWW for progesterone and FF, respectively (data not shown).

View larger version (14K): [in this window] [in a new window]   FIG. 1. Time-dependent sperm accumulation in progesterone and in diluted FF, measured by the microscopic assay in a sealed chamber. The figure represents a typical experiment among 4 (each experiment carried out with a sperm sample from a different donor). The sperm concentration was 5 x 108 cells/ml. The sperm density is expressed in spermatozoa/mm2. Closed and open symbols represent the spermatozoa near the well containing the test substance (progesterone or FF) and the well containing the substance-free medium (PVP buffer or BWW, respectively). A) Progesterone (1 µg/ml); B) 10-fold-diluted FF in BWW. The ratio between the sperm densities near the test and control wells, integrated over the whole observation period, was 1.4 and 1.5 for A and B, respectively. (When the contents of both wells are the same, the curves are superimposed and the sperm density ratio is 1 [7].)

In the microscopic assays with progesterone and diluted FF, we noticed that a significant portion of the spermatozoa acquired hyperactivation-like motility upon arrival in the accumulation zone. They swam with vigorous lateral displacement of the head and with little progressive motility. The magnitude of the portion of spermatozoa with hyperactivation-like motility—sometimes as high as one half of the spermatozoa—varied with the sperm sample.

Sperm Tracks Toward Progesterone and FF

The strictest criterion for chemotaxis is characteristic directional changes of the spermatozoa toward the source of the chemoattractant [3, 7, 14–16]. We compared the trajectories made by the spermatozoa approaching the progesterone-containing and FF-containing wells. As shown in Figure 2, which includes representative tracks, and in Table 1, which summarizes all the tracks analyzed, changes in the direction of swimming, characteristic of chemotaxis, were rare (14%) among spermatozoa approaching the progesterone-containing well, while 76% of the spermatozoa approaching the FF-containing well did so by characteristic changes in the direction of swimming. These observations suggest that progesterone is not a strong chemoattractant and that it causes sperm accumulation by virtue of a mechanism other than chemotaxis. One possibility is hyperactivation: reduction in net progressive movement leading to "trapping" of spermatozoa.

View larger version (56K): [in this window] [in a new window]   FIG. 2. Representative trajectories of spermatozoa found near wells containing BWW, progesterone, FF, and charcoal-treated FF. The trajectories were made during microscopic assays in a sealed chamber. Spermatozoa found near the well already at the beginning of the measurement were not taken into account. The trajectories shown were selected at random by a person unaware of the well content. The duration of each track is indicated in parentheses after the identification number of the track. The arrows indicate the direction of progression of each trajectory. Trajectories considered by us to exhibit directional changes toward the well are colored. Those considered by us to contain no directional changes are black. The blue quarter-circle in the lower right corner of each panel is part of the well that contained the test substance. A) BWW; B) progesterone (100 µg/ml); C) FF (10-fold diluted in BWW); D) charcoal-treated FF (10-fold diluted in BWW).

Sperm Accumulation in Progesterone-Depleted FF

To examine whether or not progesterone is the chemoattractant in FF, we removed progesterone and the other steroid constituents of FF using dextran-coated charcoal. The treatment decreased the progesterone content in the studied FF from 5.9 to 0.0004 µg/ml. In spite of the nearly complete absence of progesterone, this charcoal-treated FF retained its ability to cause sperm accumulation (Fig. 3). We also compared the activities of FF before and after charcoal treatment at an FF dilution of 1:1000 (instead of 1:10). Even at this high dilution of charcoal-treated FF, where the progesterone concentration was as low as 0.4 pg/ml, sperm accumulation was comparable to that obtained in similarly diluted, nontreated FF (Fig. 3). This suggests that progesterone is not the component of FF responsible for chemotaxis, at least not the major one. The trajectories made by spermatozoa swimming toward nontreated and charcoal-treated FFs were also similar (Fig. 2, C and D; Table 1).

View larger version (42K): [in this window] [in a new window]   FIG. 3. Relative sperm accumulation in FF before and after charcoal treatment. The gray and white columns stand for FF before and after charcoal treatment, respectively. The results shown are the mean ± SEM of 31 experiments with 15 semen samples from 5 different donors (for 10-fold-diluted FF), and of 3 experiments with 3 different semen samples from 3 different donors (for 1000-fold-diluted FF). The differences between the nontreated and charcoal-treated FFs were not significant (p = 0.07 and 0.6 for the 1:10 and 1:1000 dilutions, respectively).

Unlike nontreated FF, charcoal-treated FF did not cause hyperactivation-like motility. This observation, which endorses an earlier observation of Mbizvo et al. [21], suggests that a steroid in FF, probably progesterone, is the cause of the hyperactivation caused by FF.

Quantitative Comparison between Sperm Accumulation in Treated and Nontreated FF and Progesterone

If FF contains a chemoattractant(s) other than the hyperactivation-causing steroid (probably progesterone), and if chemotaxis is the main cause of sperm accumulation in FF, then the relative accumulation due to FF should be higher than that for an equivalent concentration of progesterone and comparable to that for charcoal-treated FF. Indications that this might be the case were obtained in the results shown in Figures 1 and 3. We used a three-chamber separation apparatus to make a more quantitative comparison. The middle chamber contained spermatozoa, one of the side chambers contained 1:1000-diluted FF, and the other side chamber contained an equivalent concentration of progesterone (0.006 µg/ml—the progesterone concentration measured in this 1000-fold-diluted FF). In agreement with the results described above, the accumulation in the diluted FF was almost twice the accumulation in progesterone (Table 2). In another set of experiments, the side chambers of the three-chamber separation apparatus contained 1:1000-diluted FF and 1:1000-diluted charcoal-treated FF, respectively. In accordance with the prediction, the accumulation in treated and nontreated FFs was similar (Table 3).

Effect of Progesterone-Receptor Antagonist

The conclusion that progesterone is not the major chemoattractant in FF is in conflict with the finding of Villanueva-Díaz et al. [11] that preincubation of spermatozoa with the progesterone-receptor antagonist RU-38486 eliminates sperm accumulation. In order to resolve the conflict, we preincubated spermatozoa with RU-38486 (0.1 mM for 10 min) and examined their response to FF and progesterone under the conditions of Villanueva-Díaz et al. Using the microscopic assay in a sealed chamber, we found that preincubation with RU-38486 did not reduce sperm accumulation in FF or in progesterone, and did not reduce the hyperactivation-like motility observed in the vicinity of the FF-containing or progesterone-containing well (data not shown; see Discussion).

	DISCUSSION

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Binding of progesterone to nongenomic receptors on the human sperm membrane [22] is known to result in a number of processes [23]. It has been suggested that one of these processes is sperm chemotaxis [11, 12]. This study demonstrates that progesterone either is not a sperm chemoattractant or is a weak chemoattractant, that the chemotactic activity of FF is not due to progesterone, and that progesterone may cause sperm accumulation by hyperactivating the cells. In addition, the study endorses earlier observations [21] that progesterone is probably the component that confers on FF the ability to cause hyperactivation.

Progesterone Is Not a Potent Sperm Chemoattractant

The conclusion that progesterone either is not a sperm chemoattractant or is a weak chemoattractant is based on the finding that most of the spermatozoa found near the progesterone-containing well appeared to reach it by coincidence rather than by changing their swimming path toward the well (Fig. 2; Table 1). On the other hand, a significant portion of the spermatozoa reached the FF-containing well by directional changes characteristic of chemotaxis (Fig. 2; Table 1; [7]). The uncertainty as to whether or not progesterone is a weak chemoattractant stems from the observation that the percentage of cells showing directional changes toward progesterone was somewhat higher than that toward the control (Table 1).

If progesterone is not a chemoattractant for human spermatozoa (or if it is a weak chemoattractant), how does it cause sperm accumulation (Fig. 1; [11])? Progesterone is well known to cause sperm hyperactivation [24–27]. We found that during approach to a progesterone-containing well, about half of the spermatozoa present in the accumulation area acquired hyperactivation-like motility. One of the characteristics of hyperactivation is a decrease in progressive motility (i.e., poor linearity) in spite of the vigorous sperm motion [28, 29]. This suggests that when the spermatozoa sense progesterone in the accumulation zone, they acquire hyperactivation-like motility and therefore remain or spend more time in this zone. Our observation that progesterone causes hyperactivation-like motility is in line with the observations that 10-min incubation of human spermatozoa with progesterone is sufficient to cause hyperactivation [24]. Both studies apparently contradict the finding of Villanueva-Díaz et al. [11] that progesterone does not cause hyperactivation during the assay interval (10 min).

Chemotactic Activity of FF Is Not Due to Progesterone

FF causes sperm accumulation [4, 30, 31] by way of chemotaxis [3, 7, 16]. On the basis of the observation that the chemotactic activity of FF is retained in the supernatant after precipitation with 90% acetone but is mainly in the pellet after precipitation with 100% acetone, Ralt et al. [7] concluded that the chemoattractant in FF is nonhydrophobic, possibly a short peptide or a similar molecule. We further found no correlation between the characteristics of the active fractions of FF (nonhydrophobic) and those of progesterone (very hydrophobic) [13], or between sperm accumulation in FF and the level of progesterone in the fluid [4]. In contrast, Villanueva-Díaz et al. [11] suggested that progesterone is the chemoattractant in FF. They based their suggestion on the observations that progesterone caused sperm accumulation, that dialysis of FF caused loss of this activity, that a lipid extract of FF caused sperm accumulation as did FF, that heat or trypsin treatment did not affect sperm accumulation by FF, and that preincubation of spermatozoa with the progesterone-receptor antagonist RU-38486 eliminated the accumulation. All these observations, except for the last one, can be explained by trapping caused by hyperactivation. The last observation could not be reproduced in our study, in agreement with earlier observations that sperm preincubation with RU-38486 does not inhibit progesterone-induced hyperactivation [24, 27], and that the high-affinity nongenomic receptor, recently found on human sperm membrane, is insensitive to RU-38486 [22]. The current study provides additional, more direct lines of evidence against the possibility that progesterone is the major chemoattractant in FF: 1) progesterone caused very few spermatozoa to acquire directional changes characteristic of chemotaxis (Fig. 2; Table 1), and 2) removal of progesterone from FF did not eliminate the chemotactic activity of the fluid (but did eliminate its hyperactivation-causing activity) (Fig. 3; Table 3). Our results therefore strongly suggest that, even though the possibility that progesterone is a weak chemoattractant cannot be ruled out, progesterone is not the major chemoattractant in FF.

Progesterone in FF Appears to Be the Reason for the Hyperactivation Caused by FF

An open question since the discovery of human sperm chemotaxis and hyperactivation by FF is whether both processes are caused by the same constituent of FF or by different constituents [7]. This study demonstrates that, even though the chemotactic activity of FF is retained after charcoal treatment, the apparent hyperactivation-causing activity is eliminated by this treatment. Since charcoal removes the steroid constituents of FF (including progesterone) [19], it can be concluded that the hyperactivation stimulated by FF is caused by a steroid constituent in the fluid, most likely progesterone. Our results and conclusion are in agreement with those of others [21, 32] who found that sperm incubation with FF results in hyperactivation, unlike incubation with charcoal-treated FF or steroid-free plasma medium.

	ACKNOWLEDGMENTS

 
We thank Dr. Anat Cohen-Dayag for her involvement in this study from its onset and for critical reading of the manuscript, Dr. Laura C. Giojalas for helpful comments, and Tomer Keren for technical assistance.

	FOOTNOTES

 
¹ M.E. is the incumbent of the Jack and Simon Djanogly Professorial Chair in Biochemistry.

² Correspondence. FAX: 972 8 934 4112; bmeisen{at}weizmann.weizmann.ac.il

Accepted: January 5, 1999.

Received: July 14, 1998.

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