Hum. Reprod. Advance Access originally published online on July 11, 2008
Human Reproduction 2008 23(10):2339-2345; doi:10.1093/humrep/den265
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The sperm chemoattractant secreted from human cumulus cells is progesterone
1 Department of Biological Chemistry, The Weizmann Institute of Science, 76100 Rehovot, Israel 2 In vitro Fertilization Unit, Department of Obstetrics and Gynecology, Barzilai Medical Center, Ben-Gurion University, 78278 Ashkelon, Israel
3 Correspondence address. Tel: +972-8-934-3923; Fax: +972-8-947-2722; E-mail: m.eisenbach{at}weizmann.ac.il
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Abstract |
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BACKGROUND: Human spermatozoa appear to be guided by chemotaxis to the oocyte in the female genital tract. While one of the sources of sperm chemoattractants is the cumulus cells that surround the oocyte, the identity of the chemoattractant secreted from them is unknown. Progesterone, recognized to be secreted from cumulus cells, was demonstrated, at the pM concentration range, to be a chemoattractant for human spermatozoa. Here, we examined whether this steroid is the cumulus-originated chemoattractant for human spermatozoa.
METHODS: Human cumulus cells were cultured, and the cultured medium was demonstrated to be chemotactically active. Progesterone was then eliminated from the medium by a specific anti-progesterone antibody, and the residual chemotactic activity was assessed.
RESULTS: The rate of progesterone secretion from the cells decreased with time. Removal of progesterone from the cumulus-cultured medium resulted in total loss of the chemotactic activity of the medium. Furthermore, the cumulus-cultured medium could substitute for progesterone in stimulating changes in the intracellular Ca2+ concentration in the spermatozoa, and the changes were very similar to those caused by measured progesterone concentrations in the medium.
CONCLUSIONS: Taken together, the results suggest that progesterone is the main, if not the sole, chemoattractant secreted by human cumulus cells.
Key words: progesterone/cumulus cells/chemoattractant/sperm chemotaxis
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Introduction |
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In mammals, spermatozoa entering the oviduct usually undergo a phase of storage at the isthmus (the oviductal part proximal to the uterus) (Suarez, 2002
). Spermatozoa that become capacitated (i.e. spermatozoa that acquire a state of readiness for fertilizing the oocyte; Jaiswal and Eisenbach, 2002) are released from this storage site (Smith and Yanagimachi, 1991; Lefebvre and Suarez, 1996) and reach the fertilization site at the ampulla (the oviductal part distal to the uterus) or the isthmic-ampullary junction within minutes (Barratt and Cooke, 1991; Overstreet and Drobnis, 1991; Hunter, 1993). This highly efficient arrival is made in spite of the tortuous passage the spermatozoa have to make in the narrow, densely ciliated and high mucus-containing isthmus (Harper, 1982). It has been suggested that this is due, at least in part, to a process of thermotaxis, guiding capacitated spermatozoa from the cooler storage site to the warmer fertilization site (see Bahat et al., 2003; Bahat and Eisenbach, 2006 for a review). However, because the oocyte is surrounded by a dense matrix of cumulus cells (Bedford, 1982), approaching the oocyte is not trivial even when the spermatozoa have already arrived at the fertilization site. The finding that both the oocyte and the surrounding cumulus cells secrete sperm chemoattractants after ovulation (Sun et al., 2005) raised the possibility that spermatozoa, which are already at (or near) the fertilization site, may be guided to the oocyte by a two-step process of chemotaxis. First, they may be guided to the oocyte–cumulus complex by the chemoattractant(s) secreted from the cumulus cells. Once within the cumulus matrix, the spermatozoa may be guided by the chemoattractant(s) secreted from the oocyte (Sun et al., 2005). The identities of the chemoattractants secreted from the cumulus cells and the oocyte are not known (see Eisenbach and Giojalas, 2006 for a review).
One of the substances secreted by the cumulus cells is progesterone (Schuetz and Dubin, 1981). This steroid has been reported to stimulate, or be involved in, a number of sperm functions, including capacitation, hyperactivation, acrosome reaction, binding to the oocyte's zona pellucida and penetration into the oocyte (Calogero et al., 2000; Yamano et al., 2004). Recently, it was demonstrated that progesterone at concentrations as low as 10–100 pM chemotactically attracts human and rabbit spermatozoa and that there is a progesterone gradient along the cumulus cell mass (Teves et al., 2006). These observations suggest that progesterone may be a chemoattractant secreted from the cumulus cells. Here, we examined whether, indeed, progesterone is the cumulus-originated chemoattractant for human spermatozoa. Our approach consisted of specific progesterone removal from cumulus exudates and examination of the remaining chemoattractive activity.
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Chemicals
Hyaluronidase, BSA, Tween 20, Protein A-Sepharose CL-4Ba, NaCl, NaHCO3, Na2HPO4 and 3,3',5,5'-tetramethylbenzidine were obtained from Sigma Medical Company, St Louis, MO, USA; Tris was from BIO LAB, Jerusalem, Israel; Na2CO3, H2O2 and citrate were from Merck, Darmstadt, Germany; Protein A magnetic beads were from Pierce, Rockford, IL USA; and Fluo-3-acetoxymethyl ester (Fluo-3-AM) was from AnaSpec, San Jose, CA, USA. Progesterone-BSA and Progesterone-Ovalbumin were kindly provided by F. Kohen (Weizmann Institute, Rehovot, Israel).
Solutions and media
P-1 medium and Serum Substitute Supplement were obtained from Irvine Scientific, Santa Ana, CA, USA; Dulbecco's phosphate-buffered saline (DPBS) was from Gibco-Invitrogen Corporation, Paisley, UK, and trypane blue solution was from Sigma. The medium used for suspending spermatozoa was Modified HTF Medium (Human Tubal Fluid buffered with HEPES; Irvine Scientific), supplemented with 0.3% human serum albumin (HSA; Irvine Scientific). The cumulus cells were cultured in Dulbecco's modified Eagle's medium nutrient mixture F-12 Ham (Sigma) supplemented with 10% fetal calf serum, charcoal stripped (Biological Industries, Beit Haemek, Israel), 2 mM L-glutamine in saline solution (Biological Industries) and a mixture of penicillin G and streptomycin (100 U/ml and 100 µg/ml, respectively; Gibco-Invitrogen Corporation). This medium is termed hereafter ‘culturing medium’ (distinct from the cumulus-cultured medium, defined below).
Antibodies
The monoclonal primary anti-progesterone antibody used for ELISA was anti-progesterone-7 thioether BSA monoclonal antibody, produced by a rat–mouse hybridoma cell line; IgG1 class, clone #2H4 (Kohen and Lichter, 1986), kindly provided by Dr F. Kohen (Weizmann Institute of Science). The secondary ELISA antibody was goat anti-mouse IgA-peroxidase antibody (Sigma). The antibody used for progesterone removal from cumulus-cultured media was anti-progesterone polyclonal antibody, produced in rabbits against progesterone-11-
-hemisuccinate-BSA (Barnard et al., 1995). The negative control was the pre-immune serum of the same rabbit. Both sera were kindly provided by F. Kohen.
Spermatozoa
Human semen samples were obtained from healthy donors after 3 days of sexual abstinence. Informed consent was obtained from each donor. Semen samples with normal sperm density, motility and morphology (according to World Health Organization, 1993 guidelines) were allowed to liquefy for 30–60 min at room temperature. The spermatozoa were separated from the seminal plasma by the migration-sedimentation technique (Hauser et al., 1992), which avoids the centrifugation stress. Briefly, the inner conical tube of the separation device and the bottom part of the larger surrounding tube were filled with 900 µl of HTF + 0.3% HSA. Semen (200 µl) was gently added to the bottom of the larger tube. The device was incubated under an atmosphere of 5% CO2 at 37°C for 1 h, after which the motile spermatozoa were collected from the inner tube. Following this procedure, the sperm concentration was adjusted to the concentration used in the chemotaxis assays, 1 x 106 cells/ml. The sperm suspensions were incubated under an atmosphere of 5% CO2 at 37°C for additional 1 h (in total, 2 h together with the separation procedure) to obtain capacitated spermatozoa (Cohen-Dayag et al., 1995).
Cumulus cell culture
Cumulus cells were obtained from oocyte–cumulus complexes, retrieved from randomly selected women undergoing transvaginal oocyte aspiration for in vitro fertilization. The women were treated with GnRH analog and gonadotrophin for ovarian stimulation. Oocyte retrieval was performed 34–36 h after human choriogonadotrophin injection. The cumulus cells were cut from the oocyte–cumulus complex, by employing two 23 Gauge syringe needles. Subsequent to oocyte removal, the sheared cumulus cells were washed twice with fresh medium of P-1 medium containing 10% Serum Substitute Supplement and 0.1% hyaluronidase by 5 min centrifugation at 800g, then dispersed in DPBS by 0.1% hyaluronidase with gentle pipetting and washed twice with DPBS by centrifugation at 300g for 10 min. The cumulus cell vitality was evaluated by trypane blue solution (Sigma). The washed cumulus cells were cultured for 10 days in 96-well tissue culture dishes in 200 µl culturing medium. Within this period, once the cumulus cells covered 70–80% of the wells' surface area, the medium (the culturing medium that now also contained secretions from the cumulus cells, termed hereafter ‘cumulus-cultured medium’) was pooled every 2–3 days from all the wells for later use in chemotaxis assays, and the cells were distributed to additional wells for further growth in fresh culturing medium. The pooled cumulus-cultured medium was divided into 10 µl aliquots and stored at –20°C for up to 2 months. Informed consent was obtained from each cumulus donor, and the study was approved by the Barzilai Hospital Committee of Clinical Research.
Estimation of the progesterone concentration in the cumulus-cultured medium
The progesterone concentration in the cumulus-cultured medium was determined by ELISA immunoassay (Crowther, 1995). The ELISA assay was preformed by coating the 96 wells immuno-plates with 200 µl of 1 µg progesterone-BSA or progesterone-ovalbumin, and by using as first antibody at 100 µl/well of anti-progesterone monoclonal antibody (1:20 000) and secondary antibody at 200 µl/well of goat anti-mouse peroxidase (1:10 000).
Progesterone removal by magnetic beads
Progesterone was removed from the cumulus-cultured medium by a Magnetic Separator (Cortex Biochem, San Leandro, USA), using protein A magnetic beads bound to anti-progesterone rabbit polyclonal antibodies. The binding of the antibodies to the protein A magnetic beads and the removal of progesterone were carried out according to the manufacture's instructions, except that the beads were washed with the culturing medium. The final concentration of anti-progesterone antibody concentration in the beads was 0.4 mg/ml. The magnetic separation was repeated three times. The negative-control beads with pre-immune serum or naked beads underwent the same treatment. Both the anti-progesterone polyclonal antibody and the pre-immune serum were pre-purified by Protein A-Sepharose CL-4Ba according to the manufacture's instructions.
Stimulated changes in the sperm's intracellular free calcium concentration
Spermatozoa (15 x 106 cells/ml), pre-washed twice by centrifugation (120g for 20 min each, instead of migration-sedimentation separation) and pre-incubated for 1.5 h at 37°C under an atmosphere of 5% CO2, were loaded (under the same conditions) with 2.5 µM Fluo-3-AM for 30 min in the dark, washed twice (120g, 20 min, room temperature) with HTF (without HSA), and the concentration was adjusted to 10 x 106 cells/ml with the same medium; this was followed by an additional 20 min incubation at room temperature in the dark. Measurements of intracellular Ca2+ (Ca2+in) were carried out using a Cary Eclipse fluorescence spectrophotometer (Varian). The temperature of the cuvette was maintained at 37°C. Excitation wavelength was set at 506 nM with the emission held at 526 nM. Slit width was set to 5 nM with a sampling rate 12.5 Hz.
Chemotaxis assay
Chemotaxis assays were performed, as described earlier (Fabro et al., 2002), at 37°C in a Zigmond chemotaxis chamber consisting of two wells separated by a wall and closed with a coverslip (Zigmond, 1977). Both wells contained spermatozoa (1 x 106 per ml) in HTF + 0.3% HSA. The right well contained cumulus-cultured medium, in 104- to 108-fold dilutions. The same dilution of the culturing medium was added to the left well and on top of the partition wall separating between the wells. Thus, the whole chamber contained an identical concentration of the culturing medium. (This is important because one of the ingredients of this medium, fetal calf serum, may contain remnants of progesterone, though at extremely low concentrations.) In the negative-control experiments, both wells contained only spermatozoa (1 x 106 per ml) in HTF + 0.3% HSA. After sealing the chamber and subsequently allowing a 10 min equilibration, the movement of spermatozoa on top of the partition wall, in the middle of the field between the two wells, was video-recorded for 5 min.
Chemotaxis assessment and statistical analysis
Chemotaxis was evaluated as described by Gakamsky et al. (2008) on the basis of distribution of the instantaneous directionality angles (
inst, the angle between the vector of the cell frame-to-frame displacement and the gradient direction; the video frequency was 25 frames/s). Two bins were defined, each 90° wide, centered on 0° and ±180° (where the direction of the chemoattractant gradient was defined as 0°; Fig. 1). Having the total numbers of angles in the 0° and 180° bins, N+ and N–, respectively, we evaluated the degree of asymmetry of the circular distribution diagram by the odds parameter, where Odds = N+/N–. The odds parameter yields values close to 1 when the swimming is random; it is >1 when the swimming is biased in the gradient direction. The strength of the chemotactic response was obtained by the odds ratio (O.R.) parameter (O.R. = Oddstreatment/Oddscontrol). The significance of the response was estimated by the 
2-test, where the critical 2 values were adjusted to the sample size (Gakamsky et al., 2008).
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Our earlier study, which identified human cumulus cells as a source of a sperm chemoattractant(s), employed conditioned media and lysates prepared from cumulus cells subsequent to their mechanical removal from the oocyte (Sun et al., 2005
). Here, to avoid the slightest possibility that the tested medium could contain remnants of oocyte secretions, we cultured cumulus cells and then tested the cultured medium (at 104- to108-fold dilutions) for chemotaxis. We employed the recently developed approach for the analysis of sperm chemotaxis, statistically assessing by the OR parameter the difference between the distributions of the instantaneous directionality angles (the angle between the vector of the cell frame-to-frame displacement and the gradient direction) made by spermatozoa swimming in a gradient of cumulus-cultured medium and in a no-gradient control (Gakamsky et al., 2008). A chemotactic response was clearly observed (Fig. 2). The dilution of cumulus-cultured medium at which the response was observed depended on the batch of the cultured cells. It was usually observed at 107 dilution, as in Fig. 2, but in another set of experiments carried out with a different batch, the response was observed at both 107 and 106 dilutions (data not shown). In accordance with the expected bell-shaped dependence of a chemotactic response on the chemoattractant concentration (Adler, 1973; Ralt et al., 1994), no response was observed at higher and lower dilutions of the cumulus-cultured medium, i.e. the O.R. values were not significantly different from the values of the negative control (Fig. 2). This chemotactic response to cumulus-cultured medium is in agreement with the published results of chemotaxis assays carried out with cumulus-conditioned media and lysates, except that in those fluids the response was observed at much lower dilutions, 103 and 104 (Sun et al., 2005).
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Knowing that the concentration range, at which progesterone is effective as a chemoattractant, is 1–100 pM (Teves et al., 2006
), we measured the progesterone concentration in the cumulus-cultured medium. We found it to be in the range 0.9–4 µM after 3 days of culturing 
105 cumulus cells in 200 µl culture medium. This means that the 107-fold diluted cultured medium, at which maximal chemotactic activity was observed (Fig. 2), contained 0.09–0.4 pM progesterone, which is at the lower edge of the chemotaxis-effective concentration range of this steroid (see Discussion). To examine whether progesterone is the cause of the chemotactic activity of the cultured medium, we wished to specifically reduce the progesterone concentration in the medium. After finding that anti-progesterone antibody totally inhibits the chemotactic response to progesterone (10 pM; O.R. = 1.24 ± 0.02 in the absence of antibody versus 0.99 ± 0.01 in the presence of antibody; P = 0.001, two experiments), we pre-incubated the cultured medium with polyclonal anti-progesterone antibody linked to magnetic beads via protein A, and then removed the beads from the medium by a magnet. This treatment clearly eliminated the ability of the cumulus-cultured medium to chemoattract spermatozoa (Fig. 3A and B). These results indicate that progesterone was the main, if not the only, chemoattractant in the cumulus-cultured medium, suggesting that the chemoattractant secreted from cumulus cells is progesterone. Interestingly, the mere incubation of the medium with the positive control beads, consisting of either naked beads (Fig. 3A) or beads linked to pre-immune antibodies of the same rabbit (Fig. 3B), somewhat reduced the progesterone concentration (Fig. 3C), and as a result, the chemotactic response was observed at lower dilutions of the cumulus-cultured medium (105–107). The reduction in the progesterone concentration by the beads alone was probably due to the non-specific adsorption of progesterone to the beads.
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This finding raised the question of whether the secretion of progesterone from the cultured cumulus cells is time-dependent. To address this question, we determined the progesterone concentration in the cumulus-cultured medium at three time periods after culturing the cells, each 3 days long. At the end of each period, we replaced the culturing medium with a fresh one, and measured by ELISA the progesterone concentration in the collected medium. We found in this way that the progesterone secretion decreased with time (Fig. 3D), even though the total number of cells doubled during the 9 days measurement (from 100 000 to 200 000 cells/ml) and the fraction of live cells, measured by Trypan blue, increased from 70% to 90%.
One of the best-studied processes known to occur in spermatozoa and known to be triggered by progesterone is the elevation of intracellular Ca2+ (Ca2+in) (Blackmore et al., 1990, 1991). Sequential, repeated stimulation with progesterone results in a decreased Ca2+in response, possibly due to receptor saturation. We, therefore, investigated whether the cumulus-cultured medium, like progesterone, stimulates a transient rise in Ca2+in and, if so, whether pre-stimulation with cumulus-cultured medium reduces the Ca2+in response to a subsequent stimulation with progesterone. Following pre-incubation of spermatozoa with the Ca2+in probe, Fluo-3-AM, we stimulated them sequentially with diluted cumulus-cultured medium, progesterone, and again with the cultured medium (Fig. 4). The observed cultured medium-stimulated changes in Ca2+in (Fig. 4A for a 1000-fold diluted medium and Fig. 4B for a 100-fold diluted medium) were very similar to those observed when the cells were stimulated with the respective measured progesterone concentrations in the cultured medium (Fig. 4C and D, respectively). Furthermore, exposure of the cells to 10–5 M progesterone prevented their response to 1000-100-fold diluted cultured medium (Fig. 4A and B, respectively) or to 10–8–10–7 M progesterone (Fig. 4C and D, respectively). These results are consistent with the possibility that the cultured medium-stimulated changes in Ca2+in were caused by progesterone.
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Discussion |
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Progesterone, at the picomolar concentration range, was recently shown to be a chemoattractant for human spermatozoa (Teves et al., 2006
). This finding taken with the observations that progesterone is secreted from cumulus cells (Schuetz and Dubin, 1981) and that it forms a concentration gradient within the cumulus mass (Teves et al., 2006), raised the possibility that it is the chemoattractant secreted from the cumulus cells (Teves et al., 2006). The aim of this study was to examine whether, indeed, progesterone is the chemoattractant secreted from these cells. The approach that we took was to substantially reduce the progesterone concentration in a medium pre-cultured with cumulus cells and examine whether the medium retained chemotactic activity. To reduce the progesterone concentration, we treated the cumulus-cultured medium with a specific, anti-progesterone polyclonal antibody (lowering the progesterone concentration in the non-diluted medium to 0.02–0.05 µM; Fig. 3C; in the chemotaxis assays, this concentration was further diluted 105- to 107-fold). The outcome was the total loss of the chemotactic activity (Fig. 3A and B), suggesting that progesterone is the main, probably the sole, chemoattractant secreted from a culture of human cumulus cells. The similarity between the stimulatory effects of progesterone and the equivalently diluted cumulus-cultured medium on changes in Ca2+in (Fig. 4) is consistent with this conclusion. We further found that the progesterone secretion from the cumulus cells is time-dependent (Fig. 3D).
A point that should be addressed is the apparent differences between the results of this study and those of Teves et al. (2006) with respect to the effective concentration range of chemotactic activity. Teves et al. (2006) reported maximal activity at 10 pM, the range of activity being at 1–100 pM progesterone. We found the activity at 107-fold diluted cumulus-cultured medium (Fig. 2), equivalent to 0.15 pM progesterone (Fig. 3C). These differences might have been the outcome of the extremely low progesterone concentrations, where any small effect (e.g. a small difference in adsorption to the vessel) may result in a large difference in concentration. Another possible cause of this difference might be the probable presence of a progesterone-carrier protein in the medium. Progesterone is a highly hydrophobic molecule, insoluble in aqueous solutions. Like any other molecule of similar solubility and activity at low concentrations, it probably has some binding protein that carries it in solution to the target receptor in the sperm cell. Indeed, cytoplasmic carrier proteins that bind steroid hormones and are confined to the oviduct have been described (e.g. Taylor and Smith, 1982). Thus, when progesterone is sequentially diluted many orders of magnitude from an organic solvent into an aqueous solution (containing just buffer, nutrients, salts and albumin), it is not improbable that a large fraction of the molecules go out of solution and that the effective concentration is much lower.
The conclusion that progesterone is the chemoattractant secreted from cumulus cells is consistent with earlier studies, which indirectly implied that cumulus cells might be a source of the sperm chemoattractant (for reviews, see Eisenbach, 1999b; Van Soom et al., 2002). For example, Bronson and Hamada (1977) found that cumulus cells secrete a substance that alters the pattern of sperm movement. Bedford and Kim (1993) found that, in spite of the dense matrix, the few first spermatozoa that enter the cumulus find the oocyte very effectively. A number of groups (reviewed in Jaiswal and Eisenbach, 2002) demonstrated that only capacitated spermatozoa, the only spermatozoa that are chemotactically responsive (Cohen-Dayag et al., 1995; Eisenbach, 1999a; Fabro et al., 2002), can penetrate the cumulus oophorus. Ito et al. (1991) provided evidence that the products of ovulation (including cumulus secretions) stimulate sperm transport in the hamster oviduct. In a number of mammals (depending on the species), cumulus removal decreases in vitro fertilization (Van Soom et al., 2002 for a review; Romar et al., 2003; Tanghe et al., 2003 for later studies).
The results of this study do not contradict earlier results of Jaiswal et al. (1999), which demonstrated that progesterone is not the major chemoattractant in human follicular fluid. Jaiswal et al. showed that progesterone at the concentration found in a chemotactically active dilution of follicular fluid is hardly active as a chemoattractant. This concentration was much higher than the chemotactically active progesterone concentration (Teves et al., 2006) and, therefore, chemotactic activity could not be observed. (In chemotaxis, chemoattractant concentrations that are too high are not effective because the chemotaxis receptors are saturated and cannot detect the chemoattractant gradient.) It, thus, appears that follicular fluid, which contains secretions from a number of different sources, includes chemoattractants additional to progesterone.
Earlier studies demonstrated that, following ovulation, both the mature oocyte and the cumulus cells secrete chemoattractants, probably distinct ones (Sun et al., 2005), and that human and rabbit spermatozoa can sense the temperature difference that exists at ovulation between the storage and fertilization sites (David et al., 1972; Hunter and Nichol, 1986; Bahat et al., 2005) and respond to it by thermotaxis (Bahat et al., 2003). These observations, taken together with the results of this study, suggest the following sequence of events in vivo (Fig. 5). Capacitated spermatozoa, released from the sperm storage site at the isthmus (Suarez, 2002), may be first guided by thermotaxis from the cooler sperm storage site towards the warmer fertilization site in the ampulla (Bahat et al., 2003). Passive contractions of the oviduct (Battalia and Yanagimachi, 1979) may assist the spermatozoa to reach the fertilization site. Near or at the fertilization site, the spermatozoa may be chemotactically guided to the oocyte–cumulus complex by the gradient of progesterone, secreted from the cumulus cells. In addition, progesterone may inwardly guide spermatozoa, already present within the periphery of the cumulus oophorus (Teves et al., 2006). Such inward guidance is not superfluous, because the cumulus oophorus is a dense milieu consisting of cells and their matrix, built from polymerized hyaluronic acid conjugated with proteoglycans and proteins (Yanagimachi, 1994). Without guidance, arrival to the oocyte through this expanded, dense matrix would be quite difficult (Bedford, 1982). Spermatozoa that are already deep within the cumulus oophorus may sense the more potent chemoattractant that is secreted from the oocyte (Sun et al., 2005) and chemotactically guide themselves to the oocyte according to the gradient of this chemoattractant. The identity of the chemoattractant secreted from the oocyte is not yet known.
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This study was supported by research grants from the Benoziyo Institute of Molecular Medicine at the Weizmann Institute of Science and from the Dr Josef Cohn Minerva Center for Biomembrane Research.
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We thank Dr Fortüne Kohen for advice and for the generous gift of antibodies, Dr Rose M. Johnstone for the suggestion of using magnetic beads and Liran Ben Yaakov for technical assistance. M.E. is an incumbent of the Jack and Simon Djanogly Professorial Chair in Biochemistry.
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Submitted on February 27, 2008; resubmitted on June 5, 2008; accepted on June 9, 2008.
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