The effect of RANTES on human sperm chemotaxis

Tetsuya Isobe1, Hiroyuki Minoura1,3, Keisuke Tanaka1, Takashi Shibahara2, Naoko Hayashi2 and Nagayasu Toyoda1

1 Department of Obstetrics and Gynecology, School of Medicine, Mie University, Edobashi 2-174, Tsu, Mie, Japan and 2 Suzuka Kaisei General Hospital, Kouchou Hoshizaka 112-1, Suzuka, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Follicular fluid (FF) is a powerful stimulator of human sperm hyperactivation and the acrosome reaction. FF causes sperm accumulation by way of chemotaxis. The chemoattractant in FF has not yet been identified in the human. It is well known that some types of chemokine such as RANTES (Regulated on Activation Normal T Expressed and Secreted Chemokine) exist in genital tract fluids (for example, FF, seminal plasma and uterine fluid). Few reports appear to exist concerning the direct effect of chemokines on the function of human sperm. METHODS: The chemotactic effect of RANTES on human sperm were investigated by capillary and double-chamber assays, while the chemokinetic effect was investigated using computer-assisted sperm analysis. RESULTS: Both the capillary and double-chamber assays demonstrated a significant chemotactic effect of RANTES on human sperm. Anti-RANTES rabbit IgG partially neutralized the chemotactic effect of FF. In contrast, no statistically significant chemokinetic effect was observed in any motility parameter. CONCLUSIONS: Here, it was demonstrated that mRNA for the RANTES receptors CCR-1 and CCR-5 are present in human sperm. Furthermore, RANTES is involved in the chemotactic effect of FF in vitro.

Key words: chemotaxis/cytokine/follicular fluid/human sperm/RANTES


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The induction of chemokinesis and chemotaxis in human sperm by follicular fluid (FF) in vitro appears to be well established (Villanueva-Diaz et al., 1990Go; Ralt et al., 1991Go, 1994Go; Makler et al., 1992Go; Eisenbach and Tur-Kaspa, 1994Go, 1999Go; Eisenbach, 1999Go). Accumulating evidence has revealed that progesterone induces sperm chemokinesis (Uhler et al., 1992Go; Sueldo et al., 1993Go; Oehninger et al., 1994Go; Parinaud and Milhet, 1996Go), though the chemoattractant in FF has not yet been identified. It has been suggested (Villanueva-Diaz et al., 1995Go) that progesterone may be the chemoattractant in FF, but others (Jaiswal et al., 1999Go) have failed to substantiate this finding.

It is well known that FF contains several types of chemokine, including RANTES (Regulated on Activation Normal T Expressed and Secreted Chemokine) (Karstrom-Encrantz et al., 1998Go). RANTES is a 68-amino acid peptide (Nelson et al., 1993Go) of 8 kDa molecular weight (Schall et al., 1990Go) that belongs to the CC subfamily of chemokines in which the first two Cys residues are continuous and bind to CCR-1, CCR-3 and CCR-5 (Wells et al., 1999Go). RANTES may be a potent chemoattractant for eosinophils, monocytes and T lymphocytes (Schall et al., 1990Go; Alam et al., 1993Go).

Human sperm are exposed to RANTES in both the male and female genital tracts before they reach the site of fertilization. RANTES exists in seminal plasma (Naz and Leslie, 2000Go), uterine fluid (Hornung et al., 1997Go) and peritoneal fluid (Khorram et al., 1993Go; Hornung et al., 2001Go) as well as FF. The level of this chemokine in genital tract fluid is elevated in diseases related to infertility, such as endometriosis (Khorram et al., 1993Go; Hornung et al., 2001Go) and male genital tract infection (Naz and Leslie, 2000Go). The mean concentration of RANTES in FF has been reported as 174 pg/ml (Machelon et al., 2000Go), while in severe endometriosis the level was 530.2 pg/ml (Khorram et al., 1993Go). RANTES is produced by granulosa cells from human pre-ovulatory follicles up-regulated by TNF-{alpha}, the level of which in FF is elevated in women with endometriosis (Machelon et al., 2000Go). RANTES is synthesized by endometriotic stromal cells (Hornung et al., 1997Go).

Few reports appear to exist concerning the direct effect of RANTES on sperm motility. The aims of this study, using RT-PCR, were to detect whether mRNAs for RANTES receptors are present in human sperm, and to determine whether RANTES induces chemokinesis and chemotaxis of human sperm.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Preparation of sperm
Human ejaculates were collected by masturbation from normal healthy donors who had fathered at least one child. Each ejaculate was allowed to liquefy at 37°C, layered onto 0.5 ml 90% Percoll in sperm washing medium (SWM; Irvine Scientific, CA, USA), and then centrifuged at 400 g for 20 min. The pellets were washed twice with SWM. The concentration of sperm was counted using a Makler chamber and adjusted to a concentration of 10–30x106 cells/ml in suitable medium for each experiment.

RNA extraction and PCR procedures
Total RNA was extracted from sperm prepared from six ejaculates by means of the acidic-guanidium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987Go), with minor modifications. Briefly, the sperm sample was homogenized in 500 µl 4 mol/l guanidium thiocyanate, 25 mmol/l sodium citrate, 0.1% sarcosyl and 1% ß-mercaptoethanol. An aliquot (55 µl) of 2 mol/l sodium acetate was added to the sperm mixture, which was extracted once with phenol-chloroform. The RNA was precipitated with the same volume of iso-propanol and washed twice with 70% ethanol. The majority (90%) of the RNA obtained was subjected to the RT reaction, and 10% was used as a negative control for the PCR. RT was performed for 60 min at 37°C in a total volume of 20 µl that contained 1st strand buffer (Amersham Pharmacia Biotech, Piscataway, NJ, USA), Moloney Murine Leukaemia Virus reverse transcriptase (200 units; Gibco-BRL, Gaithersburg, MD, USA), and a random primer (10 pmol; Amersham Pharmacia Biotech). A 2 µl portion of the RT solution was subjected to multiplex-PCR, which was performed in a 50 µl mixture containing Taq polymerase buffer, 10 nmol/l of each deoxynucleotide triphosphate, multiplex PCR primer (Maxim Biotech, Inc., South San Francisco, CA, USA), and 2.5 IU Taq DNA polymerase (Amersham Pharmacia Biotech). The positive control template was provided by the manufacturer. Amplification was achieved at 96°C for 1 min and 60°C for 4 min for two cycles, and 94°C for 1 min and 60°C for 2.5 min for 40 cycles; the final extension step consisted of heating to 70°C for 10 min. A 20 µl portion of each reaction mixture was subjected to electrophoresis in a 3% agarose gel containing ethidium bromide (0.5 mg/ml). The gel was photographed under ultraviolet light.

Follicular fluid
Human FF samples were obtained from women whose routine infertility examination revealed no abnormal findings, and who were undergoing transvaginal follicular aspiration for IVF due to male infertility. Women with endometriosis, polycystic ovary syndrome and infectious diseases were excluded. The FF obtained from follicles of >16 mm diameter were pooled, immediately centrifuged for 5 min at 1500 g, and then filtered through a 0.5 µm disc filter to remove cells and cell debris. The filtrates were divided and stored at –20°C until assayed.

Capillary assay
A capillary assay was carried out using a previously published method (Ralt et al., 1994Go), but with minor modifications. The assay was performed in a system comprising a series of Teflon tubes and polyethylene capillary tubes filled with 100 µl of sperm at a concentration of 30x106 cells/ml suspended in SWM. The capillaries were each filled with 5 µl of test solution and sealed at one end with a heated clamp. The open side of each capillary tube was inserted into the sperm solution. After varying periods of incubation at 37°C, the contents of the capillary were transferred into a Makler chamber, and the number of sperm that migrated into the capillary was counted. A recombinant RANTES (Chemicon International, Inc., Temecula, CA, USA) was used in this assay. The test solution contained SWM with or without various concentrations of RANTES (from 10 to 1000 pg/ml). Neutralization of the chemotactic effect of FF was investigated with 9 µg/ml of anti-RANTES rabbit monoclonal IgG (American Research Products, Inc., Belmont, MA, USA) and normal rabbit IgG (Marine Biological Laboratory, Massachusetts, MA, USA).

Double-chamber assay
A double-chamber assay was performed using a published method (Villanueva-Diaz et al., 1992Go), with a minor modification. The control chamber was filled with 1 ml of SWM, and the experimental chamber with 1 ml of various test solutions. A 100 µl portion of sperm suspension was deposited into the cell port. After 40 min incubation at room temperature, the liquids in each chamber were transferred separately to another tube, centrifuged at 400xg for 10 min at room temperature, and the supernatants were discarded by aspiration until only 0.1 ml remained. After being resuspended, the number of migrated sperm was counted in a Makler chamber.

Computer analysis of sperm motility
Sperm were prepared at a concentration of 10x106 cells/ml in human tubal fluid (HTF) medium (Irvine Scientific) in the presence or absence of 1000 pg/ml of RANTES, and incubated under an atmosphere of 5% CO2 in air at 37°C for 30 min. The analysis of spermatozoan kinetic parameters was performed using computer-assisted sperm analysis (CASA) (C-men; Compix, Inc. Imaging Systems, Torrance, CA, USA). The following parameters were measured: percentage of motile sperm; mean curvilinear velocity; mean linearity; and maximum-amplitude lateral head movements.

Statistical analysis
Student's t-test was used to compare normally distributed means. Statistical significance was accepted when P <= 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
RT-PCR
CC receptor 1, 2, 4 and 5 mRNA was detected by multiplex RT-PCR in human sperm total RNA (Figure 1Go). No band was generated from human sperm RNA without reverse transcription (data not shown).



View larger version (59K):
[in this window]
[in a new window]
 
Figure 1. Electrophoresis of human sperm multi-plex RT-PCR products. PCR products were separated on 3% agarose gel and visualized under ultraviolet light (315 nm) after ethidium bromide staining. The CC subfamily chemokine receptor mRNA kit is involved in the primer pairs for GAPDH (500 bp), CCR-1(363 bp), CCR-2 (163 bp), CCR-3 (318 bp), CCR-4 (287 bp)and CCR-5 (246 bp) mRNAs.

 
CASA analysis
No statistically significant difference was observed in any parameter of motility (Table IGo).


View this table:
[in this window]
[in a new window]
 
Table I. The effect of RANTES on chemokinetics of spermatozoa
 
Capillary assay
The chemotactic effect of RANTES on human sperm was examined by means of a capillary assay. The number of sperm that migrated into the capillaries containing FF within the indicated time period is shown in Figure 2Go. A plateau of migrated sperm counts into the capillary was obtained after 40 min using this apparatus; hence capillary assays were carried with an incubation time of 40 min in subsequent experiments. The number of migrated sperm increased in a dose-dependent manner (Figure 3Go).



View larger version (10K):
[in this window]
[in a new window]
 
Figure 2. Time-dependent accumulation of spermatozoa in polyethylene capillaries. A capillary assay using diluted FF and SWM as test solutions was carried out to estimate the time needed to attain a plateau. The number of sperm that migrated into the capillary within the indicated time period is shown. Each point represents the average of three determinations (± SEM). A plateau of migrated sperm counts was obtained 40 min after connecting the tube containing 30x106/ml sperm with the capillary. *, P < 0.05 between the two solutions.

 


View larger version (14K):
[in this window]
[in a new window]
 
Figure 3. Effect of RANTES on human spermatozoa accumulation in capillary assays. The capillary assay was carried out as described in Materials and methods, using varying concentrations (0, 10, 100, 1000 pg/ml) of RANTES in SWM, and with FF as a positive control. Incubation time was 40 min. Typical results for three experiments are presented; each column represents the average of three determinations (± SEM). *, P < 0.05.

 
In order to investigate the nature of the chemotactic effect of RANTES on sperm, capillary assays were carried out in a variety of combinations (ascending or descending gradients of RANTES). The presence of an ascending gradient of RANTES (SWM in tube, RANTES in capillary) induced sperm to migrate into the capillary at significantly higher concentrations than in the combination without an ascending gradient (RANTES in tube, RANTES in capillary or SWM in tube, SWM in capillary), or with a descending gradient (RANTES in tube, SWM in capillary) (Figure 4Go).



View larger version (9K):
[in this window]
[in a new window]
 
Figure 4. Spermatozoa accumulation in capillary assays with a gradient of RANTES, or no gradient. The capillary assay was carried out as described in Materials and methods, using a RANTES concentration of 1000 pg/ml. Incubation time was 40 min. Typical results for three experiments are given; each column represents the average of three determinations (± SEM). A = RANTES in tube, RANTES in capillary; B = RANTES in tube, SWM in capillary; C = SWM in tube, RANTES in capillary; D = SWM in tube, SWM in capillary. *, P < 0.05.

 
Double-chamber assay
A double-chamber assay was performed in order to confirm the chemotactic effect of RANTES on sperm. As shown in Figure 5Go, the number of sperm migrating into an experimental chamber containing 1000 pg/ml of RANTES in addition to FF was significantly higher than for sperm migrating into the control chamber (SWM).



View larger version (10K):
[in this window]
[in a new window]
 
Figure 5. Result of double-chamber assay. The assay was carried out as described in Materials and methods, using a RANTES concentration of 1000 pg/ml. Incubation time was 40 min. Typical results for three experiments are shown; each column represents the average of three determinations (± SEM). *, P < 0.05.

 
Neutralization of chemotactic effect of FF by anti-RANTES rabbit IgG
The chemotactic effect of RANTES was neutralized completely by anti-RANTES rabbit IgG (Figure 6Go), though normal rabbit IgG had no effect on sperm migration into the capillary. Anti-RANTES rabbit IgG was shown to partly neutralize the chemotactic effect of FF, but normal rabbit IgG had no neutralizing effect.



View larger version (15K):
[in this window]
[in a new window]
 
Figure 6. Neutralization of chemotactic effect of follicular fluidsby anti-RANTES rabbit IgG. The RANTES concentration was1000 pg/ml. Incubation time was 40 min. Anti-RANTES rabbit IgG (500 nl) was added to the systems. The amount of IgG in normal rabbit IgG solution was made equivalent to that of anti-RANTES rabbit IgG solution. Typical results for three experiments are presented; each column represents the mean of four determinations (± SEM). **, P < 0.01.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Results obtained in the present study showed that mRNA for RANTES receptors (CCR-1 and CCR-5) exist in human sperm total RNA, and that RANTES is one of the chemoattractants of human sperm in FF in vitro.

Sperm chemotaxis constitutes one of the mechanisms that bring the female and male gametes together. The physiological role of sperm chemotaxis in mammals is thought to be the recruitment of a selective population of capacitated sperm to fertilize the oocyte (Eisenbach, 1999Go; Eisenbach and Tur-Kaspa, 1999Go). Moreover, sperm chemotaxis is thought to be exploited as a diagnostic tool for sperm quality, and may in the future also be used as a valuable marker of therapy for male infertility (Eisenbach, 1999Go).

Human sperm chemotaxis to FF in vitro appears to be well established (Villanueva-Diaz et al., 1990Go; Ralt et al., 1991Go, 1994Go; Makler et al., 1992Go; Eisenbach and Tur-Kaspa, 1994Go, 1999Go; Eisenbach, 1999Go), although those factors in FF which act as a chemoattractant to sperm have not yet been identified. Progesterone is well known to cause sperm hyperactivation and the acrosome reaction (Uhler et al., 1992Go; Sueldo et al., 1993Go; Oehninger et al., 1994Go; Parinaud and Milhet, 1996Go; Jaiswal et al., 1999Go). These effects of FF are cAMP-dependent (Hartshorne, 1991Go) and calcium influx-dependent (Blackmore et al., 1990Go; Krausz et al., 1996Go) on, and are mediated by, intracellular progesterone receptors (Morales et al., 1992Go; Sabeur et al., 1996Go). It has been reported by one group that progesterone causes sperm chemotaxis (Villanueva-Diaz et al., 1995Go), though others (Jaiswal et al., 1999Go) have suggested that the removal of progesterone from FF with charcoal treatment abolished sperm hyperactivation-like motility, but not sperm chemotaxis. The accumulation of sperm induced by progesterone is caused primarily by physiological trapping, rather than by chemotaxis (Eisenbach and Tur-Kaspa, 1999Go). It is thought by some that progesterone is not a chemoattractant in FF (Uhler et al., 1992Go; Jaiswal et al., 1999Go), but partial fractionation of FF using a Centricon microconcentrator showed at least one of the chemotactic factors in FF to be a small (<10 kDa) molecule (Ralt et al., 1994Go).

The capillary assay revealed that RANTES had a dose-dependent chemotactic effect on human sperm (Figure 3Go), with the number of sperm migrating into the capillary being influenced by the chemokinetic effect of the test solution. RANTES was shown to have no effect on the chemokinetic parameters of human sperm (Table IGo). Indeed, the number of sperm that migrated into capillaries due to the combination of a descending gradient (SWM in capillary and RANTES in tube), as well as in the absence of any gradient (RANTES in tube and RANTES in capillary), were similar to that of the control combination (SWM in tube and SWM in capillary). Moreover, this result—which was confirmed by the double-chamber assay (Figure 5Go)—is thought to avoid any chemokinetic effect.

It has been reported that the mean concentration of RANTES in FF in normal women was 174 pg/ml (Machelon et al., 2000Go), and in severe endometriosis was 530.2 pg/ml (Khorram et al., 1993Go). The mean concentration of RANTES in FF used in the present experiments was 234 pg/ml (range 123–452 pg/ml; data not shown), and consequently the chemotactic effect of RANTES on human sperm was observed at a physiological concentration.

The chemotactic effect of RANTES was also shown to be neutralized completely by anti-RANTES rabbit IgG (Figure 6Go), whilst the anti-RANTES antibody also neutralized the chemotactic effect of FF. By contrast, normal rabbit IgG had no influence on the chemotactic effect of FF, which suggested that the neutralizing effect of anti-RANTES rabbit IgG may have been specifically targeted. Indeed, the present data indicate that RANTES contributes to the chemotactic effect of FF in vitro.

Macrophage inflammatory protein-1(MIP-1){alpha},ß, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), monocyte chemotactic protein (MCP)1{alpha}4, myeloid progenitor inhibitory factor (MPIF)-1, monocyte chemotactic and activating factor (MCAF), haemofiltration-derived CC chemokine (HCC)-1 and leukotactin (Lkn)-1 were all reported to be ligands for CCR-1, -2, -4 and -5. It has also been reported that MCP-1 was detected in both FF (Kawano et al., 2001Go) and peritoneal fluid (Zeyneloglu et al., 1998Go; Garcia-Velasco et al., 1999Go; Yih et al., 2001), and that MCAF was detected in seminal plasma (Shimoya et al., 1995Go). It is possible that these chemokines, as well as RANTES, modulate sperm motility.

As yet, the physiological significance of the present results is not clear, despite the data having demonstrated that RANTES and other ligands for CCR-1, -2, -4 and -5 directly modulate spermatozoan function. Sperm chemotaxis may serve as a key process in the transfer of sperm to the fertilization site, and in the bringing together of human gametes. It is possible that the high concentrations of RANTES in genital tract fluids observed in several diseases associated with infertility (endometriosis and infectious disease) disturb the transfer of sperm to the fertilization site. Future studies should demonstrate the intracellular localization of the RANTES receptor in sperm, and may also reveal which type of sperm are attracted by RANTES, whether or not they are capacitated, and whether sperm attracted by RANTES have a high fertilizing ability.


    Notes
 
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, School of Medicine, Mie University, 514-8507, Edobashi 2-174, Tsu, Mie, Japan. E-mail: hminoura{at}clin.medic.mie-u.ac.jp Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Alam, R., Stafford, S., Forsythe, P., Harrison, R., Faubion, D., Lett-Brown, M.A. and Grant, J.A. (1993) RANTES is a chemotactic and activating factor for human eosinophils. J. Immunol., 150, 3442–3447.[Abstract/Free Full Text]

Blackmore, P.F., Beebe, S.J., Danforth, D.R. and Alexandre, M. (1990) Progesterone and 17-ßhydroxyprogesterone: novel stimulators of calcium influx in human sperm. J. Biol. Chem., 265, 1376–1380.[Abstract/Free Full Text]

Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate phenol chloroform extraction. Anal. Biochem., 162, 156–159.[ISI][Medline]

Eisenbach, M. (1999) Sperm chemotaxis. Rev. Reprod., 4, 56–66.[Abstract/Free Full Text]

Eisenbach, M. and Tur-Kaspa, I. (1994) Human sperm chemotaxis is not enigmatic anymore. Fertil. Steril., 62, 233–235.[ISI][Medline]

Eisenbach, M. and Tur-Kaspa, I. (1999) Do human eggs attract spermatozoa? BioEssays, 21, 203–210.[ISI][Medline]

Garcia-Velasco, J.A., Seli, E. and Arici, A. (1999) Regulation of monocyte chemotactic protein-1 expression in human endometrial stromal cells by integrin-dependent cell adhesion. Biol. Reprod., 61, 548–552.[Abstract/Free Full Text]

Hartshorne, G.M. (1991) Dynamic and phagocytotic activity of human granulosa cells in vitro. Hum. Reprod., 6, 331–337.[Abstract]

Hornung, D., Ryan, I.P., Chao, V.A., Vigne, J.L., Schriock, E.D. and Taylor, R.N. (1997) Immuno-localization and regulation of the chemokine RANTES in human endometrial and endometriosis tissue and cells. J. Clin. Endocrinol. Metab., 85, 1621–1628.

Hornung, D., Bentzien, F., Wallwiener, D., Kiesel, L. and Taylor, R.N. (2001) Chemokine bioactivity of RANTES in endometriotic and normal endometrial stromal cells and peritoneal fluid. Mol. Hum. Reprod., 7, 163–168.[Abstract/Free Full Text]

Jaiswal, B.S., Tur-Kaspa, I., Dor, J., Mashiach, S. and Eisenbach, M. (1999) Human sperm chemotaxis: is progesterone a chemoattractant? Biol. Reprod., 60, 1314–1319.[Abstract/Free Full Text]

Karstrom-Encrantz, L., Runesson, E., Bostrom, E.K. and Brannstrom, M. (1998) Selective presence of the chemokine growth-regulated oncogene alpha (GRO alpha) in the human follicle and secretion from cultured granulosa-lutein cells at ovulation. Mol. Hum. Reprod., 4, 1077–1083.[Abstract]

Kawano, Y., Kawasaki, F., Nakamura, S., Matsui, N., Narahara, H. and Miyakawa, I. (2001) The production and clinical evaluation of macrophage colony-stimulating factor and macrophage chemoattractant protein-1 in human follicular fluids. Am. J. Reprod. Immunol., 45, 1–5.[ISI][Medline]

Khorram, O., Taylor, R.N., Ryan, I.P., Schall, T.J. and Landers, D.V. (1993) Peritoneal fluid concentrations of cytokine RANTES correlate with severity of endometriosis. Am. J. Obstet. Gynecol., 169, 1545–1549.[ISI][Medline]

Krausz, C., Bonaccorsi, L., Maggio, P., Luconi, M., Criscuoli, L., Fuzzi, B., Pellegrini, S., Forti, G. and Baldi, E. (1996) Two functional assays of sperm responsiveness to progesterone and their predictive values in in-vitro fertilization. Hum. Reprod., 11, 1661–1667.[Abstract]

Machelon, V., Nome, F. and Emilie, D. (2000) Regulated on activation normal T expressed and secreted chemokine is induced by tumor necrosis factor-{alpha} in granulosa cells from human preovulatory follicle. J. Clin. Endocrinol. Metab., 85, 417–424.[Abstract/Free Full Text]

Makler, A., Reichler, A., Stoller, J. and Feigin, P.D. (1992) A new model for investigating in real-time the existence of chemotaxis in human spermatozoa. Fertil. Steril., 57, 1066–1074.[ISI][Medline]

Morales, P., Llanos, M., Gutierrez, G., Kohen, P., Vigil, P. and Vantman, D. (1992) The acrosome reaction-inducing activity of individual human follicular fluid samples is highly variable and is related to the steroid content. Hum. Reprod., 7, 646–651.[Abstract]

Naz, P.K. and Leslie, M.H. (2000) Immunobiologic implication of RANTES in seminal plasma of fertile, infertile and immunoinfertile men. Am. J. Reprod. Immunol., 44, 197–204.[ISI][Medline]

Nelson, P.J., Kim, H.T., Manning, W.C., Goralski, T.J. and Krensky, A.M. (1993) Genomic organization and transcriptional regulation of the RANTES cytokine gene. J. Immunol., 151, 2601–2612.[Abstract/Free Full Text]

Oehninger, S., Sueldo, C., Lanzendorf, S., Mahony, M., Burkman, L.J., Alexander, N.J. and Hodgen, G.D. (1994) A sequential analysis of the effect of progesterone on specific sperm functions crucial to fertilization in vitro in infertile patients. Hum. Reprod., 9, 1322–1327.[Abstract]

Parinaud, J. and Milhet, P. (1996) Progesterone induces Ca-dependent 3',5'-cyclic adenosine monophosphate increase in human sperm. J. Clin. Endocrinol. Metab., 81, 1357–1360.[Abstract]

Ralt, D., Goldenberg, M., Fetterolf, P., Thompson, D., Dor, J., Mashiach, S., Gabers, D.L. and Eisenbach, M. (1991) Sperm attraction of follicular factor(s) correlates with human egg fertilizability. Proc. Natl Acad. Sci. USA, 88, 2840–2844.[Abstract]

Ralt, D., Manor, M., Cohen-Dayag, A., Tur-Kaspa, I., Ben-Shlomo, I., Makler, A., Yuli, I., Dor, J., Blumberg, S., Mashiach, S. et al. (1994) Chemotaxis and chemokinesis of human spermatozoa to follicular factors. Biol. Reprod., 50, 774–785.[Abstract]

Sabeur, K., Edwards, D.P. and Meizel, S. (1996) Human sperm plasma membrane progesterone receptor(s) and the acrosome reaction. Biol. Reprod., 54, 993–1001.[Abstract]

Schall, T.J., Bacon, K., Toy, K.I. and Goeddel, D.V. (1990) Selective attraction of monocytes and T lymphocytes of the memory phenotype by cytokine RANTES. Nature, 347, 669–671.[ISI][Medline]

Shimoya, K., Matsuzaki, N., Ida, N., Okada, T., Taniguchi, T., Sawai, K., Itoh, S., Ohashi, K., Saji, F. and Tanizawa, O. (1995) Detection of monocyte chemotactic and activating factor (MCAF) and interleukin (IL)-6 in human seminal plasma and effect of leukospermia on these cytokine levels. Am. J. Reprod. Immunol., 34, 311–316.[ISI][Medline]

Sueldo, C., Oehninger, S., Subia, E., Mahony, M., Alexander, N.J., Burkman, L.J. and Acosta, A.A. (1993) Effect of progesterone on human zona pellucida sperm binding and oocyte penetrating capacity. Fertil. Steril., 60, 137–140.[ISI][Medline]

Uhler, M.L., Leung, A., Chan, S.Y. and Wang, C. (1992) Direct effects of progesterone and antiprogesterone on human sperm hyperactivated motility and acrosome reaction. Fertil. Steril., 58, 1191–1198.[ISI][Medline]

Villanueva-Diaz, C., Vadillo-Ortega, F., Kably-Ambe, A., Dias-Perez, M.A. and Krivitzky S.K. (1990) Evidence that human follicular fluid contains a chemoattractant for spermatozoa. Fertil. Steril., 54, 1180–1182.[ISI][Medline]

Villanueva-Diaz, C., Arias-Martinez, J., Bustos-Lopez, H. and Vadillo-Ortega, F. (1992) Novel model for study of human sperm chemotaxis. Fertil. Steril., 58, 392–395.[ISI][Medline]

Villanueva-Diaz, C., Arias-Martinez, J., Bermejo-Martinez, L. and Vadillo-Oretega, F. (1995) Progesterone induces human sperm chemotaxis. Fertil. Steril., 64, 1183–1188.[ISI][Medline]

Wells, T.N., Proufoot, A.E. and Power, C.A. (1999) Chemokine receptors and their role in leukocyte activation. Immunol. Lett., 65, 35–40.[ISI][Medline]

Yih, S., Katabuchi, H., Araki, M., Matsuura, K., Takeya, M., Takahashi, K. and Okamura, H. (2001) Expression of monocyte chemoattractant protein-1 in peritoneal endometriotic cells. Virchows Arch., 438, 70–77.[Medline]

Zeyneloglu, B.H., Senturk, M.L., Seli, E., Oral, E., Olive, L.D. and Arici, A. (1998) The role of monocyte chemotactic protein-1 in intraperitoneal adhesion formation. Hum. Reprod., 13, 1194–1199.[Abstract]

Submitted on October 1, 2001; accepted on January 16, 2002.