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
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Abstract |
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Key words: chemotaxis/cytokine/follicular fluid/human sperm/RANTES
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Introduction |
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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., 1998). RANTES is a 68-amino acid peptide (Nelson et al., 1993
) of 8 kDa molecular weight (Schall et al., 1990
) 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., 1999
). RANTES may be a potent chemoattractant for eosinophils, monocytes and T lymphocytes (Schall et al., 1990
; Alam et al., 1993
).
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, 2000), uterine fluid (Hornung et al., 1997
) and peritoneal fluid (Khorram et al., 1993
; Hornung et al., 2001
) 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., 1993
; Hornung et al., 2001
) and male genital tract infection (Naz and Leslie, 2000
). The mean concentration of RANTES in FF has been reported as 174 pg/ml (Machelon et al., 2000
), while in severe endometriosis the level was 530.2 pg/ml (Khorram et al., 1993
). RANTES is produced by granulosa cells from human pre-ovulatory follicles up-regulated by TNF-
, the level of which in FF is elevated in women with endometriosis (Machelon et al., 2000
). RANTES is synthesized by endometriotic stromal cells (Hornung et al., 1997
).
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.
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Materials and methods |
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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, 1987), 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., 1994), 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., 1992), 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.
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Results |
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Discussion |
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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, 1999; Eisenbach and Tur-Kaspa, 1999
). 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, 1999
).
Human sperm chemotaxis to FF in vitro appears to be well established (Villanueva-Diaz et al., 1990; Ralt et al., 1991
, 1994
; Makler et al., 1992
; Eisenbach and Tur-Kaspa, 1994
, 1999
; Eisenbach, 1999
), 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., 1992
; Sueldo et al., 1993
; Oehninger et al., 1994
; Parinaud and Milhet, 1996
; Jaiswal et al., 1999
). These effects of FF are cAMP-dependent (Hartshorne, 1991
) and calcium influx-dependent (Blackmore et al., 1990
; Krausz et al., 1996
) on, and are mediated by, intracellular progesterone receptors (Morales et al., 1992
; Sabeur et al., 1996
). It has been reported by one group that progesterone causes sperm chemotaxis (Villanueva-Diaz et al., 1995
), though others (Jaiswal et al., 1999
) 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, 1999
). It is thought by some that progesterone is not a chemoattractant in FF (Uhler et al., 1992
; Jaiswal et al., 1999
), 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., 1994
).
The capillary assay revealed that RANTES had a dose-dependent chemotactic effect on human sperm (Figure 3), 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 I
). 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 resultwhich was confirmed by the double-chamber assay (Figure 5
)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., 2000), and in severe endometriosis was 530.2 pg/ml (Khorram et al., 1993
). The mean concentration of RANTES in FF used in the present experiments was 234 pg/ml (range 123452 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 6), 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),ß, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), monocyte chemotactic protein (MCP)1
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., 2001
) and peritoneal fluid (Zeyneloglu et al., 1998
; Garcia-Velasco et al., 1999
; Yih et al., 2001), and that MCAF was detected in seminal plasma (Shimoya et al., 1995
). 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.
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Notes |
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References |
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Submitted on October 1, 2001; accepted on January 16, 2002.