Human Reproduction Vol.19, No.2 pp. 409±414, 2004
DOI: 10.1093/humrep/deh085
Expression of fractalkine in the Fallopian tube and of
CX3CR1 in sperm
Qing Zhang, Koichiro Shimoya1, Kumiko Temma, Tadashi Kimura, Tomoko Tsujie,
Mitsunori Shioji, Kenshi Wasada, On Fukui, Shusaku Hayashi, Takeshi Kanagawa,
Toru Kanzaki, Masayasu Koyama and Yuji Murata
Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita City,
Osaka 565-0871, Japan
1
To whom correspondence should be addressed. E-mail: [email protected]
BACKGROUND: Fractalkine is a CX3C chemokine that has chemoattractant activity for T cells, monocytes and
natural killer (NK) cells. The objective of this study was 2-fold: to evaluate (i) the presence of fractalkine in the
Fallopian tube and (ii) the existence of CX3CR1 (fractalkine receptor) in ejaculated sperm. METHODS AND
RESULTS: Western blot analysis revealed that fractalkine protein was detected as a 95 kDa band in the isthmus,
the ampulla and the infundibulum of the Fallopian tube. Immunohistochemistry revealed positive staining of
epithelial cells in the Fallopian tube. RT±PCR demonstrated that fractalkine transcripts were expressed in all parts
of the Fallopian tube. RT±PCR also revealed that CX3CR1-positive cells were present in the Fallopian tube.
CX3CR1-positive cells were present in the stroma of the Fallopian tube. The villi of the ciliated cells were positively
stained. To determine the function of fractalkine in the Fallopian tube, we examined whether CX3CR1 was present
in ejaculated sperm. RT±PCR demonstrated that CX3CR1 transcripts were expressed in the ejaculated sperm.
Immunohistochemistry demonstrated positive staining of the tail of the spermatozoa. CONCLUSIONS: The present
®ndings suggest that fractalkine in the Fallopian tube contributes to the immunodefence mechanism during
fertilization and to the sperm motion in the oviduct.
Key words: CX3CR1/Fallopian tube/fractalkine/sperm
Introduction
Chemokines are small proteins that stimulate the migration of
leukocytes and mediate in¯ammation. These proteins are
classi®ed into subgroups according to characteristic cysteine
signature motifs (Baggiolini, 1998). CXC molecules, such as
interleukin (IL)-8, target neutrophils and, to some degree,
lymphocytes; CC molecules, such as monocyte chemotactic
and activating factor (MCAF), target monocytes, lymphocytes,
basophils and eosinophils with variable selectivity; and the Cchemokine seems to act only on lymphocytes (Rollins, 1997).
A chemokine bearing a CX3C cysteine motif has been cloned
(Bazan et al., 1997; Pan et al., 1997). In contrast to other
chemokines, this chemokine, named fractalkine, displays
potent chemoattractant activity for T cells, natural killer
(NK) cells and monocytes, but not neutrophils, and is of nonhaemopoietic origin (Bazan et al., 1997). Fractalkine is
produced by endothelial cells and neurons and occurs as a
cell surface-bound, as well as a cleaved soluble protein
(Harrison et al., 1999; Papadopoulos et al., 1999;
Muehlhoefer et al., 2000). The extracellular domain of
fractalkine is released into the supernatant of transfected
cells as a 95 kDa glycoprotein (Bazan et al., 1997; Pan et al.,
1997). In the reproductive system, this molecule is present in
the seminal plasma and is associated with sperm motility
(Zhang et al., 2002). We have already reported that fractalkine
contributes to the immunodefence mechanism during pregnancy (Shimoya et al., 2003). The expression of fractalkine has
been reported to be upregulated by in¯ammatory signals
(Schall, 1997). Fractalkine may be responsible for the accumulation of lymphocytes in regions of in¯ammation. The
receptor for fractalkine, named CX3CR1, has been characterized (Imai et al., 1997; Jiang et al., 1998). CX3CR1 is
expressed on the surface of NK cells, monocytes and CD8+ T
cells (Imai et al., 1997). CX3CR1-mediated signal transduction
presumably plays a role in cell migration and adhesion (Imai
et al., 1997; Jiang et al., 1998).
In the Fallopian tube, several important steps, such as
gamete transport, maturation, fertilization and early embryogenesis, take place. It follows, therefore, that the environment
in the Fallopian tube represents the optimal conditions for these
and other important developmental processes. In addition, the
mechanisms of chemotaxis and thermotaxis of the sperm have
been revealed in several studies (Bahat et al., 2003; Spehr et al.,
2003). The Fallopian tube contains various factors, such as
Human Reproduction vol. 19 no. 2 ã European Society of Human Reproduction and Embryology 2004; all rights reserved
409
Q.Zhang et al
growth factors and cytokines (Buhi et al., 1999; Ota et al.,
2002). However, little is known about the chemokines in the
Fallopian tube. The aim of this study was to investigate the
expression of fractalkine in the Fallopian tube and to clarify the
expression of fractalkine receptor (CX3CR1) in the spermatozoa.
Determination of fractalkine levels in various parts of the
Fallopian tubes by densitometric analysis of western blotting
To measure the fractalkine levels in various parts of the Fallopian
tubes among six cases, the expression of fractalkine protein was
quanti®ed and analysed using the NIH image software program
[developed and provided by the Research Services Branch (RSB) of
the National Institute of Mental Health (NIMH)].
Materials and methods
RNA extraction
RNA was extracted from Fallopian tube and amniotic membrane
samples of 0.5 g wet weight by acid guanidine thiocyanate±phenol±
chloroform extraction according to the method of Chomczynski and
Sacchi (1987). The concentrations of RNA were determined by their
absorbance at 260 nm.
Reagents
Goat anti-human fractalkine polyclonal antibodies and recombinant
fractalkine were purchased from R&D systems (Minneapolis, MN).
Normal goat IgG for use as a control in the histochemical analysis was
purchased from Zymed Laboratories (San Francisco, CA). A human
CX3CR1 antibody (rat IgG2b), ¯uorescein isothiocyanate (FITC)labelled human CX3CR1 antibody (rat IgG2b) and FITC-conjugated
rat IgG2b were purchased from MBL (Nagoya, Japan). Bisbenzimide
Hoechst 33258 ¯uorochrome was purchased from Nacalai Tesque,
Inc. (Kyoto, Japan).
Samples
Twelve samples of Fallopian tubes were obtained from gynaecological patients who underwent total hysterectomy and bilateral
oophorectomy. The age range of the patients was 29±45 years old.
Patients with complications of venereal infection were excluded from
the study. For the control of the analysis, amniotic membranes were
obtained from pregnant women in the third trimester. This study was
approved by the local ethics committee of the Department of
Obstetrics and Gynecology, Osaka University Graduate School of
Medicine. Informed consent was obtained from each patient. Semen
samples were obtained from three proven fertile men. The fertile men
had fathered at least one child and had no recent history of venereal
infection. Semen was obtained by masturbation after 5 days of
abstinence. Samples were collected in a sterile container and
examined within 1 h after ejaculation.
Tissue preparation for western blot analysis
The homogenizing buffer for protein extraction from the Fallopian
tubes and amniotic membranes consisted of 0.5 M Tris±HCl pH 6.8,
10% sodium dodecyl sulphate (SDS), 6% b-mercaptoethanol and 1%
bromophenol blue. The 0.5 g sample of Fallopian tubes and amniotic
membranes were homogenized in a 2 ml volume. Homogenates were
centrifuged at 4°C for 30 min at 14 000 g to remove debris. Following
protein determinations, the samples were aliquoted and subjected to
polyacrylamide gel electrophoresis (PAGE).
Western blot analysis of Fallopian tubes
To examine fractalkine protein in the Fallopian tubes, we performed
western blotting analysis using an anti-human fractalkine polyclonal
antibody. A 10 mg aliquot of oviductal protein was electrophoresed on
a 15% SDS±polyacrylamide gel and transferred onto a nitrocellulose
membrane (0.45 mm; Schleicher and Schuell, Dassel, Germany). The
membrane was incubated with 5% dried milk protein followed by antihuman fractalkine polyclonal antibody. The primary antibody was
used at a ®nal concentration of 1.0 mg/ml. The fractalkine
immunoreactivity was visualized using an enhanced chemiluminescence (ECL) western blotting analysis system (Amersham, Aylesbury,
UK).
Protein assay
Protein levels were determined with Bio-Rad (Hercules, CA) Protein
Determination Reagent, according to the method of Bradford (1976).
410
RT±PCR ampli®cation
RT±PCR was performed using an RT±PCR high kit (TOYOBO Co.,
Osaka, Japan). The reaction was carried out in a mixture containing of
Moloney murine leukaemia virus reverse transcriptase (RTase) and 1
mg of RNA sample in 13 RTase buffer, random hexamers and dNTP
mix for 40 min at 42°C. PCR ampli®cation was performed using the
reverse transcription mixture after the incubation described above
(10 ml), with sequence-speci®c primers for human CX3CR1 (5¢TTGAGTACGATGATTTGGCTGA-3¢/ 5¢-GGCTTTGGCTTTCTTGTGG-3¢) (GenBank accession No. U28934) or human fractalkine
(5¢-ACTCTTGCCCACCCTCAGC-3¢/5¢-TGGAGACGGGAGGCACTC-3¢) (GenBank accession No. U84487). PCR was carried out for
35 cycles using a thermal cycler (Perkin-Elmer/Cetus, Norwalk, CT).
Each cycle consisted of denaturation at 94°C (40 s), annealing at 52°C
(40 s) and extension at 72°C (40 s). Ampli®cation using CX3CR1speci®c primers yielded a 653 bp DNA product with a sequence that
matched the published sequence of the CX3CR1 gene (Muehlhoefer
et al., 2000), while ampli®cation using the fractalkine-speci®c primers
yielded a 597 bp DNA product that matched the published sequence of
the fractalkine gene (Muehlhoefer et al., 2000). A mock reverse
transcription was performed to detect possible contamination of RNA
samples by genomic DNA. A 20 ml aliquot of a 50 ml PCR mixture was
electrophoresed on a 1% agarose gel and stained with ethidium
bromide, and ampli®ed products were visualized by UV illumination.
Molecular sizes were estimated by comparison with a 100 bp DNA
ladder. All primers were obtained from Life Technologies (Tokyo,
Japan).
Immunohistochemical staining of fractalkine in the Fallopian
tubes
To determine the localization of fractalkine in the Fallopian tube, we
performed immunohistochemical staining by using an avidin±biotin
peroxidase complex method kit (OminiTags Universal Streptavidin/
Biotin Af®nity Immunostaining Systems, Lipshaw, Pittsburg, PA).
Paraf®n sections of the Fallopian tube were incubated in 0.3%
hydrogen peroxide to block endogenous peroxidase and covered with
2% goat IgG to minimize non-speci®c binding. The 1000-fold diluted
goat polyclonal anti-fractalkine antibody (R&D Systems) or control
pre-immune goat serum for the control was applied at room
temperature and left for 1 h. After the sections were rinsed with
phosphate-buffered saline (PBS) solution, they were incubated further
for 30 min with biotin-labelled goat anti-mouse IgG, and then with
avidin±peroxidase complex at 4°C. Peroxidase activity in the sections
was visualized with 0.1% 3,3-diaminobenzidinine tetrahydrochloride
containing 0.02% hydrogen peroxide in 0.1 mol/l Tris buffer pH 7.2.
The brown colour was determined to be positive. The slides were
counterstained with Mayer's haematoxylin.
Fractalkine expression in the Fallopian tube
Immunohistochemical staining of CX3CR1 in the Fallopian tubes
The method for the determination of the localization of CX3CR1 in the
Fallopian tube was as described above for fractalkine, except that a
1000-fold diluted goat polyclonal anti-CX3CR1 antibody from MBL
(Nagoya, Japan) was used
Preparation of motile sperm
Semen specimens were obtained after 5 days of abstinence. After
liquefaction at room temperature, the semen was examined to
determine the sperm count and motility using a Makler Counting
Chamber (Seti-Medical Instruments, Haifa, Israel). The absence of
leukospermia (polymorphonuclear cells >106/ ml) in the collected
samples was veri®ed. Motile sperm were obtained by the swim-up
method in addition to the Percoll gradient method (World Health
Organization, 1999).
Immunohistochemical staining of CX3CR1 in the sperm
To determine the localization of CX3CR1 in the spermatozoa, we
performed ¯uorescence staining with anti-CX3CR1 antibody. The
spermatozoa were washed twice with PBS and co-incubated with
FITC-labelled anti-human CX3CR1 (rat IgG2b) and control IgG2b for
1 h at 37°C in 5% CO2. The spermatozoa were washed three times
with PBS and suspended in 95% ethanol for 10 min. An aliquot was
dried on a glass slide. The slide was incubated with 0.8 mg/ml
bisbenzimide Hoechst 33258 ¯uorochrome (H33258) for 2 min. The
slide was rinsed with distilled water and observed with an Olympus
BH2 epi¯uorescence microscope (Tokyo, Japan) with an excitation
maximum of 490 nm and an emission maximum of 520 nm to detect
FITC staining.
Statistical analysis
The data were subjected to one-way ANOVA using the Statview
statistics package (Abacus Concepts, Inc., Berkeley, CA). P < 0.05
was considered signi®cant.
Results
To detect fractalkine protein in the Fallopian tubes, we
performed western blot analysis. As shown in Figure 1A,
fractalkine protein was detected as a 95 kDa band in all parts of
the Fallopian tube. To determine fractalkine levels in various
parts of the Fallopian tubes, we analysed the densitometric
intensity of the bands observed in western blot analysis using
the software `NIH image'. The fractalkine levels in various
parts of the Fallopian tubes of six samples were analysed by
western blot analysis. The intensity of fractalkine protein in the
ampulla of the tube (lane 4) looked stronger than that in the
infundibulum (lanes 5 and 6). However, there were no
signi®cant differences in the expression levels of fractalkine
in the isthmus, ampulla and infundibulum among six cases
(P > 0.05). Fractalkine protein was present at similar levels in
the isthmus, ampulla and infundibulum.
RT±PCR was then performed to examine the expression of
the fractalkine gene in the Fallopian tube. Figure 1B shows that
fractalkine transcripts were present in all parts of the Fallopian
tube. To identify the origin of fractalkine, we performed
immunohistochemical staining of sections of the Fallopian tube
using an anti-fractalkine polyclonal antibody. The epithelial
cells in the Fallopian tube were stained (Figure 2). The
intensity of staining in the infundibulum of the Fallopian tube
Figure 1. (A) Western blot analysis of fractalkine protein in the
Fallopian tube. Approximately 10 mg of Fallopian tube protein were
electrophoresed on a 15% SDS±polyacrylamide gel and transferred
onto a 0.45 mm nitrocellulose membrane. The fractalkine signal was
detected as described in the text. Molecular weight is demonstrated
in the left panel (kDa). rFRK: recombinant fractalkine (10 ng).
Tissue lysate from the isthmus of the Fallopian tube of case 1
(lane 1) and case 2 (lane 2) with myoma uteri. Tissue lysate from
the ampulla of the Fallopian tube of case 1 (lane 3) and case 2
(lane 4). Tissue lysate from the infundibulum of the Fallopian tube
of case 1 (lane 5) and case 2 (lane 6). (B) Fractalkine mRNA
expression in the Fallopian tube. Agarose gel electrophoresis of
RT±PCR-ampli®ed fractalkine cDNA. Lane 1: cDNA from the
isthmus of the Fallopian tubes of a woman with myoma uteri (case
1). Lane 2: cDNA from a mock RT sample of the isthmus of the
Fallopian tubes of case 1. Lane 3: cDNA from the ampulla of the
Fallopian tubes of case 1. Lane 4: cDNA from a mock RT sample
of the ampulla of the Fallopian tubes of case 1. Lane 5: cDNA from
the infundibulum of the Fallopian tubes of case 1. Lane 6: cDNA
from a mock RT sample of the infundibulum of the Fallopian tubes
of case 1. Lane 7: cDNA from the control sample of the amniotic
membranes. Lane 8: cDNA from a mock RT sample of the control
sample of the amniotic membranes.
was slightly weaker than that in other parts. Both the ciliated
and non-ciliated epithelial cells were stained (Figure 2G). The
positive staining seemed to be mainly nuclear. To determine
whether CX3CR1-positive cells were present in the Fallopian
tube, we performed RT±PCR using speci®c primers for
CX3CR1. As shown in Figure 3A, RT±PCR demonstrated the
presence of CX3CR1 mRNA expression in the Fallopian tube.
The expression of CX3CR1 mRNA in the spermatozoa was
also examined by RT±PCR. As shown in Figure 3B, CX3CR1
mRNA was detected in the spermatozoa.
The localization of CX3CR1-positive cells in the Fallopian
tube is demonstrated in Figure 4. There were several cells in the
stroma of the Fallopian tube stained with CX3CR1 antibodies
(Figure 4A and B). In addition to the positively stained cells in
the stroma of the Fallopian tube, the villi of ciliated epithelial
cells were also positively stained (Figure 4C). To determine
411
Q.Zhang et al
Figure 2. Immunohistochemical staining of fractalkine-producing cells in the Fallopian tube. The cells in the Fallopian tube were stained by
the avidin±biotin complex method with a goat polyclonal anti-fractalkine antibody (A, C and E) or control goat serum (B, D and F). (A and
B) The isthmus of the Fallopian tube. (C and D) The ampulla of the Fallopian tube. (E and F) The infundibulum of the Fallopian tube. The
epithelial cells in the Fallopian tube were stained. (G) High magni®cation of the ampulla of the Fallopian tube.
whether CX3CR1 molecule was present on spermatozoa, we
performed immunocytochemical staining. About half of
spermatozoa were stained by anti-CX3CR1 antibody conjugated with FITC in Figure 5A. As shown in Figure 5C,
immunocytochemical analysis using an anti-CX3CR1 antibody
conjugated with FITC demonstrated positive staining of the
tails of the spermatozoa. To demonstrate the localization of
CX3CR1 we used bisbenzmide Hoechst 33258 ¯uorochrome.
The localization in the sperm head is shown in Figure 5B and
D.
Discussion
Human semen contains a certain amount of fractalkine, and an
association between sperm motility and the fractalkine concentration in the seminal plasma has been observed (Zhang
et al., 2002). Recently, several studies revealed the mechanism
of chemotaxis and thermotaxis of the sperm (Bahat et al., 2003;
Spehr et al., 2003). Isobe et al. (2002) demonstrated that
mRNAs for the RANTES (regulated on activation normal T
expressed and secreted chemokine) receptors CCR-1 and CCR5 are present in human sperm. RANTES has a chemotactic
effect on human sperm but does not affect sperm motility
(Isobe et al., 2002). In the present study, we demonstrated that
CX3CR1 mRNA and protein were present in sperm. Taken
together, these ®ndings indicate that fractalkine might play a
412
role in maintaining the motility of spermatozoa and regulating
sperm chemotaxis.
Human oviductal cells produce factors that are important for
the maintenance of sperm motility in vitro (Yao et al., 2000).
Several studies using animal models demonstrated that the
oviduct had important roles in the fertilization process, such as
the penetration of eggs by sperm, capacitation of the sperm and
the reservoir for spermatozoa during the period from mating to
ovulation (Hunter, 1984; Smith et al., 1987; Parrish et al.,
1989; Boatman and Magnoni, 1995). In the isthmus of the
Fallopian tube, spermatozoa bind to the oviduct epithelium.
This interaction may represent part of the in vivo capacitation
process (Smith et al., 1987). Because the fractalkine receptor
(CX3CR1) is present on the surface of sperm, fractalkine in the
isthmus might play an important role in maintaining the
motility of spermatozoa and their ability to undergo the
acrosome reaction while sperm are stored until ovulation.
Fractalkine is one of the chemokines with a CX3C cysteine
motif. In the present study, we demonstrated the expression of
fractalkine protein and mRNA in the Fallopian tube. The
present ®ndings suggest that fractalkine is constitutively
present in all parts of the Fallopian tube. Our immunohistochemical analysis using anti-fractalkine polyclonal antibody
showed that the epithelial cells of the Fallopian tube were
stained, suggesting that these epithelial cells are the main
source of fractalkine in the Fallopian tube. The reason why the
Fractalkine expression in the Fallopian tube
Figure 3. (A) CX3CR1 mRNA expression in the Fallopian tube.
Agarose gel electrophoresis of RT±PCR-ampli®ed CX3CR1 cDNA.
Lane 1: cDNA from the isthmus of the Fallopian tubes of a woman
with myoma uteri (case 1). Lane 2: cDNA from a mock RT sample of
the isthmus of the Fallopian tubes of case 1. Lane 3: cDNA from the
ampulla of the Fallopian tubes of case 1. Lane 4: cDNA from a mock
RT sample of the ampulla of the Fallopian tubes of case 1. Lane 5:
cDNA from the infundibulum of the Fallopian tubes of case 1. Lane 6:
cDNA from mock RT sample of the infundibulum of the Fallopian
tubes of case 1. Lane 7: cDNA from the control sample of the
amniotic membranes. Lane 8: cDNA from a mock RT sample of the
control sample of the amniotic membranes. (B) CX3CR1 mRNA
expression in the spermatozoa. Motile sperm were obtained by the
Percoll gradient method and swim-up method. Agarose gel
electrophoresis of PCR-ampli®ed DNA of CX3CR1. cDNA from the
spermatozoa of donor 1 (lane 1), donor 2 (lane 2) and donor 3 (lane 3).
Lane 4: cDNA from the control sample of the amniotic membranes.
Figure 4. Immunohistochemical staining of CX3CR1-positive cells
in the Fallopian tube. The cells in the Fallopian tube were stained
by the avidin±biotin complex method with a rat monoclonal antiCX3CR1 antibody (A±C) or control goat serum (D±F). (A and
D) The ampulla of the Fallopian tube at low magni®cation. (B and
E) The stroma of the ampulla of the Fallopian tube at high
magni®cation. (C and F) The epithelium of the ampulla of the
Fallopian tube at high magni®cation.
Figure 5. Fluorescence staining of CX3CR1 in the spermatozoa. (A and C) The spermatozoa were stained with the FITC-conjugated
anti-CX3CR1 antibody. (A) Low and (C) high magni®cation. (B and D) Bisbenzmide Hoechst 33258 ¯uorochrome staining. (C) Low and (D)
high magni®cation. The arrows represent the positive staining in the tail of spermatozoa.
413
Q.Zhang et al
positive staining seemed to be mainly nuclear is unknown.
Further investigations would be necessary to reveal this. The
Fallopian tubes have bacteriostatic and bactercidal mechanisms that protect against infection through the Fallopian tubes
into the peritoneal cavity. It is well known that in¯ammatory
cytokines are increased at in¯ammation sites. Several in¯ammatory mediators enhance the fractalkine mRNA levels and the
production of fractalkine (Muehlhoefer et al., 2000). Large
numbers of leukocytes in the Fallopian tube are associated with
oviductal infection. In the present study, we detected the
presence of CX3CR1 mRNA in the cells of the Fallopian tube.
Immunohistochemistry revealed that CX3CR1-positive cells
were present in the Fallopian tube. Fractalkine has an important
role in Th1 type cell function and the in¯ammatory response
(Cockwell et al., 2001; Nishimura et al., 2002). Such
lymphocytes might contribute to the immunodefence system
in the oviduct. Fractalkine might protect the Fallopian tube
epithelium against infection in the oviduct. This molecule
might contribute to homeostasis of the immunodefence system
in the oviduct in cases of salpingitis, for example in chlamydia
infection. Further investigations will be necessary to examine
the relationship between the fractalkine level and genital tract
infections such as salpingitis. The villi of the ciliated cells were
also positively stained by CX3CR1 antibody. These results
demonstrated that the epithelial cells of the Fallopian tube
might be regulated by fractalkine in the autocrine mechanism.
Acknowledgements
This work was supported, in part, by Grants-in-Aid for Scienti®c
Research (Nos 13671712, 13671713, 13877273, 12671596, 15209054
and 15591746) from the Ministry of Education, Science, and Culture
of Japan (Tokyo, Japan), and Akaeda Medical Research Foundation.
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Submitted on March 20, 2003; resubmitted on August 27, 2003; accepted on
October 20, 2003