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From the * Laboratory of Zoology, Graduate School
of Agriculture, Kyushu University, Higashiku Hakozaki, Fukuoka, Japan; and the
Department of Anatomy and Developmental
Biology, Graduate School of Medicine, Chiba University, Chiba, Japan.
| Correspondence to: Dr Hiroshi Iida, Laboratory of Zoology, Graduate School of Agriculture, Kyushu University, Higashiku Hakozaki 6-10-1, Fukuoka 812-8581, Japan (e-mail: iidahiro{at}agr.kyushu-u.ac.jp). |
| Received for publication March 5, 2009; accepted for publication September 10, 2009. |
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Abstract |
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Key words: Sperm, epididymis, maturation, BPI
The epididymis is functionally specialized for transport, maturation, and storage of spermatozoa. Usually, it is divided anatomically into 3 regions: caput, corpus, and cauda. The composition of luminal fluid, which is produced by secretory proteins expressed in a region-specific manner, is highly complex and changes progressively along the epididymal duct (Turner, 1991; Hinton and Palladino, 1995). Some of these proteins seem to be related to the epididymal maturation of spermatozoa. For example, cysteine-rich secretory protein (Crisp-1) expressed predominantly in corpus and cauda epididymis regions might play some roles in regulation of sperm capacitation, as well as interaction with oocytes (Roberts et al, 2006). Bin1b expressed in the caput region might initiate the acquisition of sperm motility (Li et al, 2001; Zhou et al, 2004). A region-specific environment produced along epididymal ducts is believed to play an essential role in controlling or inducing biochemical changes of spermatozoa in epididymis. To understand the molecular mechanisms of epididymal maturation, it is effective to identify region-specifically expressed genes in epididymis as well as their gene products secreted into the lumen of epididymal ducts.
In this study, we isolated by differential display a gene encoding a bactericidal permeability-increasing protein (BPI) that was highly expressed in the caput of rat epididymis. It is a member of the lipid transfer/lipopolysaccharide–binding protein gene family and is related to 2 mammalian lipid transport proteins: cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP; Beamer et al, 1999). We describe here the expression pattern of the rat BPI gene and immmunolocalization of BPI protein in epididymis and epididymal spermatozoa of rats and mice.
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Materials and Methods |
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Differential Display
Differential display (Liang and Pardee,
1992; Blanchard and Cousins,
1996) was carried out with the Delta Differential Display Kit
(Clontech Laboratories, Palo Alto, California). RNA isolated from caput and
cauda epididymides of 8-week-old Wistar rats was reverse transcribed with
oligo-dT primers anchored to the beginning of the poly(A) tail. The resulting
complementary DNA (cDNA) was amplified by polymerase chain reaction (PCR) with
T-primers and P-primers (arbitrary primers) in the kit. The cycling parameters
were as follows: 40 cycles at 94°C for 30 seconds, 40°C for 2 minutes,
and 72°C for 30 seconds. The amplified cDNA was separated on 6%
urea-polyacrylamide gels, fixed, and stained by the silver sequence system
(Promega, Madison, Wisconsin). cDNA fragments, whose expression levels were
regionally specific, were recovered directly by cutting out the gel slices.
After elution by boiling the gel slices in distilled water for 15 minutes,
cDNA fragments were reamplified by using the same primers used in the initial
PCR for differential display. The cDNA fragments were then purified by
electrophoresis, cloned into the pGEM easy T-vector (Promega), and sequenced
with the use of a DNA sequencer (Applied Biosystems, Foster City,
California).
Reverse Transcription PCR
cDNA strands were synthesized from 2 µg of total RNA by using a
first-strand synthesis kit (GE Healthcare BioScience) with random primers. The
reverse-transcribed cDNA was used as a PCR template to synthesize a rat
BPI gene. The primers used to amplify the full-length rat
BPI gene were 5'-ATG GCC TGG GGC CCT GAC AAC-3' (forward)
and 5'-TCA GGT CCG GTG TAA ATC CGC C-3' (reverse). The
PCR-amplified DNA of 1449 bp length was cloned into pGEM-T easy vector and
sequenced with a DNA sequencer. The PCR products were examined by agarose gel
electrophoresis. The PCR primers for glyceraldehyde-3-phosphate dehydrogenase
(G3PDH) were 5'-TGA AGG TCG GTG TCA ACG GAT TTG GC-3'
(forward) and 5'-CAT GTA GGC CAT GAG GTC CAC CAC-3' (reverse).
In Situ Hybridization
DNA of BPI (611-nucleotide length) was PCR-amplified and ligated into
pGEM-T–easy vector (Promega). DNA (611 bp) was cut out from the vector
by PvuII, purified by agarose gel electrophoresis, and used to make probes.
Primers used to amplify the 611-bp-length DNA fragment were
5'-CAGTCACATCAACAGCGTCCG-3' (forward) and
5'-GATCCCTCTCTTCCTGCTTGG-3' (reverse). Sense and antisense RNA
probes were transcribed in vitro by digoxigenin (DIG)-labeled UTP and T7 or
SP6 RNA polymerase, as described in the manufacture's manual (DIG RNA Labeling
Kit; Roche Diagnostics, Penzberg, Germany). In situ hybridization was carried
out as previously reported (Iida et al,
2001; Doiguchi et al,
2002b). In brief, frozen sections of adult rat epididymis were
preincubated for 30 minutes at 42°C in a hybridization buffer (20 mM
Tris-HCl [pH 8.0], 0.3 M NaCl, 2 mM EDTA, 50% formamide, 1 mg/mL bovine serum
albumin, 0.02% Ficoll, 0.02% polyvinylpyrolidone, 1 mg/mL herring sperm DNA)
and hybridized for 5 hours at 42°C in the hybridization buffer containing
a DIG-labeled sense or antisense RNA probe of 611 nucleotides. After
hybridization, the sections were washed for 1 hour in 2 x SSC with 50%
formamide at 42°C and incubated for 30 minutes at 37°C with RNaseA (20
mg/mL), and bound complementary RNA (cRNA) was detected with the use of the
anti-DIG alkaline phosphatase–conjugated antibody (1:500 dilution, Roche
Diagnostics) and visualized with nitroblue
tetrazolium-5-bromocresyl-3-indolyl-phosphate (NTB-BCIP, Roche
Diagnostics).
Antibody Production
The peptide used for raising antibody is derived from the hydrophilic
region of rat BPI (SGDFKIKHLGKG), which corresponds to the amino acid residues
64–75 of rat BPI (482–amino acid length). The peptide was coupled
to keyhole limpet hemocyanin (KLH; Pierce, Rockford, Illinois). The peptide
coupled to KLH (1 mg total dose) was dissolved in 1 mL of saline, emulsified
with 1 mL of Freund complete adjuvant, and injected at multiple sites on the
back of a rabbit as described previously
(Katafuchi et al, 2000). The
antiserum was collected within 2 weeks of the third injection. Affinity
purification of the antibody was carried out over a matrix of the peptide
coupled to formyl-cellulofine (Seikagaku Kohgyo, Japan) as described
previously (Iida et al, 2001).
This antibody not only reacted with rat BPI but also recognized mouse BPI,
probably because only 1 amino acid residue D within the peptide sequence of
rat BPI is replaced by V in mouse BPI (SGVFKIKHLGKG). Affinity-purified
anti-BPI antibody (7 µL) was incubated with the synthetic peptide (1 mg)
for 16 hours at 4°C, followed by centrifugation at 15 000 x
g for 20 minutes. The resultant supernatant was used as absorbed
antibody.
Preparation of Glutathione S-Transferase Fusion Proteins
Rat BPI has an open reading frame of 1446 nucleotides encoding 482 amino
acids. Rat BPI (167 amino acid length, amino acids 
8–174) was
PCR-amplified and cloned in-frame to the COOH terminus of glutathione
S-transferase (GST) with a pGEX-4T-1 system (GE Healthcare).
Molecular size of GST-rat BPI is 43 kd. N-terminal 98 amino acid–length
(amino acids 1–98) of mouse BPI comprising 483 amino acids was
similarly fused to the COOH terminus of GST. Molecular size of GST mouse BPI
is 35 kd. Recombinant proteins were expressed in Escherichia coli and
purified onto glutathione-Sepharose (GE Healthcare) as previously described
(Iida et al, 2001). GST-fused
recombinant proteins Rab3A, Rab3D, Rab6, Spergen3, Spetex1, and Syntaxin2 were
similarly produced and purified. These recombinant proteins were used for
immunoblot analysis.
Transfection
The nucleotide encoding the myc-tag peptide (EQKLISEEDL) was engineered to
fuse in-frame with the C-terminus of full-length rat BPI cDNA by PCR as
reported previously (Doiguchi et al,
2002b). The sequence of PCR product was confirmed by a DNA
sequencer. Myc-tagged BPI was subcloned into the pCI-neo expression vector
(Promega) for transfection into COS7 cells cultivated in Dulbecco-modified
Eagle medium supplemented with 10% fetal bovine serum. Transfection of the
plasmids into COS7 cells was performed with Lipofectamine 2000 reagent
(Invitrogen, Carlsbad, California) following the manufacture's instructions.
After 24 hours of culture, transfected cells were directly dissolved in a
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample
buffer. After centrifugation for 15 minutes at 10 000 x g,
clarified materials (total lysate) were processed for immunoblot analysis.
Nontransfected COS7 cells were used for immunoblot as a control.
Immunoblot Analysis
Caput and cauda epididymides taken from ether-anesthetized adult Wistar
rats and ddY mice were washed in phosphate-buffered saline (PBS) at 4°C.
Rat testes were immersed in PBS on ice, and capsule (tunica albuginea) was
torn away by tweezers. Seminiferous tubules released from testes were
untangled and gently agitated in several washes of PBS at 4°C to remove
interstitial cells. The tissues were cut into small pieces and homogenized in
RIPA buffer (10 mM Tris-Cl, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.1% SDS, 0.1%
Na deoxycholate, pH 7.4) with protease cocktail (Roche Diagnostics). The
samples were centrifuged for 15 minutes at 10 000 x g to remove
undissolved materials, and resultant supernatants were used for
electrophoresis. Spermatozoa, which were collected from caput or cauda
epididymides of adult rats and mice, were purified separately by Percoll
density gradient centrifugation as reported previously
(Doiguchi et al, 2002a).
Purified spermatozoa were solubilized directly in a SDS-PAGE sample buffer.
Proteins prepared for SDS-PAGE were separated on 12% acryl amide gel, and
separated proteins were either stained with Coomassie brilliant blue or
transferred to nitrocellulose sheets. Proteins (12–15 µg) were
loaded for each lane. The sheets were incubated for 2 hours with the anti-BPI
antibody diluted 1:10 000 with a blocking buffer (PBS containing 5% nonfat
milk and 0.1% Tween-20), followed by incubation with horseradish peroxidase
(HRP)–conjugated goat anti-rabbit immunoglobulin G (IgG; BioRad,
Richmond, California) diluted 1:2000 in the same buffer. For detection of
myc-tagged BPI, the sheets were incubated for 2 hours with the anti-myc
antibody (Medical and Biological Laboratories Co, Nagoya, Japan) diluted 1:10
000 with a blocking buffer. Antigen-antibody complexes were visualized with an
ECL-plus detection kit (GE Healthcare).
Immunohistochemistry
Caput and cauda epididymides as well as testis of adult rats and mice were
fixed in 4% paraformaldehyde in PBS at 4°C for 4 hours, washed 3 times in
PBS, incubated in PBS containing 50 mM NH4Cl for 30 minutes, and
then rinsed in PBS. After infiltration of 20% (wt/vol) sucrose in PBS, the
samples were immersed in Optimal Cutting Temperature (OCT) compound
(Tissue-Tek, Miles, Inc, Elkhart, Indiana) and immediately frozen by liquid
nitrogen. Frozen sections of 10 µm thickness were cut by a cryostat
(CM1850; Leica, Nussloch, Germany). The sections were washed in PBS, incubated
for 1 hour with the anti-BPI antibody diluted 1:200 with the blocking buffer,
followed by incubation for 1 hour with goat anti-rabbit IgG conjugated with
Cy3 (GE Healthcare) diluted 1:3000 with the blocking buffer. In some cases,
the sections were incubated overnight with the anti-BPI antibody. For DNA
staining, immunostained samples were incubated for 30 minutes with PBS
containing SYTOX Green (1:10 000 dilution, Molecular Probes, Eugene, Oregon).
The samples were then washed with PBS and examined by a confocal laser
scanning microscope (Olympus LSM-GB 2000, Tokyo, Japan). For controls, the
primary antibody was replaced by preimmune serum.
Spermatozoa released from epididymis of rats and mice were fixed immediately and processed for immunocyto-chemistry as described above. Spermatozoa attached to poly-L-lysine–coated glass slides were immunostained by the anti-BPI antibody and Cy3-labeled secondary antibody, followed by incubation with SYTOX green to label nuclear DNA. For double immunostaining, rat spermatozoa were stained for 1 hour by the anti-BPI antibody and monoclonal MN-7 antibody diluted 1:100 with the blocking buffer, followed by incubation for 1 hour with Cy3-labeled anti-rabbit IgG and fluorescein isothiocyanate (FITC)–labeled anti-mouse IgG diluted 1:100 with the blocking buffer. Monoclonal MN-7 antibody recognizes Acrin1, an intra-acrosomal protein (Saxena and Toshimori, 2004). Mouse spermatozoa immunostained by the anti-BPI antibody were further stained either with SYTOX green to label nuclei or with pure Arachis hypogaea lectin conjugated with FITC (FITC-PNA, 1mg/mL, EY Lab, San Mateo, California) to label acrosome.
Acrosome Reaction Assays
Mouse acrosome reaction assays were conducted essentially as previously
described (Iida et al, 1999).
Sperm isolated from the cauda of mature male mice (ddY strain) were
capacitated for 1 hour at 37°C in M199 medium (Gibco-BRL) supplemented
with 25 mM HEPES (pH 7.4), 30 mg/mL sodium pyruvate, and 4 mg/mL BSA.
Capacitated sperm were treated for 30 minutes either with 1 mM calcium
ionophore A23187 (Sigma Chemical Co) or with vehicle (0.1% dimethyl
sulfoxide). After incubation at 37°C in a humidified atmosphere of 5%
CO2 in air, the samples were fixed with 3% paraformaldehide for 15
minutes, washed in PBS, and dried onto poly-L-lysine–coated
glass slides. Spermatozoa that were immunostained for BPI were further stained
either with FITC-PNA or with SYTOX green. These samples were examined by a
confocal laser scanning microscope or a fluorescence microscope (Olympus
BX-40, Tokyo, Japan). Spermatozoa without PNA-positive acrosomes were scored
as acrosome-reacted cells. Acrosome reaction assays were done independently 3
times and acrosome-reacted spermatozoa were counted in duplicate in each
experimental group. More than 100 sperm per slide were evaluated for acrosomal
status. Data represent ± SD.
Castration
Bilateral castrations were done in pentobarbital-anesthetized Wistar rats
(250–300 g body weight). After a recovery period of 7 days, the
epididymides were removed and either fixed for immunohistochemistry or
dissolved directly in SDS-PAGE sample buffer for immunoblot analysis, as
described above.
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Expression of Rat BPI Messenger RNA
We performed reverse transcription PCR (RT-PCR) analysis to verify that the
BPI gene is expressed in caput but not in cauda of rat epididymis. As
shown in Figure 1B (left
panel), the full-length transcript of rat BPI was detected in caput
epididymis, but it was undetectable in cauda. BPI transcript seemed
to be developmentally expressed because it was not detected in caput
epididymis of 12- and 30-day-old rats
(Figure 1B, right panel). To
examine the expression of the BPI gene in various tissues besides
epididymis, we performed RT-PCR analysis. Expression of the BPI gene
was detected in testis but was undetectable in other organs examined
(Figure 1C). RT-PCR was
performed to examine the developmental expression of BPI in rat testis. It was
first detectable at 3 weeks during postnatal development of testis and
continued to be expressed at up to 8 weeks
(Figure 1D).
To examine the in situ localization of rat BPI messenger RNA (mRNA), we carried out in situ hybridization. Frozen sections prepared from caput and cauda of adult rat epididymis were hybridized either with an antisense probe or with a sense probe as a control. In caput region, strong signals for BPI mRNA was detected in the epithelium of epididymal ducts surrounded by the connective tissues showing relatively weak signals (Figure 2A). Hybridization with a sense probe (control) produced faint or no signal (Figure 2B). In the cauda region, both antisense and sense probes failed to produce distinct signals for BPI mRNA (Figure 2C and D). These data indicate that the rat BPI mRNA is expressed in the epithelium of caput epididymis.
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We performed immunoblot analysis to examine whether BPI protein is expressed in rat epididymis. Presence of BPI on rat spermatozoa was also examined because proteins secreted from epididymis often reveal binding activity to spermatozoa. Proteins were extracted from caput and cauda epididymides as well as spermatozoa purified from caput and cauda epididymides. The proteins were separated on SDS-PAGE and transferred to membranes for immunoblot analysis with the anti-BPI antibody. A major band migrating at 25 kd was detected in all samples, with concomitant appearance of minor bands migrating at 34 and 22 kd (Figure 3D, center panel). The size of a major protein (25 kd) recognized by the antibody is smaller than the predicted molecular mass of BPI (53 kd). Gray et al (1989) has reported that human BPI undergoes proteolytic cleavage, which results in production of an approximately 25-kd N-terminal fragment that is responsible for antimicrobial activity. The 25-kd protein recognized by the antibody in epididymis and spermatozoa probably represents the cleaved N-terminal BPI. The reason for the appearance of immunoreactive 22- and 34-kd bands on the blot was not clear at present. Replacement of the anti-BPI antibody with the antibody that was adsorbed by the synthetic peptide used for immunization (adsorbed antibody) resulted in abolishment of immunodetectable bands on the blot (Figure 3D, right panel). No distinct immunoreactive band was seen on the blot when preimmune serum was used for immunoblot analysis (not shown).
We also examined whether BPI is present in epididymis and spermatozoa of mice. Proteins extracted from caput and cauda epididymis and spermatozoa derived from caput and cauda epididymis were separated on SDS-PAGE, and separated proteins were subjected to immunoblot analysis. In both samples, the anti-BPI antibody recognized a protein migrating at 45 kd on the blot, not cross-reacted with proteins of lower molecular mass (Figure 3E). It suggests that BPI might be not cleaved in mouse.
BPI Localization in Epididymis, Spermatozoa, and Testis
To examine the localization of BPI visually in epididymis, confocal laser
scanning microscopy was carried out on cryosections of caput and cauda
epididymides stained with the anti-BPI antibody. BPI was visualized by
Cy-3–conjugated anti-rabbit IgG (red color), and nuclear DNA was
counterstained by CYTOX-Green dye (green color) after immunostaining. In rat
epididymis, the immunosignal for BPI appeared on the granulelike structures in
the duct lumen of both caput (Figure
4A) and cauda (Figure
4B). They measured about 0.5–5 µm in diameter, sometimes
close to 10 µm. In optimum sections incubated with the anti-BPI antibody
for prolonged time (16 hours), BPI was also detected at the apical
surface region of the epididymal epithelium
(Figure 4C, arrow) as well as
on the head region of spermatozoa (Figure
4D) at high magnification. Weak signal for BPI was seen on the
amorphous structures in the lumen of the initial segment
(Figure 4E). Replacement of the
BPI antibody with preimmune serum gave no specific staining
(Figure 4F). In mouse
epididymis, BPI-positive granulelike structures were also detected in the
lumen of mouse epididymal ducts (Figure
4G).
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Expression and localization of BPI in rat testis was examined by immunoblot analysis and immunohistochemistry with a confocal laser scanning microscope. On the blot to which proteins extracted from seminiferous tubules of rat testis were transferred, the anti-BPI antibody detected a major band migrating at 25 kd, as well as minor bands migrating at 30 and 34 kd (Figure 4H). In testis sections immunostained by the anti-BPI antibody, BPI-positive aggregations were seen in the lumen of the seminiferous tubules (Figure 4I, left panel), whereas no signal was seen in the control (Figure 4I, right panel). These outcomes suggested that BPI might be secreted not only from epididymis but also from the seminiferous tubules. These results are consistent with the observation that BPI gene is expressed in testis as well as in epididymis (Figure 1).
Because BPI immunosignals were detected in spermatozoa (Figures 3D and E, 4D), we performed confocal laser scanning microscopy to examine whether BPI is actually associated with epididymal spermatozoa. Rat spermatozoa released from both caput and cauda epididymis were fixed immediately and processed for BPI immunolabeling. Confocal laser scanning microscopy revealed that in most spermatozoa (>95%) of caput (Figure 4J) and cauda (Figure 4K), immunoreactive BPI was associated with the sperm heads covering the acrosome region. To confirm the localization of BPI over the acrosome region, spermatozoa were double-stained by the anti-BPI antibody and monoclonal MN-7 antibody that recognizes Acrin1 in acrosome. Colocalization of BPI and Acrin1 (Figure 4L) indicated that BPI is localized over the acrosome region of sperm heads. We also examined the distribution of BPI on spermatozoa released from rat testis. Distinct BPI immunosignal was not detected on testis-derived spermatozoa (not shown).
Mouse spermatozoa released from cauda epididymis were similarly processed for BPI immunostaining. We observed that BPI was localized at the sperm heads covering the acrosome region (Figures 4M and N). In mouse spermatozoa stained with the anti-BPI antibody followed by labeling with FITC-PNA, a marker for the acrosome, BPI was found to colocalize with PNA at the acrosome region (Figure 4O). These observations suggest that BPI is localized over the acrosome region of mouse spermatozoa as well.
Acrosome Reaction
We next examined the effects of acrosome reaction on the distribution of
BPI in mouse spermatozoa. Spermatozoa released from mouse cauda epididymis
were incubated for 1 hour in the capacitation medium followed by further
incubation for 30 minutes in the presence or the absence (control) of 1 mM
calcium ionophore A23187 (Ca2+ ionophore) that induces the acrosome
reaction in vitro (Iida et al,
1999). The acrosome reaction of spermatozoa were monitored by
staining with FITC-PNA.
We observed that PNA was seen at the acrosomal region in a large part of control spermatozoa (82.9 ± 5.6%) that were incubated for 30 minutes in the absence of Ca2+ ionophore, whereas Ca2+ ionophore–exposed spermatozoa with PNA on the acrosome region was reduced to 19.2 ± 5.6%. Spermatozoa with BPI immunosignals over the acrosome region in the controls (Figure 5A) and the Ca2+ ionophore treatment (Figure 5B) were 79.0 ± 9.9% and 14.6 ± 6.2%, respectively. By double-staining spermatozoa with the anti-BPI antibody and FITC-PNA, we confirmed that disappearance of PNA was always accompanied by loss of BPI immunosignals at the acrosome region. These data indicate that immunodetectable BPI on sperm head disappears during the acrosome reaction.
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Discussion |
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In addition to BPI-related proteins, other antimicrobial proteins such as cysteine-rich secretory protein 3 (Udby et al, 2002), hCAP-18 (Malm et al, 2000), and defensins (Com et al, 2003) are expressed in male reproductive tracts. It has recently been reported that beta-defensin plays an important role in induction of sperm motility by inducing Ca2+ uptake (Zhou et al, 2004). Hence, antimicrobial proteins can have other specific properties when expressed in various tissues. Indeed, BPI has been implicated in biological functions besides those related directly to antibactericidal effects; for example to act as an endogenous inhibitor of angiogenesis (Van Der Schaft et al, 2000), to neutralize the inflammatory effects of LPS (Katz et al, 1996), or to stimulate phagocytosis by complement activation (Nishimura et al, 2001).
In this study, we isolated rat BPI by differential display as a caput-specific expressed gene. BPI mRNA was also expressed in testis but was not detectable in other organs examined. Northern blot analysis performed by Lennartsson et al (2005) and Eckert et al (2006) has showed that mRNA of BPI is expressed in testis and epididymis of mice, which is consistent with the findings of this study. In addition, this study disclosed the following new aspects of BPI expressed in epididymis: 1) The observation that BPI transcripts were not seen in epididymides of 12- and 30-day-old rats suggests that the expression level of the BPI gene is developmentally up-regulated toward adulthood. 2) Because BPI mRNA was detected in the epithelium of caput, but not in the cauda epididymis, BPI protein seems to be synthesized in the caput epithelium and secreted into its lumen. 3) Expression of BPI in epididymis might be regulated by some factors from testis because of the disappearance of BPI in epididymides of castrated rats. 4) A part of secreted BPI appeared to be associated with the granulelike structures in caput epididymis. They might be transported to caudal epididymis, coupled with spermatozoa. 5) BPI that is synthesized in epididymis is cleaved into 25-kd fragment in rats, but not in mice. It seems to bind to the plasma membrane covering the acrosomal region of both rat and mouse spermatozoa. 6) BPI localized on the sperm surface covering the acrosome region disappeared during the acrosome reaction.
Epididymal maturation provides spermatozoa with motility and ability to fertilize oocytes (Cooper,1995; Jones, 1998; Toshimori, 2003). These events are mediated mainly by the secretory activities of the epididymal epithelium (Cooper, 1998; Dacheux et al, 2003). In addition to the classical secretory exocrine process of the tissues, it has been reported that some proteins are secreted from the epididymal epithelium by apocrine secretion (Aumuller et al, 1999). This secretory process is defined by protrusions or bleb emissions from the apical cytoplasm of the epithelium that form vesicular structures called exosomes, aposomes, or epididymosomes circulating in the lumen of the genital tract (Aumuller and Seitz, 1990). Some epididymosome-associated proteins, such as CD52, glutathione peroxidase type 5, ubiquity, and macrophage migration inhibitory factor (MIF), are selectively transferred to spermatozoa during epididymal transit (Eickhoff et al, 2001; Sullivan et al, 2005). Hence, epididymosomes, which are characterized by their surrounding plasma membrane, are thought to be involved in the modification of spermatozoa during epididymal transit. We performed electron microscopy to examine whether epididymosome-like structures are present in the lumen of the caudal epididymis. Although we observed amorphous structures that were homogeneous in electron density with no apparent plasma membrane and cell organelles, we failed to observe membrane-bound epididymosome-like structures in their lumen (not shown). Thus, BPI-positive granules might correspond to the "dense bodies" reported by Asquith et al (2005), rather than epididymosomes.
We present the evidence showing that BPI is localized at the sperm heads covering the acrosome region in spermatozoa freshly released from both caput and cauda epididymides, but not associated with sperm flagella. Association of BPI with the acrosome region was strengthened by the fact that the BPI signal on the acrosome region was lost after the acrosome reaction. Binding of BPI to the sperm heads covering the acrosome region might be due to the cationic properties of BPI, especially a large amount of basic amino acid residues in the N-terminal half of the protein (Gray et al, 1989). These data suggest that a part of BPI secreted into the epididymal lumen binds to the sperm plasma membrane covering the acrosome region. BPI located over the acrosome region of spermatozoa is retained during their passage from caput to cauda epididymis. Spermatozoa in testis, on the other hand, did not show BPI immunoreactivity despite the synthesis and secretion of BPI into the lumen of the seminiferous tubules. Because the antibody against the short peptide sequence of BPI might not detect all populations of BPI, we cannot exclude the possibility that a lack of BPI signal on testicular spermatozoa might mean BPI is in a different conformation that the antibody cannot access. It is also probable that BPI on testicular spermatozoa is obscured by some molecules that prevent the antibody from reacting with the antigen.
Association of BPI with the sperm head covering the acrosome region is intriguing in the following points. Some epididymal proteins bound to spermatozoa have been reported to act as inhibitory molecules, or decapacitation factors (Fraser et al, 1990; Fraser, 1998). Nixon et al (2006) reported that the existence of such decapacitation factors on the acrosomal region of spermatozoa suppresses irrelevant acrosome reactions and their binding ability to zona pellucida. It is probable that BPI might be involved in some process of sperm maturation during epididymal transit, such as premature suppression of the acrosome reaction. Because BPI is present at the sperm heads covering the acrosome region, it is also likely that BPI is directly or indirectly involved in sperm-egg interaction.
The present study suggests that BPI is synthesized in the epithelium of caput epididymis, secreted into its lumen, and binds to not only the granulelike structures but also the sperm surface covering the acrosome region and that BPI over the acrosome region disappears during the acrosome reaction. Although physiological functions of BPI remain to be determined, further studies of BPI should provide new insight into the roles of epididymal proteins in sperm maturation and fertilization.
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Footnotes |
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These authors contributed equally to this study. 
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