From the Institute of Biochemical Sciences, College
of Science, National Taiwan University, Taipei 106, Taiwan and
the ¶ Institute of Biological Chemistry, Academia Sinica,
Taipei 106, Taiwan
Received for publication, August 2, 2000, and in revised form, December 11, 2000
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ABSTRACT |
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SVS VII, one of seven major proteins in mouse
seminal vesicle secretion, was purified to homogeneity. Neither
glycoconjugate nor free thiol group was detected in the protein. The
primary structure deduced from the corresponding cDNA was confirmed
using amino acid sequence determination, which supported the
finding that SVS VII consists of 76 amino acid residues with
five disulfide bridges. Accordingly, it has a theoretical molecular
mass of 8538, which was proven using the mass spectrum of SVS VII. The
CD spectrum of SVS VII in 50 mM phosphate buffer at
pH 7.4 appeared as one negative band arising from the The fertilization capabilities of spermatozoa are not permanent
but rather transient (1). It is well known that mammalian sperm display
an intriguing sense of timing to undergo some modification during their
transit in the reproductive tract before encountering an egg. This
involves multiple steps, and their molecular mechanisms are far from
understood. Identifying the molecular event(s) associated with the cell
modifications becomes a prerequisite to unravel the puzzle. In this
regard, studying how the materials in the lumen of the reproductive
tract affect sperm function is an important subject.
Seminal plasma of mammals is a complex biological fluid formed from the
mixing of various fluids in the male reproduction tract. Factors that
affect sperm motility have been reported in the seminal plasma of
various mammals including boar (2-4), bull (5), mouse (6), and human
(7). The fluid secreted from the seminal vesicle, an accessory
reproductive gland in most male mammals, accumulates in the lumen of
this reproductive gland after puberty. Upon ejaculation, seminal
vesicle secretion (SVS)1 is
discharged to constitute the major portion of seminal plasma. It was
found that extirpation of the seminal vesicle from a mouse greatly
reduced fertility (8, 9), thus manifesting the importance of SVS in the
sperm modification. Since rodents have proven to be good experimental
animals for the molecular study of mammalian reproduction, attempts
have been made to isolate the proteins involved in the cell
modification from mouse SVS that contains several minor proteins and
seven well defined major proteins designated SVS I-VII in decreasing
order of molecular size according to their motilities in SDS-PAGE (10).
Recently, we demonstrated two of the minor proteins, a caltrin-like
trypsin inhibitor/P12, which suppressed the Ca2+-uptake of
sperm (11), and a seminal vesicle autoantigen, which served as a
decapacitation factor (12, 13). Here we present the protein structure,
cDNA cloning, and function of SVS VII purified from mouse SVS.
Materials--
Bovine pancreatic chymotrypsin (type II) and
trypsin (type III); chlortetracycline; phosphatidylcholine (PtdCho),
lysophosphatidylcholine, and phosphatidylethanolamine (PtdEtn) from egg
yolks; phosphatidic acid and lysophosphatidic acid from egg yolk
lectin; phosphatidylinositol from pig livers, phosphatidylserine
(PtdSer), and sphingomyelin from bovine brains; and fatty acid-free BSA
were purchased from Sigma. Goat anti-rabbit IgG conjugated with
horseradish peroxidase, fluorescein-conjugated donkey anti-rabbit IgG,
Percoll, and Sephadex G-50 (superfine) were procured from Amersham
Pharmacia Biotech. C4 300A column was from Waters Co.
(Bedford, MA). The BCA protein assay reagent and IODO-BEADs were
obtained from Pierce. The Ultraspec-II RNA isolation kit was purchased
from Biotecx (Houston, TX). The Oligotex mRNA minikit was from
Qiagen GmbH (Hilden, Germany). The cloning systems including the
cDNA synthesis kit, ZAP-cDNA synthesis kit, and ZAP-cDNA
gigapack III cloning kit were obtained from Stratagene (La Jolla, CA).
pGEM-T, Taq polymerase, T4 DNA ligase, the
Prime-a-gene labeling system, and restriction enzymes were purchased
from Promega (Madison, WI). Freund's adjuvants were from Life
Technologies, Inc. Aluminum-backed silica gel TLC plates and GF/C glass
microfiber were from Whatman.
Fractionation of Mouse SVS and Preparation of
Spermatozoa--
Outbred CD-1 mice were purchased from Charles River
Laboratories (Wilmington, MA) and were maintained and bred in the
animal center at the College of Medicine, National Taiwan University. Animals were treated according to the institutional guidelines for the
care and use of experimental animals. They were housed under controlled
lighting (14 h of light, 10 h of dark) at 21-22 °C and were
provided with water and NIH 31 laboratory mouse chow ad
libitum. Adult mice (8-12 weeks) were humanely killed by cervical dislocation.
The seminal vesicles were carefully dissected to free them from the
adjacent coagulating glands, and the secretions collected from 50 mice
were expressed directly into 50 ml of ice-cold 5% acetic acid. After
stirring for 30 min at 4 °C, the supernatant was collected and
fractionated initially by 35% saturation of ammonium sulfate
precipitation at pH 2.0. The precipitate was removed using
centrifugation at 8000 × g for 20 min, and the
supernatant was dialyzed against 0.5% acetic acid and lyophilized. The
dry sample was redissolved in a minimum volume of PBS and loaded onto a
Sephadex G-50 column (1.5 × 120 cm) preequilibrated with PBS. The
column was washed with PBS at a flow rate of 6 ml/h. Fractions (2 ml)
were collected, and their absorbance at 280 nm was recorded (see Fig.
1A). The peak III fractions were resolved further using HPLC
on a Waters C4 300 A column (3.9 × 300 mm, 15µ). The column was
eluted with a linear gradient of 15-60% acetonitrile in 0.1% (v/v)
trifluoroacetic acid at a flow rate of 1.0 ml/min for 40 min (see Fig.
1B). SVS VII was identified in peak 2 using SDS-PAGE.
In accordance with a method previously used (14), a modified Tyrode's
buffer, which consisted of 124.7 mM NaCl, 2.7 mM KCl, 0.5 mM MgCl2, 0.4 mM NaH2PO4, 5.6 mM
glucose, 0.5 mM sodium pyruvate, 15 mM
NaHCO3, 10 mM HEPES, 100 IU/ml penicillin, and
100 µg/ml streptomycin, was adjusted to pH 7.3-7.4 by aeration with
humidified air/CO2 (19:1) in an incubator for 48 h at
37 °C before use. The epididymides were removed and immersed in the
medium. After they had been carefully dissected from the connective
tissue, spermatozoa were extruded from the distal portion of the
tissues for 10 min at 37 °C. The cells were gently filtered through
two layers of nylon gauze, layered on top of a linear gradient of
20-80% Percoll (v/v), and centrifuged at 275 × g for
30 min at room temperature (15, 16). Three distinct cell layers formed.
The lowest layer, which contained cells with progressive motility, was
washed in three volumes of the medium and collected using
centrifugation at 60 × g for 10 min at room
temperature. The sperm were resuspended and centrifuged similarly twice
more. The cell pellets were resuspended, and CaCl2 was
added to the culture medium at a final concentration of 1.8 mM before the sperm was assayed.
Protein Blotting and Proteolysis in the
Polyacrylamide--
Antisera against SVS VII were raised in New
Zealand White rabbits. Proteins were resolved using SDS-PAGE on a 15%
gel slab (6.5 × 10.5 × 0.075 cm) by the method of Laemmli
(17). The proteins on the gel were stained with Coomassie Brilliant
Blue or transferred to a nitrocellulose membrane. After transfer, the
protein blots were immunodetected using Western blot procedures; the
SVS VII-induced antisera was the primary antibody, and goat anti-rabbit
IgG conjugated with horseradish peroxidase was the secondary antibody.
The proteolysis of protein bands in the polyacrylamide gel was carried
out following the method of Hanspal et al. (18). After
resolution of proteins using SDS-electrophoresis on a gel slab of 15%
polyacrylamide copolymerized with 0.1% gelatin, the gel was gently
washed three times in 100 ml of 2.5% (w/v) Triton X-100 for 45 min
each to remove SDS. The gel was washed in distilled water for 45 min
and incubated for 15 h at 37 °C in a solution containing
chymotrypsin (1 N-benzoyl-L-tyrosine ethyl
ester unit/ml) or trypsin (50 N-benzoyl-L-arginine ethyl ester
units/ml) in 0.1 M Tris-HCl and 20 mM
CaCl2 at pH 8.0. The gel was washed similarly as above and
stained with Coomassie Brilliant Blue.
Preparation of Sperm Lipid and Phospholipid Liposomes--
The
total lipids of spermatozoa were extracted using a modified method of
Folch et al. (19). The cells were suspended in 1.0 ml of
0.05 M KCl, sonicated in a Branson ultrasonic water bath
(model B-52) for 5 min at room temperature, and extracted twice with
3.0 ml of chloroform/methanol (2:1, v/v). The lipid extract was dried
by flushing with N2 and redissolved in a suitable organic
solvent for TLC. Meanwhile, the sperm lipid was dissolved in ethanol
and injected through a 26-gauge needle into PBS (ethanol/PBS, 1:20,
v/v). The suspension was repeatedly forced through the needle, sonicated for 1 h at room temperature, centrifuged at 18,000 × g, and used immediately in the study of the inhibitory
effects on SVS VII-sperm binding.
The pure phospholipids in the chloroform solution were evaporated under
N2 and formed thin films in glass tubes. Liposomes of
phospholipids were prepared following the method of Genge et al. (20). A sufficient volume of incubation buffer composed of 10 mM HEPES and 100 mM KCl at pH 7.4 was added to
give a final concentration of 2 mg of lipid/ml. The mixture was
sonicated at full power for 10 min until the lipids formed a faintly
opalescent suspension.
Binding Assay--
A modified method developed by Markwell (21)
was followed to prepare 125I-SVS VII. In brief, 10 µl of
Na125I (1.0 mCi) from a commercial source was mixed with 50 µg of SVS VII in 90 µl of PBS in the presence of IODO-BEADs. The
radiolabeled protein was separated from free Na125I through
a PD-10 column preequilibrated with PBS. 125I-SVS VII
showed a single band and was indistinguishable from its parent protein
by migration on SDS-PAGE.
Spermatozoa (2.5 × 106 cells/ml) and 100 nM 125I-SVS VII in 10 mM HEPES
containing 138 mM NaCl and 3 mM KCl at pH 7.4 were incubated at specified conditions. The cells were collected on a
Whatman GF/C glass microfiber filter using rapid filtration at a
pressure of 50.66 kilopascals (0.5 atm). In accordance with the method described previously (11, 22), the filter was blocked with 5% (v/v)
nonfat skimmed milk in the buffer for 30 min and washed with the same
ice-cold buffer before use to minimize the background. The filter was
washed with six changes of 0.2 ml of the same ice-cold buffer,
air-dried, and counted using a
Sperm lipids and purified lipids were chromatographed on
aluminum-backed silica gel TLC plates in chloroform/methanol/water (65:25:4, v/v/v). The plates were treated according to the method developed by Desnoyers and Manjunath (23). The plates were overlaid with 125I-SVS VII (100,000 cpm/ml) in a blocking buffer of
PBS containing 5% nonfat skimmed milk (100 µl/cm2),
incubated for 90 min at room temperature, washed five times each for
1-2 min with cold PBS, and then dried and exposed to x-ray film for
18-36 h. The lipids on duplicate plates were sprayed with
phosphomolybdic acid solution to allow us to detect phospholipids (24).
According to a modified method by Glenney (25), 10 µg of protein and
200 µg of liposomes in 200 µl of incubation buffer were gently
mixed with or without 1.8 mM CaCl2 for 45 min
at room temperature. Liposomes became sediment at 100,000 × g for 30 min, and equivalent fractions of pellet and
supernant were analyzed using SDS-PAGE.
Cytological Observation and Assay of Sperm Motility--
The
chlortetracycline staining method developed by Ward and Storey (26) was
employed to score the population of mouse spermatozoa in the
uncapacitated, capacitated, and acrosome-reacted stages. Briefly, a
5-µl sample of the sperm suspension was mixed on a slide with 5 µl
of buffer containing 130 mM NaCl, 5 mM
cysteine, 1 mM chlortetracycline, and 20 mM
Tris-HCl at pH 7.8. After 30 s, 1.0 µl of buffer containing
1.25% glutaraldehyde, 130 mM NaCl, 1.8 mM
CaCl2, and 20 mM Tris-HCl at pH 7.8 was added.
The samples were kept in a light-shielded environment until they were
seen under a fluorescence microscope (AH3-RFCA; Olympus, Tokyo, Japan).
Freshly prepared spermatozoa (106 cells/ml) were
preincubated in a blocking solution (3% nonfat skimmed milk in PBS)
for 30 min at room temperature. The cells were further incubated with 25 µM SVS VII for another 30 min. At the end of
incubation, the cells were centrifuged, and the cell pellets were
washed with PBS to remove the unbound ligands. The cells were air-dried
on a glass slide and washed twice with PBS. The slides were incubated with the SVS VII-induced antiserum diluted 1:250 in the blocking solution for 30 min. The slides were washed three times with PBS to
remove excess antibodies before they were incubated with
fluorescein-conjugated donkey anti-rabbit IgG diluted to 1:100 in the
blocking solution for 30 min. All of the slides were washed with PBS,
covered with 50% (v/v) glycerol in PBS, and photographed with a
microscope equipped with epifluorescence.
Sperm motility was determined using computer-assisted sperm assays with
a sperm motility analyzer (IVOS version 10; Hamilton-Thorne Research,
Beverly, MA). A 7.0-µl sample was placed in a 10-µm-deep Makler
chamber at 37 °C. The analyzer was set as follows: negative phase-contrast optics and recording at 60 frames/s, minimum contrast 40, minimum cell size 4 pixels, low size gate 0.2, high size gate 1.5, low intensity gate 0.5, high intensity gate 1.5, nonmotile head size
29, nonmotile head intensity 76, medium average path velocity 50 µm/s, low path velocity 7.0 µm/s, slow motile cells yes, and
threshold straightness (STR) greater than 80%. Ten fields were
assessed for each sample.
Analytical Method--
The concentration of SVS VII was
determined using the BCA protein assay (27) according to the
manufacturer's instructions. The amino acid sequence was determined
using automated Edman degradation with a gas phase sequenator (492 protein sequencer with on line 140 C analyzer, (PerkinElmer Life
Sciences). DNA sequencing was carried out by an ABI PRISM 377-96 DNA
sequencer using the ABI PRISM BigDye Terminator cycle sequencing ready
reaction kit (PerkinElmer Life Sciences).
Spectral Measurements--
The CD spectra were measured with a
Jasco J-700 spectropolarimeter under constant flushing with
N2 at room temperature. The mean residue elipticity,
[
SVS VII dissolved in 50% acetonitrile containing 1% acetic acid at a
final concentration of 10 µM was analyzed in the ESI source of a Finnigan LCQ ion trap mass spectrometer (Finnigan MAT
Instruments Inc., San Jose, CA). Data were acquired using Bioworks software.
RNA Isolation, cDNA Construction, Cloning, and Northern
Blotting--
Total cellular RNA was prepared from fresh tissue
homogenates, using the Ultraspec-II RNA isolation kit. The
polyadenylated fraction of total RNA was isolated using the procedures
recommended for the Oligotex mRNA minikit. Preparation of
double-stranded cDNAs, construction of orientation-specific
cDNAs in Uni-ZAP XR vectors with the
EcoRI/XhoI terminus, and packaging of the
constructed vectors into phages generally followed the instructions of
Stratagene. The recombinant phages were transfected into the bacterial
host XL1-Blue MRF' strain. The cDNA library efficiency was 2.5 × 105 plaque-forming units/µg of cDNA. Plaques from
the cDNA library in the vectors were induced by
isopropyl-1-thio-
In Northern blotting, total RNA samples were analyzed by separation in
a denaturing 1.5% agarose/formaldehyde gel (28) followed by capillary
transfer to a nylon membrane and hybridization to a
32P-labeled nucleotide probe. 32P-labeled
random-primed probe for the glyceraldehyde-3-phosphate dehydrogenase
gene was prepared with a Promega Prime-a-gene labeling kit using a
cDNA segment of the mouse glyceraldehyde-3-phosphate dehydrogenase
gene inserted into a pGEM-T vector as a template. According to the
method developed by Lee et al. (29), incorporation of
[ Purification of SVS VII and Establishment of the Protein
Sequence--
SVS VII was purified from SVS through a series of
isolation steps. The peak 2 sample in Fig.
1B shows that a single 8-kDa band has the same mobility as SVS VII on SDS-PAGE (cf.
lanes 1 and 2 in Fig.
2), which indicates that the protein was
purified to homogeneity and was distinct from the peak 1 sample, which gave a single 6-kDa band that corresponds with P12 reported previously (30). The antiserum against SVS VII showed high immunoaffinity to the
antigen (Fig. 2, lane 4). Among the protein
components of mouse SVS, the antiserum only immunoreacted with SVS VII
(Fig. 2, lane 3), thus showing the high
specificity of the SVS VII antibody in the antiserum. Therefore, we
used the antiserum for the immunodetection of SVS VII throughout the
study.
The recombinant phages of the cDNA library were screened using the
antiserum against SVS VII, and the positives were excised into
phagemids. The SVS VII cDNA in the phagemid was sequenced to
establish the gene structure that included a 5'-untranslated region of
5 base pairs, an open reading frame of 297 base pairs, which encoded 99 amino acid residues, and a 3'-untranslated region of 183 base pairs,
which ended with a polyadenylated region (Fig. 3A). Automated Edman
degradation of SVS VII for 18 cycles gave reliable data, which
indicated Leu as the N-terminal residue and the amino acid sequence,
LICNSCEKSRDSRCTMSQ, which was identical with the corresponding
cDNA-deduced peptide sequence in all positions. Apparently, the
post-translational cleavage occurred at the Gly-Leu peptide bond in the
signal peptide that had 23 amino acid residues to produce a mature
protein of 76 amino acid residues containing 10 cysteines.
Protein Characterization and Gross Conformation of SVS VII--
We
found that SVS VII was not reactive to Ellman's reagent (31),
indicative of the absence of a free thiol group on the protein
molecule. The protein also did not react with Ellman's reagent when
the experiment was performed in the presence of 6.0 M urea.
Apparently, there were no free cysteines that were partially or fully
buried in the protein molecules. In addition, the SVS VII band in the
polyacrylamide gel did not stain with periodic acid-Shiff reagent,
which revealed that it was not a glycoprotein. Accordingly, the
theoretical molecular mass was estimated to be 8538 from the
cDNA-deduced protein sequence. This was proven in the electrospray
mass spectral profile of SVS VII (Fig.
4).
The CD spectrum of SVS VII in 50 mM phosphate buffer at pH
7.4 had at least seven bands in the wavelength region of 200-300 nm.
Bands I-VI in the near-UV region arose from nonpeptide chromophores (Fig. 5, right
side), and band VII in the UV region was mainly due to
peptide chromophores (Fig. 5, left side). Band
VII was negative with a minimum at 217 nm. In addition, a positive band would appear as the CD profile extended below 200 nm. The spectral profile had some resemblance to that of the
There were two tyrosines, two phenylalanines, and five cystines
but no tryptophan in the SVS VII molecule. According to the fine CD
structures of nonpeptide chromophores of a protein near UV (39, 40),
bands I-III arose from tyrosine residue(s), and band V, which appeared
as a shoulder between the positive band VI and the negative band IV,
may be attributable to phenylalanine residues, since the CD spectrum of
this amino acid in a protein is usually weak. Band VI was the most
prominent band among the CD fine structures of SVS VII. Based on the
study of Beychok and Breslow (40), bands VI and IV were assigned to
cystine residue(s). The description of bands I-VI to
specific residues may be fortuitous. The CD data from site-specific
mutagenesis for the protein may support the assignment in the future.
The secondary structure of SVS VII was stable over a wide range of pH
values. Even at pH 3 or 10, the profiles of CD band VII as well as the
CD fine structures in the near-UV region remained virtually the same as
in neutral solution (not shown). After heating the protein solution for
10 min at 90 °C, the protein conformation changed remarkably as
evidenced by the disappearance of CD bands VI and VII of native protein
and the appearance of a strong negative band below 200 nm
(cf. solid and dashed lines
of Fig. 5), which was the characteristic CD of a protein in a
completely unordered form. The protein sample after heat treatment is
referred to as SVS VIIh hereafter.
The mouse SVS proteins resolved in a slab gel of polyacrylamide
copolymerized with gelatin were digested with protease. The protein
band of SVS VII remained in the gel after either the chymotrypsin- or
trypsin-mediated proteolysis. According to previous methods (41, 42),
we measured no inhibitory effects of SVS VII on the proteolytic
activity of either chymotrypsin or trypsin (data not shown), suggesting
that the cross-linkages of five disulfide bridges constrained this
rather small protein molecule to hamper the proteolytic digestion
or/and the release of peptide segment(s) that remained in the protein
core after proteolytic degradation.
Characteristics of SVS VII-Sperm Binding--
Fig.
6 displays the sperm micrographs examined
using the indirect fluorescence staining technique. No fluorescence
appeared on the epididymal spermatozoa after they immunoreacted
successively with the SVS VII antiserum and fluorescein
isothiocyanate-conjugated anti-rabbit IgG, manifesting the lack of SVS
VII on the cell surface. When spermatozoa were preincubated with 25 µM SVS VII in the blocking solution at room temperature
for 30 min, the fluorescein fluorescence was visible around acrosome,
middle piece, and principal tail. No fluorescence was seen when the
antiserum was replaced with the normal serum. Apparently, sperm had SVS
VII-binding sites that covered the entire cell surface.
Fig. 7 shows the data from one
representative determination for 125I-SVS VII-sperm
binding. The radiolabeled 125I-SVS VII bound to the cell
surface was greatly inhibited by a 100-fold excess of the unlabeled SVS
VII, indicating the specificity of SVS VII-sperm binding. A similar
situation was also observed by the replacement of unlabeled SVS VII
with SVS VIIh in the assay. Apparently, after heat
treatment, the protein did not lose its sperm binding ability. The
125I-SVS VII-sperm binding was slightly reduced by the
presence of 1.8 mM Ca2+. Increased levels of
Ca2+ to 6.0 mM during the incubation period
occurred in more than 75% of the total SVS VII bound to sperm. The
presence of the dispersed sperm lipids during the cell incubation also
suppressed the binding of SVS VII to the cell surface; thus, SVS VII
bound to the sperm decreased as the quantity of dispersed sperm lipids
increased.
The lipid extract of epididymal spermatozoa and purified phospholipids
were chromatographed on silica gel-coated aluminum plates (Fig.
8A). As in a previous report
(23), the mouse sperm phospholipids were well separated into six major
components and several minor components in the developing solvent
employed except that PtdCho and PtdCho plasmalogens migrated together
(Fig. 8A, lane 1). Based on the
Rf values of purified lipids, the minor components remained unidentified, and five of the main components were
identified as neutral lipid, PtdEtn, PtdCho/PtdCho plasmalogen, PtdSer,
and sphingomyelin. The results of a TLC overlay binding assay (Fig.
8B) showed that 125I-SVS VII bound to purified
PtdSer and PtdEtn but did not interact with phosphatidic acid,
PtdCho/PtdCho plasmalogen, phosphatidylinositol, lysophosphatidic acid,
sphingomyelin, or lysophosphatidylcholine. Among the sperm lipids,
125I-SVS VII bound to PtdEtn and PtdSer but did not
interact with the other phospholipids (Fig. 8B,
lane 1). PtdSer is in a relative small amount of
sperm phospholipid (43). This may account for the weak radioactivity of
125I-SVS VII bound to sperm PtdSer on the TLC plate.
We found that PtdSer liposomes in 10 mM HEPES and 100 mM KCl at pH 7.4 slowly settled to form solid phase
aggregates during their incubation in the presence of SVS VII, SVS
VIIh, or Ca2+. On the contrary, PtdCho
liposomes did not settle in the presence of the proteins or
Ca2+ during incubation. As shown in Fig.
9, both proteins appeared only in the
pellet fractions of PtdSer liposomes after centrifugation of the
incubation mixture, suggesting that they bound to PtdSer liposomes.
Incubation in the presence of 1.8 mM Ca2+
partially retarded the cosedimentation of each protein with PtdSer liposomes, revealing that Ca2+ might be able to release the
protein bound to PtdSer liposomes, which can chelate Ca2+
(44). SVS VII as well as SVS VIIh did not bind to PtdCho
liposomes as evidenced from the observation that they did not
cosediment with the phospholipid vesicles and appeared only in the
supernatant fraction after incubation.
Enhancement of Sperm Motility by SVS VII--
We examined the
distribution of SVS VII and its RNA message in the tissue homogenates
of reproductive glands, such as the seminal vesicle, epididymis,
testis, coagulating gland, vas deferens, uterus, ovary, prostate,
vagina, and nonreproductive organs, including lungs, kidney, brain,
spleen, liver, pancreas, and heart. SVS VII was immunodetected in the
seminal vesicle only, and the mRNA was abundant in the seminal
vesicle and a trace in coagulating gland and vas deferens but was not
detected in the other tissues (not shown).
Most spermatozoa freshly retrieved from mouse caudal epididymis in
modified Tyrode's buffer were motile with tail beating even after
incubation for 120 min at 37 °C. Since BSA has often been used to
study sperm capacitation in vitro, we compared BSA with SVS
VII and SVS VIIh in the effects on the sperm motility and
capacitation. These two kinds of sperm function were assayed after the
cell incubation in the modified Tyrode's solution containing 1.8 mM CaCl2 at several conditions. As shown in
Fig. 10A, both 45 µM BSA and 40 µM SVS VII in the cell
culture relative to the motility of control cells greatly enhanced the
sperm motility at any incubation time. SVS VIIh at a
concentration of 40 µM in the culture medium retained the
ability to enhance the sperm motility before 10 min of
incubation, but the cells became immobile and stuck in clusters
thereafter. At 90 min of incubation, more than 90% of the control
cells remained uncapacitated, and almost no acrosome-reacted cells
appeared (Fig. 10B, panel a). The
addition of SVS VII to the incubation medium showed very slight effects on the cellular stage (Fig. 10B, panel
b). The population of capacitated cells increased remarkably
after similar incubation in the presence of BSA. Around 50% of the
BSA-treated cells were capacitated, but acrosome-reacted cells
constituted less than 15% of the total (Fig. 10B,
panel c). The BSA-induced capacitating sperm were
not influenced as the cells were exposed to BSA and SVS VII together (Fig. 10B, panel d).
Previously, Lardy and co-workers (45) purified an 8-kDa caltrin
from mouse SVS and established its primary structure of 75 amino acid
residues containing seven cysteines. Aligning the protein sequence of
SVS VII with that of caltrin revealed a very high degree of similarity
with 71 identical residues, except that Cys67,
Cys68, Cys73, and Ser75 of SVS VII
were replaced by Phe67, Gly68,
Met73, and Phe75 of caltrin (Fig.
3A). Since SVS VII and caltrin were secreted from the same
reproductive gland and have very similar molecular size, they were
assumed to be identical molecules. The SVS VII primary structure
established from our present work is reliable, considering our
demonstration that the molecular mass of SVS VII determined from the
electrospray mass spectrometry conformed to the theoretical value
estimated from the cDNA-deduced protein sequence consisting of 76 amino acid residues in which five disulfide bonds were considered.
Several lines of evidence suggested the incorrect assignment for the
phenylthiohydantoin-derivatives stated by Lardy et al. (45),
which were different from the corresponding amino acid residues in SVS
VII, after the automated Edman degradation of caltrin. First, a
molecular mass of 8470 for caltrin would have appeared in the profile
of the mass spectrum, taking into account their claim that a free thiol
group may be present in the protein molecule. However, our results
shown in Fig. 4 do not prove its presence. Second, their suggestion of
dimer formation through the intermolecular disulfide bond was not
proven. Among the protein components of mouse SVS that were resolved
using a nonreducing SDS-PAGE, we did not find a 16-kDa band. We only
found a 8-kDa band that was immunoreactive to the antiserum against SVS
VII in the Western blot analysis.
Of the 10 cysteines in the SVS VII molecule, six residues, namely
Cys3, Cys6, Cys14,
Cys21, Cys29, and Cys47 were
sterically restricted in the predicted Cytochemical observations revealed that sperm surface has SVS
VII-binding sites that cover the entire cell surface (Fig. 6). The
demonstration that the dispersed sperm lipids were able to inhibit SVS
VII-sperm binding (Fig. 7) supports the notion that the SVS VII-binding
sites are lipids in nature. In addition, the binding assay indicated
that the active conformation of SVS VII for the binding to sperm could
be maintained, although the protein unfolded during heat treatment.
125I-SVS VII blotting of the membrane phospholipid of
spermatozoa on TLC plates (Fig. 8B) suggests that PtdEtn and
PtdSer might constitute the major SVS VII-binding sites on the cell
surface. The ability of SVS VII to bind to these two phospholipids was also confirmed by both the ligand blotting of purified lipids on the
TLC plate (Fig. 8B) and the interaction of SVS VII with PtdSer liposomes (Fig. 9). Together, these two phospholipids constitute less than 15% of the total lipid in the plasma membrane of mouse spermatozoa (43). SVS VII itself is a sperm motility enhancer, but in
its unfolded form, on the other hand, it immobilizes sperm. PtdSer
liposomes alone can undergo fusion with cell membrane (46, 47) and
enhance sperm motility (48, 49) under physiological conditions.
More studies are needed to unravel the complexity of interaction among
PtdSer liposomes, SVS VII, and sperm.
The phospholipid-binding proteins in the reproductive tract have
received attention recently. Several bovine seminal plasma (BSP)
proteins, which are the major secretory products of the seminal
vesicle, have been purified and identified (50, 51). They belong to a
family of closely related acidic proteins, designated BSP-A1, BSP-A2,
BSP-A3, and BSP-30 kDa. It has been suggested that they have some role
in the membrane modification of spermatozoa that occurs during
capacitation and/or acrosome reaction through the interaction with the
membrane phospholipids (52-54). In mouse SVS, we demonstrated the
presence of the seminal vesicle autoantigen (SVA). SVA is a 19-kDa
phospholipid-binding glycoprotein that shows no significant
similarities to the protein sequences of BSP proteins and exhibits the
ability to suppress mouse sperm motility (12, 13). In comparison with
the specificity of SVS VII-phospholipid binding, SVA did not interact
with PtdSer but bound to the choline-containing phospholipids such as
PtdCho/PtdCho plasmalogen and sphingomyelin. Together, these
phosphocholine-containing lipids make up more than 70% of total lipid
in the plasma membrane of mouse spermatozoa (43). The primary structure
of SVS VII showed no significant similarities to protein sequences of
both BSP proteins and SVA. Furthermore, SVS VII was not related to any
other phospholipid-binding proteins such as perforin (55), phosphocholine-binding protein (56), factor V (57), factor VIII (58,
59), factor IX (60), p65 (61), pulmonary surfactant protein
(62), C-reactive proteins (63), apolipoproteins A-I, A-II, and A-IV
(64), or lipid transfer proteins (65-67). Therefore, SVS VII
represented a novel phospholipid-binding protein. Interestingly, the
differences in specificity between SVA-lipid and SVS VII-lipid binding
result in their extremely different effects on the sperm function,
although they are secreted from the same reproductive gland.
SVS VII was exclusively secreted from mouse seminal vesicle, and the
SVS VII-sperm binding that took place upon ejaculation enhanced the
sperm motility without leading to sperm capacitation. Apparently, the
SVS VII effects did not cause the maturation of spermatozoa at any time
earlier than the sperm-egg encounter but helped the ejaculated
spermatozoa pass through the cervix into the uterus after coitus of the
rodent. Lardy et al. (45) reported the ability of mouse
caltrin to affect the Ca2+ uptake of guinea pig sperm.
Determination of whether this kind of event is relevant to the
stimulation of mouse sperm motility by SVS VII awaits the completion of
future studies.
form at 217 nm and several fine structures due to nonpeptide chromophores including
a prominent band for the disulfide bond at 250 nm. This,
together with the predicted secondary structures, indicated no helices
but a mixture of
form,
turn, and unordered form in SVS VII. A
cytochemical study illustrated the presence of the SVS VII-binding
region on the entire surface of mouse sperm. The SVS VII-sperm binding
was inhibited by the dispersed sperm lipids. The results of TLC overlay assay for the binding of 125I-SVS VII to phospholipids and
the interaction between SVS VII and phospholipid liposomes demonstrated
a specific binding of this protein to both phosphatidylethanolamine and
phosphatidylserine. The SVS VII-sperm binding greatly enhanced sperm
motility but did not induce sperm capacitation. Heating the protein
solution for 10 min at 90 °C unfolded the protein molecule, and the
unfolded SVS VII immobilized the sperm.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-counter.
], was estimated from the mean residue weight, which was
calculated from the primary structure.
-D-galactopyranoside and
immunochemically screened using the antiserum against SVS VII. Randomly
chosen positives from different pools were purified. They were excised
into phagemids using ExAssist interference-resistant helper phages and
transformed into bacterial SOLRTM strain for DNA sequencing.
-32P]dATP to DNA was carried out using PCR, which was
designed to amplify the whole reading frame of SVS VII cDNA in the
constructed vector.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Purification of SVS VII from mouse SVS.
A, fractionation of the soluble proteins of mouse SVS from
35% ammonium sulfate saturation at pH 2.0 by Sephadex G-50 column
chromatography. B, resolution of the peak III sample by
reverse phase of HPLC on a C4 column. The broken
line indicates a linear gradient of acetonitrile (see
"Experimental Procedures" for details).
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Fig. 2.
Protein identification by SDS-PAGE and
specificity of SVS VII-induced antiserum. The proteins of mouse
SVS (15 µg, lanes 1 and 3) and peak
2 of Fig. 1B (5 µg, lanes 2 and
4) were subjected to SDS-PAGE on a 15% polyacrylamide. The
gels of lanes 1 and 2 were stained
with Coomassie Brilliant Blue to reveal the protein bands. The proteins
in the gels of lanes 3 and 4 were
transferred to nitrocellulose membranes and immunodetected using
Western blotting with the SVS VII-induced antiserum, diluted 1:2000 in
the blocking solution.
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Fig. 3.
The primary structure and the predicted
secondary structure of SVS VII. A, the nucleotide
sequences of a 500-base pair SVS VII cDNA were determined to deduce
amino acid sequences. The initial and stop codons of the open reading
frame are underlined. The deduced protein sequence and the
amino acid sequences determined directly from the protein analysis
agree in all positions. The latter is indicated by a dashed
underline. The cleavage point for the generation of mature
peptide is indicated by an arrow. The cDNA-deduced amino
acid sequences of SVS VII are identical to all positions of the protein
sequences of a mouse caltrin, except that Cys67,
Cys68, Cys73, and Ser75 of SVS VII,
which are denoted by asterisks, disagree with
Phe67, Gly68, Met73, and
Phe75 of the caltrin molecule, which are listed in
parentheses. B, the secondary structure of SVS
VII was predicted using Chou and Fasman algorithm. B or
b, strong or weak form former; T or
t, strong or weak
turn former; h, weak helix
former.
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Fig. 4.
Electrospray mass spectrum of SVS VII.
SVS VII dissolved in 50% acetonitrile in the presence of 1% acetic
acid was analyzed in the ESI source mass spectrometer. A,
original spectrum. B, computer deconvolution of the peaks in
A.
form of protein conformation (32-35). Using the criteria of the Chou and Fasman algorithm (36, 37) or the GOR algorithm (38), we predicted the
potentials for the formation of secondary structures in SVS VII (Fig.
3B). The helical formation by weak helix formers along the
two peptide segments of residues 51-56 and 60-70 was
overpredicted in view of our CD results. The predicted secondary
structure, together with the position, sign and magnitude of band VII
of SVS VII (Fig. 5), strongly supported the presence of some ordered structure other than the helix, probably a mixture of
-form,
-turn, and unordered form in the SVS VII molecule.
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Fig. 5.
Circular dichroism of SVS VII. The
protein was in 50 mM phosphate buffer at pH 7.4 at room
temperature (solid line). The protein solution
was heated for 10 min at 90 °C, cooled for 10 min at 4 °C, and
kept at room temperature for 10 min before the optical measurement
(dashed line).
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Fig. 6.
Demonstration of the SVS VII binding zone on
the spermatozoa. Fresh cells were incubated with SVS VII as
described under "Experimental Procedures." The SVS
VII-binding zone on the cells was immunolocalized by the indirect
fluorescence method using the SVS VII antiserum and
fluorescein-conjugated anti-rabbit IgG. The slides were observed by a
light microscope (A) or a fluorescence microscope
(B). Bar, 10 µm.
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Fig. 7.
The specific binding of SVS VII to sperm
lipid. Spermatozoa (2.5 × 106 cells/ml) in HEPES
buffer containing 1% (w/v) nonfat skimming milk were incubated for
1 h at room temperature in the presence of 100 nM
125I-SVS VII and a 100-fold excess of unlabeled SVS VII
(column b) or SVS VIIh
(column c). The 125I-SVS VII-sperm
binding was also assayed in the presence of 1.8 mM
Ca2+ (column d), 6 mM
Ca2+ (column e), or the dispersed
sperm lipids prepared from 2.5 × 108 cells
(column f), 5 × 108 cells
(column g), and 1.25 × 109
cells (column h). Results are the percentage of
125I-SVS VII-sperm binding of the control
(column a) expressed as means ± S.D. *,
p < 0.01 in the paired statistical comparison with the
corresponding control values are evaluated using one-way
analysis of variance.
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Fig. 8.
Binding of 125I-SVS VII to
phospholipids separated by TLC. Lipids extracted from 2.5 × 107 spermatozoa (lane 1) and 30 µg
of each of the pure phospholipids (lanes 2-9)
were chromatographed on silica gel TLC plates. A,
phospholipids were detected with phosphomolybdic acid spray.
B, autoradiograms for the binding of radiolabeled SVS VII to
separated phospholipids were obtained after a TLC overlay binding
technique described under "Experimental Procedures."
lyso PA, lysophosphatidic acid; lyso PC,
lysophosphatidylcholine; PA, phosphatidic acid;
PC, phosphatidylcholine; PE,
phosphatidylethanolamine; PS, phosphatidylserine.
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Fig. 9.
Interaction of SVS VII or SVS
VIIh with various phospholipid liposomes. Each
liposome (200 µg) was mixed with 10 µg of each protein in 200 µl
of 10 mM HEPES buffer containing 100 mM KCl and
1.8 mM CaCl2 (+Ca2+) or in
the absence of Ca2+ ( Ca2+) and
incubated for 45 min at room temperature. After centrifugation, the
protein in the supernatant (S) and pellet (P)
fraction was determined using SDS-PAGE.
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Fig. 10.
Analysis of sperm motility and capacitation
under the influence of SVS VII and BSA. A, freshly
prepared mouse spermatozoa in modified Tyrode's solution
(106 cells/ml) in the presence of 1.8 mM
CaCl2 was incubated for 0-60 min in the presence of 40 µM SVS VII ( ), 40 µM SVS
VIIh (
), or 45 µM BSA (
) at 37 °C.
The cell motility determined at each specified incubation was expressed
as a percentage of control cell motility (
) measured at zero time
incubation. Points are means ± S.D. for 10 determinations.
B, the chlortetracycline fluorescence method described under
"Experimental Procedures" was exploited to score the
population of uncapacitated cells (open bars),
capacitated cells (striped bars), and
acrosome-reacted cells (solid bars) after the
cell incubation alone (panel a) or in the
presence of 40 µM SVS VII (panel
b), 45 µM BSA (panel c),
or a combination of 40 µM SVS VII and 45 µM
BSA (panel d) for 90 min at 37 °C. The data
represent the means of eight individual trials counting 200 cells/treatment/trial. The error bars represent
the S.D. of the mean. *, p < 0.01 in the paired
statistical comparison with the corresponding control values are
evaluated using one-way analysis of variance.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
form/
turn (Fig. 3B). The chirality of a disulfide bond is relevant to its
skewed conformation. Heating SVS VII caused a great diminution of the CD band VI due to the chiral disulfide bond and the disappearance of CD
band VII arising from
form/
turn at the same time (Fig. 5),
manifesting that the configuration of the disulfide conformers in the
protein molecule is important to confine the ordered structure and
vice versa.
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FOOTNOTES |
---|
* This work was supported in part by National Science Council, Taipei, Taiwan (grants NSC 89-2311-B-002-038 and NSC 89-2311-B-001-106).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF134204.
§ Some of the work described in this paper forms part of a dissertation submitted in partial fulfillment of the requirements for a Ph.D. at the National Taiwan University.
To whom correspondence should be addressed: Institute of
Biochemical Sciences, College of Science, National Taiwan University, P.O. Box 23-106, Taipei 106, Taiwan. Tel.: 886-2-2362-0261; Fax: 886-2-2363-5038; E-mail: mrlab@ccms.ntu.edu.tw or
bc304@gate.sinica.edu.tw.
Published, JBC Papers in Press, December 15, 2000, DOI 10.1074/jbc.M006954200
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ABBREVIATIONS |
---|
The abbreviations used are: SVS, seminal vesicle secretion; BSA, bovine serum albumin; BSP, bovine seminal plasma; PAGE, polyacrylamide gel electrophoresis; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; PtdSer, phosphatidylserine; PtdIns, phosphatidylinositol; SVA, seminal vesicle autoantigen; PBS, phosphate-buffered saline.
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