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From the * Department of Biochemistry, North
Carolina State University, Raleigh, North Carolina;
Departments of Biology and Chemistry, Norfolk
State University, Norfolk, Virginia; Laboratory
for the Study of Reproductive Biochemistry and Molecular Biology, Innovative
Reproductive Technologies, Virginia Beach, Virginia.
Present address: Department of Genetics, Cell
Biology & Development, University of Minnesota, 321 Church St. SE/4-135
Jackson, Minneapolis, MN 55455-0217.
| Correspondence to: Joseph C. Hall, Department of Chemistry, Norfolk State University, Norfolk, VA (e-mail: jchall{at}nsu.edu ). |
| Received for publication August 16, 2001; accepted for publication January 14, 2002. |
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Abstract |
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120.0 kd and 130.0 kd, respectively, on the sperm surface. In vitro
treatment of epididymal spermatozoa with phosphatidylinositol
specific-phospholipase C revealed the release of protein D/E molecules over
the head region but not the tail region of spermatozoa. Indirect
immunofluorescence experiments using polyclonal antibodies generated against a
highly purified protein D/E preparation demonstrated that protein D/E
molecules were bound to the surface of spermatozoa recovered from the
epididymal and female reproductive tracts, even after 7 hours. These results
indicate that protein D/E molecules interact with specific membrane proteins,
and is subsequently covalently bound to the surface of spermatozoa via a
glycosyl-phosphatidyl inositol linkage. In addition, protein D/E molecules
remain covalently bound to spermatozoa after deposition in the female
reproductive tract, an observation that is consistent with the proposed
physiological function of the protein in the fertilization process.
Key words: Epididymal secretory protein, interaction, sperm plasma membrane
Several major secretory proteins of the rat epididymis have been isolated and biochemically characterized (Brooks and Higgins, 1980; Brooks, 1982; Wong and Tsang, 1982). However, of all the major secretory proteins of the rat epididymis that have been studied to date, only protein D/E has been shown to be involved in the fertilization process (Cuasnicu et al, 1984; Fournier-Delpech et al, 1985; Rochwerger et al, 1992; Cohen et al, 1996; Hall et al, 1997; Ellerman et al, 1998). Previous studies have shown protein D/E molecules to be synthesized by principal cells of the epididymal epithelium and to bind to the surface of spermatozoa during passage through the epididymal duct (Lea et al, 1978; Faye et al, 1980; Kohane et al, 1980, 1983; Brooks and Tiver, 1983; Turner et al, 1994). Protein D/E molecules also have been shown to be a member of the cysteine-rich secretory protein (CRISP) family, a group of proteins containing 16 conserved cysteine residues (Eberspaecher et al, 1995; Kratzschmar et al, 1996). A current emerging hypothesis is that protein D/E molecules function in sperm-egg membrane fusion (Rochwerger et al, 1992; Cohen et al, 1996, 2000, Ellerman et al, 1998). Although the specific functional roles of some epididymal secretory proteins in the fertilization process, such as protein D/E are being elucidated, how these proteins become bound to the surface of spermatozoa as they mature in the epididymis remains uncertain.
Therefore, the objective of the present study was to further our understanding of how protein D/E molecules interact with and become bound to the surface of epididymal spermatozoa. Indirect immunofluorescence (IIF) studies, Western blot analysis, and in vitro photoactivated cross-linking experiments were used to investigate the interaction of protein D/E molecules with epididymal spermatozoa and to assess the topology of the surface of spermatozoa before and after deposition in the female reproductive tract.
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Materials and Methods |
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Collection of Epididymal Spermatozoa
Epididymides with the vas deferentia attached were trimmed of extraneous
adipose tissue and sectioned into 4 anatomical segments: proximal caput,
distal caput, corpus, and cauda. These regions approximate the anatomical
areas of the rat epididymis previously described (Jones et al, 1980). Three
different buffers were used to isolate, wash, and resuspend epididymal
spermatozoa. These buffers were selected because they approximate the ion
composition of the intraluminal contents of the caput, corpus, and cauda
regions of the epididymis (Setchell and Maddocks, 1994). The intraluminal
contents of the caudal segment were flushed out through an incision in the
distal caudal region of epididymis and by backflushing the attached vas
deferens with 0.5 mL of caudal buffer. Caput and corpus segments of the
epididymis were minced several times with a single-edged razor blade.
Spermatozoa released into the caput and corpus buffers were collected with a
Pasteur pipette. Epididymal spermatozoa were pelleted and washed in their
corresponding buffers by centrifugation (1000 x g, for 10
minutes at room temperature). Sperm concentration was estimated by
hemocytometric count. Spermatozoa recovered from each segment of the
epididymis were contaminated by less than 0.1% somatic cells, as assessed by
phase-contrast microscopy. Two to 4 rats were used for each experiment (n = 3
experimental replicates).
Collection of Epididymal Sperm After Deposition in the Female
Reproductive Tract
Mating between female and male rats was initiated and observed after sunset
by illumination using a red photographic light. A male of proven fertility
(ie, a progeny producer, as assessed by Hilltop Lab Animal, Inc) was placed in
a holding cage with 4 females. When the male was observed to mount a female,
both animals were then transferred to a separate holding cage and allowed to
continue mating for an additional 15 minutes (ie, typically 8 to 10 mounts by
a male). Female and male rats were allowed to mate for 15 minutes, 1 hour, and
7 hours and then the females were killed. The fallopian tubes were removed and
gently squeezed using small metal forceps to recover the spermatozoa.
Spermatozoa recovered from the fallopian tubes were suspended in phosphate
buffered saline (PBS pH 7.38) and pelleted by centrifugation (1000
x g for 5 minutes at room temperature). The concentration of
spermatozoa and percentage of contamination by other cell types was estimated
by hemocytometric count. The preparation of sperm was contaminated by less
than 0.1% somatic cells, as revealed by phase-contrast microscopy. The mating
experiment at each time interval was repeated twice (n = 3 separate mating
experiments).
Purification of Protein D/E
Protein D/E molecules were purified from a "crude" epididymal
tissue homogenate prepared from 30 rats using a procedure described previously
(Tubbs et al, 2001). The
homogeneity and molecular mass of the protein D/E preparation used in this
study was assessed by conventional size-exclusion chromatography,
two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) under reducing and denaturing conditions as previously described
(Hall and Killian, 1989), and
electrospray ionization mass spectrometry.
Preparation and Analysis of Antiprotein D/E Polyclonal
Antibodies
Polyclonal antibodies against purified protein D/E were raised in 2 female
New Zealand white rabbits and chickens as previously described
(Hall et al, 1997;
Tubbs et al, 2001). The final
titers for rabbit antiprotein D/E immunoglobulin (Ig) G and chicken
antiprotein D/E IgY antibodies were 1:40 000 and 1:8800,
respectively. Both the rabbit and chicken antiprotein D/E antibodies were
determined to be monospecific, as assessed by a Western immunoblot competition
assay. Briefly, protein D/E samples were subjected to electrophoresis on 10%
uniform onedimensional SDS-PAGE slab gels, transferred to a nitrocellulose
membrane, and immunoblotted with antiprotein D/E antibodies. The immunoblotted
nitrocellulose membrane was incubated with either rabbit or chicken
antiprotein D/E antibodies that had been preabsorbed with 100-fold to
1000-fold concentrations of the purified protein. The major 32-kd protein band
identified using the antiprotein D/E antibody was abolished, indicating that
the polyclonal antibodies were specific for protein D/E.
SDS-PAGE and Western Blot Analysis
Protein samples were dissolved in 50 mM Tris/HCl pH 6.8 containing 10%
ß-mercaptoethanol (v/v; Sigma Chemical Company, St Louis, Mo), 10% SDS
(w/v; Sigma), 30% glycerol (v/v), and 0.1% (w/v) bromophenol blue (Fisher
Scientific Company, Atlanta, Ga), and then incubated at 40°C for 30
minutes to prevent the formation of high-molecular-weight protein aggregates.
SDS-PAGE analysis was performed under reducing and denaturing conditions by
use of the buffering system of Laemmli
(1970) as previously described
(Hall et al, 1997).
Photoactivated Cross-Linking of Purified Protein D/E to the Surface
of Epididymal Spermatozoa
The cross-linking of protein D/E molecules to the surface of epididymal
spermatozoa was performed in 2 stages. First, a purified sample of protein D/E
was dialyzed against phosphate buffered saline (PBS), resuspended at a
concentration of 1.0 µg/µL, and conjugated to the trifunctional
cross-linking reagent,
sulfo-succinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido)
hexano-amido] ethyl-1,3' dithioproionate (Sulfo-SBED, Pierce Chemical
Company, Rockford, Ill). The cross-linker, which has a biotin handle that is
transferable, was conjugated to protein D/E at a molar excess ratio of 5:1
(3.6 nmol of Sulfo-SBED to 0.727 nmol of protein). Before coupling the
cross-linking reagent to the protein, a working stock solution of the
cross-linking reagent was prepared at a concentration of 0.1 µg/µL by
dissolving 1 mg of Sulfo-SBED into 10 mL of dimethyl sulfoxide (DMSO; Sigma
Chemical Company, St Louis, Mo). Soybean trypsin inhibitor protein was
subjected to C18 reverse-phase high-pressure liquid chromatography
to assess its purity and then used for competition experiments as a
nonspecific competitor protein. The "stock" Sulfo-SBED (3.2 µg)
and purified protein D/E (0.632 µg) were added to a siliconized 1.7-mL
microfuge tube, dissolved in 300 µL of 25 mM sodium phosphate buffer (pH
7.2), and allowed to react at room temperature for 1 hour. The
Sulfo-SBED-conjugated protein was then transferred to a Tube-O-Dialyzer (Geno
Technologies, Inc, St Louis, Mo) and dialyzed for 12 hours at 4°C to
remove uncoupled cross-linking reagent.
During the second stage, spermatozoa (1.0 x 108 total cells) were incubated for 30 minutes in 25 mM phosphate buffer (pH. 7.5) containing 400 mM NaCl (Wong and Tsang, 1982) and washed twice in PBS by centrifugation at 1000 x g for 5 minutes at room temperature to remove noncovalently linked protein D/E molecules from the sperm surface. The sperm samples were then placed in clear, siliconized glass test tubes (13 x 75 mm) with buffer and other reactants. To each test tube, 0.632 µg (36 pmol) of the Sulfo-SBED-conjugated protein were added in the dark or under a red photographic light using a safelight filter (Eastman Kodak Company, Rochester, NY); the total reaction volume was 750 µL. Each test tube was mixed by gentle handvortex action and incubated for 1 hour at 33°C in the dark. To assess whether the interaction of protein D/E molecules with the surface of spermatozoa was specific and competitive, sperm cells were incubated in the presence of a constant amount of Sulfo-SBED-conjugated protein D/E, 100 molar excess of soybean trypsin inhibitor protein, and varying concentrations (ie, 50, 200, 500, and 1000 molar excess) of unconjugated protein D/E ("cold protein D/E"). After 1 hour, the test tubes were placed in a Spectronics Ultraviolet Viewing Cabinet Box (Spectronics Inc, Westbury, NY) and irradiated with short (ie, 254 nm) UV light for 15 minutes at room temperature. The sperm samples were brought to a final volume of 1.5 mL with PBS, centrifuged at 1000 x g for 10 minutes at room temperature to pellet the spermatozoa, and the pellet was resuspended in 100 µL of 50 mM Tris/HCl (pH 7.5) containing 50 mM NaCl, 1 mM ethylene diamene tetraacetic acid, and 0.1% NP-40 detergent (solubilization buffer). The pelleted sperm samples were solubilized overnight by vortex action at room temperature and centrifuged at 12 000 x g for 10 minutes at room temperature. The pelleted material (ie, large pieces of nucleic acids, undissolved mitochondria, sperm tails, etc) was discarded. To assess the efficiency of the solubilization procedure, the membrane-supernatants were centrifuged at 20 000 x g for 30 minutes at 4°C, and no pelleted material was observed. The proteins in the membrane supernatant were then resolved by one-dimensional SDS-PAGE using an 8% uniform acrylamide slab gel under reducing and denaturing conditions as previously described earlier.
Treatment of Spermatozoa with Phosphatidylinositol-Specific
Phospholipase C
Caudal spermatozoa (x1.0 x 108 total cells) were
suspended in 25 mM phosphate buffer (pH 7.5) containing 400 mM NaCl and
incubated in a 1.7-mL plastic, siliconized microcentrifuge test tube for 30
minutes at room temperature. The sperm cells were resuspended in PBS, washed
twice in PBS by centrifugation (ie, 1000 x g for 5 minutes at
room temperature), and reacted with 5 units of phosphatidylinositol-specific
phospholipase C (PSPC) for 30 minutes, 1 hour, and 2 hours at 37°C, with
gentle agitation in a rotating water bath. A control experiment consisted of
incubating a sperm sample for 2 hours at 37°C in the absence of enzyme
(ie, the PSPC solution was replaced with an equal volume of PBS). After the
various incubation periods, the sperm samples were centrifuged at 1000 x
g for 5 minutes and the supernatant solutions were removed and stored
at -20°C until further analysis. The sperm pellets were suspended in PBS
and washed 3 times in PBS by centrifugation at 1000 x g for 5
minutes at room temperature. The sperm pellets were then resuspended in 100
µL solubilization buffer, vortexed overnight at room temperature, and
centrifuged at 12 000 x g for 10 minutes at room temperature.
The proteins in the supernatant solutions (ie, the 1000 x g and
12 000 x g centrifugations) were resolved by one-dimensional
SDS-PAGE under reducing and denaturing conditions on an 8% uniform acrylamide
slab gel as described earlier.
Immunofluorescent Localization of Protein D/E Molecules on Sperm
Surface
All immunofluorescent microscopic procedures were performed at room
temperature. Spermatozoa recovered from epididymides and female reproductive
tracts were suspended in PBS, washed 3 times in PBS by centrifugation at
700 x g for 5 minutes, and prepared for immunofluorescence
at 3.0 x 107 total cells. The sperm samples were
incubated for 30 minutes in Bouin fixative, which was prepared fresh for each
sperm isolation. The fixed spermatozoa were washed 4 to 5 times in PBS to
remove excess fixative and pelleted by centrifugation at 700 x
g. The pelleted spermatozoa were resuspended in PBS containing 1%
bovine serum albumin (BSA; used as a blocking reagent) for 1 to 2 hours at
4°C.
Spermatozoa were centrifuged at 700 x g for 5 minutes
and resuspended in normal donkey serum for 30 minutes. After the incubation
period, spermatozoa were centrifuged at 700 x g for 5
minutes and washed 3 times in PBS. The pellet was resuspended in 198 µL of
5% nonfat milk/PBS (w/v) and 2 µL of chicken antiprotein D/E IgY antibodies
for 1 hour; this yielded a 1:100 dilution of the primary antibodies. After
incubation with the primary antibodies, the spermatozoa were resuspended in
PBS, washed 3 times by centrifugation at 700 x g at room
temperature, and the supernatant was discarded. A drop of gel mount solution
was placed on a glass microscope slide; 25 µL of the sperm pellet was
injected directly into the gel mount, and a coverslip was placed on the
microscope slide. Control experiments to assess nonspecific antibody staining
included 1) replacement of the immune antibodies with preimmune chicken IgY
fraction, 2) preabsorption of the immune antibodies with purified antigen, 3)
dilution or elimination of immune antibodies, 4) dilution or elimination of
the secondary antibodies, and 5) secondary antibodies in the absence of
antiprotein D/E serum. To reduce internal bias, the staining procedures were
performed 3 separate times (n = 3 separate mating and sperm isolations). The
results from each immunofluorescent staining experiment were viewed by 2
different scientists working in the laboratory to verify the immunofluorescent
staining intensity and patterns. Only results that were consistent between
replicates and scientists were taken as representative data. Sperm were
examined using a Nikon LabPhot microscope equipped with a fluorescein
isothiocyanate epifluorescence apparatus, and photomicrographs were taken
using Kodak T-Max 400 black/white film (400 ASA).
Protein Assay
Soluble protein was measured using the method of Lowry
(Lowry et al, 1951), with BSA
as the standard protein. Samples were assayed in duplicate with the
concentration of BSA ranging from 10 to 80 µg/mL in 10-µg
increments.
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Results |
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32 kd and a minor band corresponding
to 34 kd. Densitometric scanning indicated that the 32kd protein
band represented >98% of the total protein associated with the final
preparation. A single polypeptide spot corresponding to 32 kd was
revealed by two-dimensional SDS-PAGE analysis
(Figure 1B), suggesting a
highly purified protein D/E preparation. To further assess the purity of the
final protein D/E preparation and to obtain structural information about
protein D/E, mass spectral analysis was performed
(Figure 1C). Mathematical
deconvolution of the mass spectra indicated a highly purified protein D/E
sample and confirmed a previous finding of carbohydrate heterogeneity
associated with protein D/E (Lea et al,
1978). The protein has a molecular mass of 27.4 kd, with
carbohydrates representing 6% of the total mass.
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Immunodetection of Protein D/E Molecules in the Protein Extract of
Epididymal Tissues and Spermatozoa
To assess the specificity of chicken antiprotein IgY antibodies, a
competition assay was performed, and the result of the assay is presented in
Figure 2A. No protein D/E
immune-positive staining was detected when the antibodies were preabsorbed
with 100-fold excess purified protein D/E
(Figure 2A, lane 2). As shown
in Figure 2B, protein D/E
molecules were detected in the protein extracts of tissue and spermatozoa
obtained from the distal caput, corpus, and caudal regions, but not in tissues
and spermatozoa obtained from the proximal caput region of epididymides.
Densitometric scanning of the Western blots revealed that the amount of
protein D/E molecules detected increased from the distal caput to the caudal
regions (Figure 2B).
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Photoactivated Cross-linking of Purified Protein D/E Molecules to the
Surface of Epididymal Spermatozoa
An in vitro "competition" experiment to assess the interaction
of exogenously added protein D/E molecules with the surface of epididymal
spermatozoa in vitro is presented in Figure
3. Unconjugated (ie, cold) protein D/E molecules at 50-, 200-,
500-, and 1000-fold molar excess in the presence of a constant amount of
Sulfo-SBED-conjugated protein D/E molecules eliminated the banding intensity
of 2 proteins exhibiting a molecular mass of 130 kd and 120 kd,
respectively (Figure 3).
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Treatment of Epididymal Spermatozoa with PSPC
The effect of PSPC treatment for various periods of time on the release of
protein D/E molecules from the surface of epididymal spermatozoa is presented
in Figure 4. In comparison to
the control (Figure 4D),
spermatozoa treated for 30 minutes, 1 hour, and 2 hours with PSPC exhibited a
marked decrease in the fluorescence intensity over the head region of
spermatozoa, but not the regions covering the mid piece and tail
(Figure 4, A-C).
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The release of protein D/E molecules from the surface of epididymal spermatozoa into the supernatant by PSPC treatment, as assessed by one-dimensional SDS-PAGE followed by Western blot analysis, is presented in Figure 5. After only 30 minutes of PSPC treatment, protein D/E molecules were observed to be released into the supernatant from the surface of spermatozoa when compared to those of the control supernatant (Figure 5, lanes b—e).
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Immunolocalization of Protein D/E Molecules on the Surface of
Epididymal Spermatozoa Before and After Deposition in the Female Reproductive
Tract
Figure 6 shows the IIF
localization of protein D/E molecules on the surface of spermatozoa recovered
from different anatomical regions of the epididymis. In comparison to the
control (Figure 6D), regional
differences were observed in the surface fluorescent staining pattern of
epididymal spermatozoa. Whereas a more generalized fluorescent staining
pattern was detected over the entire plasma membrane of caput and corpus
spermatozoa (Figure 6, A and
B), the fluorescent staining pattern was more specialized and
limited to the head and mid piece regions of caudal spermatozoa
(Figure 6C, arrows).
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Figure 7 shows the IIF localization of protein D/E molecules on the surface of ejaculated spermatozoa deposited immediately (ie, within minutes) in the female vaginal cavity and after being deposited in the female vaginal cavity for 1 and 7 hours. Spermatozoa recovered immediately from the vaginal cavity exhibited intense fluorescent staining that was localized over the entire surface of the spermatozoa (Figure 7, ejaculate). In contrast, an intense fluorescent staining pattern was primarily localized to the head and mid piece regions of spermatozoa recovered from the vaginal cavity after 1 hour (Figure 7, 1 hour). After 7 hours of being deposited in the female reproductive tract (Figure 7, 7 hours), the fluorescent staining intensity on the surface of the spermatozoa was comparable to that of the control samples.
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Discussion |
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Protein D/E is a major secretory protein in the caput region of the epididymis, binds to caput spermatozoa (Lea et al, 1978; Faye et al, 1980; Kohane et al, 1980, 1983; Brown et al, 1983; Brooks, 1987), and remains bound to spermatozoa during incubation in capacitating media (Cameo et al, 1986). Antibodies against protein D/E preparations have been used to demonstrate the involvement of this protein in the fertilization process (Cuasnicu et al, 1984; Fournier-Delpech et al, 1985; Cameo et al, 1986; Cohen et al, 1996, 2000; Ellerman et al, 1998). Few studies have presented data verifying the purity of the protein D/E preparation used to generated the antiserum. In addition, one study (Olson and Hinton, 1985; Vreeburg et al, 1992) has suggested that rat epididymal protein D/E may consist of various components of different charges and sizes, and that only some of these variants bind to spermatozoa. Given the findings of previous studies (Cuasnicu et al 1984; Fournier-Delpeach et al, 1985; Cameo et al, 1986; Cohen et al, 1996, 2000; Ellerman et al, 1998) and the emerging concept for a functional role of protein D/E molecules in the fertilization process, we wanted to verify the homogeneity of the protein D/E preparation used in the present study. As shown in Figure 1, two-dimensional SDS-PAGE and mass spectral analysis indicated a highly purified protein D/E sample.
Earlier studies (Wong and Tsang, 1982; Hall and Hadley, 1990; Hall et al, 1997) have suggested that the interaction of protein D/E molecules with the surface of epididymal spermatozoa may be a simple "reversible" receptor-ligand-mediated interaction. However, our finding demonstrating that protein D/E molecules specifically interact with at least 2 membrane proteins (Figure 3) suggests that the interaction may be more complex than a simple receptor-ligand type interaction, and that it is perhaps a two-step process. The initial step could involve the cysteine-rich region in the C-terminus of protein D/E molecules, which may serve to direct or orient protein D/E molecules toward their intended receptor-like molecules. In other cell systems, attachment of the protein moiety to a glycosylphosphatidyl inositol (GPI) anchor has been shown to be "directed" by a signal at the C-terminus of the polypeptide (Low, 1989; Fraser, 1995). The second step of the binding process may involve an enzyme-mediated covalent linkage of protein D molecules to a GPI anchor found in association with the head region.
Only a few studies to date have provided "direct" experimental evidence for the binding of specific secretory proteins to the surface of rat epididymal spermatozoa. One such study (Vreeburg et al, 1992) has shown that only a few proteins become bound to caput spermatozoa.
However, in the corpus region of the epididymis, 5 major proteins with an estimated molecular mass range of 25 to 100 kd were observed to specifically bind to the surface of corpus spermatozoa. With the exception of one of these proteins (eg, the 25-kd protein), all the other major proteins were not observed to be bound to caudal spermatozoa. This experimental observation suggests that extensive modification of the sperm surface was occurring as they traversed the epididymis. The experimental observation of this study (Vreeburg et al, 1992) is consistent with the data in the immunofluorescent studies of the present study (Figure 6), particularly if these major proteins of the corpus region of the epididymis share some common antigenic determinants with protein D/E molecules. This may explain the intense immunofluorescent staining pattern over the entire surface of corpus spermatozoa compared with that of caudal spermatozoa.
To assess whether the observed in vitro reversible binding of protein D/E molecules that had been reported previously (Wong and Tsang, 1982; Hall and Hadley, 1990) would persist in vivo, the topology of the surface of spermatozoa was assessed before and after deposition into the female reproductive tract (Figures 6 and 7). Previous studies (Rochwerger and Cuasnicu, 1992; Rochwerger et al, 1992) have shown that rat oocytes exhibit protein D/E complementary binding sites on their surface and that protein D/E molecules were restricted to the sperm head after in vitro and in vivo capacitation. These experimental observations taken together suggest but do not conclusively prove that protein D/E molecules are present on the surface of in utero spermatozoa as they travel toward the oocyte. These observations also raise an important scientific question: if protein D/E molecules bind to spermatozoa reversibly as they mature in the epididymis, how do the molecules remain associated with spermatozoa after ejaculation and deposition in the female reproductive tract? Clearly, the data in the present study (Figures 4 and 7) provide strong experimental evidence that protein D/E molecules are bound to the surface of spermatozoa through a GPI anchor. We hypothesize that the function of the GPI anchor on the surface of mammalian spermatozoa is to retain a specific population of protein D/E molecules, perhaps only protein "D" molecules or other molecules that participate in the fertilization process on the surface of spermatozoa until they reach the oocyte. Whether the covalently linked protein D/E molecules on the surface facilitates motility of spermatozoa, or spermegg binding, or both (Fournier-Delpech et al, 1985; Hall et al, 1997); or sperm-egg membrane fusion (Rochwerger and Cuansnicu, 1992; Rochwerger et al, 1992; Cohen et al, 1996, 2000), or a combination of these membrane-mediated fertilization processes, remains to be elucidated and awaits further investigation.
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Acknowledgments |
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Footnotes |
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V.P.C. is a Dozoretz National Institute for Minorities in Applied Science Scholar, Norfolk State University, Norfolk, Virginia.
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Brooks DE. Purification of rat epididymal proteins "D" and "E," demonstration of shared immunological determinants, and identification of regional synthesis and secretion. Int J Androl. 1982;5:513 -524.[Medline]
Brooks DE. Androgen-regulated epididymal secretory proteins associated with post-testicular sperm development. Ann N Y Acad Sci. 1987;513:179 -194.[Abstract]
Brooks DE, Tiver K. Localization of epididymal secretory proteins on rat spermatozoa. J Reprod Fertil.1983; 69:651 -675.
Brown CR, Von Glos KI, Jones R. Changes in plasma membrane
glycoproteins of rat spermatozoa during maturation in the epididymis.
J Cell Biol.1983; 96:256
-264.
Cameo MS, Echeverria FG, Blaquier JA, Burgos MH. Immunochemical localization of epididymal protein DE on rat spermatozoa. Gamete Res. 1986;15:247 -257.
Cohen DJ, Munuce MJ, Cuasnicu PS. Mammalian sperm-egg fusion: the development of rat oolemma fusibility during oogenesis involves the appearance of binding sites for sperm protein "DE." Biol Reprod. 1996;55:200 -206.[Abstract]
Cohen DJ, Ellerman DA, Cuasnicu PS. Mammalian sperm-egg fusion:
evidence that epididymal protein DE plays a role in mouse gamete fusion.
Biol Reprod.2000; 63:462
-468.
Cuasnicu PS, Echeverria FG, Piazza AD, Cameo MS, Blaquier JA. Antibodies against epididymal glycoproteins block fertilizing ability in rat. J Reprod Fertil.1984; 72:467 -471.
Dacheux JL, Paquignon M. Relations between the fertilizing ability, motility and metabolism of epididymal spermatozoa. Reprod Nutr Dev. 1980;20:1085 -1099.
Eberspaecher U, Roosterman D, Kratzschmar J, et al. Mouse androgendependent glycoprotein CRISP-1 (DE/AEG): isolation, biochemical characterization, and expression in recombinant form. Mol Reprod Dev. 1995;42:157 -172.[Medline]
Echeverria FG, Cuasnicu PS, Piazza A, Pineiro L, Blaquier JA. Addition of an androgen-free epididymal protein extract increases the ability of immature hamster spermatozoa to fertilize in vivo and in vitro. J Reprod Fertil.1984; 71:433 -437.
Ellerman DA, Brantua VS, Martinez SP, Cohen DJ, Conesa D, Cuasnicu
PS. Potential contraceptive use of epididymal proteins: immunization of male
rats with epididymal protein DE inhibits sperm fusion ability. Biol
Reprod. 1998;59:1029
-1036.
Faye JC, Duguet L, Mazzuca M, Bayard F. Purification, radioimmunoassay, and immuno-histochemical localization of a glycoprotein produced by the rat epididymis. Biol Reprod.1980; 23:423 -432.[Abstract]
Fournier-Delpech S, Courot M, Dubois MP. Decreased fertility and
motility of spermatozoa from rats immunized with a prealbumin
epididymal-specific glycoprotein. J Androl.1985; 6:246
-250.
Fournier-Delpech S, Hamamah S, Tananis-Anthony C, Courot M, Orgebin-Crist M-C. Hormonal regulation of zona-binding ability and fertilizing ability of rat epididymal spermatozoa. Gamete Res.1984; 9:21 -30.
Fraser L. Ionic control of sperm function. Reprod Fertil. 1995;7:905 -925.
Hall JC, Tubbs CE, Li Y, Ashraf S, Lamarche MD. Affinity purification and characterization of a 32 Kd glycoprotein from the rat sperm plasma membrane that is required for egg zona pellucida binding. IJBC. 1997;3:155 -176.
Hall JC, Hadley JA. Kinetics of receptor-mediated binding of a 32 Kd epididymal glycoprotein to the surface of testicular and epididymal sperm. Mol Androl.1990; 1:232 -240.
Hall JC, Hadley J, Doman T. Correlation between changes in rat
sperm membrane lipids, proteins, and the membrane physical state during
epididymal maturation. J Androl.1991; 12:76
-87.
Hall JC, Killian GJ. Two-dimensional gel electrophoretic analysis
of rat sperm membrane interaction with cauda epididymal fluid. J
Androl. 1989;10:64
-76.
Kohane AC, Cameo MS, Pineiro L, Garberi JC, Blaquier JA. Distribution and site of production of specific proteins in the rat epididymis. Biol Reprod.1980; 23:181 -187.[Abstract]
Kohane AC, Pineiro L, Blaquier JA. Androgen-controlled synthesis of specific protein in the rat epididymis. Endocrinology.1983; 112:1590 -1596.[Abstract]
Kratzschmar J, Haedler B, Eberspaecher U, Rootsterman D, Donner P, Schleuning WD. The human cysteine-rich secretory (CRISP) family; primary structure and tissue distribution of CRISP-1, CRISP-2 and CRISP-3. Euro J Biochem.1996; 236:827 -836.[Medline]
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature Lond.1970; 227:680 -685.[Medline]
Lea OA, Petrusz P, French FS. Purification and localization of acidic epididymal glycoprotein (AEG): a sperm coating protein secreted by the rat epididymis. Int J Androl. 1978;(suppl 2): 592-607.
Low MG. Glycosyl-phosphatidylinositol: a versatile anchor for cell surface proteins. FASEB J.1989; 3:1600 -1608.[Abstract]
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement
with the folin phenol reagent. J Biol Chem.1951; 193:265
-275.
Moore HDM, Hartman TD. In-vitro development of the fertilizing ability of hamster epididymal spermatozoa after co-culture with epithelium from the proximal cauda epididymis. J Reprod Fertil.1986; 78:347 -352.
Morrissette J, Kratzschmar J, Haendler B, el-Hayek R,
Mochca-Morales J, Martin BM, Patel JR, Moss RL, Schleuning WD, Coronado R.
Primary structure and properties of helothermine, a peptide toxin that blocks
ryanodine receptors. Biophys J.1995; 68:2280
-2288.
Nikolopoulou M, Soucek DA, Vay JC. Changes in the lipid content of boar sperm plasma membranes during epididymal maturation. Biochim Biophys Acta. 1985;814:486 -498.
Olson G, Hinton B. Regional differences in luminal fluid polypeptides of the rat testis and epididymis revealed by two-dimensional electrophoresis. Androl J.1985; 6:20 -34.
Orgebin-Crist M-C, Jahad N. The maturation of rabbit epididymal spermatozoa in organ culture: stimulation by epididymal cytoplasmic extracts. Biol Reprod.1979; 21:511 -515.[Abstract]
Parks JE, Hammersted RH. Developmental changes in the lipids of ram epididymal spermatozoa plasma membrane Biol Reprod.1985; 32:653 -668.[Abstract]
Quill TA, Ren D, Clapham DE, Garbers DL. A voltage-gated ion
channel expressed specifically in spermatozoa. Proc Natl Acad Sci
USA. 2001;98:12527
-12531.
Rochwerger L, Cohen DJ, Cuasnicu PS. Mammalian sperm-egg fusion: the rat egg has complementary sites for a sperm protein that mediates gamete fusion. Dev Biol.1992; 153:83 -90.[Medline]
Rochwerger L, Cuasnicu PS. Redistribution of a rat sperm epididymal glycoprotein after in vitro and in vivo capacitation. Mol Reprod Dev. 1992;31:34 -41.[Medline]
Setchell BP, Maddocks S, Brooks DE. Anatomy, vasculature, innervation, and fluids of the male reproductive tract. In: Knobil E, Neill JD, Greenwald GS, Marker CL, Pfaff DW, eds. The Physiology of Reproduction. New York: Raven Press; 1994:1063 -1147.
Tubbs CE, Hall JC, Scott RO, Copeland C, Cooper R. A rapid and efficient method for purification of protein D/E from the rat epididymis: biochemical characterization of a protein involved in the fertilization process. IJBC.2001; 6:33 -50.
Turner TT, Avery EA, Sawchuk TJ. Assessment of protein synthesis and secretion by rat seminiferous and epididymal tubules in vivo. Int J Androl.1994; 17:203 -213.
Vreeburg JTM, Holland MK, Orgebin-Crist M-C. Binding of epididymal proteins to rat spermatozoa in vivo. Biol Reprod.1992; 47:588 -597.[Abstract]
Wong PYD, Tsang AYF. Studies on the binding of a 32K rat epididymal protein to rat epididymal spermatozoa. Biol Reprod.1982; 27:1239 -1246.[Abstract]
Xu W, Hamilton DW. Identification of the rat epididymis-secreted 4E9 antigen as protein E: further biochemical characterization of the highly homologous epididymal secretory proteins D and E. Mol Reprod Dev. 1996;43:347 -357.[Medline]
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