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From the Departments of * Biochemistry and
Molecular and Cell Biology and Animal
Pathology, School of Veterinary Medicine, University of Zaragoza, Zaragoza,
Spain.
| Correspondence to: Dr T. Muiño-Blanco, Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Veterinaria, C/ Miguel Servet, 177, 50013 Zaragoza, Spain (e-mail: muino{at}unizar.es). |
| Received for publication October 3, 2005; accepted for publication March 12, 2006. |
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Abstract |
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Key words: Seminal plasma proteins, seminal vesicles, cold shock, ram spermatozoa
We recently reported the biochemical characterization and partial amino acid sequence of 2 ram seminal plasma proteins of approximately 14 (P14) and 20 (P20) kDa, which protect spermatozoa against cold shock (Barrios et al, 2005). The P14 sequenced fragment contained the fibronectin domain type II (FN2) domain (Greube et al, 2001), as with bovine PDC-109 (Esch et al, 1983), also called bull seminal plasma proteins (BSP) A1/A2 (Manjunath and Sairam, 1987). FN2 domain is a collagen-binding domain that binds to different extracellular matrix and cytoskeleton components to stabilize the extracellular matrix and determine the shape of the cell and cytoskeleton organization. However, the sequence of the P20 fragment was not homologous with any reported protein. The results of immunocytochemical detection and Western blotting analysis of P14 and P20 content on sperm before and after in vitro induction of capacitation and acrosome reaction confirm that the induced membrane alterations account for a decrease in the content as well as migration and redistribution of both proteins to the equatorial and postequatorial regions. Our results suggest that the protective effect of these proteins could be related to their decapacitating role (Barrios et al, 2005).
The aim of the present work was to investigate the expression of P14 and P20 in the ram reproductive tract tissues to determine their origin.
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Materials and Methods |
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Extraction of Proteins from Ram Reproductive Tract Tissues
Proteins from ram reproductive tract tissues were extracted by homogenizing
1–2 g with 1 mL 10 mM sodium phosphate buffer containing 10% of a
protease inhibitor cocktail (Sigma Chemical Co, St Louis, Mo) in an ice bath.
After centrifuging at 5000 x g for 10 minutes at 4°C, the
supernatant was recovered and stored at -20°C. Protein concentration was
determined according to the method described by Bradford
(1976).
Polyclonal Antibodies
Polyclonal antibodies were raised against the whole seminal plasma fraction
6 (F6) isolated by exclusion chromatography in Sephacryl-100
(Barrios et al, 2000), and the
purified P14 and P20 bands were recovered by electroelution from a
nondenaturing gel (Barrios et al,
2000) by rabbit immunization with 500 µg (F6) and 300 µg
(P14 and P20) of protein in Freunds complete adjuvant. After 15 days they were
reimmunized with the same amount dissolved in Freunds incomplete adjuvant. The
antiserum was obtained 15 days after the second immunization by centrifugation
of 10–20 mL of blood from each rabbit and purification by protein G
affinity chromatography.
Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis and Western Blotting
Immunoblots were carried out by a 7%–22% sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of 25-to 100-µg
proteins transferred for 2 hours onto a polyvinyldene difluoride membrane with
a Hoefer TE70 Semiphor Semidry-Transfer Unit (Pharmacia-Biotech, Uppsala,
Sweden). Non-specific sites on the membranes were blocked for 1 hour with 5%
bovine serum albumin (BSA) in blocking buffer (Tris-HCl 10 mM pH 8; NaCl 120
mM, 0.05% Tween 20). The proteins were immunodetected by incubating for 3
hours at 20°C with the polyclonal antibodies diluted at 1:2000 (P14) and
1: 500 (P20) in blocking buffer that contained 0.17% BSA. After exhaustive
washing, the blots were incubated with a secondary goat anti-rabbit
alkaline-phosphatase–conjugated IgG (Sigma) at a dilution 1:30 000 for 2
hours. After 4 washings of 5 minutes each, the membranes were incubated with
66 µg/mL 5-bromo-4-chloro-3-indolyl phosphate and 111 µg/mL nitro blue
tetrazolium in Tris 0.19 M, MgCl2 1 mM until color appeared. The
image was scanned and a densitometric analysis was carried out. The Gel Doc
System with Molecular Analyst software (Bio Rad, Hercules, Calif) was used to
quantify the changes in intensity of various bands. Replacing the antiserum
with preimmune serum was used as a negative control to rule out nonspecific
binding to the transferred proteins.
Indirect Immunofluorescence
Tissue sections (7 µm thick) were cut on a Surgipath microtome (American
Instrument Exchange, Inc, Haverhill, Mass), air dried, and fixed in acetone
for 20 minutes. Nonspecific binding sites were blocked with 5% BSA in PBS for
30 minutes at room temperature in a humidified chamber. After blocking, the
slides were washed 3 times with PBS, and 50 µL of primary polyclonal
antibodies diluted in PBS with 1% BSA at 1:250 (P14) and 1:150 (P20) were
added. The preparations were incubated for 1 hour at 37°C in the wet
chamber, washed 3 times, and incubated again with 250 µL of anti-rabbit IgG
(Alexa Fluor 488, Molecular Probes (Leiden, The Netherlands); 1:900 diluted in
PBS with 1% BSA) for 1
hours at 37°C in the dark. The slides were
rinsed with PBS. Finally, the preparations were covered with coverslips,
sealed with colorless enamel, and visualized through a Nikon Eclipse E-400
microscope (Tokyo, Japan) under epifluorescence illumination with a
fluorescein filter at 10x and 40x. Negative controls were
performed by omission of the primary antibodies and also by replacing the
primary antibody with normal rabbit serum.
Immunohistochemical Techniques
Tissue samples were fixed in Bouins solution for 90 minutes and dehydrated
in a graded series of ethanol and embedded in paraffin. Sections (7 um thick)
were cut on a Surgipath microtome. The sections were exposed to
immunohistochemical staining by an avidin-biotin complex technique (Vector,
Burlingame, Calif). Endogenous peroxidase was inactivated with 1.7% hydrogen
peroxide (H2O2) in 100% ethanol for 30 minutes.
Subsequently, the sections were washed in PBS pH 7.4 and then incubated with
normal goat serum (blocking reagent) (Vector) for 20 minutes, followed by
incubation with the specific antiserum (primary antibody, dilution 1:125 for
P14 and 1:200 for P20) overnight at room temperature. After washing, the
slides were incubated with biotinylated anti-rabbit antiserum (Vector) for 30
minutes. An avidin-biotin-peroxidase complex (Vector) was then applied for 40
minutes. The binding sites of the primary antibodies were visualized by
diaminobenzidine (DAB) and H2O2 solution (0.12 g DAB in
240 mL of PBS pH 7.4 containing 3 % H2O2) for 7 minutes.
Slides were contrasted with Carazzi hematoxylin and mounted with 1,3
diethyl-8-phenylxanthine. As a negative control, samples were incubated with
normal goat serum instead of the primary antibody, with the remaining
procedure being the same.
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The images obtained by these techniques were taken with a microscope digital camera system (Sony 2CCd Colour Video Camera, and Sony Digital Still Recorder, Carson, Calif) and saved and edited with Visilog 5.1 Software (Noesis S.A, Orsay, France).
RNA Isolation and Reverse Transcriptase–Polymerase Chain Reaction
Total RNA was extracted from seminal vesicles and vas deferens by the
guanidinium thiocyanate/phenol extraction method
(Chomczynski and Sacchi, 1987;
Chomczynski, 1993) by
homogenization in 1 mL of TRI REAGENT (Sigma) per 200 mg of tissue. RNA
concentration was estimated by reading the absorbance at 260 nm and checked
for purity at 280 nm on a Hitachi U-2001 spectrophotometer (Lexington, Ky).
Samples were stored at -80°C until used.
Between 200 and 250 ng of total RNA from seminal vesicles was reverse transcribed by using poly (dT) primer and the SuperScript III RT enzyme (Invitrogen Inc, Carlsbad, Calif). One tenth of reverse-transcribed sample was used in the polymerase chain reaction (PCR) amplifications (SuperScript III First-Strand Synthesis System for RT-PCR, Invitrogen Inc).
Amplification of Partial cDNA Sequences Encoding P14 and P20
Degenerate PCR primers (see Table) for P14 and P20 were designed on the
basis of the primary sequence of the proteins
(Barrios et al, 2005) in the
5' region. Primers P14-5' and P20-5' were based on amino
acids residues 1–6. The 3' primer in both cases was Oligo
(dT)20. Another primer pair left/right (LP/RP, respectively) was
designed by Primer3 Software
(http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi).
PCR was performed with 2 µL of cDNA. Using these primers, 35 PCR cycles were carried out on reverse-transcribed RNA from ram seminal vesicles. Cycling conditions consisted of 45-second denaturation at 94°C, 1-minute and 30-second annealing (57°C for P14-5', 53°C for P20-5', and 66°C for LP/RP), and 3-minute primer extension at 72°C. A 1-minute denaturation step at 94°C preceded cycling; at the end, a final 10-minute primer extension at 72°C was performed, followed by a 24-hour soak at 4°C. The "hot start" procedure was accomplished by adding 1 U of Platinum Taq DNA polymerase high fidelity (Invitrogen Inc) to each reaction tube.
cDNA Sequences
PCR fragments covering the complete P14 and P20 cDNA were sequenced. This
was performed on both strands with either LP/RP for P20 or P14-5'/Oligo
(dT)20 primers for P14. DNA was sequenced and analyzed on an ABI
Prism 3700 sequencer (Applied Biosystems, Foster City, Calif).
PCR products were separated on 1% agarose gel in 1 x Tris-borate-EDTA buffer containing 0.4 ng/µL ethidium bromide (Sigma-Aldrich) and were visualized under ultraviolet (UV) light. The intensity of each band was assessed by densitometry using an image analysis program (Molecular Analyst software, Bio Rad). Molecular size was stimated by using Step Ladder 50 bp (Sigma).
PCR products were purified by using the GENECLEAN Turbo Nucleic Acid Purification kit (Q-BIO gene, Morgan Irvine, Calif) according to the manufacturer's instructions.
Northern Blot Analysis
Five micrograms of total RNA were denatured at 75°C for 15 minutes and
electrophoresed in a 1.2% agarose gel containing 7% formaldehyde. After
electrophoresis, the RNAs were transferred to a nylon membrane (Hybond-N,
Amersham, Arlington Heights, Ill) for at least 18 hours. The membrane was
hybridized in a solution consisting of 50% formamide, 3 x SSC (0.45 M
NaCl, 45 mM sodium citrate), 1 x Denhardts solution, 5% dextran
sulphate, 2% SDS, 20 µg/mL denatured salmon sperm DNA, and
32P-labeded DNA probes at 42°C. The membrane was washed twice
in 3 x SSC and 0.5% SDS at room temperature and twice in 3 x SSC
and 0.5% SDS at 50°C for 60 minutes. After drying, the membrane was
cross-linked by exposure to UV light.
The P20 and P14 probes were synthesized by reverse transcriptase–PCR
(RT-PCR) from seminal vesicle total RNA. A mouse 18S probe obtained from
plasmid pUC 830 (752-bp EcoRI/BamHI fragment) was used to normalize the amount
of RNA loaded on the gel. Both probes were labelled using
[
-32P]-dCTP and Rediprime (Amersham). Filters were exposed
to Biomax film (Kodak, Amersham) and analyzed by Molecular Analyst software
(Bio Rad).
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Results |
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Indirect Immunofluorescence Analysis
To discard the possibility that the vas deferens signaling was due to
contamination from the seminal vesicle while obtaining the sample, we used
immunofluorescence to determine the presence of both proteins on slides of
seminal vesicle, vas deferens, and corpus and cauda of epididymis. Only the
seminal vesicle sample showed fluorescence labeling, restricted to the
secretory cells, with the antibodies raised against P14
(Figure 2a) or P20
(Figure 2b). Immunofluorescence
of vas deferens and corpus and cauda of epididymis samples was negative with
both antisera (anti-P14 or anti-P20) (data not shown). Replacing the antiserum
with preimmune serum abolished fluorescence detection (data not shown).
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P14 and P20 Expression Analyzed by RT-PCR and Northern Blot
To further confirm that P14 and P20 are specifically expressed in seminal
vesicles, we used Northern blot analyses to investigate the expression of both
proteins in both seminal vesicles and vas deferens.
P20 and P14 were first analyzed by RT-PCR with the primer pairs deduced from the analyzed peptide sequence. Specific primers (P14-5' for P14 and P20-5' for P20) were designed according to our previous results (Barrios et al, 2005) and their paired primers were Oligo(dT)20. When the RT-PCR products were analyzed by 1% agarose gel electrophoresis, a single band of approximately 450 bp (P14) and 500 bp (P20) (assessed as indicated in "Materials and Methods") was amplified (data not shown). Both target amplicons were absent in the RT-PCR products from vas deferens samples (data not shown).
Northern blot analysis of total RNAs isolated from seminal vesicles and vas deferens of the adult ram was carried out with the 300-bp fragment of P20 (obtained from specific primers left/right, synthesized by Primer3 Software, on the basis of nucleotide sequence of the former RT-PCR product) and the 450-bp fragment of P14 cDNA as probes. We found a 515-bp mRNA signal for P14 (Figure 5a) and a 600-bp mRNA signal for P20 (Figure 5b) in the seminal vesicles. No signal was found in the vas deferens (Figure 5). These results indicate that the positive reaction with the antibody found in the vas deferens (Figure 1) could be because of contamination from the seminal vesicles during collection of the tissues or because of the presence of spermatozoa in the lumen (Figure 3), as we have already shown that P14 and P20 are adsorbed onto the sperm surface (Barrios et al, 2005).
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Discussion |
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In the present study, we examined the localization and distribution of P14 and P20 in testis, prostate, seminal vesicles, vas deferens (the distal fraction including the ampullae), efferent ductules, bulbourethral glands, and 3 epididymal sections (caput, corpus, and cauda) of the ram. Western blot analyses showed that both proteins were detected only in the seminal vesicles and vas deferens. As these 2 parts are very close to each other, the signal detected in the vas deferens could be due to contamination with the SVS while the samples were taken. Results of immunohistochemical analysis by means of indirect immunofluorescence and avidin-biotin complex technique confirmed this hypothesis, as reactivity was exclusively found in seminal vesicles. Consistent findings were obtained by Northern blot analyses, as P14 and P20 were undetectable in vas deferens but present in seminal vesicles. These results demonstrate that the ram P14 and P20 genes are restrictedly expressed in the seminal vesicles, suggesting the important role of these genes in vesicular functions.
Furthermore, the immunohistochemical staining by the avidin-biotin complex technique showed that both proteins were expressed in the cytoplasm of the secretory epithelial cells, though not all the cells were in the same active state. The fact that some cells in contact with the lumen did not show reactivity in the cytoplasm suggests that they are resting cells from the secretory cycle. Indeed, transition forms were observed between highly active and inactive cell types.
The fusion of secretory membrane vesicles with the sperm plasma membrane may be one of the mechanisms involved in the transfer of some seminal plasma proteins to the sperm surface (Frenette and Sullivan, 2001). Using specific antibodies, Aumüller and Scheit (1987) identified the bovine seminal vesicles as the source of different secretory proteins of bull seminal plasma. One of these proteins, the PDC 109, was shown to specifically bind to the midpiece of epididymal spermatozoa that was interpreted as initiating the onset of sperm motility (Scheit et al, 1988).
Results of this investigation indicate that 2 seminal plasma proteins of approximately 14 (P14) and 20 (P20) kDa that protect ram spermatozoa against cold shock (Barrios et al, 2005) are secreted specifically in the seminal vesicles. On the basis of these results, we suggest referring to these 2 proteins as RSVP14 and RSVP20, respectively, according to their origin and molecular weight. Because of the high homology found between RSVP14 and bovine PDC 109 (both containing the FN2 domain) and the obtained evidence that P14 is partially released from the sperm membrane during capacitation and is redistributed over the sperm surface (Barrios et al, 2005), as reported for BSP proteins (Thérien et al, 2001), we have recently suggested that RSVP14 may take part in the protein structure surrounding the spermatozoa in a similar way as fibronectin, stabilizing membrane phospholipids and cytoskeleton (Barrios et al, 2005). We have found out by partition in a 2-phase system with Triton X-114 (Sigma) that these proteins are highly hydrophobic (data not shown), and it was confirmed by the obtained results of partial amino acid sequencing, especially P20 (Barrios et al, 2005). These hydrophobic domains might account for their fusion with the sperm surface. Furthermore, we have reported seasonal differences in the ability of ram seminal plasma proteins to recover membrane integrity of cold-shocked sperm, with an important decrease in several seminal plasma proteins of low molecular weight during the nonbreeding season (Pérez-Pé et al, 2001b).
These results should increase the understanding of the ram SVS composition and its putative role in the sperm functionality. Studies to define the role that RSVP14 and RSVP20 play in influencing ram fertility are currently under way.
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Acknowledgments |
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Footnotes |
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These authors contributed equally to this article and share senior
co-authorship. 
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