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From the Department of Biological Sciences, University of Delaware, Newark, Delaware.
| Correspondence to: Dr Patricia A DeLeon, Department of Biological Sciences, University of Delaware, Newark, DE 19716-2590 (e-mail: pdeleon{at}udel.edu). |
| Received for publication June 11, 2002; accepted for publication August 15, 2002. |
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Abstract |
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Key words: GPI-anchor, insoluble particles, epididymosomes
Because secretory epididymal proteins are involved in the morphological, physiological, and biochemical changes that mammalian sperm undergo during epididymal transit when they acquire motility and fertilizing capability (Orgebin-Crist, 1967; Cooper, 1990; Kirchhoff et al, 1998), it is possible that ES may play a role in sperm maturation. This may occur if ES binds to sperm either as a unique isoform or for the purpose of enhancing Spam1 of testicular origin. The aim of this study was to further characterize ES by comparing it with TS, and to determine whether it is released with its lipid anchor intact, a form in which it can bind to sperm.
For the comparison we investigated both the protein and the transcript, where we focused on the 3' untranslated region (UTR) and the poly(A) tails, which are known to play an important role in messenger RNA (mRNA) stability (Wickens et al, 1997). To study the protein we used two-dimensional gel electrophoresis and lectins to investigate the diversity of the associated oligosaccharides in the two tissues, as well as on sperm. Treatment with phosphatidylinositol-specific phospholipase C (PI-PLC) or Triton X-100 was carried out to determine whether ES in the luminal fluid is associated with membrane particles and is secreted with its GPI anchor. Our results indicate that the mRNA transcripts are significantly shorter in the epididymis, and that TS and ES have different N-linked glycosylation patterns with the latter showing glycans identical to those in caudal sperm. Our findings also reveal that ES is secreted with its lipid anchor, a form in which it can bind to sperm.
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Materials and Methods |
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Preparation of Total RNAs From Epididymis and Testis
Total RNAs from testes, and corpus and caudal epididymides were extracted
using Tri-reagent according to the manufacturer's protocol. RNA samples were
further treated with RNase-free DNase (Boehringer-Mannheim, Indianapolis, Ind;
final concentration 5-10 U/mL) for 2-4 hours at 37°C, followed by
phenol/chloroform extraction and ethanol precipitation.
Rapid Amplification of the 3' Complementary DNA End
Amplification of the 3' Spam1 complementary DNA (cDNA) end
was done using the GeneRacer Kit (Invitrogen, Carlsbad, Calif). Briefly, cDNA
was generated using avian myeloblastosis virus reverse transcriptase and
GeneRacer Oligo(dT) primer from 3 µg of total RNA. One microliter of
diluted (twofold) cDNA was subjected to the polymerase chain reaction (PCR) to
amplify the 3' end using a forward gene-specific primer designed from
the mouse Spam1 cDNA sequence (nt 1896-1920; Gen-Bank accession
number U33958) and the GeneRacer 3' primer provided with the kit. Nested
PCR, using the same gene-specific primer and GeneRacer 3' nested primer,
was performed. After gel-purification, the PCR product was cloned into pCR
4-TOPO TA vector according to the manufacturer's instructions (Invitrogen).
Several clones were isolated, plasmid DNA was prepared, and inserts were
subjected to automated sequencing in our core facility.
Preparation of Protein Extracts From Sperm, Testis, Epididymal
Tissues, and Luminal Fluids
Protein extracts from testis, caudal sperm, and epididymal tissues were
prepared according to the methods described by Cherr et al
(1996) and Deng et al
(2000).
Sperm— To collect sperm, cauda epididymides were thoroughly minced and incubated in 2 mL of sperm suspension buffer (50 mM Tris, 20 mM EDTA, 1 mM p-hydroxy-mercurobenzenzoate, 5 mM N-ethylmaleimide, and 1 mM benzamidine pH 7.2) to disperse the sperm and allow them to swim out. The sperm suspension was centrifuged at 500 x g for 2 minutes to pellet the tissues, and sperm were collected by centrifuging at 1000 x g for 10 minutes. Protein extracts were prepared by lysing sperm with a solubilization buffer (62.5 mM Tris-HCl, 10% glycerol, 1% sodium dodecyl sulfate [SDS] pH 6.8) at 4°C at a final sperm concentration of 1 x 105 sperm/µL. The suspension was vigorously vortexed for 3 minutes and then centrifuged at 10 000 x g for 10 minutes, and the supernatant containing proteins was collected.
Testicular and Epididymal Tissue— Testes were minced and the proteins were extracted with solubilization buffer. Minced epididymal tissues from which sperm were previously collected were washed a total of six times with Whittingham buffer containing 1 mM phenylmethylsulphonyl fluoride (PMSF) (centrifuged at 500 x g for 2 minutes) to remove adhering sperm until the sperm concentration in the final supernatant (5 mL) was less than 100 sperm/µL, and the proteins were extracted with the solubilization buffer. Tissues from this washing procedure were previously shown to harbor 103 sperm, which is 2 orders of magnitude lower then the 105 cells required to provide a band in Western analysis (Deng et al, 2000).
Luminal Fluids— Collection of epididymal luminal fluids was performed according to our previous procedure (Zhang and Martin-DeLeon, 2001) with some modifications. Briefly, the epididymides were isolated and divided into caput, corpus, and cauda. Each segment was minced into 2 mL of Whittingham buffer containing 1 mM PMSF and gently shaken to permit dispersal of the luminal contents. After the tissue pieces were allowed to settle, the upper fraction containing sperm and luminal fluid was centrifuged at 1000 x g for 3 minutes to remove cells. The supernatant obtained was then centrifuged at 3500 x g for 20 minutes at 4°C to remove cellular fragments. The proteins in the supernatant were precipitated with three volumes of acetone and recovered in sample buffer.
Two-Dimensional Polyacrylamide Gel Electrophoresis and Western Blot
Analysis
First-dimensional electrophoresis was performed using the IPGphor
Isoelectric Focusing System (Amersham Pharmacia Biotech) according to the
manufacturer's instructions. Briefly, protein samples were solubilized in a
focusing solution containing 8 M urea, 2% CHAPS (w/v), 0.5% IPG Buffer type pH
3-10L (Amersham Pharmacia Biotech) and 18 mM dithiothreitol (DTT). Solubilized
samples were then loaded on pH 3-10 linear gradient 7 cm IPG dry strips
(Amersham Pharmacia Biotech) and isoelectric focusing was performed. Following
this, the strips were subjected to 15% SDS-polyacrylamide gel electrophoresis
(PAGE), and transferred to nitrocellulose membrane according to standard
protocols. Western blotting was performed with the WesternBreeze
Chemiluminescent Immunodetection Kit (Invitrogen) according to the
manufacturer's protocol. In brief, the membrane was blocked for 30 minutes at
room temperature, then probed with rabbit antipeptide Spam1 antiserum
(specific for Spam1; Deng et al,
2000), diluted 1:1000 in blocking solution. After washing, the
membranes were incubated with antirabbit immunoglobulin G (IgG)
biotin-conjugated (1:150 000 in blocking buffer). The protein was visualized
by the chemiluminescence substrate provided with the kit.
Lectin Blotting
Reduced protein samples (heated at 99°C for 4 minutes in the presence
of 100 mM DTT) were subjected to 15% SDS-PAGE and transferred to
nitrocellulose membranes. The membranes were blocked in 3% w/v bovine serum
albumin (BSA) in TTBS (50 mM Tris-HCl, 0.3 M NaCl, and 0.02% v/v Tween-20 pH
7.5) at room temperature overnight. The blots were then incubated for 2 hours
separately with the following nine biotinylated lectins: concanavalin A (Con
A), peanut agglutinin (PNA), Pisum sativum agglutinin (PSA), Lens
culinaris agglutinin (LCA), Phaseolus vulgaris erythroagglutinin
(PHA-E), Jacalin, Griffonia simplicifolia lectin I (GSL I),
Lycopersicon esculentum lectin (LEL), and Solanum tuberosum
lectin (STL) (Vector Laboratories, Burlingame, Calif) at a concentration of 1
µg/mL in TTBS containing 3% BSA and 10 mM Ca2+ and 10 mM
Mn2+ at room temperature. After thorough washing with TTBS,
membranes were incubated with alkaline phosphatase antibiotin antibodies
(1:1000 in blocking buffer) (Vector Laboratories) for 1 hour. Positive
staining was developed using the 1-Step NBT/BCIP (Pierce, Rockford, Ill).
Preincubation of proteins extracted from caudal sperm and epididymal tissue
with rabbit antipeptide Spam1 antiserum or preimmune serum was used to
determine the identity of an N-linked band.
O-Linked Deglycosylation
O-Linked deglycosylation was performed as described by Anakwe et
al (1991) with some
modifications. In brief, protein extracts were incubated with 80 mU/mL of
O-glycosidase for 20 hours at 37°C in 50 mM sodium phosphate
supplemented with 1 mM PMSF pH 6.0. Control samples were incubated in the same
conditions without O-glycosidase. The deglycosylated proteins were
then used for Western blot analysis.
Preparation and PI-PLC Treatment of Insoluble Particles in Luminal
Fluid
Insoluble particles were prepared from epididymal luminal fluid and treated
with PI-PLC as described by Frenette and Sullivan
(2001). In brief, epididymal
luminal fluid was collected from 20 mice, subjected to 2 centrifugations as
described above, and then ultracentrifuged at 120 000 x g for 2
hours. Pellets of insoluble particles were resuspended in 150 mM NaCl with or
without 5 U/mL of PI-PLC for 15 minutes at 30°C, or treated with 1% Triton
X-100 for 10 minutes at 4°C. The insoluble particles were then pelleted by
ultracentrifugation at 120 000 x g for 2 hours. Both pellets
and supernatants (from equivalent volumes of luminal fluid) were subjected to
SDS-PAGE and immunoblotting analysis.
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Results |
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Analyses of Two-Dimensional PAGE and Immunoblotting Revealed
Different Spam1 Isoforms in Epididymis, Testis, and Caudal Sperm
With two-dimensional PAGE and immunoblotting, one to three isoforms for
epididymal Spam1 were observed in proteins from tissues as well as luminal
fluid in all three regions. For all these, the isoelectric point (pI) ranged
from 7.3 to 9.0. On the other hand, for TS there were four isoforms in a pI
range of 6.6 to 9.0. Caudal sperm revealed two isoforms with the pI ranging
from 7.6 to 9.0 (Figure 2).
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Oligosaccharides of Spam1 in Epididymis, Testis, and Caudal
Sperm
Information about oligosaccharides in the epididymis, testis, and caudal
sperm was obtained by probing Western blotted proteins with lectins having
known carbohydrate specificities. Of the 9 different lectins tested, PHA-E,
LEL, and STL, which all bind to N-linked side chains, recognized a 67
kd molecular mass in the epididymis and caudal sperm, but not in testis
(Figure 3). After preincubating
proteins from the epididymis and caudal sperm with anti-Spam1 antiserum or
preimmune serum, the LEL staining disappeared with anti-Spam1, but not with
the control preimmune serum, indicating that Spam1 is responsible for the
presence of the staining. Further, lectin blotting (using LEL and STL) of
two-dimensional SDS-PAGE revealed a staining at 67 kd with a pI of 7.6 to 8.3
in epididymal tissue and luminal fluid (data not shown) where Spam1 protein
was identified (Figure 2).
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PNA, which preferentially binds to O-linked side chains, recognized the 67 kd molecular mass in both epididymis and testis as well as caudal sperm (Figure 3). On the other hand, Con A, GSL I, Jacalin, LCA, and PSA did not recognize the 67 kd molecular mass in any of the protein extracts (data not shown).
Enzymatic O-Linked Deglycosylation of Spam1 Suggests That the Protein
Contains a Functional O-Linked Site
To date Spam1 has not been known to contain an O-linked sugar,
although in a previous study we used computer analysis of the cDNA sequence to
demonstrate the presence of a potential O-linked glycosylation site
at position Thr379 (Deng et al,
1999). To determine whether the 67 kd protein that was positive
for a PNA glycan in all samples (Figure
3) could be Spam1, O-linked degly-cosylation was
performed on extracts from testis, epididymis, and caudal sperm. After
deglycosylation, proteins from all three regions show a 3 kd reduction in
molecular weight (Figure 4),
confirming that the potential O-linked site is functional. This
suggests that the 67 kd protein that is positive for PNA in testis,
epididymis, and sperm is likely to be Spam1.
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ES Is Released Predominantly as Insoluble Particles in the Luminal
Fluid and Contains its Lipid Anchor, as Revealed by Treatment With PI-PLC or
Triton X-100
To determine whether released ES is soluble in the luminal fluid or
associated with membranous particles, components of the luminal fluid were
separated by ultracentrifugation and Spam1 antibody was used to detect the
protein in the supernatant and the insoluble fractions. Using antipeptide
Spam1 antibody, Spam1 in epididymal luminal fluid was predominantly associated
with insoluble particles, and to a lesser extent with the supernatant
(Figure 5, B and A, respectively). When the insoluble particles were treated with PI-PLC, which is
known to cleave PI-anchored plasma membrane proteins from sperm
(Phelps et al, 1988;
Thaler and Cardullo, 1995; Legare et al, 1999), more than
half (
60%) of Spam1 was released
(Figure 5, E and F).
Conversely, the same protocol applied without PI-PLC did not remove any Spam1
from the insoluble particles (Figure 5, C
and D). Furthermore, 1% Triton X-100 was able to extract all the
Spam1 from the insoluble particles (Figure
5, G and H), confirming that the protein is released in a
membrane-bound form.
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Discussion |
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Although a correlation between a specific number of residues in the poly(A) tract and translational activity has not been reported for epididymal mRNAs, it is known that androgens exert a positive effect on "short" (but not "long") poly(A) tails of CD52, a glycoprotein secreted by the epididymis and found on the surface of epididymal sperm (Pera et al, 1997). Thus it is possible that the maintenance of epididymal Spam1 mRNAs with short tails might be regulated by androgens, which would not be the case for spermatidal mRNAs, because germ cells are without androgen receptors. The tissue difference in poly(A) tail length seen in this study may therefore well reflect regulation of expression (Curtis et al, 1995).
Oligosaccharides of Spam1 Show Tissue Differences in Epididymis and
Testis, and Sperm and Epididymis Have Similar Glycan Structures
In the present study, Western blot analysis of two-dimensional SDS-PAGE was
used to further identify biochemical characteristics of epididymal Spam1. Our
results indicate that different patterns of isoforms of Spam1 exist in the
testis and the epididymis. This correlates with the finding that Spam1 is
synthesized independently in the testis and epididymis
(Deng et al, 2000;
Zhang and Martin-DeLeon,
2001), and suggests that its expression in the latter may not be
redundant. There was more diversity in the testis which has four discrete
isoelectric variants from 6.6 to 9.0 compared to the one-to-three isoforms in
the epididymis spanning 7.3-9.0. The two isoforms with pI ranging from 7.6 to
9.0 observed in caudal sperm are within the range of those found in the testis
and epididymis. This suggests that populations of Spam1 on sperm may be
derived from both testis and epididymis. The pI variants are likely to be a
result of different carbohydrate components in the glycan structure of Spam1
in the two tissues. It is well known that there is tissue-specificity of
glycosyl-transferases and glycosidases
(Kobata, 1992).
PNA, which preferentially binds to O-linked side chains that are present on sperm plasma membranes (Navaneetham et al, 1996) and apical cells (Calvo et al, 2000) recognized a 67 kd molecular mass in the epididymis, testis, and in caudal sperm. Evidence that this 67 kd membrane protein may be Spam1 was obtained by showing that a potential O-linked glycosylation site at Thr379 in mouse Spam1 that we identified from a computer analysis of Spam1 cDNA sequence (Deng et al, 1999) is functional (Figure 3). It is noteworthy that recently, Baker et al (2002) identified a 68 kd PNA-staining sperm membrane protein that localizes to the acrosomal crescent and the principal piece where we have localized Spam1 (Deng et al, 1999).
Our results demonstrate that the lectins PHA-E, LEL, and STL, which bind to N-linked side chains and which were also shown to stain PH-20 from macaque sperm (Li et al, 2002), identified a 67 kd protein molecular mass in epididymis and caudal sperm, but not in testis. Whereas LEL and STL have an affinity for N-acetyl glucosamine, PHA-E has a specificity for complex structures. Thus the findings in this study indicate that testicular and epididymal Spam1 glycoforms, which contain an identical peptide core, are differentially glycosylated: in the epididymal glycoform N-acetyl glucosamine is a major sugar within or at the terminal end of one or more linked glycans. Differential glycosylation has been reported for at least two other epididymal proteins: CD52 (SAGA-1), in which the difference is between lymphocytes and epididymis (Diekman et al, 1997); and clusterin (Ahuja et al, 1996), in which the difference is between testicular and epididymal glycoforms.
The 67 kd band for the different N-linked lectins was verified to be Spam1 by two methods. First, lectin blot analysis of two-dimensional SDS-PAGE of protein in epididymal tissue and luminal fluid identified isoforms at 67 kd molecular mass with pIs ranging from 7.6 to 8.3. This correlates with the results from Western blot analysis of two-dimensional SDS-PAGE in epididymal tissue and luminal fluid. Second, the LEL bands at 67 kd in epididymis and sperm were confirmed to be Spam1-specific with the use of anti-Spam1 serum. After preincubating protein extracts from epididymis and caudal sperm with preimmune serum or anti-Spam1 serum, the lectin bands disappeared in the latter indicating Spam1 specificity. It must be pointed out that Spam1 N-linked sites (Asn46, 165, 293, and 401) are evenly spaced (Deng et al, 1999) and that the antipeptide Spam1 that was used is generated from a 15-mer at positions 381-395, which would be between Asn293 and Asn401. It is possible that steric hindrance from the antibody binding could be responsible for occluding the N-linked chains at Asn293 and Asn401.
N-glycans have been shown to mediate apical sorting of GPI-anchored proteins (Benting et al, 1999), a finding consistent with Spam1 location on sperm and on the apical surface of the epididymis from where it was shown to be released in vivo and in vitro (Deng et al, 2000; Zhang and Martin-DeLeon, 2001). Our finding that three lectins specific for N-linked glycans stain a protein identified as Spam1 from the epididymis and caudal sperm, but not testis, suggests the following: 1) TS on sperm may undergo deglycosylation during epididymal transit to generate N-linked sites not found on the protein in the testis (Deng et al, 1999), and 2) ES may be directly involved in the N-linked sites on sperm by either functioning as the glycosidase (in the luminal fluid) involved in the deglycosylation process or by binding to sperm. It is note-worthy that Baker et al (2002) identified a 68 kd mouse sperm membrane protein that stained with LEA (LEL) and showed it to be located on intact sperm in a pattern distinctly different from that of PNA. It was seen on the anterior crescent and the posterior head where we have localized Spam1 (Deng et al, 1999). The similarities in molecular weights, staining, and localization of Spam1 and that of the protein described by Baker et al (2002), strongly argue that the latter is Spam1. The staining distribution reported by Baker et al (2002) was seen on some sperm but not on others, suggesting that it may be associated with epididymal maturational states and implicating the involvement of ES.
Epididymal Spam1 Is Released in Epididymosomes (Exosomes) With its
Lipid Anchor
After ultracentrifugation of the luminal fluid, Spam1 was found to be
present in both the supernatant and the pellet although preferentially in the
latter. This indicates that there are two populations of this membrane
protein, as is the case for epididymal protein DE in rats
(Cohen et al, 2000) and for
prostasome-like particles in rat epididymal fluid
(Fornes et al, 1995). However,
it is possible that there may be two sources of Spam1 in the luminal fluid:
molecules that come from sperm and those that are released from the
epididymis. Because ES was observed in vesicles that were seen in the process
of being released in the lumen (Deng et
al, 2000), it is likely to reside in the insoluble pellet. This
insoluble membranous material, which is similar to prostasomes, has been
referred to as epididymosomes and serves as a means of transferring proteins
to the sperm surface (Rooney et al,
1996; Yeung et al,
1997).
We have demonstrated that 60% of the Spam1 from the insoluble
particles obtained after the initial ultracentrifugation of the luminal fluid
was released into the medium following 15 minutes of exposure to PI-PLC
(Figure 5, E and F). This
confirms that ES is secreted into the lumen via lipid vesicles or
epididymosomes, probably as a complex of GPI-linked proteins and signaling
molecules. Our inability to release the lipid anchor in 100% of Spam1
molecules might result from the inaccessibility of some of the molecules to
the enzyme, or it could indicate the presence of a population of Spam1 that is
either not PI-anchored or has a GPI anchor that is insensitive to the
particular PI-PLC (Cooper,
1998).
GPI-anchored proteins are often known to be resistant to detergent extraction (Triton X-100) and are referred to as detergent-insoluble glycolipids, which float on top of sucrose gradients (Primakoff et al, 1988; Cherr et al, 2001). However, 100% of the Spam1 from the insoluble particles was extracted by exposure to 1% Triton X-100 in our study (Figure 5, G and H), supporting the evidence that ES is released with its lipid anchor, a form in which it can bind to sperm.
Our studies, which provide further evidence that Spam1 is produced independently in the testis and epididymis, reveal that TS and ES are different glycoforms and that the latter shares identical glycans with caudal sperm. There is therefore evidence to suggest that ES may not be a redundant protein. The findings of this study implicate an interaction between sperm and ES, similar to other GPI-anchored proteins such as CD52 (Rooney et al, 1996) and P26h, P34H, and P25b (Frenette and Sullivan, 2001).
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Footnotes |
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 G. Frenette, J. Girouard, and R. Sullivan Comparison Between Epididymosomes Collected in the Intraluminal Compartment of the Bovine Caput and Cauda Epididymidis Biol Reprod, December 1, 2006; 75(6): 885 - 890. [Abstract] [Full Text] [PDF]
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H. Chen, G. Griffiths, D. S. Galileo, and P. A. Martin-DeLeon Epididymal SPAM1 Is a Marker for Sperm Maturation in the Mouse Biol Reprod, May 1, 2006; 74(5): 923 - 930. [Abstract] [Full Text] [PDF]
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H. Zhang, C. R. Morales, H. Badran, M. El-Alfy, and P. A. Martin-DeLeon Spam1 (PH-20) Expression in the Extratesticular Duct and Accessory Organs of the Mouse: A Possible Role in Sperm Fluid Reabsorption Biol Reprod, October 1, 2004; 71(4): 1101 - 1107. [Abstract] [Full Text] [PDF]
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C. M Hardy, G. Clydesdale, K. J Mobbs, J. Pekin, M. L Lloyd, C. Sweet, G. R Shellam, and M. A Lawson Assessment of contraceptive vaccines based on recombinant mouse sperm protein PH20 Reproduction, March 1, 2004; 127(3): 325 - 334. [Abstract] [Full Text] [PDF]
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A. I. Yudin, T. L. Tollner, M.-W. Li, C. A. Treece, J. W. Overstreet, and G. N. Cherr ESP13.2, a Member of the {beta}-Defensin Family, Is a Macaque Sperm Surface-Coating Protein Involved in the Capacitation Process Biol Reprod, October 1, 2003; 69(4): 1118 - 1128. [Abstract] [Full Text] [PDF]
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H. Zhang and P. A. Martin-DeLeon Mouse Spam1 (PH-20) Is a Multifunctional Protein: Evidence for Its Expression in the Female Reproductive Tract Biol Reprod, August 1, 2003; 69(2): 446 - 454. [Abstract] [Full Text] [PDF]
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