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From the * Department of Gamete Immuno Biology,
National Institute for Research in Reproductive Health, Mumbai, India; and the
Laboratory of Cellular and Developmental
Biology, NIDDK, National Institute of Health, Bethesda, Maryland.
| Correspondence to: Dr Vrinda V. Khole, Department of Gamete Immuno Biology, National Institute for Research in Reproductive Health, J M Street, Parel, Mumbai-400012, India (e-mail: vrindakhole{at}hotmail.com). |
| Received for publication February 24, 2005; accepted for publication June 17, 2005. |
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Abstract |
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Key words: Vasectomy, testicular auto antigen, acrosomal matrix, capacitation
Because of the low titers of the sperm autoantibodies and the polyclonal nature of the serum, it has been extremely difficult to determine the nature and the number of sperm autoantigens involved in vasectomy-induced autoimmunity. Therefore, we decided to raise monoclonal antibodies to identify and characterize functionally relevant sperm antigens using a vasectomized mouse. Identification and characterization of a 67-kd sperm autoantigen using a similar approach has been reported (Nakamura et al, 1994). In the present study, using the vasectomized mouse, we have generated a number of monoclonal antibodies to sperm autoantigens. Using one of the antibodies, D5E5, we have identified a testicular protein of approximately 70 kd (TSA70) that is post-meiotically expressed, localized on acrosome and principal piece of the tail, and is conserved across the species.
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Materials and Methods |
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Vasectomy
Twelve adult Balb/C male mice of inbred strain were procured from the
animal house of the institute. The animals were divided into 2 groups: group I
and group II, comprising 6 animals each. The animals from group I (Vx)
underwent bilateral vasectomy performed according to Handley and coworkers
(Handley et al, 1988). Briefly, animals were anesthetized by ether inhalation. Under aseptic
conditions, a midventral vertical transabdominal cut was made; the vas
deferens on one side was dissected out and double-ligated approximately 0.5 cm
apart by a silk thread. The vas was then cut between the ligatures. The
procedure was repeated on the other side. The wound was closed with 4-0 gut
thread for musculature and, similarly, with 4-0 silk thread for the skin.
Animals from group II (So) underwent sham operation. Sham operation mimicked
the surgical procedure except that the vas was not ligated or cut.
Collection of Postvasectomy Sera
Serum was collected from all the animals before vasectomy. The sera from
both vasectomized and sham-operated mice were obtained every 10 days starting
from day 0 up to day 60 (ie, at 10 days, 20 days, 30 days, 40 days, 50 days,
and 60 days). The blood was drawn from the retro-orbital plexus; blood samples
were allowed to clot at 4°C overnight. The sera were separated,
centrifuged at 400 x g for 15 minutes and stored at -20°C
until further use.
Titration of Antisperm Antibodies in the Postvasectomy Sera
The antisperm antibody titers in the sera of Vx as well as So animals were
detected by enzyme-linked immunosorbent assay (ELISA), as described by Khole
and coworkers (Khole et al,
2000). Mouse caudal sperm obtained from Swiss mice were coated at
a concentration of 0.5 x 106 sperm/100 µL/well onto a
96-well microtiter plate (Nunc, Denmark) by overnight incubation at 37°C.
The plates were then fixed with glutaraldehyde (0.025%, vol/vol) and washed in
phosphate-buffered saline (PBS; 0.01 M, pH 7.4). The nonspecific binding sites
were blocked using 2% (wt/vol) nonfat dry milk (NFDM) in PBS for 1 hour at
37°C (300 µL/well). Antisera from Vx and So animals diluted 1:50 in 1%
(wt/vol) NFDM + PBS were incubated at 37°C for 1 hour (100 µL/well).
The plates were then washed 4 times with PBS containing 0.05% (vol/vol)
Tween-20 (PBST20). The secondary antibody horseradish peroxidase
(HRP)-conjugated goat anti-mouse (Jackson ImmunoResearch Laboratories, West
Grove, Penn) diluted 1:5000 in 1% (wt/vol) NFDM was added and incubated for 1
hour at 37°C (100 µL/well). Finally, plates were washed with PBS-T20
before incubation with 200 µL of substrate solution (8 mg
ortho-phenylenediamine dihydrochloride [OPD] + 0.03% [vol/vol]
H2O2 in 0.1 M citric acid and 0.2 M disodium hydrogen
orthophosphate). The reaction was terminated by addition of 100 µL of 4 N
H2SO4, and the color intensity was measured at 492 nm on
Universal Micro plate Reader (Bio-Tek Instruments Inc, Winooski, Vt). Each
sample was assayed in duplicate.
Generation of Monoclonal Antibodies
The monoclonal antibodies (mAb) were produced according to the method
described by Köhler and Milstein
(1975). Based on the antisperm
antibody titer detected by ELISA, the vasectomized animal showing the greatest
response was selected for carrying out fusion. The splenocytes were fused with
mouse myeloma cell line SP2/0-Ag14 (procured from the National Center for Cell
Science, Pune, India) in a ratio of 2:1 using 50% (vol/vol) polyethylene
glycol (PEG-1500; Sigma Chemical Co, St Louis, Mo) as a fusogen. The clonal
specificity for sperm antigens was detected by screening the culture
supernatant against the mouse caudal sperm coated onto 96-well microtiter
plates as described earlier. Positive clones following secondary and tertiary
screening were subcloned further by limiting dilution to ensure monoclonality.
Based on its high titer, a clone, D5E5, was chosen for detailed
characterization of the sperm antigen.
Immunochemical Characterization of TSA70 Antigen
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis—
Proteins from mouse sperm and testis and from 3 different regions of
epididymis, namely caput, corpus, and cauda, were extracted in 1% (wt/vol)
sodium dodecyl sulfate (SDS) and estimated using the Folin-Lowry method
(Lowry et al, 1951). In brief,
decapsulated testis and caput, corpus, and cauda regions of epididymis were
teased separately in 0.01 M PBS and then allowed to stand for 15 minutes at
37°C. The supernatant containing the sperm and the settled tissue was
collected and centrifuged at 400 x g for 10 minutes. In each
case, the pellet was washed twice with PBS and finally suspended in 1% SDS
(wt/vol in 0.01 M PBS, pH 7.4) overnight at 4°C. The next day the pellet
was sonicated and spun down to collect the supernatant. Fifty micrograms of
each protein was loaded per well and electrophoretically separated by 12%
SDS-polyacrylamide gel electrophoresis (PAGE) in the presence of
ß-mercaptoethanol. The extracts were diluted 50% (vol/vol) with 2x
Laemmli sample buffer (Laemmli,
1970) and boiled for 5 minutes at 95°C before each run. The
gels were run on a minigel electrophoresis apparatus (Miniprotean II, Bio-Rad,
Hercules, Calif). To check the tissue specificity of the cognate antigen,
detergent-extracted proteins from different somatic tissues, such as liver,
heart, brain, kidney, and spleen, as well as accessory reproductive tissues
such as the vas deferens and seminal vesicle, were also separated on 12%
SDS-PAGE.
Western Blot Analysis
SDS-PAGE-separated proteins were transferred onto nitrocellulose membrane
(Amersham, Buckinghamshire, United Kindgom) according to the method of Towbin
et al (1979) for 1 hour at 100
V. The blots were then stained with Ponceau S (0.5 mg Ponceau in 0.01%
[vol/vol] acetic acid) and destained with distilled water to determine the
quality of SDS-PAGE separation of the proteins. Each blot was then blocked in
5% (wt/vol) NFDM in PBS for 1 hour at room temperature to reduce nonspecific
binding in subsequent incubations. The blot was then incubated overnight at
4°C with undiluted culture supernatant of the mAb D5E5. The culture
supernatant from myeloma cells (SP2/0-Ag14) served as control. The blots were
then washed 4 times with PBS-T20 and were then incubated at 37°C for 2
hours with 1:20 000 dilution of horseradish peroxidase-conjugated goat
anti-mouse immunoglobulin G (IgG; Jackson ImmunoResearch Laboratories) diluted
in 0.5% (wt/vol) NFDM in PBS. Blots were again washed 4 times with PBS-T20 as
described earlier and developed using 8 mg 3,3-diamino benzidine (DAB; Sigma)
and 0.03% (vol/vol) H2O2 in 10 mL PBS for 15 minutes.
The reaction was stopped with distilled water; next blots were rinsed, blotted
dry, and scanned.
Immunohistochemistry
Testes and whole epididymides obtained from the Swiss mice were fixed in
Bouin solution and embedded in paraffin. The protocol standardized in our
laboratory (Khole et al, 2000)
was used for immunohistochemical localization using the mAb D5E5. Briefly,
slides containing 5-µm sections were deparaffinized. The slides were
subsequently hydrated through alcohol grades; deparaffinized sections were
treated with 0.3% (vol/vol) H2O2 in methanol for 30
minutes to block endogenous peroxidase activity. To block the nonspecific
binding, 5% (wt/vol) NFDM in PBS was used for 1 hour at room temperature. Then
slides were incubated with mAb (undiluted culture supernatant) overnight at
4°C in a humidified chamber. Slides were washed 4 times with PBS and then
incubated for 2 hours at room temperature (RT) with HRP-conjugated goat
anti-mouse diluted 1:1000 in 1% (wt/vol) NFDM + PBS (Jackson Immuno Research
Laboratory). The slides were then washed as described above. Color reaction
was carried out using DAB + 0.03% (vol/vol) H2O2 (10 mg
DAB + 10 µL H2O2 in 10 mL PBS). The reaction was
terminated with distilled water; slides were then counterstained with
hematoxylin. Following dehydration, the slides were mounted in DPX mountant.
Controls were incubated with culture supernatant from myeloma cells.
Immunohistochemical localization using the mAb D5E5 was also performed on rat
testicular sections to check the expression pattern of protein during rat
spermatogenesis.
Indirect Immunofluorescence
The spermatozoa from the testis and 3 regions of the epididymis (caput,
corpus, and cauda) were obtained from Swiss male mice. The sperm were fixed
with 4% (wt/vol) paraformaldehyde in PBS for 5 minutes at 4°C and then
washed twice in PBS. The sperm then suspended in PBS at a concentration of 1
x 105 were smeared on a glass slide. Slides were incubated at
37°C for 1 hour in blocking buffer consisting of 5% (wt/vol) bovine serum
albumin (BSA; Sigma) in PBS. Excess of blocking buffer was removed before
incubation with the mAb. The slides were incubated overnight at 4°C with
200 µL of culture supernatant of the mAb D5E5 in a humidified chamber. The
culture supernatant from myeloma cells served as control. Slides were washed 4
times with plain PBS and then incubated with 200 µL of fluorescein
isothiocyanate (FITC)-conjugated rabbit anti-mouse (Fab'2
specific) (Dako, Denmark) diluted 1:40 in 2% (wt/vol) BSA in PBS for 2 hours
at RT in the dark. Slides were washed with PBS as described previously and
then viewed under an epifluorescence microscope (Axioskop 2plus; Zeiss,
Germany).
Conservation of the Protein Across the Species
To study the conservation of the cognate protein across the species,
indirect immunofluorescence (IIF) was performed on rat, bull, marmoset, and
human sperm. In brief, rat and marmoset (Callithrix jaccus) caudal
sperm were washed in PBS and smeared on glass slides at a concentration of 1
x 105 sperm/10 µL. For bull and human sperm, seminal
plasma was separated and the sperm washed with PBS 2-3 times. The sperm
samples were fixed with 4% (wt/vol) paraformaldehyde in PBS for 5 minutes at
4°C, as described above. Following fixation, the slides were further
processed for immunofluorescence labeling with D5E5, as described above.
The conservation of the protein was further confirmed by SDS-PAGE Western blot analysis. Briefly, the sperm proteins from rat, bull, marmoset, and human were run on 10% SDS-PAGE, and the transferred proteins were then probed with the D5E5, as described above.
IIF Localization on Capacitated and Acrosome-Reacted Sperm
The capacitation and acrosome reaction was carried out according to the
protocol standardized in our laboratory. The caudal sperm were obtained from 4
Swiss mice; the sperm were retrieved in Biggers, Whitten, and Whittingham
(BWW) (1971) medium and
centrifuged at 500 x g for 10 minutes. The pellet was then
resuspended in fresh BWW medium, and the sample was divided into 3 equal
aliquots for normal, capacitated, and acrosome-reacted sperm. To obtain
capacitated spermatozoa, sperm were incubated in BWW containing 3 mg/mL of
BSA-fraction V (Sigma) for 3-4 hours at 37°C in 5% CO2 in
screw-cap tubes. After capacitation, the third aliquot was used for acrosome
reaction (AR). The AR was induced by Ca++ ionophore A23187. The
sperm were incubated with 5 µM Ca++ ionophore A23187 (Sigma) at
37°C in 5% CO2 for 45 minutes. The sperm were washed and fixed
with 4% (wt/vol) paraformaldehyde in PBS for 5 minutes at 4°C after the
completion of capacitation and/or acrosome reaction. The sperm were then
smeared on glass slides for the IIF localization using mAb D5E5, as described
above. The success of AR was assessed using FITC-labeled Pisum
sativum agglutinin (Sigma), as described by Cohen and Wassarman
(2001).
Isolation of Sperm Head and Tail Proteins
Rat caudal sperm were washed in 0.01 M PBS. The head and tail were isolated
using the protocol described by Oko and colleagues (1988). In brief, the sperm
were sonicated at 4°C at 100% output for four 15-second bursts at
30-second intervals. This treatment assured 95% decapitation of spermatozoa,
which was verified by the phase contrast microscopy. After sonication, 10 mL
of PBS was added to the suspension and given a wash at 500 x g
for 10 minutes. The head and tail fractions were then isolated by layering
onto sucrose gradients of 65%, 70%, and 75% (8 mL of each) prepared in 0.02 M
PBS and spun at 100 000 g for 70 minutes in a Sorvall Pro 80 swinging
bucket rotor. The sperm tails were collected at the 65%-70% interface, whereas
the isolated heads were collected from the bottom.
The isolated heads and tails were given a wash with 0.01 M PBS, and to the pellet we added 0.1% Triton X-100 in a PBS-containing cocktail of protease inhibitors. The suspensions were then vortexed for 5 minutes at RT and then centrifuged at 500 x g for 10 minutes. The supernatant contains the soluble fraction, while the pellet contains the particulate fraction. Following extraction with the SDS sample buffer, the protein was resolved on 10% SDS-PAGE. The resolved proteins were then immuno-blotted with mAb D5E5 as described above.
Assessment of Sperm Motility and Viability
The effect of the mAb on mouse sperm motility and viability was studied
using the protocol described by Martinez and coworkers
(1995). To study the effect of
the mAb on sperm motility, the motile sperm were incubated for 1 hour at
37°C with the culture supernatant of D5E5 and the culture supernatant from
SP2/0 as a control. At 15-minute intervals, 10-µL aliquots of sperm
suspension were placed on prewarmed slides. The percentage of motile sperm was
determined by manually counting 150 sperm per sample using a phase contrast
microscope (Axioskop 2plus).
For viability assessment, 10 µL of the above sperm suspension incubated with the culture supernatants of the mAb and SP2/0 were stained with 0.5% (wt/vol) prewarmed eosin (Sigma) in saline solution, and the incorporation of the dye was determined by light microscopy. The percentage of viability was calculated as the number of sperm that did not incorporate the dye over the total number of sperm counted.
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Molecular Weight of the Antigens in Tissues and Sperm From Testis and Different Regions of Epididymis
The Western blot analysis using sperm and tissue proteins from the testis
and epididymis (Figure 1A) was
done to see if there was any change in the molecular weight of the protein
during epididymal sojourn. The antigen identified by the mAb D5E5 is of
testicular origin and is present on both the testicular as well as the
epididymal sperm, but no reactivity was observed in tissues of any epididymal
regions. The antibody recognized the number of proteins in the range of
approximately 65-80 kd in the testicular tissue extract. The mAb identified a
single band of approximately 70 kd in testicular sperm as well as in sperm
from 3 different regions of the epididymis. Myeloma supernatant did not show
any reactivity (Figure 1B). The
antibody did not react with any of the somatic tissues tested as well as other
accessory reproductive tissues (data not shown). Since the mAb D5E5 reacted
with an approximately 70-kd protein expressed in testis, the cognate antigen
is henceforth referred to as testis-specific autoantigen (TSA70).
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TSA70 Is Expressed in Stage-Specific Manner During Spermatogenesis
Immunochemical localization of D5E5 reactive antigen in testis and
epididymis is illustrated in Figure
1C. A stage-specific expression of the antigen was observed. In
mouse testis, the expression of the antigen starts from step 8 of spermatids
and is subsequently expressed by the fully formed spermatozoa during
spermiogenesis. However, no acrosomal staining was observed during
spermiogenesis. In the epididymis, the antigen was immunolocalized only on the
spermatozoa from all of the 3 regions, and no reactivity was observed in
epididymal epithelium of caput, corpus, or cauda
(Figure 1C, lane 1).
In rat testis, the antigen is localized in the seminiferous tubules postmeiotically, beginning with spermatid step 8, and is present thereafter on the fully formed spermatozoa in the lumen of the seminiferous tubule. A distinct pattern of antigen localization was observed. The localization of the antigenic determinant on the sperm head was observed from the step 8 of spermatids and continued on the fully formed spermatozoa, while the localization of the antigen on the sperm tail started from step 16 of spermatids onward until spermiation (Figure 1C, lane 3). No staining was observed with the myeloma cell culture supernatant (Figure 1C, lane 2).
TSA70 Shows Identical Domain Specificity in Spermatozoa From Testis and All the Regions of Epididymis
When domain specificity of the cognate antigen on mouse caudal sperm was
studied using IIF with D5E5, it identified the antigen on the principal piece
and end piece of the sperm tail and also on the tip of the acrosome. The
fluorescence pattern was identical for sperm from testis and those from all 3
regions of epididymis (Figure
2).
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Discussion |
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In the present study, a monoclonal antisperm antibody, D5E5, generated using vasectomized mouse, was used for immunochemical characterization of sperm-specific autoantigen. The 70-kd protein identified by the mAb D5E5 seems to be acquired by the sperm in the testis. D5E5 strongly reacted with the epididymal sperm from caput, corpus, and cauda regions but not with epididymal tissue. The absence of reactivity with somatic as well as accessory reproductive tissues included in the study substantiates the testis-restricted expression of the protein. The multiple protein bands identified in the Western blot analysis of testicular tissue may originate from germ cells at different stages of development. This may be indicative of the processing of the protein as germ cells differentiate to form a spermatozoon. A mature/processed form of the protein may then be expressed by the testicular sperm, as reflected by a single band identified by the mAb. These observations indicate that the protein is synthesized, processed, and acquired by sperm in the testis. Similar observations have been made for a major mouse fibrous sheath protein (Carrera et al, 1994; Fulcher et al, 1995) as well as sp56 (Kim et al, 2001).
From immunohistochemistry data, it appears that the antigen is abundantly expressed postmeiotically by the elongating spermatids in a stage-specific pattern. The expression of the antigenic determinant on the sperm head is coincident with the acrosome phase of spermiogenesis, when the acrosome biogenesis has already occurred and the acrosome no longer grows but conforms to the changing shape of the spermatid head to attain the species-specific form. The sperm tail antigen is found to be expressed in the final phases of the spermiogenesis, during which the major activity is the assembly of the accessory components to the sperm axoneme (Oko, 1998).
The localization of TSA70 on head and tail regions of both testicular as well as epididymal sperm indicates that there are no alterations during epididymal transit. The localization of the determinants on both the tip of the acrosome and the sperm tail indicates sharing of epitope between these 2 regions. A similar pattern of localization has been reported for testis-specific protein 1 (tpx-1), a member of the cysteine-rich secretory protein family (O'Bryan et al, 2001). Concurrent with sperm head formation, tpx-1 is found to be incorporated into the developing spermatid tail, indicating its functional significance in the process of head development and tail function. This may also be the case with the protein under investigation.
Presence of TSA70 on spermatozoa from different species studied indicates the conserved nature of the antigen. This again is indicative of the possibility that TSA70 may have an important role to play in sperm function. Interestingly, we observed variation in the domain specificity of the mAb with spermatozoa from different species. This differential localization could have occurred during the process of evolution.
The presence of TSA70 on the acrosome led us to study its fate following capacitation and AR. Relocation of surface molecules during capacitation and the AR has already been reported for certain other sperm proteins, such as 2B1 (Jones et al, 1990), PH20 (Myles and Primakoff, 1984), and MC31 (Saxena and Toshimori, 2004). It might be correct to speculate that the increased reactivity seen following capacitation could be due to destabilization of the acrosomal membrane resulting in shuffling/migration of certain molecules and/or unmasking of certain hidden epitopes as a result of a change in membrane integrity/architecture. The loss of this antigen after AR could be due to the exocytosis phenomenon known to occur during AR.
Next we investigated whether TSA70 is a soluble protein or an acrosomal matrix protein. TSA70 solubility was checked following Triton X-100 extraction under conditions that block proteolysis (Huang et al, 1985). It has been suggested that the position and solubility of a specific acrosomal protein may govern its function during the course of AR and thereafter (Kim et al, 2001). A component of acrosomal matrix is predicted to be associated with the sperm head for a longer period of time than would a soluble protein (Kim et al, 2001). Retention of TSA70 at the tip of the acrosome following AR in mouse sperm and on the head of rat sperm following Triton X-100 extraction strengthens the possibility of TSA70 being an acrosomal matrix protein. The shift in localization of TSA70 over the acrosome during capacitation raises 2 possibilities: 1) Does TSA70 exist in 2 forms—as both a soluble and matrix protein?; and 2) Does the "transitional state" of remodeling/remodifying the plasma membrane during capacitation contribute to this phenomenon? The persistence of the antigen at the tip of the acrosome even after the AR indicates that the antigen could have a physiological role post-AR, one which needs to be elucidated further.
The localization of TSA70 on the tail is very important from the viewpoint of its function, and, therefore, studies on the effect of mAb on motility were undertaken. In vitro incubation with D5E5 led to a sharp, time-dependent decline in the forward progressive motility. Forward progressive motility is an essential requirement for successful unassisted fertilization, and since the antibody against the cognate antigen affects the same, it could be hypothesized that the autoantigen has a functional role in sperm motility. In support of the significance of sperm tail autoantibodies in reproductive failure, Witkin and Chaudhary (1989) found a strong correlation between the presence of anti-tail IgG antibodies and a history of recurrent spontaneous abortion in women. Although the mAb had an effect on sperm motility, no apparent effect was seen on viability.
Interestingly, TSA70 was localized to the tip of the acrosome as well as on the tail of sperm. This indicates that either it could be the same protein localized to different domains or they could be different proteins with a shared epitope with which the mAb identifies. Considering the first possibility, could TSA70 be attributed to a class of "moonlighting proteins"? The concept of moonlighting proteins first described by Jeffery (1999) refers to the observations that the same protein can perform 2 different functions in 2 different locations within the cell. This concept has emerged from the fact that the prokaryotes have multifunctional proteins, so as to save a great deal of energy in growth and reproduction. It is likely that sperm also harbor such moonlighting proteins to conserve the energy.
The protein TSA70 identified in the present investigation is localized on important domains and is conserved across the species. Further cloning and overexpression of the protein would give us a probe to understand the molecular mechanism in mammalian fertilization as well the physiological role of the cognate antigen. Considering that the relevance of capacitation and AR in the interactions between spermatozoa and egg-identifying sperm proteins involved in these processes is of great importance to understanding the mechanism of fertilization. Such an understanding may also contribute a great deal to the development of new diagnostic assays, which may eventually help in the treatment of male infertility and help in identifying new targets for contraception.
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Acknowledgments |
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