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From the Center for Research in Contraceptive and Reproductive Health, University of Virginia, School of Medicine, Charlottesville, Virginia.
| Correspondence to: John C. Herr, Department of Cell Biology, University of Virginia Health System School of Medicine, P.O. Box 800732, University of Virginia, Charlottesville, VA 22908 (e-mail: jch7k{at}virginia.edu). |
| Received for publication May 10, 2006; accepted for publication September 18, 2007. |
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Abstract |
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Key words: Antisperm antibodies, sperm alloantigens, immune infertility, spermatogenesis, acrosome, fertilization
Recently, in conjunction with a comprehensive human sperm proteome (Naaby-Hansen et al, 2002), two-dimensional (2D) Western blots were employed to assess the concanavalin A (Con-A) binding capabilities of human sperm proteins. This led to the discovery of a novel 349–amino acid Con-A binding protein, Equatorial Segment Protein (ESP; Wolkowicz et al, 2003). ESP localizes to the equatorial segment of ejaculated human sperm and is encoded by a 2-exon gene (SP-ESP) located on chromosome 15 at q22. Northern blot analysis demonstrated that ESP is abundantly expressed in the testis and is also detected as an expressed sequence tag transcript (EST) in placenta. However, immunocytochemical staining of placental tissue failed to detect the protein. Immunofluorescent observations on human sperm using polyclonal antibodies to recombinant human ESP (rec-h-ESP) revealed that ESP delineates an equatorial segment domain during early acrosome biogenesis. ESP first appeared in the nascent acrosomal vesicle in early round spermatids and subsequently segregated to the periphery of the expanding acrosomal vesicle, thereby defining a peripheral equatorial segment compartment within flattened acrosomal vesicles, and it localized as well in the acrosomes of early and late cap phase, elongating, and mature spermatids. Thus, ESP serves as a marker for the specification of the equatorial segment as early as the Golgi phase of acrosome biogenesis and allows the equatorial segment to be traced through all subsequent phases of acrosomal biogenesis.
The equatorial segment of the acrosome is of considerable functional importance to fertilization because it 1) remains intact following the acrosome reaction, 2) underlies the domain of the plasma membrane involved in fusion with the egg membrane, and 3) is the site where breakdown of the sperm nuclear envelope is initiated after fertilization (Yanagimachi and Noda, 1970). The present immunologic studies focused on two main questions: 1) Is ESP immunogenic in humans? and 2) Is ESP involved in fertilization events mediated by the equatorial segment? Western blots (1D and 2D) of human sperm (Naaby-Hansen et al, 1997) were examined with ASA-positive sera from infertile patients (Shetty et al, 1999), and the immunoreactive proteins were compared to blots of sera from fertile individuals, revealing that ESP is alloantigenic. Sera from patients with antisperm antibodies also recognized recombinant ESP. ESP persisted on capacitated, acrosome-reacted human sperm as well as on sperm bound to hamster oocytes, and in accord with this retention of ESP, antisera raised to recombinant ESP inhibited the binding and fusion of human sperm in the hamster egg penetration assay. The results indicate that ESP is involved in key events of fertilization and may be useful in the diagnosis of antibody-mediated infertility and in understanding its underlying mechanisms.
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Materials and Methods |
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Electrophoretic transfer of separated proteins to nitrocellulose membranes was performed in transblot buffer (25 mM Tris-HCl, pH 8.3, 192 mM glycine, and 20% methanol) for 1 A/h. The membranes were either used intact for 2D gels or, in the case of standard 1D protein gels, cut into strips, blocked with 5% fat-free dry milk in phosphate-buffered saline–Tween 20 (1x PBS [pH 7.4], 0.05% Tween 20), and then incubated in rat antisera to ESP for 1 hour at 22°C. Immunodetection was performed with horseradish peroxidase–conjugated goat anti-rat IgG (Jackson ImmunoResearch, West Grove, Pennsylvania) at a 1:5000 dilution in blocking buffer and visualized with diaminobenzidine (Sigma, St Louis, Missouri) in H2O2 or TMB (Kierkegard and Perry Laboratories, Gaithersburg, MD). Rat monospecifc polyclonal antisera to ESP was prepared as previously described (Wolkowicz et al, 2003) by cloning the human ESP cDNA, expressing the recombinant protein in E coli, purifying the protein, injecting the material into virgin female Lewis rats, and collecting the immune sera. Western blots of 2D separated human proteins also were screened with a 1:2000 dilution of sera from infertile patients containing ASAs (Shetty et al, 1999) as determined by the immunobead binding test (IBT; Bronson and Tung, 1992) as well as with fertile control sera (1:2000 dilution). Bound ASAs were detected by incubation of thrice-washed blots for 1 hour at 20°C with secondary enzyme-conjugated goat anti-human immunoglobulin G (IgG) + IgM antibodies (Jackson ImmunoResearch) diluted 1:5000 in PBS containing 0.05% Tween 20. Horseradish peroxidase conjugates were visualized by enhanced chemiluminescence using the manufacturer's protocol (Amersham, Buckinghamshire, United Kingdom). Sequencing by LC-MS analysis was performed by the W. M. Keck Foundation Center for Biomedical Mass Spectrometry at the University of Virginia on an LC-Q apparatus (Finnigan, Austin, Texas) according to the manufacturer's instructions and as described previously (Wolkowicz et al, 2002).
Serum From Infertile Patients
Serum samples were obtained from infertile men and women with unexplained
infertility. The infertile subjects did not have any previously diagnosed
hormonal, infective, or physical causes for their infertility. The male
subjects had not undergone vasectomy. The ASAs in the serum samples from
infertile subjects were detected by direct or indirect immuno-bead binding as
previously described (Bronson and Tung,
1992). All the serum and sperm samples were tested for the
presence of IgG-, IgM-, and IgA-specific ASAs, and included samples from 15
infertile male subjects, 5 infertile female subjects, 5 fertile male subjects,
and 5 fertile female subjects. All of the control fertile subjects were
individuals with proven fertility and were ASA negative based on the
immunobead test. Serum samples from infertile subjects with more than 60% of
the spermatozoa binding beads, indicative of IgG- and/or IgM-specific ASAs,
were used for 2D Western blotting. All of the selected samples contained
antibodies directed against the sperm head or the entire spermatozoa.
Hamster Egg Penetration Xeno-Assay
Sperm-egg interaction was assessed in a binding and fusion assay using
zona-free hamster eggs. Biggers, Whitten and Whittingham (BWW) media (2 mL;
Irvine Scientific, Santa Ana, California) containing 5 mg/mL human serum
albumin (HSA) was underlayed with human sperm ejaculate, and the sperm were
allowed to swim up for 1.5–2 hours. The swim-up sperm were collected,
and 8 mL BWW and 5 mg/mL HSA were added before centrifugation at 600 x
g for 8 minutes at 22°C. This wash was repeated before the pellet
was suspended in 50 µL BWW containing 30 mg/mL HSA. Sperm were counted and
incubated overnight in BWW and 30 mg/mL HSA at a concentration of 2 x
106 sperm/mL. Hamster eggs were collected by injecting female
hamsters intraperitoneally with 30 IU pregnant mare serum gonadotropin (PMSG),
followed 72 hours later by 30 IU human chorionic gonadotropin (hCG). After
14–16 hours, the hamsters were sacrificed, oviducts were collected in
BWW media containing 5 mg/mL HSA, and cumulus cells were removed with 1 mg/mL
hyaluronidase. Eggs were washed and zona pellucidae removed with 1 mg/mL
trypsin before final thorough washing and incubation. Diluted sperm were
incubated with preimmune or immune sera to ESP (1:10 and 1:50 dilutions were
tested) in paraffin oil–covered microdrops for 1 hour before addition of
the collected hamster eggs. Preimmune and immune rat sera against the
intra-acrosomal protein C113 were used as a second control at 1:10, 1:50, and
1:100 dilutions.
Gametes were coincubated for 3 hours, and sperm and egg binding was assessed after washing unbound and loosely bound sperm by serial passage through 5 (50 µL) wash drops. Eggs then were stained by 5–15 seconds of exposure to 1 mM acridine orange/3% DMSO in bovine serum albumin (BSA)/BWW (30 mg/mL), washed through 4 (50 µL) wash drops, and mounted under 22-mm coverslips. Under ultraviolet illumination, unexpanded heads of oolemma-adherent sperm were counted, as were sperm that had penetrated the ooplasm and exhibited expanded green heads. All in vitro assays were repeated 3 times. Data for experimental and control groups were reported as means ± SD. Student's t test was used to identify differences between treated and control samples in the number of bound or fused spermatozoa. P < .05 was considered to be statistically significant.
Immunostaining of Human Sperm/Hamster Eggs
Mature ova were collected from the oviducts of superovulated hamsters 15 to
17 hours after intraperitoneal injection of hCG. The cumulus cells were
removed by treating the eggs for 3 minutes with 0.1% hyaluronidase in BWW
media containing 5 mg/mL HSA. The zonae pellucidae were removed with 0.1%
trypsin treatment for 2–3 minutes. The washed zona-free eggs were placed
in BWW medium in 100-µL drops under mineral oil. Human semen from healthy
donors was obtained and allowed to liquefy for 30 minutes at 22°C. Semen
(500 µL) was underlain with 2 mL BWW media containing 5 mg/mL HAS, and
sperm were allowed to swim up for 2 hours at 37°C and 5% CO2.
Following the swim-up period, sperm were removed and added to 8 mL BWW
containing 5 mg/mL HSA. The sample was washed 2 times by centrifugation at 600
x g for 8 minutes at 22°C. The pellet then was resuspended
in BWW containing 30 mg/mL HSA. Sperm were capacitated overnight at 20 x
106 sperm cells/mL at 37°C and 5% CO2. The sample
was diluted to 2 x 106 sperm/mL in 20-mL drops, and 10
zona-free eggs were added to each drop. Gametes were coincubated for 3 hours
at 37°C and 5% CO2. Subsequently, the eggs were washed gently
in BWW medium and fixed in 4% paraformaldehyde for 20 minutes at 22°C.
Following fixation, eggs were incubated overnight with 15 µg/mL of 3C6 IgG
overnight at 4°C. The 3C6 is a monoclonal antibody that we prepared that
recognizes the various isoforms of ESP essentially as demonstrated and
described in Wolkowicz et al
(2003). Eggs then were washed
and incubated with a 1:100 dilution of Texas Red–conjugated donkey
anti-mouse IgG for 1 hour at room temperature. The eggs then were washed
extensively (4 x 10-minute washes), placed in 0.4 mg/mL RNase in PBS
with 1% BSA for 30 minutes, and stained with 20 nM Sytox (Molecular Probes,
Eugene, Oregon) for 10 minutes. The eggs were again extensively washed, placed
in slow fade (Molecular Probes) equilibration media for approximately 1
minute, and then mounted in slow fade mounting media. Scanning confocal
microscopy images were obtained on a Zeiss 410 Axiovert 100 microsystems LSM
confocal microscope (Carl Zeiss GmbH, Jena, Germany). Attenuation, contrast,
brightness and pinhole aperture remained constant. Four-second scans were
averaged 4 times per line using ax63 oil lens.
Dual Staining of Acrosome-Reacted Sperm
To determine the relationship between acrosomal status and the presence of
ESP, capacitated, acrosome-reacted sperm were dually stained with PSA lectin,
to assess acrosome reactivity, and with antibody to ESP. Sperm were prepared
by modifying the procedure of Garcia and Meisel
(1999). Briefly, liquefied
ejaculates were pooled and 0.25 mL semen was layered under 2 mL BWW medium
with 34 mM NaHCO3 in 10 x 75 mm round-bottom tubes. Sperm
were allowed to swim up into the medium for 1 hour at 37°C in a 5%
CO2 atmosphere. Swim-up sperm were aspirated and washed 1x in
BWW by centrifugation at 700 x g for 10 minutes. Sperm were
resuspended in BWW with 30 mg/mL fatty acid–free BSA (Sigma). Aliquots
of these "noncapacitated" sperm were washed twice in PBS at 600
x g for 8 minutes and then spotted on microscope slides and
allowed to air dry. The remaining sperm were allowed to capacitate for 6 hours
in Petri dishes in BWW medium with 30 mg/mL BSA and at 37°C with 5%
CO2 with penicillin (100 U/mL) and streptomycin (100 µg/mL;
Gibco/BRL, Rockville, Maryland) present. To one group of capacitated sperm, a
1:500 dilution of 2.5 mM stock progesterone (Sigma) solution in ethanol was
added for a final concentration of 5 µM progesterone. A control group
received an equal volume of ethanol alone at a 1:500 dilution. After
incubation for 20 minutes at 37°C in 5% CO2, both groups were
washed twice in PBS at 600 x g for 8 minutes and then spotted
on microscope slides.
Previously, live staining of human sperm with the 
–rec-h-ESP
polyclonal serum demonstrated no reactivity, indicating that the ESP protein
was not present on the surface of the sperm
(Wolkowicz et al, 2003).
Acrosome-reacted sperm were detected by adapting the procedure of Cross et al
(1986). Air-dried sperm on
microscope slides were blocked for 15 minutes with 1% BSA in PBS and incubated
for 1 hour at 22°C with undiluted supernatant from a murine cell line
(3C6), producing a monoclonal antibody to ESP. Another slide was incubated
with 1% BSA in PBS as a negative control. After washing with PBS, the cells
were incubated for 1 hour at 22°C with TRITC-conjugated F(ab)2
fragments of donkey anti-mouse IgG (Jackson ImmunoResearch) diluted 1:50 in
PBS with 1% BSA. After washing with PBS, the sperm were fixed for 15 minutes
with 4% paraformaldehyde in PBS, washed with PBS, and incubated with 50
µg/mL FITC-conjugated PSA lectin (Sigma). The dual-stained cells were
observed with a Zeiss Axiovert 200 microscope equipped for epifluorescence.
Photos were taken of identical fields with fluorescein and rhodamine
excitation filters using a Diagnostic Instruments 4.1 digital camera and Spot
Insight software (Diagnostic Instruments Inc, Sterling Heights, Michigan).
Sperm were scored visually as acrosome intact or acrosome reacted according to
the degree of staining with PSA lectin, and the same sperm were scored for the
presence or absence of ESP immunoreactivity.
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Results |
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In addition to common spots immunoreactive with sera from both fertile and infertile males and females, the ASA-positive infertile male individuals recognized several alloantigenic proteins (Figure 1B) that were not stained on immunoblots reacted with control sera from fertile males (Figure 1A). An oblique train of spots at approximately 36–38 kDa and 5.1 pI, designated C7 and C8, was bound by ASA-positive infertile male sera (Figure 1B, arrows) but was not recognized by the fertile male sera pool (Figure 1A). In all, 15 sera from infertile men were assayed separately by 2D Western blots, and 4 (26.7%) of the 15 recognized these same spots with identical patterns on Western blots. Concomitantly with the above experiments, 5 ASA-positive sera from 5 infertile female individuals as well as sera from 5 fertile females also were assayed in a similar manner vs human sperm proteins, and 2 of the samples recognized C7 and C8 spots (data not shown). Both infertile and fertile female sera recognized some of the same patterns of proteins and ubiquitous areas as the male sera to varying degrees (data not shown). However, the area important to the present study was recognized by a pool of infertile female sera and not recognized at all by a fertile sera pool.
Probing 2D gels with Con-A (Figure 2) also proved useful in identifying the precise location of ESP on 2D gels. In Figure 2, the Western blot was first stained with Ponceau S to ascertain both the efficiency of transfer and to yield a traceable pattern of sperm proteins for subsequent comparison (Figure 2A). After removal of the stain, the blot was incubated with horseradish peroxidase–conjugated Con-A to reveal glycosylated human sperm proteins (Figure 2B). Con-A stains a limited subset of proteins, including the area at approximately 36–38 kDa and 5.1 pI, that encompasses the area and configuration revealed by the probing with ASA-positive sera. Microsequencing of the spots indicated in Figure 1B at the top and bottom of this protein train (indicated by C7 and C8) yielded microsequences (AATVFNTLK, LYEYLDLK, SLALAAAAEHK) contained within the recently cloned sperm protein ESP (Wolkowicz et al, 2003), thus confirming by mass spectometry that the glycosylated protein spots were ESP.
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Acrosome Reaction and In Vitro Fertilization
A substantial amount of literature points to the equatorial segment's
involvement in sperm-egg adhesion and membrane fusion
(Yanagimachi and Noda, 1970;
Yanagimachi, 1994; Arts et al,
1994,
1997). To test the possible
role of ESP in these events, the fate of ESP was studied after in vitro
fertilization. Capacitated human sperm and zona-free hamster eggs were
incubated with ESP antiserum, and the numbers of sperm bound and those fused
were determined and compared to control samples incubated with preimmune serum
(Figure 4). The results
indicate a 42% reduction in sperm binding
(Figure 4A) in the presence of
antiserum to ESP (P 
.05). More importantly, we observed a 68%
inhibition in the fusion of sperm with eggs (P .05;
Figure 4B). When the data on
fused sperm were expressed as a ratio of fused/bound + fused to eliminate the
effect of binding on fusion (Figure
4C), it was noted that there was a statistically significant 52%
reduction in fusion (P .01). No effect on either sperm binding
or fusion was observed with the rat antiserum to the intra-acrosomal protein
C113 at any dilution.
The observation that antibody to ESP interfered with sperm-egg binding and
fusion led to a prediction that ESP would persist to some degree on
acrosome-reacted sperm. Since fertilization is a 2-step process of sequential
sperm binding and then fusion of the equatorial segment, if ESP plays a role
in fusion, then it should be retained and exposed in acrosome-reacted sperm.
Dual staining with PSA lectin and rat –rec-h-ESP antibodies was
performed as illustrated in Figure
5. The loss of the PSA-positive principal segment of the acrosome
and retention of a PSA-positive equatorial segment was observed
(Figure 5), indicating
acrosome-reacted sperm also stained positively for, and retained, ESP
(Figure 5). Sperm cells from
this dual staining procedure were counted to determine the numbers of
acrosome-reacted sperm and those acrosome-reacted sperm that retained ESP
staining (Table). A total of 17.8% of human sperm underwent spontaneous or
induced acrosome reaction when treated with progesterone, and 89% of these
were positive for ESP staining in an equatorial band. These results indicate
that a significant population of sperm retain ESP in their equatorial segment
after the progesterone-induced acrosome reaction. This finding is in concert
with the prediction from the observation of 68% inhibition of sperm-egg fusion
that ESP is retained and exposed on acrosome-reacted sperm.
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Direct Observation of ESP in Bound Sperm
The finding that antibody to the ESP reduced both sperm-egg binding and
fusion (Figure 4) and that a
subpopulation of sperm retained ESP in the equatorial segment after an in
vitro acrosome reaction (Table) led us to a direct observation of the fate of
ESP on sperm already bound to the egg
(Figure 6). Capacitated human
sperm and hamster eggs were incubated together for 3 hours, washed thoroughly,
fixed, and double stained. Examination by scanning confocal microscopy of the
egg in Figure 6, for example,
demonstrated 6 bound sperm in different planes of focus, as depicted in
Figure 6, 1A through D and 2A through
D. Sytox was employed to highlight condensed chromatin in the
sperm head (Figure 6, 1A and
2A; green fluorescence). All 6 sperm bound to the oolemma (eg,
Figure 6, 1A and 2A), showed
immunofluorescent equatorial bands when stained with the
–rec-h-ESP sera (Figure 6, 1B
and 2B; red immunofluorescence). Overlaying the 2 sets of
fluorescent images onto the phase microscopy image
(Figure 6, panels 1D and 2D)
demonstrates a yellow fluorescent band at the approximate location of the
equatorial segment in each case. In addition, attached sperm stained with the
control preimmune sera were negative (data not shown). Of 78 sperm observed on
10 eggs, 100% were ESP positive, indicating that ESP is retained after tight
binding of human sperm to hamster
eggs.
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Discussion |
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It is particularly interesting that all the human sperm that tightly bound to the hamster oocyte surface were ESP positive. One tentative interpretation of this observation may be that an intact equatorial segment may be required to achieve tight binding. Now that a probe specific for the human equatorial segment is available, it may be important to extend such observations to human oocytes.
Regions of ESP protein sequence with homology to other molecules (Figure 7) suggest functionalities that relate both to the location of ESP in the equatorial segment (Wolkowicz et al, 2003) and to binding and fusion events. ESP's N-terminal portion is homologous to the cytolysin family of proteins, which function in oligomerization, membrane insertion, cholesterol binding, and pore formation (Palmer, 2001). A C-terminal domain in ESP homologous to a bacterial type II membrane-binding protein is overlaid on top of a portion of the osteoglycin domain. Type II membrane-binding proteins are used by bacteria in attaching their chromosome to the interior wall of the cell. Lastly, the C-terminus of ESP is homologous to a 68–amino acid region of osteoglycin. Osteoglycin belongs to a large family of leucine-rich repeat proteoglycans that includes decorin, keratocan, fibromodulin, and biglycan (Matsushima et al, 2000) that act through protein-protein interactions and appear to mediate such diverse cellular processes as signal transduction, cell adhesion, and recombination. In fact, ESP does contain a conserved serine residue (ESP amino acid 306) thought to enable covalent cross linking (osteoglycin amino acid 101). This may be important for the final binding and fusion steps of fertilization and may serve to anchor and hold the 2 opposing gametes together, facilitating membrane fusion.
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Mechanisms of action of ASA-positive seminal fluids in males and females following ejaculation have been well described (Bronson, 1999). In intact sperm, ESP lies within the acrosomal matrix (Wolkowicz et al, 2003). However, acrosome-reacted sperm may reveal exposed ESP in the cleft between inner and outer acrosomal membranes, thus providing sufficient antibody binding to cause the inhibitory effects observed if antibodies to ESP are present in oviductal fluids. Likewise, spontaneous acrosome reactions in both the male and female reproductive tracts may carry some intra-acrosmal ESP onto the plasma membrane. Anti-ESP antibodies present in seminal plasma may be predicted to affect fertility through this mechanism. Finally, the zona pellucida is known to be permeable to IgG. ASAs present in the zona and perivitelline space of sufficient titer may exert immunologic effects that are localized at the egg following the zona-induced acrosome reaction. Fine structural immunolocalization studies to date have not been successful in simultaneously preserving membranes and retaining immunogenicity of ESP sufficient to allow its precise localization with respect to the plasma membrane, inner and outer equatorial segment membranes, and matrix following the acrosome reaction and after egg binding.
ESP: Relationship to Other Equatorial Segment Proteins
Several proteins found to be associated with the equatorial region have
been hypothesized to be involved in fertilization. One group thought to be
involved in the final steps of fertilization is the ADAM family of proteins
(Primakoff and Myles, 2000).
This family, a subset of which is testis specific, contains both disintegrin
and metalloprotease domains and therefore is potentially involved in both cell
adhesion and protease activities. Indeed, antibodies to the testis-specific
ADAM 2 (fertilin β) protein
(Primakoff et al, 1987) and
peptides representing the disintegrin domain of ADAM 2 (RGD motif;
Evans et al, 1995) and ADAM 3
(cyritestin; Yuan et al, 1997)
were found to block the adhesion and fusion steps of fertilization. More
recently, knockout experiments with both ADAM 2
(Cho et al, 2000) and
cyritestin (Nishimura et al,
2001) have demonstrated greatly reduced sperm-egg adhesion but no
apparent involvement in the fusion step. ADAM 2 has been localized to the
posterior of the head (Hunnicutt et al,
1997), not the equatorial segment.
The cysteine-rich secretory protein (CRISP) family of acidic proteins is thought to regulate calcium channels (Kirchoff, 1998), and some members are transcribed and secreted by the epididymis, where they associate with maturing spermatozoa. One family member, rat epididymal glycoprotein DE (named for isoforms "D" and "E"), associates with the dorsal sperm surface but migrates to the equatorial segment upon capacitation (Rochwerger and Cuasnicu, 1992). Antisera raised against recombinant forms of the mouse and human orthologs (Cohen et al, 2001) of this protein appear to inhibit sperm-egg fusion. However, unlike ESP's immunolocalization, the human ortholog reveals staining of the head as well as the principal and midpieces of the tail (Hayashi et al, 1996). Recently, targeted deletion of the intra-acrosomal protein IZUMO has been shown to result in complete infertility, with sperm entering the perivitelline space without difficulty but unable to fuse with the oolemma (Inoue et al, 2005). Antiserum to human IZUMO blocks human sperm fusion with the oolemma in the hamster egg model.
Previous investigations have provided evidence for subcompartments of the
acrosomal matrix and membranes. During mouse spermiogenesis, the Golgi markers
giantin, βCOP, golgin 97, and mannosidase II are localized to an
acrosomal subcompartment that surrounds the region occupied by acrosin within
the acrosomal granule (Ramalho-Santos et
al, 2001). Similarly, the vSNARE, VAMP, and tSNARE syntaxin are
present in a subdomain surrounding the acrosomal granule in mouse sperm
(Ramalho-Santos et al, 2000)
and are concentrated in human and rhesus sperm in the equatorial segment of
non–acrosome-reacted sperm and persist on the equatorial segment
following the acrosome reaction
(Ramalho-Santos et al, 2000).
Likewise, the cystatin-related CRES gene product has been shown to be present
in the equatorial segment of human sperm
(Wassler et al, 2002).
Coimmunoprecipitation experiments are currently underway using the
–rec-hum-ESP serum to determine whether ESP is complexed or
associated with any other equatorial segment proteins and to identify other
constituents of the specific equatorial segment compartment that ESP
occupies.
Useful ASA reagents have been generated to study sperm subcompartments (Bronson and Tung, 1992; Noor and Moore, 1999; Auer et al, 2000). Monoclonal antibodies HS1A.1 (Villaroya and Scholler, 1986) and MA1-3 (Isahakia and Alexander, 1984) recognize proteins residing only in the principal segment of the human acrosome, whereas D3 (Hinrichsen-Kohane et al, 1985) and 21D3 (Le et al, 1984) bind antigens located only in the equatorial segment. Olson et al (1998) have demonstrated immunohistochemically that 2 major acrosomal matrix proteins, AM22 and AM29, are excluded from the equatorial segment of mouse sperm, whereas the MN9 monoclonal antibody (Toshimori et al, 1992) recognizes 38- and 48-kDa proteins in mouse that are segregated specifically to the equatorial segment. Other researchers reported that the M1 (Noor and Moore, 1999) and P36 (Auer et al, 2000) monoclonal antibodies, which recognize sperm protein bands of 38 and 34 kDa in hamster and humans, respectively, localize to the equatorial segment and inhibit fertilization. However, while the identities and functions of the proteins corresponding to these immunoreagents remain unknown, there is a striking similarity in molecular weight to isoforms of ESP.
Clinical Implications for Infertility
ASAs have been identified in 10%–15% of men experiencing infertility
and 15%–20% of women with unexplained infertility
(Ghazeeri and Kutteh, 2001).
ASAs persist for years following vasectomy and are thought to be one reason
for the infertility that may follow vasectomy reversal (for reviews, see
Bronson et al, 1994; Marshburn and Kutteh,
1994; Bronson,
1999). The presence of ASAs in clinical cases of infertility,
therefore, indicates a possible etiology for the inability to conceive on the
part of either the male or female partner. The present experiments demonstrate
that ASA-positive infertile male and, to a lesser degree, infertile female
sera reacted positively with the 36- to 38-kDa ESP region on 2D gels of human
sperm protein extracts, indicating that this antigen is both accessible to and
immunogenic for the human immune system. Our live staining,
immunofluorescence, and electron microscopy observations
(Wolkowicz et al, 2003) have
shown ESP to be present within the acrosome, bringing into question how the
ESP protein could elicit an immune response in males. It will be noted that
some sperm undergo a spontaneous acrosome reaction, thus releasing their
acrosomal contents prematurely. Indeed, exposure of the male immune system to
the acrosomal contents in the urethra or elsewhere in the postepididymal male
reproductive tract may cause the production of antibodies and contribute in
some cases to an antifertility effect in the male. Likewise, this same
mechanism of premature, spontaneous acrosome reaction could elicit antibodies
in the female's lower reproductive tract and infertility via vaginal secretion
of antibodies. Endogenously generated ASA-positive sera recognized both native
and recombinant ESP on Western blots, indicating the rec-h-ESP retains
sufficient primary and secondary structure to be recognized by antibodies to
the native antigen. Polyclonal immune sera raised against the cloned
recombinant ESP showed that endogenous ESP was localized to the equatorial
segment, where it was retained in acrosome-reacted sperm. This same anti-ESP
antiserum significantly blocked sperm binding and, more importantly, fusion
when tested in vitro. This result is in accord with the finding that ESP is
retained in the acrosomes of all sperm tightly bound to the oolemma.
Taken together, these data suggest ESP is an antigen that may be involved in immune infertility in humans and that recombinant ESP may be useful to monitor infertility-associated antibody responses. Intracytoplasmic sperm injection (ICSI) is now the standard of care in many cases of ASA as well as other forms of male infertility. Presently, the incidence of transmission of infertilities of genetic origins (such as motility defects) to the male offspring after ICSI is poorly understood, because the first ICSI males are only now reaching puberty. Interest in differential diagnosis of the causes of male immunoinfertility, such as ASA, may become more important in the future if the acceptability of ICSI in treating some forms of infertility were to wane due to a high rate of transmission of infertilities of genetic origin. In the future, ICSI might continue to be the management tool of choice in the case of ASA, whereas it might not be the management tool of choice for certain infertilities with transmissible genetic origins. A differential diagnosis of immunoinfertile patients with ESP and other molecules would be helpful in deciding which patients should be treated with ICSI therapy. Moreover, ESP's testis-abundant expression, presence on acrosome-reacted sperm and on sperm bound to eggs, as well as the inhibition of fertilization by antibodies to recombinant ESP, indicate that ESP may also be a candidate contraceptive vaccinogen. Currently, we have a number of sperm proteins, including ESP, undergoing testing in macaques to ascertain their immunogenic properties (Herr, 1996; Diekman and Herr, 1997; Kurth et al, 2007).
In addition to previous strategies for establishing the identities of the target antigens recognized by ASAs (Herr et al, 1985; Tsuji et al, 1988; Diekman et al, 1999; Li et al, 2000), the present report shows that screening of 2D Western blots with ASA-positive sera and mass spectrometry can result in the characterization of relevant antigens with key roles in fertilization and demonstrates that proteomic strategies are a powerful approach to dissect the molecular mediators of immunoinfertility.
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Footnotes |
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References |
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