National Institute of Immunology, Aruna Asaf Ali Road, New Delhi 110067, India
* Author for correspondence (e-mail: cshaha{at}nii.res.in )
Accepted 12 February 2002
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Summary |
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These data therefore demonstrate the strategic location of catalytically active defensive enzymes on the sperm surface that also act as zona-binding proteins. Therefore, sperm-surface GSTs serve as bifunctional molecules in a transcriptionally inactive cell whose requirement for cellular defense and economy of molecules that it can carry is greater than that of any somatic cell type.
Key words: GST, Plasma membrane, Sperm, Zona binding
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Introduction |
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GSTs are cytosolic proteins, except for in one cell type, namely the
hepatocyte, which is reported to express GSTs of 17.2 kDa on the plasma
membrane (Sies et al., 1998).
We present clear evidence that GSTs of 24 kDa are present on mature cauda
epididymal sperm plasma membrane and are attached to it by peripheral
non-covalent interactions. Spermatozoal plasma-membrane-associated GST
activity is significantly higher than that of plasma membrane from somatic
cells such as brain cells, spleenocytes, ventriculocytes and hepatocytes. Two
GST isoforms, namely GST Mu and Pi are present on the sperm plasma membrane,
and both are capable of binding to the ZP. This binding appears to be mediated
primarily via the N-terminii of these molecules. Interestingly, both the
isoforms bind to the same site on ZP although GST Pi shows a higher affinity
than GST Mu. Goat ZP resolves into three components of which only the ZP3-like
component binds to biotinylated sperm plasma membrane GSTs. Solubilised ZP
(SZP) or higher concentrations of anti-GST antibodies leads to aggregation of
sperm-surface GSTs, resulting in acrosin release. In contrast, saturation of
sperm GSTs by lower concentrations of anti-GST antibodies or their Fab
fragments does not induce aggregation and therefore prevents ZP binding to
sperm, resulting in inhibition of acrosome reaction. This inhibition appears
to be caused by reduction of the intracellular Ca2+ increase that
normally occur in response to ZP binding. Having a detoxification enzyme as a
zona-binding protein would give a great survival advantage to a cell that is
highly streamlined, vulnerable to oxidative stress and exposed to a variety of
environments en route from the testis to the oocyte in the fallopian tube.
Taken together, the data provide a comprehensive view of the functional role
of sperm GSTs as ZP-binding proteins that add to the importance of
multifunctional molecules in a transcriptionally inactive cell such as
sperm.
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Materials and Methods |
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Materials
Goat anti-rabbit IgG antibodies conjugated to fluorescein isothiocyanate
(FITC) were procured from Jackson Laboratories (West Grove, PA). The
bicinchoninic acid protein assay reagent was from Pierce Chemical Company
(Rockford, IL). The GST purification module and Protein G Sepharose Fast Flow
were obtained from Pharmacia Biotech (Uppsala, Sweden). ProblottTM
membranes and reagents for synthesis and conjugation of peptides were from
Applied Biosystems Inc. (Foster City, CA). Other chemicals, unless specified,
were purchased from Sigma Chemical Co. (St. Louis, MO). Na[125I]
was purchased from NEN Life Science Products (Boston, MA).
Peptides and antibodies
A total of four peptides designed from two different regions of domain I of
the GST Pi and Mu were synthesised and characterised as described previously
(Gopalakrishnan et al.,
1998b). Two peptides representative of the N-terminal sequences of
the two GST isoforms were GST PiN (Pro-Pro-Tyr-Thr-Ile-Val-Tyr-Phe-Pro-Val)
and GST MuN (Pro-Met-Thr-Leu-Gly-Tyr-Trp-Asp-Ile). Two other peptides
representing a region that is involved in glutathione (GSH) binding
(Manoharan, 1992
) were GST PiC
(Gln-Leu-Pro-Lys-Phe-Gln-Asp-Gly-Asp-Leu-Thr-Leu-Tyr) and GST MuC
(Asn-Leu-Pro-Tyr-Leu-Ile-Asp-Gly-Ser-His-Lys-Ile-Thr). The peptides were
conjugated to diptheria toxoid by the glutaraldehyde method
(Avrameas, 1969
).
The conjugated peptides were used to raise antisera in rabbits using
standard protocols (Vaitukaitis et al.,
1971). Characterisation of the antisera was done as described
previously (Gopalakrishnan et al.,
1998b
). The specificity of the antisera was judged by (a)
immunoprecipitation of GSTs from sperm extracts, (b) the reactivity of
antisera to their respective peptides and the absence of cross reactivity
towards other peptides (Gopalakrishnan et
al., 1998b
), (c) the inability of the antisera to crossreact with
any other proteins from total sperm extracts on western blots, (d) the ability
of antigen pre-adsorption to abolish immunorecognition and (e) reproducibility
of the results with antisera from different animals.
Fab fragments were prepared from IgG purified from 50% ammonium sulfate
precipitates of the immune sera prepared using Protein G Sepharose 4B Fast
Flow column. Papain (25 µg) activated with ß-mercaptoethanol was used
to digest 500 µg of purified IgG, and Fab fragments were purified on a
Protein Pak column DEAE-5PW [Semiprep 21.5 mmx150 mm (Waters, USA)]
equilibrated with 10 mM Tris-HCl (pH 8.0) on a gradient of 0-100% of 10 mM
Tris-HCl, 1 M NaCl (pH 8.0) (Jungbauer et
al., 1989).
Circular dichroism
Circular dichroism (CD) experiments were carried out as described
previously (Kaur et al., 1997)
on a JASCO 710 spectropolarimeter with a 2.0 nm band width, 1 nm resolution
and 1 second response time. The peptide concentrations of GST PiN and MuN was
30 µM in 10 mM Tris-HCl (pH 7.0).
SDS-PAGE and western blots
Electrophoresis was carried out using the buffer system of Laemmli
(Laemmli, 1970) on 10%
polyacrylamide gels. Western blotting was performed following a modification
of the method of Towbin (Towbin et al.,
1979
) as described previously
(Aravinda et al., 1995
).
Preparation of capacitated spermatozoa, gametes, embryos and
solubilised zonae
Sperm preparation and capacitation, recovery and maturation of oocytes were
carried out as described previously
(Gopalakrishnan et al.,
1998b). Embryos were allowed to develop in TC199 medium
supplemented with 20% fetal calf serum (FCS).
For the preparation of SZP, oocytes matured in vitro with cumulus oophorous were treated with 5 mg/ml hyaluronidase for 5 minutes to remove the cumulus cells. ZP were then removed from cumulus-free eggs mechanically by forcing through a narrow-bore micropipette. Isolated zonae were washed thoroughly to remove any adherent egg cytoplasm and were subsequently heat solubilised. The purity of this SZP preparation was checked routinely by SDS-PAGE. SZPs were drop frozen and stored in liquid nitrogen until use.
Preparation of plasma membranes
Germ cells from goat testes were prepared by digestion with
collagenase/dispase in Ham's F-12 media as described previously
(Aravinda et al., 1995) and
crude membranes prepared according to Scott et al.
(Scott et al., 1993
). Liver
and brain tissues were digested with collagenase/dispase (3 mg/ml) and 0.25%
trypsin (Ferreira and Loomis,
1998
), respectively. Spleens were mashed between two frosted
slides to prepare spleenocytes (Coligan et
al., 1992
), and ventriculocytes
(Springhorn, 1998
) were
prepared from heart ventricular tissue by trituration in Krebs-Henseleit
buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.25 mM
CaCl2, 1.2 mM KH2PO4, 25 mM
NaHCO3, 11 mM glucose) followed by treatment with 3 mg/ml
collagenase and 0.25% trypsin. The resulting tissue masses were filtered
through cheesecloth, and cell suspensions were separated on 40% percoll
gradients. The cells were homogenised in STM (10 mM Tris-HCl, 1 mM EDTA, 0.25
M sucrose, pH 7.4) in the presence of a protease inhibitor cocktail (1 mM
EDTA, 1 mM PMSF, 1 mM EGTA 0.17 units/ml aprotinin, and 1 mM leupeptin).
Unbroken cells were removed at low speed and the nuclear pellet collected
after spinning at 600 g for 10 minutes at 4°C. The mitochondrial
fraction was separated out by spinning the supernatant at 10,000 g
for 10 minutes. Crude membrane fraction was obtained after centrifugation of
the above supernatant at 100,000 g for 60 minutes in a Beckman TL-100
ultracentrifuge with an SN863 rotor (Beckman Inc. Palo Alto, CA). Crude plasma
membrane was further purified on a 25% linear Percoll gradient, and purity was
checked by assaying for plasma membrane marker enzyme 5'-nucleotidase
(Glastris and Pfieffer,
1974
).
Study of mode of membrane attachment
Purified sperm plasma membranes were treated with different concentrations
of proteases, trypsin type III and pronase A (0.1 mg/ml, 0.5 mg/ml, 1 mg/ml
for different time periods, 10 and 20 minutes), and protease inhibitors were
used to stop reactions. To detach peripherally attached proteins, membranes
were incubated with 10 mM, 20 mM HCl and 1 M NaCl for 30 minutes at 4°C.
To remove GPI-anchored proteins, phosphotidylinositol-specific phospholipase C
(PIPLC, 0.05 units/ml, for 10 minutes)
(Shur and Neely, 1988) was
used. Release of GSTs from the plasma membranes after treatment was checked in
the 100,000 g supernatants by GST activity assay with
1-chloro-2,4-dinitrobenzene (CDNB) as the substrate. Purification of GSTs from
peripherally attached proteins was done as described below and run on SDS
PAGE, western blots of which were probed with anti-GST antibodies.
Purification of GSTs, GST assay and identification of isoforms
GSTs from NP-40 extracts of sperm plasma membrane preparations and extracts
of total blood cells were purified by GSH affinity chromatography as described
earlier (Aravinda et al.,
1995). The GST assay was carried out according to Warholm
(Warholm et al., 1985
) with
slight modifications as described previously
(Aravinda et al., 1995
;
Mukherjee et al., 1999
).
Reverse-phase high performance liquid chromatography (RP-HPLC) was used to
purify GST isoforms from the total sperm plasma membrane GSTs using a Vydac
C18 column (Type 218TP54, Waters, USA) equilibrated with 60%
Solvent 1 (0.08% Trifluoroacetic acid, TFA in water) and 40% Solvent 2 (0.08%
TFA in 80% acetonitrile) (Gopalakrishnan
et al., 1998b). N-terminal sequencing of the purified peaks was
carried out as described in an earlier report
(Gopalakrishnan et al.,
1998b
).
Radiolabelling of cells and proteins
To label the spermatozoal surface, we selectively used a highly motile
population of spermatozoa that was purified by centrifugation on Percoll
gradient (90-45%). Iodination was carried out according to Spencer and
Nicoloff (Spencer and Nicoloff,
1980). Briefly, 107 Percoll-purified sperm per vial
were mixed with 200 µM lactoperoxidase, 0.5 mCi Na[125I] and
0.03% H2O2 on ice. After gentle mixing for 5 minutes, 20
µM lactoperoxidase and 0.03% H2O2 were added followed
by three additions of 0.03% H2O2 on ice. The cells were
thoroughly washed with 15 mM NaI and then tested for viability by trypan blue
exclusion and the motility pattern of the cells. Vials with cell viability
above 95% were homogenised to break open the cells, and plasma membrane
fractions were separated as mentioned above. GSTs were purified from 0.1%
NP-40 extracts of these fractions, and other remaining fractions followed by
autoradiography that was carried out after running these extracts on SDS-PAGE.
The sperm GSTs and SZP (50 µg of protein) were iodinated. Iodinated
proteins were purified on a Sephadex G-75 column to remove free iodine.
Immunocytochemistry
For visualising the binding of GST MuN and PiN peptides to oocytes and
embryos, oocytes were incubated with GST PiN and MuN peptides (10 µg/ml)
for 30 minutes at 37°C followed by treatment with rabbit anti-GST PiN and
MuN antibodies (1:100) and subsequently treated with anti-rabbit IgG (1:200)
conjugated to FITC. Oocytes were mounted in hanging drop preparations and
observed under a Nikon Optiphot fluorescence microscope. To detect any
staining of oocyte cytoplasmic GSTs, inner cytoplasmic masses were pushed out
just before the oocytes were observed. Embryos were also treated
similarly.
To visualise the aggregation of sperm-surface GSTs, capacitated sperm were incubated with SZP (10 µg/ml/107 sperm), anti PiN, anti MuN antibodies (1:10) or their Fab fragments at 4°C followed by incubation at 37°C. Aliquots of sperm were fixed at different time points in 4% buffered paraformaldehyde. The movement of the GST molecules was visualised by using FITC-labelled second antibody (1:500). Pisum sativum agglutinin (PSA) conjugated to rhodamine (1:100) was used to stain the same samples to visualise the status of the acrosome. A minimum of 200 sperm were scored according to the following classification: (a) staining for the total acrosome; (b) patchy staining implying ongoing aggregation - patching; c) aggregation towards the tip of the acrosome - capping and d) unstained sperm.
Sperm GST-SZP binding
To check the binding of sperm GSTs to SZP, 5 µg each of BSA, sperm
plasma membrane and blood cell GSTs were dotted on nitrocellulose sheet and
blocked with 2% BSA-PBS. The dot blots were probed with
125I-labelled SZP for 16 hours at 4°C and exposed to x-ray
films or phosphorimager screens for autoradiography. For specificity studies,
dot blots were preincubated with unlabelled SZP followed by incubation with
the iodinated material. Quantitation of binding of SZP to the dot blots was
done with a Molecular Dynamics Phosphorimager (Sunnyvale, CA) with Imagequant
software package.
To check the binding of SZP with the GST PiN and MuN peptides, binding studies were performed with different concentrations of GST PiN and MuN peptides immobilised on microtitre plates and probed with 50 Ng of I125-SZP. For competition studies, excess unlabelled SZP (200 ng) or competitive peptides (50 µg) was used.
In addition to these studies, GST Mu and Pi purified by RP-HPLC from plasma membrane GST were radiolabelled and used for binding studies with SZP (1 µg) immobilised onto microtitre plates. Each individual well of the microtitre plate was transferred to a vial, and readings were taken in a 1275 Minigamma counter (Wallac Inc., Turku, Finland).
For equilibrium-binding studies, SZP (1 µg/well) was immobilised on microtitre plates and blocked with 2% BSA in PBS before allowing concentration-dependent equilibrium binding (3 hours at 37°C) of 125I labelled GST (*GST). Unbound *GST was washed out with 0.05% Tween-20 in PBS. Non-specific binding was determined as the binding of *GST to zona after GST sites on zona were blocked with 100 mM cold GST. Affinity constant (Kd) of the GSTs was calculated by using Langmuir and Scatchard plots of specific binding. For competition experiments, 600 nM *GST was used to bind to immobilised SZP in the presence of different concentrations of GST PiN and GST MuN peptides. IC50 was calculated from normalised fractional binding curves.
Identification of ZP components binding to sperm GSTs
To determine the specific ZP components that the sperm GSTs bind to, ZP (10
µg) was run on SDS-PAGE and transferred onto nitrocellulose. These blots
were probed with biotinylated sperm plasma membrane GSTs (1 µM) for 3 hours
at room temperature, and binding was visualised by avidin linked to HRP. The
plasma membrane GSTs were biotinylated using NHS-biotin as described earlier
(Rao and Shaha, 2001). For
identification of the component of the ZP that the biotinylated sperm GSTs
were binding to, similar blots were probed with two monoclonal anti-ZP3
antibodies. One was raised against an epitope conserved among species (MA 451)
(Kaul et al., 1997
), and
another was raised against the N-terminus of the porcine ZP (MA 455/467)
(Gupta et al., 1995
). Further
characterisation of the component band was carried out by N-terminal
sequencing as described previously
(Gopalakrishnan et al.,
1998b
).
In vitro sperm agglutination, immobilisation and sperm-oocyte binding
assay
The agglutination assay was carried out as described previously
(Shaha et al., 1988). Briefly,
106 sperm were placed in a 50 µl droplet of media under mineral
oil, and antibodies were added at dilutions of 1:25, 1:50 and 1:100, following
which observations were made under an inverted microscope.
To directly determine the effects of the antibodies bound to sperm on their
capability to bind to oocytes, antibody-treated (1:100 dilution, anti-GST-PiN,
MuN, PiC, MuC and a mixture of anti-GST MuN and PiN) capacitated spermatozoa
were washed and allowed to interact with mature oocytes (106
sperm/10 oocytes) in fertilisation media for 30 minutes at 37°C under
mineral oil (Aravinda et al.,
1995). To check if saturation of the GST-binding sites on oocyte
ZP would inhibit sperm-oocyte binding, oocytes were exposed to total sperm
plasma membrane GSTs and relevant peptides for 30 minutes at 37°C prior to
interaction with capacitated spermatozoa. In both the above experiments, the
number of sperm bound to each oocyte was scored in a standard plane of focus
that passed approximately through the center of the oocyte as described
earlier (Shaha et al.,
1991
).
Acrosome reaction and acrosin assay
Binding to the multimeric ZP induces acrosome reaction in sperm
(Yanagimachi, 1981). To
determine the effect of binding of anti-GST antibodies to sperm on
zona-induced acrosome reaction, sperm were capacitated in the presence of
anti-PiN and anti-MuN antisera (1:100), their Fab fragments (10 µg/ml) and
preimmune sera (1:100) for 3 hours and incubated with SZP (10 µg/ml). SZP
preadsorbed with GST PiN and MuN was used to induce an acrosome reaction in
untreated capacitated spermatozoa to check if saturation of GST-binding sites
on SZP would render the SZP unable to induce acrosome reactions. Acrosin
release was measured by a spectrophotometric assay using BAEE (Benzoyl
Arginine Ethyl Ester) as substrate
(Polakoski and Zaneveld,
1977
).
To assess specifically if crosslinking of sperm-surface GSTs could initiate intracellular signaling events for acrosome reaction, capacitated sperm were treated with various concentrations of antibodies [1:10, 1:25, 1:50 of anti-GST-MuN and PiN, total IgG or Fab fragments (50 µg/ml)] for 60 minutes at 37°C, and acrosin release was measured as described above. In groups treated with higher dilutions (1:50) of anti-GST antisera, secondary antibody (1:100) was used to precipitate immunoaggregation.
Measurement of intracellular levels of Ca2+
Intracellular Ca2+ was measured according to Gorczynska and
Handelsman (Gorczynska and Handelsman,
1993) with slight modifications. Briefly, spermatozoa
(107) capacitated in the presence or absence of anti-GST antibodies
were loaded with 1 µM of Ca2+-binding dye Fura 2-AM at 37°C
for 40 minutes. Acrosome reaction was induced with SZP (10 µg/ml), and
subsequently cells were lysed with 0.1% digitonin. After centrifugation at 750
g for 30 minutes, fluorescence in the supernatant was measured
using a spectrophotofluorimeter (RF-1501, Shimadzu Inc., Tokyo, Japan) keeping
the
excitation at 340 nm and
emission at
400-500 nm. For microscopic estimation, the number of Fura 2-AM-positive sperm
was scored under a Nikon Optiphot fluorescence microscope.
Statistical analyses
Data from various treatment groups were compared using unpaired two-tailed
student's t-test. Tadpole III software (Elsevier Biosoft, Cambridge,
UK) was used for the analysis.
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Results |
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Having established that GSTs are associated with the sperm plasma membrane,
we tried to determine whether they are exposed to the extracellular side of
the cell. In case of an extracellular side orientation, GSTs should
theoretically be labelled when cell surface proteins were radioiodinated. We
purified radiolabelled GSTs of apparent molecular mass of 24 kDa from extracts
of spermatozoa that were radioiodinated in live conditions
(Fig. 2A). Radiolabelling of
internal proteins was ruled out by autoradiography of sperm proteins after
their plasma membranes have been removed. No significant labelling was
observed in these extracts (Fig.
2A, lane a). Therefore, it can be inferred that GSTs are present
on the sperm surface. This observation further supports our previous report
that spermatozoa excluding propidium iodide stain positive for both GST Pi and
Mu when immunostained with anti-GST antibodies
(Gopalakrishnan et al.,
1998b).
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To confirm the molecular mass, purified GSTs from sperm plasma membrane were run on SDS-PAGE, which revealed two protein bands of apparent molecular mass of 25.6 and 23.4 kDa (Fig. 2B). The identities of these bands was further confirmed by immunoprecipitation of plasma membrane extracts with anti GST Pi and Mu antisera that showed precipitation of proteins of molecular masses between 23 and 26 kDa (Fig. 2C).
Distinct identification of the GST proteins was established by N-terminal sequencing and western blots. Affinity-purified plasma membrane GSTs resolved into three peaks on RP-HPLC (Fig. 2D). Peak three showed a 100% sequence identity to bovine GST Pi N-terminii over 10 amino acids (Pro-Pro-Tyr-Thr-Ile-Val-Tyr-Phe-Pro-Val). Attempts to sequence the N-terminii of peaks one and two were not successful, implying that the N-terminii may be blocked. The identity of the two peaks was confirmed by western blots with antibodies against GST-M1 that reacted to the first two peaks, whereas anti-GST Pi antisera did not recognise either of them (Fig. 2E). Attempts to sequence endo-protease glu-C digests of peaks one and two were not successful owing to limitations in the starting material. GSTs purified by RP-HPLC from goat blood resolved into one distinct peak that shared 100% sequence identity at the N-terminus with sperm plasma membrane GST Pi (data not shown).
The above results provide evidence that plasma-membrane-associated GSTs are present on spermatozoa. Two GST isoforms, namely GST Pi and Mu, are present on the plasma membrane and have a molecular mass range between 23 and 26 kDa. Plasma-membrane-associated GST activity was highest on sperm - higher than on the four types of somatic cells tested.
GSTs are attached to the sperm surface by non-covalent
interactions
To determine the mode of attachment of GSTs, plasma membranes were exposed
to high salt, low pH buffer, PIPLC or subjected to controlled trypsinisation.
All three treatments except PIPLC yielded GST activity in the supernatants
(Fig. 3), indicating
non-covalent interactions as the possible mode of attachment. Absence of GST
activity in the supernatants of the treatments using PIPLC rules out the
possibility of GPI anchoring. No activity could be detected in the pronase
digest supernatants. Anti-GST antibodies on western blots of 1M NaCl extract
of plasma membranes recognised a 24 kDa band
(Fig. 3, inset, lane b).
Immunorecognisable 24 kDa GSTs could be purified from these supernatants
by GSH affinity chromatography as well
(Fig. 3, inset, lane c).
Trypsinisation of live spermatozoa yielded GST activity in the supernatant
(data not shown); however, this experiment could not be carried out with 1 M
NaCl as the sperm lost membrane integrity, and therefore there was a
possibility that cytosolic proteins including sperm GSTs might have leaked
out. In trypsinisation experiments, time points and concentrations were chosen
where sperm membrane integrity was intact and sperm were viable after
treatment.
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The above data suggested that the mode of attachment of GSTs to the sperm plasma membrane was primarily through non-covalent interactions.
Sperm GSTs bind to oocyte ZP and are involved in sperm-oocyte
binding
Considering that the above data suggested a plasma membrane localisation of
sperm GSTs, with them facing the extracellular side, and our earlier data on
the ability of anti-GST antibodies to reduce fertilisation, it seemed possible
that sperm GSTs might take part in the process of primary binding between the
gametes. We therefore, explored several ways to determine if sperm GSTs bound
to molecules on the ZP.
Purified sperm plasma membrane GSTs immobilised on the nitrocellulose membrane was able to bind to radiolabelled SZP that could be displaced by preincubation of the blots with unlabelled SZP (Fig. 4Aa1,a2, Fig. 4Ba1,a2). GST Pi derived from goat blood cells was also capable of binding to labelled SZP specifically (Fig. 4Ab1,b2, Fig. 4Bb1,b2). BSA was used as a negative control (Fig. 4Ac1,c2; Fig. 4Bc1,c2). Both GST isoforms, GST Mu and Pi purified from sperm plasma membrane by RP-HPLC and subsequently labelled with [125I] could bind to immobilised SZP on microtitre plates (Fig. 4C). Binding of GST Mu and Pi to SZP (Fig. 4Cc,e) could be successfully competed out if these isoforms were preadsorbed with SZP prior to incubation with immobilised zona (Fig. 4Cd,f). Total sperm plasma membrane GSTs were used as a positive control (Fig. 4Ca) and SZP preadsorption could displace binding (Fig. 4Cb).
|
Equilibrium saturation binding experiments where SZP was immobilised and subsequently probed with radiolabelled GSTs showed a specific binding (Fig. 5) with an equilibrium constant of (Kd) 11.23 Mx10-6 calculated from Scatchard plots. When GST PiN and MuN peptides were immobilised and subsequently probed with radiolabelled SZP, peptides could also bind to radiolabelled SZP in a typical Langmuir pattern (Fig. 6A,B). This binding could be competed out with unlabelled SZP, showing the specificity of the binding. Unlabelled PiN could compete out the binding of SZP to MuN and vice versa (Fig. 6A,B). Competitive binding curves (Fig. 6C) show that both peptides can compete for zona binding with sperm GSTs. IC50 calculated from normalised binding curves reveals high affinity of GST PiN (IC50, 1.067 µM) for SZP binding compared to MuN peptide (IC50, 6.5 µM) (Fig. 6C). To resolve whether or not the differential affinity of the binding of PiN and MuN was caused by differences in the structure they adopt in solution, circular dichroism (CD) spectra of both peptides were scanned. It showed that the PiN peptide had a broad minima around 206 nm, a characteristic of polyproline II structure, suggesting that the majority of the PiN peptide assumes an extended conformation. MuN peptide has a minima at around 200 nm by CD, which is closer to representing a random coil structure (Fig. 6D).
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Since total SZP bound to sperm GSTs, it was of interest to determine the
component of ZP that was primarily involved in ZP-GST binding. Goat ZP from
mature oocytes separated predominantly into three bands in reducing SDS-PAGE
(Fig. 7, lane a). Densitometric
analysis of the bands from several zona preparations showed a slight variation
in band density (band 1, 1362±47; band 2, 1874±114; band 3,
1999±103, n=6). Biotinylated sperm GSTs could bind to only
band three (Fig. 7, lane b).
This binding could be displaced by preincubation of the labelled GSTs with
excess SZP (data not shown). Anti-ZP3 antibody directed towards a region of
porcine ZP that is conserved among species (EEEKLVFSLRLM) recognised band
three (Fig. 7, lane c);
however, monoclonal antibodies towards a N-terminal peptide of porcine ZP
(PQPVWQDQGQRL) did not recognise band three (data not shown). This suggested
that sperm GSTs could bind to the ZP3-like component of goat ZP. N-terminal
sequencing of this band gave a sequence of DVT/PA/NGPKPQMG. This had extremely
low homology with the N-terminus of known ZP3 sequences from other species. It
is of interest that the N-terminii of ZP3 is the least conserved region among
the species (Zhu and Naz,
1999).
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The above experiments suggested that sperm GSTs bound specifically to ZP in a saturable fashion. The next question was if sperm GSTs are able to bind to ZP, can specific blocking of binding sites for GSTs on the ZP or for the ZP on the sperm GSTs reduce the extent of sperm-oocyte binding. Prior to the blocking experiments, we labelled oocytes with the peptides and visually tested peptide binding. Distinct labelling of the oocytes treated with GST PiN and MuN peptides or sperm plasma membrane GSTs was visible (Fig. 8A-C). Inner cell masses of the oocytes were pushed out to rule out any staining of oocyte cytoplasmic GSTs contributing to the staining of the oocyte ZP. Both the peptides and plasma membrane GSTs did not bind to the embryos. A representative figure is shown (Fig. 8D).
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We blocked sites on the sperm GSTs using antibodies directed against two different regions of GST Pi and Mu. When sperm were treated with anti-GST PiN, MuN, PiC and MuC during capacitation, before incubation with mature oocytes, antibodies against GST PiN and MuN could reduce sperm oocyte binding by 87% and 85%, respectively, in comparison to those treated with preimmune sera (Table 1A). Antisera directed against the peptides (MuC and PiC) designed from the protein region involved in GSH binding (Table 1A) did not interfere with sperm-oocyte binding. No agglutination or immobilisation of the spermatozoa after treatment with the antibodies was observed; hence the reduction in binding was not caused by interference with sperm motility. When oocytes were preincubated with GST PiN and MuN peptides to block sperm GST-binding sites and subsequently incubated with sperm, a decreased level of sperm binding to oocytes (a reduction of 86% and 88%, respectively) was noted (Table 1B). PiC and MuC could not influence sperm-oocyte binding. Preincubation of oocytes with sperm GSTs also blocked sperm-oocyte binding.
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Collectively, the above data suggested that reactive sites on the N-terminus of sperm GSTs were able to interact with counterparts in the ZP. Any blocking of the interacting molecules on either of the gametes reduced sperm-oocyte binding. The binding pattern of sperm GSTs to SZP followed typical saturation kinetics. The data also suggested ZP3 as the component on goat ZP required for binding to sperm GSTs.
GST aggregation either by SZP or anti-GST antibodies induce acrosin
release
Multimeric ZP functionally activates the sperm by crosslinking its
receptors on the surface of spermatozoa
(Leyton and Saling, 1989).
Since the above experiments indicate that sperm GSTs are involved in ZP
binding, we checked whether crosslinking of GSTs on sperm could be
precipitated by SZP. When capacitated sperm were incubated with SZP,
aggregation of both GST-Pi and Mu occurred in a time-dependent manner
(Table 2). Increase in the
number of unlabelled sperm at the end of 2 hours shows the completion of
acrosome reaction. The intactness of the acrosome during the process of
patching and capping was checked by double labelling the same sperm with
Pisum sativum agglutinin linked to rhodamine
(Fig. 9). Treatment with
anti-GST antibodies mimics the action of ZP and induces the aggregation of
sperm membrane GSTs to form a cap-like structure on the acrosomal tip
(Fig. 10A-D). Aggregation
induced by anti-GST Pi antibodies is represented in
Fig. 10. Anti-GST Mu shows
similar pattern (data not shown). The aggregation of GSTs by anti-GST
antibodies also induced the release of acrosin
(Fig. 10E). No crosslinking
and aggregation was observed with Fab fragments of the antibodies (data not
shown). At higher dilutions (1:50), where there was no aggregation of GSTs, no
acrosin release was observed (Fig.
10Ee,i). In these groups, crosslinking was accomplished with the
help of secondary antibodies and thus the release of acrosin was observed
(Fig. 10Ef,j). Acrosin release
by SZP was used as a positive control. Insignificant acrosin release was
observed in second antibody control - the data being similar to group `a'
(data not shown).
|
|
|
When SZP was used to induce acrosin release in sperm treated with antibodies (above 1:100 dilution) or the Fab fragments, acrosin release was inhibited as compared with the controls (Table 3). If SZP was preincubated with the GST peptides and then allowed to interact with the sperm, it could not induce an acrosin release (Table 3) presumably because of occupancy of GST-binding sites on zona showing that the acrosin release is very much dependent on ZP-GST binding.
|
Ca2+ increase is affected by the inhibition of GST-zona
binding
One of the prerequisites for the acrosome reaction to occur is the increase
in intracellular Ca2+ (Fraser,
1998). When sperm capacitated in the presence or absence of
anti-GST PiN and MuN were loaded with the Ca2+-binding dye Fura-2AM
and subsequently treated with SZP, it was observed that the post zona binding
increase in intracellular Ca2+ was significantly less in the
antibody-treated groups than in the controls
(Fig. 11A). Groups where
antibodies were adsorbed with excess peptide (GST PiN) prior to incubation
with sperm, had antibodies that did not affect Ca2+ accumulation.
Saturation of GST-binding sites on SZP with PiN and MuN peptides also
inhibited the zona-induced increase in intracellular Ca2+ in
capacitated sperm. Data obtained microscopically showed a similar pattern
(Fig. 11B).
|
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Discussion |
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In this paper, we make five conclusions: (1) GST activity is associated with the sperm plasma membrane, and this membrane has significantly higher associated GST activity than other somatic cell plasma membranes; (2) the sperm plasma membrane GSTs bind to the ZP3-like component of goat ZP; (3) the GST pool of sperm plasma membrane contain two isoforms, namely GST Pi and Mu; (4) both isoforms show saturable binding to the ZP; and (5) interference with sperm GST-ZP binding leads to functional impairment of spermatozoa, resulting in reduced sperm-oocyte binding.
GSTs are conventionally known as cytosolic enzymes; therefore, the
observation pertaining to the presence of catalytically active GSTs on the
sperm plasma membrane is of great importance. We provide evidence for the
plasma membrane localisation of GSTs by several observations: (1)
immunolocalisation of surface GSTs on live spermatozoa shown by immunostaining
and FACS analysis (Gopalakrishnan et al.,
1998b); (2) recovery of labelled GSTs from the surface iodinated
live spermatozoa (Fig. 2A); (3)
the sperm plasmamembrane had associated GST activities
(Fig. 1); (4) inhibition of in
vitro sperm-zona interaction by anti-GST antibodies
(Table 1); (5) successful
removal of sperm-surface GSTs by controlled trypsinisation of live spermatozoa
(data not shown); (6) the presence of GSTs in high salt buffer extracts of
sperm plasma membrane preparations (Fig.
3); and (7) translocation of around 10% of freshly synthesised
GSTs to plasma membrane shown by 35S-methionine incorporation in
germ cells (data not shown).
The above data provide a clear indication of the plasma membrane
association of GSTs on sperm. This association is particularly valuable to a
cell such as sperm from the point of view of cellular defense. A
detoxification enzyme on the sperm surface would be a great asset, as this
particular cell is exposed to a variety of environments during its passage
from the testis to the fallopian tube
(Yanagimachi, 1981). In
addition, the sperm plasma membrane is rich in oxidation-prone polyunsaturated
fatty acids owing to its fusible nature
(Aitken, 1999
). This feature in
combination with the deficiency of defensive enzymes in sperm owing to the
presence of meagre cytoplasmic space
(Russell et al., 1990
) makes
this cell more vulnerable to oxidative stress in comparison with any other
cell type. It is an established fact that functional incompetence of sperm
owing to lipid peroxidation of their membranes is a major cause of infertility
in the male (Aitken, 1999
).
Since GSTs detoxify products of lipid peroxidation, their association with the
plasma membrane is strategic, as the process of lipid peroxidation is
initiated in lipid-containing structures such as membranes. One report
identifies plasma-membrane-associated GSTs with a molecular mass of
17
kDa (Horbach et al., 1994
) in
hepatocytes, a mass that is close to that of microsomal GSTs. However, the two
isoforms of GSTs that we identify on the sperm plasma membrane have molecular
masses of around 23.4 and 25.6 kDa. It is noteworthy that in our studies the
activity of sperm plasma-membrane-associated GSTs was found to be around
threefold higher than that of hepatic cell plasma membrane GSTs.
Since the mode of attachment of GSTs to the sperm plasma membrane was found to be peripheral, with no covalent interactions, it would be reasonable to speculate that the GSTs are anchored to the plasma membrane through an integral membrane protein. If this is true then the role of GSTs in sperm-oocyte interaction would be solely to recognise and bind to the ZP and help the integral protein to initiate signal transduction. Once bound to the ZP, the hypothetical integral membrane protein may transmit the signal required to trigger membrane fusion and acrosome reaction. Unpublished data from this laboratory with testicular germ cells that are progenitor cells of sperm show that around 10% of total cellular GSTs synthesised are transported to the plasma membrane. Sperm would have acquired the proteins during earlier stages of differentiation, as sperm themselves are transcriptionally inactive.
The orientation of the GSTs on the extracellular side of the plasma
membrane, as shown by the recovery of labelled GSTs from live surface
iodinated spermatozoa, together with our earlier observations of a reduced
fertilisation rate when sperm were treated with anti-GST antibodies, prompted
us to hypothesise that ZP has binding sites for sperm GSTs. Sperm GSTs
fulfilled the essential criteria for any ZP-binding molecule, such as
orientation towards the extracellular side of the apical region of the sperm
head plasma membrane. A quantitative assessment of binding with steady-state
equilibrium binding studies showed that both GST isoforms were able to bind to
ZP, and, since one could compete out the binding of the other, it appears that
both isoforms bind to the same site. The higher affinity of PiN peptides
compared with MuN peptides for the ZP could be explained by their CD profiles.
MuN peptides assume no distinct secondary structure and are randomly coiled or
highly flexible, therefore, binding in this case would be an induced-fit type.
In contrast, PiN peptide has an extended structure (polyproline II like) that
is similar to the secondary structure of this peptide sequence in the original
GST crystal structure (Oakley et al.,
1997). In other words, PiN has a definitive structure, unlike MuN
peptide, and that is possibly the reason for the higher binding affinity of
PiN to ZP compared with MuN. Since the N-terminal sequences are well conserved
within the GST classes and we found that the N-terminal sequence of goat blood
cell GST is similar to goat sperm GSTPi, it was not surprising that blood cell
GSTs could also bind to ZP, although this is biologically inappropriate.
Therefore, GSTs have the structural requirements to bind to the oocyte ZP. It
is possible that during evolution the system might have used this property to
its advantage to meet the unusual demands of the spermatozoa.
We used total SZP in binding studies, as it is known that components other
than the primary binding component assist in ZP binding to sperm receptors
(Yurewicz et al., 1998);
however, it was of interest to identify the primary component on ZP that binds
to sperm GSTs. The binding of sperm GST to the ZP3 component of the goat ZP
indicates similarity with other species, such as mice, where ZP3 acts as the
primary sperm receptor (McLeskey et al.,
1998
). Unpublished data from this laboratory shows that mannose
and N-oligosaccharides do not play a role in ZP-GST binding unlike in the
bovine system, which is close to goat in phylogeny, where N-oligosaccharides
are involved in sperm-ZP interactions
(Nakano et al., 1996
). This
however, does not rule out the possibility of sperm binding to ZP through
mannose residues that are involved with other sperm proteins. Since the
characterisation of goat ZP is yet to be done, the types of oligosaccharide
present in goat zona and their role in GST binding have to be carefully
investigated.
Given the importance of ZP binding to sperm, which brings about functional
changes in the male gamete (Breitbart et
al., 1997), the inhibition of ZP-sperm GST binding should
theoretically interfere with the required changes in sperm and consequently
disrupt the sperm-oocyte interaction. Since the in vitro fertilisation system
provides an excellent tool to dissect the phases of fertilisation
(Frayne and Hall, 1999
), we
used this system and several reagents that either bound to GSTs on sperm or
GST recognition sites on the ZP to decipher the precise phase of fertilisation
in which sperm GSTs were involved. Blocking of GST-zona binding by either
anti-GST antibodies or N-terminal peptides could functionally impair
spermatozoa in terms of acrosin release and binding to the oocyte, showing
that sperm GST-ZP binding was a necessary event for successful fertilisation.
This was in contrast to the inability of peptides (GST PiC and MuC), designed
from a region of the GST Pi and Mu involved in GSH binding or antibodies
against them, to block the gamete interaction. The inability of these
antibodies to block such events was not caused by inaccessibility of the
antibodies to the desired region as they recognised live sperm in indirect
immunofluorescence (data not shown). Therefore, it is possible that the GSH
binding site has no role in sperm-oocyte binding. The above observations have
clearly demonstrated the importance of the N-terminal regions of GST Pi and Mu
in the gamete recognition process. The inability of the antibodies to induce
agglutination or immobilisation of spermatozoa after treatment with antibodies
confirms that reduced binding is not caused by lack of motility or cell
agglutination. It is known that once the oocyte is fertilised, embryos become
refractory to molecules involved in primary binding
(Yanagimachi, 1981
);
therefore, binding of the GSTs and peptides to the oocyte but not the embryos
further confirm the possibility of sperm GSTs as primary recognition molecules
in sperm-oocyte interactions.
As SZP could aggregate sperm GSTs and prevention of binding of SZP to sperm
led to the inhibition of acrosin release, sperm GSTs must have a role in post
zona binding functions of sperm. This was further corroborated by the observed
inhibition of the intracellular Ca2+ increase that is a
prerequisite for acrosome reaction
(Publicover and Barratt, 1999)
when anti-GST PiN and MuN antibodies were used to block the sperm GST-zona
interaction. An interpretation of this data would be that initiation of
acrosome reaction was inhibited owing to the inability of the cell to acquire
the required level of intracellular Ca2+, which is caused by
inhibition of ZP binding to sperm GSTs.
In the past decade, there has been a considerable effort to identify the
proteins and pathways utilised during gamete recognition, and the molecular
bases of this process are beginning to be understood. Zona-binding candidates
identified include beta 1, 4-galactosyltransferase
(Miller et al., 1992;
Miller et al., 1993
) and sperm
adhesins (Topfer-Petersen and Calvete,
1995
), in addition to some others
(McLeskey et al., 1998
). The
existence of multiple receptors indicate that sperm-zona binding may involve
either a complex of receptors, consisting of different members, or redundancy
built into the system because of the important nature of the event. It is also
possible that a species-specific preference for a particular molecule may
exist that justifies the presence of a molecule on sperm of several species
but may be functionally important only in selected groups. One such example is
beta galactosyltransferase, which is present on the surface of sperm of
multiple species, but it is functionally important in only a few
(Rebeiz and Miller, 1999
). The
multiple sperm-surface components that have been implicated in zona binding
may or may not function in concert with sperm GSTs.
The ability of the cells to make multifunctional proteins is of fundamental importance to the course of evolution. As sperm cells are terminally differentiated and possess a variety of functional domains, it is possible that GSTs have evolved to function in the very unique structural environment of this cell. Therefore, the ability of GSTs to serve as receptors for cell-cell interactions, which is evident from this study, and as protective enzymes observed in our earlier studies might reflect the unusual demands the transcriptionally inactive sperm meets in different parts of the reproductive tract to reach the oocyte. It is possible that the presence of two isoforms on the surface gives the cell a larger flexibility in terms of dealing with toxins. The delineation of dual function of GSTs in sperm opens an interesting prospect for future studies into the mechanisms of infertility. Defects in either zona-binding capacity or the catalytic function of these molecules may be responsible for certain forms of infertility.
In summary, our studies clearly demonstrate that GSTs on sperm membranes serve very important functions. In addition to being enzymatically active, they mediate the binding of ZP to sperm, which brings about important prefertilisation changes.
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Acknowledgments |
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