1 Centre of Dermatology and Andrology, Justus Liebig Universität Giessen, Gaffkystr. 14, 35385 Giessen, Germany and 2 The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, 601 Colley Avenue, Norfolk, Virginia 23507, USA
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Abstract |
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Key words: hemizona assay/oocyte/synthetic peptide antisera/zona pellucida/ZP3
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Introduction |
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The reported number of discrete glycoproteins comprising the ZP varies among different species (Dunbar et al., 1981; Timmons and Dunbar, 1988
). The relations of these proteins across species is unclear. Their biochemical analysis is difficult due to problems in isolating large quantities of pure protein and the relatively large amount of glycosylation of the peptide backbone. With advances in molecular biology, it became apparent that a more direct approach to answer the questions about the composition and functions of the ZP was to clone the genes that code for the ZP proteins. Recombinant technology and its application can be used now for the study of spermoocyte interaction (Chapman and Barratt, 1997). Recently, full length cDNA clones of ZP3 for different mammalian species (including mouse, hamster, human, rabbit, marmoset, pig, cat, cow and dog) have been isolated (for review see Harris et al., 1994
). However, there is confusion in the literature as to the nomenclature of ZP proteins. The names ZP2, ZP1 and ZP3 usually refer to molecular mass, although the terms ZPA, ZPB and ZPC have been coined to express functions and respective genes coding for these proteins (Harris et al., 1994
). Genes encoding ZP2 and ZP3 have been shown to be conserved among mammals and the DNA sequences of ZP3 cDNA coding regions show extensive homology between species studied so far. Despite these genetic similarities, ZP3 proteins from different species are heterogeneous with respect to their physical, chemical, structural, and immunological properties due to post-translational modifications. In particular, different forms of ZP glycosylation are responsible for this observation (Timmons et al., 1990
; Dunbar et al., 1991
).
It has been well established that the immunogenic and antigenic properties of ZP glycoproteins are very complex and vary among species (Dunbar et al., 1981, 1994
). These observations have become crucial for the development of contraceptive vaccines using ZP protein as well as synthetic ZP3 peptide antigens from various species (Timmons et al., 1990
; Afzalpurkar et al., 1997a
,b
; Gupta et al., 1997
; Talwar, 1997
; Zhang et al., 1997
). For comparative studies and for the evaluation of the molecular mechanisms of spermZP interaction in general, efforts have been made to elucidate the structures and physiological properties of mammalian ZP3 proteins (Dunbar et al., 1991
). These results were important for the identification of functional domains of the ZP. For example, recent data showed that a functionally active murine ZP3 domain is located at the carboxy-terminal half of the polypeptide (Rosiere and Wassarman, 1992
).
The aims of the present study were: (i) to identify and localize ZP3 protein in human and mouse oocytes in ovary sections (using immunohistochemistry and transmission electron microscopy) and in immunoblots from isolated oocytes. Additionally, we examined native hemizonae pellucidae obtained after microbisection of isolated, denuded human oocytes (using immunochemistry). Antibodies generated against synthetic ZP3 peptides (anti-ZP3 antisera) were used for ZP3 immunolocalization; and (ii) to assess the capacity of the anti-ZP3 antisera to influence human spermZP binding in homologous assays. A competitive hemizona assay was used to test the effect of the anti-ZP3 antibodies on human spermZP interaction. Results demonstrated that the anti-ZP3 antisera can be used as highly specific markers for defined human and mouse ZP3 epitopes in ovarian tissue and in isolated oocytes and provide evidence for the use of the antisera in the evaluation of functional ZP3 protein domains.
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Materials and methods |
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Post-mortem human ovaries and surplus human oocytes from women undergoing IVF therapy were used in the competitive hemizona assays following institutional review board approval (IRB) from Eastern Virginia Medical School. Denuded human oocytes used for immunoblots were from patients participating in the in-vitro fertilizationembryo transfer programme at the department of Obstetrics and Gynaecology, University of Bonn (Bonn, Germany) following the appropriate IRB approval. These specimens represented material that was not used in the programme and which would normally have been discarded. All human oocytes used in the studies were recovered from antral follicles; these oocytes were fully grown and were at the prophase I stage of nuclear development. Previous studies have demonstrated the functional competence of these oocytes in spermZP binding studies (Oehninger et al., 1992).
Anti-ZP3 antisera
Defined ZP3 peptide sequences were used to synthesize peptides in order to generate specific antisera. The sequences of the synthetic ZP3 peptides used in these studies were deduced from cDNA clones coding for mouse and human ZP3 (Kinloch et al., 1988, 1990
; Chamberlin and Dean, 1990
). The sequence of the ZP3-9 peptide (a mouse specific sequence) is C-S-N-S-S-S-S-Q-F-Q-I-H-G-P (ZP3 amino acid numbers 327340, Chamberlin and Dean, 1990
), whereas the sequence of the ZP3-14 peptide (human specific) is C-G-T-P-S-H-S-R-R-Q-P-H-V-M (ZP3 amino acid numbers 327340, Chamberlin and Dean, 1990
). The amino acid sequences are given in the one-letter code. Antisera were generated against these peptides and designated as AS ZP3-9 and AS ZP3-14. In addition, two other anti-ZP3 antisera generated against common (conserved) peptide sequences (synthetic peptides ZP3-5 and ZP3-6) and designated as AS ZP3-5 and AS ZP3-6 were used (Hinsch et al., 1994a
). The methodologies employed for peptide synthesis and antisera generation have been described previously (Hinsch et al., 1994a
; Hinsch and Hinsch, 1996
). Polyclonal chicken antibodies against purified porcine ZP3ß (Sinowatz et al., 1995
) used as controls in competitive hemizona assays were kindly provided by Dr E.Töpfer-Petersen (Institut für Reproduktionsmedizin, Hannover, Germany). These antibodies are directed against a fraction of 5565 kDa porcine ZP proteins that was digested with endo-ß-galactosidase from E. freundii.
Immunohistochemistry
For immunohistochemical studies, ovaries were cut into small cubes, fixed in Methacarn fixative (60% methanol, 30% chloroform, 10% acetic acid), dehydrated with alcohol and subsequently embedded in paraffin. Sections of 4 µm were deposited on glass slides, deparaffinized with xylene, rehydrated through a graded sequence of alcohols and rinsed with distilled water. Sections were subsequently treated with anti-ZP3 antibodies or control antibodies. The binding sites were visualized with anti-rabbit Ig antibodies (dilution 1: 100) (DAKO, Hamburg, Germany), the peroxidaseantiperoxidase method (DAKO) and 3,3'-diaminobenzidine (DAB) (Sigma, Deisenhofen, Germany). The specificity of the reaction was determined by applying mouse-specific ZP3 peptide antibodies on human ovary sections or using human-specific ZP3 peptide antibodies on mouse ovary sections. Furthermore, the corresponding pre-immune sera were used as negative controls. Finally, sections were counter-stained with haematoxylin (Sigma).
Immunochemical detection of ZP3 in human hemizonae
Detection of anti-ZP3 antibodies bound to human hemizonae was essentially performed as described before (Hinsch et al., 1994b; Oehninger et al., 1996
). Hemizonae were prepared and mounted to glass slides and air dried over night. For immunochemical studies, hemizonae were treated with antisera at appropriate dilutions. Specific binding of anti-ZP3 antibodies was visualized with biotinylated anti-chicken-Ig antibodies (dilution 1:100) and peroxidase labelled streptavidin (dilution 1:100) (Vector, Peterborough, UK) or with anti-rabbit-Ig antibodies (dilution 1:100) (DAKO) and the peroxidaseantiperoxidase method (DAKO). DAB (Sigma) was used as colour reagent.
Electron microscopy
Mouse and human ovaries with ripe follicles were fixed in 0.1% (v/v) glutaraldehyde and 1.25% (w/v) paraformaldehyde in cacodylate buffer (100 mM, pH 7.3). Samples were washed in cacodylate buffer (100 mM, pH 7.3), dehydrated with graded alcohols and then embedded in LR-White. LR-White resin was from Plano (Wetzlar, Germany), and glutaraldehyde was purchased from Serva (Heidelberg, Germany). Sodium cacodylate was obtained from Merck (Darmstadt, Germany). Ultrathin sections were cut on an LKB ultramicrotome equipped with a diamond knife, and sections were collected on nickel grids. Subsequently, sections were pre-treated with 0.1% (w/v) glycine in Tris-buffered saline solution (TBS, 150 mM NaCl, 20 mM Tris, pH 7.4) for 20 min, followed by three washes with TBS. Sections were then incubated with 10% normal goat serum in TBS for 30 min at room temperature. Thereafter, sections were exposed to antisera (1:5 dilution in TBS) or pre-immune sera (1:5 dilution in TBS) for 1 h at room temperature. The specimens were subsequently incubated with goat anti-rabbit IgG antibodies conjugated with colloidal gold (particle size of 30 nm) (Bio Cell, Cardiff, UK) at a 1:10 dilution for 45 min. Finally, sections were stained for 20 min with 2% (w/v) uranyl acetate, viewed and photographed with a Philips 201 G transmission electron microscope operated with 80 kV.
Gel electrophoresis and immunoblotting
SDSPAGE was performed according to Laemmli (1970) with some modifications. Denuded oocytes were dissolved in sample buffer. Solubilized proteins were loaded onto a BioRad mini-Protean gel electrophoresis system (BioRad, München, Germany). Slab gels were composed of 10% (w/v) acrylamide and 0.21% (w/v) bisacrylamide. SDSPAGE was performed under reducing conditions at room temperature and at a constant voltage of 100 V per gel for 45 min. Immunoblotting was essentially performed as described before with some modifications (Hinsch and Hinsch, 1996). Briefly, proteins resolved by SDSPAGE were transferred to PVDF membranes (Millipore, Eschborn, Germany) at constant voltage (35 V for 70 min). Membranes were air-dried, stained with Ponceau S and blocked with 5% (v/v) teleostean gelatine (Sigma) in phosphate-buffered saline (pH 7.4). Thereafter, filters were incubated for 1 h with antisera at appropriate dilutions. After extensive washing, membranes were incubated with 1:3000 diluted peroxidase-conjugated anti-rabbit IgG for 1 h. Specific binding of antibodies was visualized by the enhanced chemiluminescence Western blotting detection reagents (ECL) (Amersham Buchler, Braunschweig, Germany) and subsequent exposition to X-ray films (Fuji Photo Film, Düsseldorf, Germany) as recommended by the supplier. The specificity of reaction was determined by pre-absorption of the antiserum with the peptide employed as immunogen. Antisera were pre-incubated with 10 µg of peptide/ml for 1 h at room temperature and subsequently assayed in immunoblotting.
Competitive hemizona assay (HZA)
The competitive-HZA was used to assess the capacity of anti-ZP3 antisera to interfere with the process of spermZP binding. The assay was carried out following the HZA that was previously described by Burkman and co-workers (1988) and used in our previous studies (Oehninger et al., 1996). Denuded salt stored human oocytes were microbisected into matching hemizonae using Narishige micromanipulators (Narishige, Tokyo, Japan). Hemizonae were separated from oocyte cytoplasmic particles by micropipetting. Matching hemizonae were incubated with test (anti-ZP3 antisera) sera or control sera (pre-immune sera) (diluted 1:30) for 2 h in an incubator at 37°C in 5% CO2 in air. Human spermatozoa were collected by fertile men and the motile sperm fractions were obtained using a swim-up technique effected in Ham's F-10 (Gibco Laboratories, Grand Island, New York, USA) and capacitated in the presence of 0.3% (w/v) human serum albumin (Sigma) for 1 h in an incubator at 37°C in 5% CO2 in air. Thoroughly washed hemizonae were placed in a sperm suspension containing 0.5x106 motile spermatozoa/ml for 4 h. Following spermhemizona co-incubation in separate 100 µl droplets under oil and at 37° C in 5% CO2 in air, each hemizona was rinsed to remove loosely attached spermatozoa. The number of motile spermatozoa tightly bound to the outer surface of each hemizona was counted, and the hemizona index (HZI) was calculated (HZI = [number of spermatozoa bound for test hemizona/number of spermatozoa bound for control hemizona]x100).
Statistical analysis
Results are presented as means ± SEM. On the assumption that an HZI of 100 reflects no difference in spermZP binding, statistical analysis of HZI was carried out using the one sample t-test.
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Results |
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Antiserum AS ZP3-14, directed against a human ZP3-specific peptide, recognized human ZP. As shown in a human ovary section (Figure 1,C), human ZP protein was strongly recognized by AS ZP3-14. Positive staining was seen as a dark ring enclosing the oocyte. This reaction appeared more homogeneous, and no bilamellar structure was apparent as in Figure 1
,A. In human oocytes, antigenic ZP3 material was also recognized in the ooplasm, although the deposition of colour substrate was even less intense as compared to AS ZP3-9 reaction in the mouse. No reaction was detectable within cells surrounding the oocyte. However, extensions of ZP protein at the outer ridge of the ZP were observed. This reaction appeared to involve the apical part of cumulus cells as indicated by the arrow. With the exception of apically stained cumulus cells the antiserum did not react with other cells of the ovary or of any other human tissues tested (not shown). Antisera AS ZP3-9 and AS ZP3-14 did not detect any other antigen in various mouse tissues that were tested, including the oviduct, uterus, kidney, adrenal, liver, heart, skeletal muscle, brain, spleen, lung, stomach, small intestine, and gut (not shown).
Transmission electron microscopy results
The ultrastructure of the mouse and human ZP3 was investigated with immune electron microscopy using the mouse ZP3-specific antiserum AS ZP3-9 and the human ZP3-specific antiserum AS ZP3-14 respectively (Figures 2 and 3). In ultra-thin sections of mouse ovaries, the sites of immunoreaction of AS ZP3-9 were almost exclusively found in the ZP (Figure 2A
,C). The ZP appeared to be the only structure that was intensely labelled, but occasionally areas almost free of gold particle deposition were also observed (Figure 2B and D
, asterisk). The distribution of gold particles in the ZP matrix appears uniform and homogeneous. However, careful examination of distribution of gold particles revealed specific binding to dense, filamentous material. As demonstrated in a higher magnification, gold particles were arranged in a circular network of cord-like structures (Figure 2C
, arrowheads). Immunoreactive material was also detected as extension-like gold particle deposition towards the adjacent cumulus cells (Figure 2C
, arrows). Only a few gold particles, but above background deposition of particles, were found in the ooplasm. Cumulus cells that were seen in direct contact with the ZP were found almost negative to immunolabelling.
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Comparable results, but with a less intense deposition of gold particles, were observed with the human specific antiserum AS ZP3-14 when assayed with ultra-thin sections of human ovaries (Figure 3B and D). Immunoreaction was clearly seen in the ZP (arrowheads). Immunoreactive material was homogeneously distributed throughout the ZP without any circular arrangement, as was demonstrated in the mouse ZP. A few gold particles (barely above background) were observed in the ooplasm and almost none in the adjacent cumulus cells (not shown). Only very few gold grains were observed in control sections that were treated with mouse ZP3 specific antiserum AS ZP3-9 (Figure 3A and C
).
Immunoblotting studies
For biochemical characterization of the antigen detected by the AS ZP3-9 and AS ZP3-14, immunobiochemical studies were carried out. Characterization of the antigens detected by AS ZP3-9 and AS ZP3-14 was performed by Western blotting of mouse and human oocyte proteins followed by immunodetection of the respective antigen. In mouse oocytes, antiserum AS ZP3-9 recognized a polypeptide that appeared as a broad band with an apparent molecular mass of ~6997 kDa (Figure 4, lane a). An additional well-focused band with an apparent molecular mass of ~66 kDa was also visible. As demonstrated in Figure 4
, lane b, AS ZP3-9 did not cross-react with antigens in human oocytes. The reaction of antibodies with the above described immunoreactive oocyte proteins was blocked when the antisera were pre-incubated with their respective synthetic ZP3 peptide used as antigen (not shown).
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Competitive HZA studies
The competitive-HZA was developed to assess the capacity of anti-ZP3 peptide antisera to interfere with the process of human spermZP binding. As depicted in Table I, human test hemizonae pre-incubated with AS ZP3-5 and AS ZP3-6 revealed an average of 31 and 89 human spermatozoa respectively that were bound to the outer surface, whereas the corresponding control hemizonae yielded a mean value of 32 and 89 tightly bound spermatozoa respectively. The calculated hemizona indices (113 for AS ZP3-5 and 104 for AS ZP3-6) as well as the statistical analysis revealed no significant inhibition of sperm binding to the ZP by antisera directed against conserved ZP3 sequences. Similar results were obtained with AS ZP3-14, an antiserum generated against a human ZP3 amino acid sequence. The number of spermatozoa bound tightly to test hemizona (31 sperm cells) did not substantially differ from the number bound to control hemizona (30 cells). The hemizona index of 107 indicated no interference of antibodies bound to the hemizonae with the capacity of sperm to bind to the ZP. On the other hand, treatment of hemizonae with anti porcine ZP3ß antibodies did substantially influence spermzona interaction. After incubation of hemizonae with anti porcine ZP3ß only 6 spermatozoa bound to the test hemizonae, whereas 83 sperm cells on average bound to control hemizonae. The calculated hemizona index (seven on average) as well as the statistical analysis revealed a significant inhibition of sperm binding to the ZP by anti porcine ZP3ß as compared to controls (P < 0.0001).
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Treatment of human hemizonae with the human ZP3 specific antiserum AS ZP3-14 and subsequent immunoperoxidase detection of bound antibodies resulted in a clear deposition of substrate throughout the hemizona (Figure 5, no. 3). The periphery of the hemizona revealed a dark and ragged staining pattern that became less intense towards the outer border of the bisected ZP.
Incubation of human hemizonae with polyclonal chicken antibodies against purified porcine ZP3ß resulted in an intense staining of the ZP (Figure 5, no. 4). A dark ragged, partially discontinuous ring of substrate deposition was observed at the border of the hemizona, whereas the central portion of the hemizona exhibited a more smooth and homogeneous labelling with intensely stained fern-like structures. Almost no staining was observed with control hemizonae pre-treated with the mouse ZP3 specific antiserum AS ZP3-9 (Figure 5
, no. 5) or without pre-treatment with antibodies (Figure 5
, no. 6).
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Discussion |
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Immunohistochemical studies revealed that AS ZP3-9 (generated against a mouse specific sequence) specifically recognized mouse ZP. AS ZP3-9 exhibited a similar bi-lamellar staining pattern of the ZP as described for other anti-ZP3 synthetic peptide antisera in the mouse (Hinsch et al., 1994a). The significance of this bilamellar composition of ZP is not clear. Earlier reports have demonstrated that ZP exhibit distinct layers that have different physical and biochemical properties (Dunbar et al., 1991
, 1994
) indicating a highly organized extracellular matrix synthesized by the oocyte and/or its surrounding cells. When human ovary sections were probed with AS ZP3-14 (generated against a human specific sequence), the human ZP protein was recognized. The lack of cross-reaction with mouse ZP indicates species specificity of antibody recognition.
The ultrastructural studies with AS ZP3-9 and AS ZP3-14, however, did not reveal any bi-lamellar structure of mouse ZP or human ZP respectively. Occasionally, we found areas within the ZP that were free of gold particle deposition, which might be caused by cumulus cell bodies or branches that are directly attached to the oocyte. Extensions of ZP3 material towards the surrounding cumulus cells were observed. As has been demonstrated in an earlier study using light microscopy, at high magnifications, extension-like staining was evident in human and rat ovaries (Hinsch et al., 1994a). We propose that ZP protein branches could facilitate the penetration of the spermatozoon through cumulus masses to recognize the oocyte and to bind to the ZP. Another possible role of ZP3 protein spreading towards cumulus cells could be the stabilization of the cumulus oophorus. Rankin and co-workers (1996) demonstrated that an insertional mutation in the mouse ZP3 gene results in a lack of ZP matrix and in disorganization of the cumulus oophorus. They suggested that the absence of ZP matrix in which ZP processes tether the cells of the corona radiata might hinder proper cumulus organization.
Immunoelectron optical studies with AS ZP3-9 depicted a circular, network-like deposition of gold particles throughout the mouse ZP. This pattern of gold particle distribution was not observed in the human ZP. This may be due to a lower affinity of the AS ZP3-14 antibodies. Circular filamentous structures of ZP were previously described with monoclonal antibodies directed against hamster oviductal ZP antigen (Kan et al., 1989). The antigen was only detectable after ovulation and has not been detected in hamster ovarian tissue. Using anti-ZP3 peptide antibodies, we also identified immunoreactive material in the mouse ooplasm and, to a lesser extent, in the human ooplasm. In a recent study, we demonstrated that in oocytes of most of the species that were tested and particularly in the porcine oocyte, high amounts of immunoreactive ZP3 were detected in the ooplasm (Hinsch et al., 1994a
). Takagi et al. (1989) obtained similar results with monoclonal antibodies against porcine ZP proteins showing immunoreactive material in both the ZP and oocyte cytoplasm. Because de novo synthesis of ZP proteins occurs during oogenesis (Bleil and Wassarman, 1980b
; Shimizu et al., 1983
), it is not surprising to detect antigenic ZP3 material inside the cell which probably reflects ZP3 protein synthesis and transport of ZP3 during oogenesis.
In addition to the ZP and ooplasm, considerable amounts of mouse ZP3 protein were detected in the first two layers of granulosa cells surrounding the mouse oocyte. The finding that ZP3 material is present in murine cumulus cells might be inconsistent with data published recently. Others have reported on the oocyte-specific expression of mouse ZP3 (Roller et al., 1989; Epifano et al., 1995
). Additionally, it has been shown that the mouse ZP3 gene directs expression of firefly luciferase specifically to growing oocytes in transgenic mice providing evidence that expression of genes encoding mouse ZP3 protein is oocyte-specific (Ringuette et al., 1986
; Lira et al., 1990
); at the protein level, this observation was supported by electron microscopy studies investigating rat and porcine ovaries (Kang, 1974
; Takagi et al., 1989
). However, the precise site of ZP3 synthesis in the follicle is still controversial because varying results were published for different species. Recent studies investigating human and rhesus monkey ovaries with antibodies against recombinant human ZP3 clearly demonstrated the presence of ZP3 in primordial follicles and in granulosa cells (Grootenhuis et al., 1996
). Wohlgemuth et al. (1984) and Prasad et al. (1996) showed that rabbit ZP originates from both the oocyte and the follicular cells. Using in-situ hybridization techniques, porcine ZP3 alpha mRNA was localized in the oocyte and in follicle cells (Kolle et al., 1996
). The authors observed that during folliculogenesis ZP3 alpha mRNA was synthesized in both cell types in sequence. Further studies in the rabbit demonstrated that granulosa cells cultured in vitro transcribe mRNA encoding a 55 kDa ZP protein that contains sperm receptor activity and that those cells also synthesize, glycosylate and secrete the 55 kDa ZP protein (Lee and Dunbar, 1993
; Dunbar et al., 1994
).
Discrepancies in the site of ZP3 synthesis may be ascribed to species-specific particularities and/or to differences in the specificity of antibodies or RNA-probes. ZP3 antigen located in mouse cumulus cells as has been shown in this study could also represent phagocytosed or degraded ZP3 protein that was originally synthesized by the oocyte. In summary, the site of ZP synthesis in various mammals remains controversial. A combined approach of immunological studies with highly specific antibodies and advanced techniques of molecular biology will probably allow clarification of this issue.
Here, the antisera were also applied for immunobiochemical detection of ZP3 protein in isolated mouse and human oocytes. AS ZP3-9 antibodies detected mouse ZP3 protein in mouse oocytes as a broad and smeary band between 69 and 97 kDa. An additional, well focused band of about 66 kDa was evident. The origin of this low molecular mass ZP3 protein is not clear; it might represent proteolytic degradation of ZP3 polypeptides or ZP3 protein that originates from the ooplasm and has not been completely modified post-translationally (e.g. different pattern of glycosylation). Our results are consistent with the range of apparent molecular masses for mouse ZP3 that were published before by various investigators (for synopsis, see Yanagimachi, 1994). The human specific AS ZP3-14 reacted with a polypeptide of ~6365 kDa. This polypeptide detected by the antibodies is most probably identical with human ZP3 protein because Shabanowitz and O'Rand (1988) estimated the apparent molecular mass of human ZP3 between 57 and 73 kDa. For the evaluation of antibody specificity, important control experiments were performed. It could be demonstrated that no reaction with ZP proteins was observed when mouse oocyte or human oocyte proteins were tested with the human-specific ZP3 antiserum AS ZP3-14 and the mouse-specific ZP3 antiserum AS ZP3-9 respectively.
Our results show that anti-ZP3 peptide antibodies react with ZP3 protein present in oocyte preparations. The data demonstrate that in the view of the similarity of human and mouse ZP3 proteins (67% protein homology as estimated by Harris et al., 1994), anti-ZP3 synthetic antisera can be used as highly specific markers for ZP3 proteins of different species.
Having demonstrated the specificity of anti-ZP3 synthetic peptide antisera, we sought to investigate if anti-ZP3 antibodies may allow us to unveil linear defined ZP3 epitopes of physiological relevance, particularly domains for primary sperm binding. The human competitive HZA was used to assess the capacity of anti-ZP3 peptide antibodies to interfere with the process of human spermZP binding. We applied antisera against a human ZP3 specific synthetic peptide (AS ZP3-14) and against two different amino acid sequences that are identical or very similar (conserved epitopes) in most mammalian ZP3 proteins (AS ZP3-5 and AS ZP3-6). It is of interest to note that studies with the antiserum AS ZP3-6 showed that this antiserum can be used as a clinical marker for the functional integrity of the human ZP (Oehninger et al., 1996). Moreover, manipulation of oocytes such as cryopreservation in the presence of 1,2 propanediol resulted in less binding of anti ZP3-6 synthetic peptide antibodies to hemizona pellucida, whereas spermZP binding potential of those oocytes was not influenced (Hinsch et al., 1994b
).
Applying the human competitive-HZA, we found no significant decrease of sperm tight binding to hemizonae that were treated with anti-synthetic ZP3 peptide antibodies. In contrast, binding of sperm was strongly affected when hemizonae were incubated with antibodies against porcine ZP3ß protein. In fact, spermhemizona interaction was almost completely blocked by antibodies against purified partially deglycosylated porcine ZP3ß protein. This observation indicates that polyclonal antibodies against the porcine ZP3ß polypeptide detect one or more human ZP epitopes that are of physiological significance for sperm binding. However, the particular epitope(s) of physiological relevance is (are) still unknown; it is not clear at all if remaining glycosylated ZP3 sites or if protein domains are affected by antibody binding. It has to be noted that the epitope(s) detected by anti-porcine ZP3ß antibodies might not be identical with the domain of true physiological importance. Steric hindrance of antibody molecules could influence other human ZP3 domains which are different from but closely located to the epitopes detected by the antibodies. The data obtained with three different anti-ZP3 synthetic peptide antibodies (AS ZP3-5, AS ZP3-6 and AS ZP3-14) indicate that the domains detected by them are not relevant for spermZP binding. Because only two conserved ZP3 domains (AS ZP3-5 and AS ZP3-6) and one human specific domain (AS ZP3-14) were evaluated, it cannot be concluded from these data that the ZP3 protein backbone is not relevant for spermZP recognition and binding. Interestingly, antibodies against a synthetic peptide corresponding to bonnet monkey ZP3 protein were reported to inhibit human spermoocyte binding in vitro (Afzalpurkar et al., 1997b). This synthetic ZP3 peptide was designed on the basis of epitope mapping data of the bioactive monoclonal antibody in the murine model (Millar et al.; 1989
); it consisted of 24 amino acids and included a sequence of 13 amino acids that was identical with the ZP3-14 peptide. Thus, antiserum AS ZP3-14 is a sensitive tool; the antibodies detect a particular epitope that is close to a ZP3 region involved in spermZP interaction but they do not prevent primary binding.
Finally, we evaluated whether the antibodies bind to intact ZP3 protein which has not been chemically modified (e.g. after Western blotting procedures or immunohistochemical treatment). This information is important for the application of anti-ZP3 antisera in in-vitro tests. In order to postulate a role of the antibodies on the interference of spermoocyte interaction, it has to be demonstrated that the antibodies in fact bind to the intact ZP. Immunochemical studies with intact rehydrated salt stored human ZP revealed that hemizonae incubated with anti-ZP3 antisera that were generated against a human specific sequence (AS ZP3-14) and against common or conserved epitopes (AS ZP3-5 and AS ZP3-6) displayed intense immunoperoxidase reactions. The results show that the antibodies bind to human hemizonae with high affinity (most probably interacting with their defined linear ZP3 domains) and suggest that the antisera recognize functionally intact human ZP protein.
In summary, we have demonstrated that anti-ZP3 synthetic peptide antibodies react in a specific manner with mouse and human ZP within ovarian tissue and can be localized by immunohistochemistry and transmission immunoelectron microscopy. In addition, human specific antibodies detect denatured and native human ZP3 protein in immunoblotting and in microbisected hemizonae from isolated oocytes. The human ZP3 peptide sequences tested in this study do not reflect ZP3 domains of physiological importance for spermZP binding. Antibodies directed against purified porcine ZP3ß protein, on the other hand, reacted with human ZP epitopes that seem to be relevant for spermZP binding. We conclude that our studies may contribute to the understanding of crucial events that occur in spermZP interaction leading to fertilization and we estimate that the use of antibodies against synthetic ZP3 peptides may become useful for the investigation of the molecular mechanisms of spermoocyte interaction.
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Acknowledgments |
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Notes |
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References |
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Submitted on January 20, 1998; accepted on October 13, 1998.