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Gamete Biology |
Department of Molecular Genetics, Institute of Medical Biochemistry, Max F. Perutz Laboratories, Medical University of Vienna, Biocenter, A-1030 Wien, Austria
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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fertilization, follicle, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The primary structures of the zona pellucida (ZP) proteins have been conserved throughout vertebrate evolution. This alone suggests that the species specificity of fertilization does not reside solely in the primary sequence of these proteins, but rather that posttranslational modifications such as glycosylation, or three-dimensional arrangements of the matrix, are involved, and experimental evidence indeed supports this idea [6]. Thus, carbohydrates have been shown to be important in sperm-egg interactions. The nature of these oligosaccharides varies between species. In the mouse, O-linked sugars on the ZP3 protein are essential for sperm binding [7], whereas in other animals, N-linked sugars appear to be required [8]. In pigs, some reports showed dependence of binding on the presence of N-linked carbohydrates [9], but others could not find a requirement for any glycosylation at all [10]. In some species, initial interactions between sperm and egg do not involve just ZP3 alone. In the pig, sperm-binding activity resides in a heterodimer between the ZP1 and the ZP3 homologues [10], and the ZP1 homologue ZPB has been shown to play a major role in sperm binding to the bovine zona [11]. Likewise, Escherichia coli-produced recombinant bonnet monkey ZPB protein has been shown to bind to spermatozoa [12, 13]. Similarly, in rabbits, the ZP1 homologue r55 can bind to the anterior surface of the sperm acrosome, and antibodies against it prevent sperm binding to the ZP [14].
Variations between species are also apparent in the origin of the ZP proteins. In the mouse [15] and in Xenopus laevis [16], the ZP proteins are expressed exclusively in the oocyte. In teleost fish, expression is either ovarian or hepatic [17, 18]; in medaka, ZP proteins derive from both tissues [19]. In rabbits, the synthesis of the ZP1 homologue is not restricted to the oocyte, but also takes place in granulosa cells [20], as has been suggested for cats [21], cows [22], and humans [23]. In cows, ZP2 has been detected in the follicle cells surrounding the oocyte [24].
In birds, the structure analogous to the ZP, the perivitelline membrane (PVM), contains only two major components, which have been shown to be homologues of the mammalian ZP1 and ZP3 proteins [25, 26], and are called here chkZP1 and chkZPC, respectively. Because of an apparent lack of a ZP2 homologue, the three-dimensional structure of the PVM has to be different from that of the mouse ZP. It is unclear how PVM assembly is regulated because synthesis of the avian ZP proteins takes place in distinct tissues. Thus, the liver of a laying hen produces large amounts of chkZP1 under the influence of estrogen [26], whereas chkZPC is secreted from follicular granulosa cells in the ovary [27] and is not induced by estrogen (unpublished data).
To begin to understand the biochemistry of sperm-egg interactions, we took advantage of the fact that, because of the enormous size of the avian oocyte, chkZP1 is synthesized in large quantities in extrafollicular tissues and that cultured granulosa cells readily secrete chkZPC into the medium. These circumstances allowed us to isolate large quantities of native ZP components by immunoaffinity chromatography and use these preparations in sperm-binding experiments.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Thirty- to 40-wk-old Derco brown laying hens and roosters (Heindl Co., Vienna, Austria) were used as source of eggs, follicles, and tissues, except where indicated. Semen was collected by abdominal massage [28]. All animals were kept in the animal care facilities of the Vienna Biocenter, approved by the Ethics Commission of the Medical University of Vienna, and supervised by the Department of Animal Experimentation of the Austrian Ministry of Health and Women.
Purification of chkZP1
For use in affinity chromatography, rabbit IgG directed against the entire PVM-derived chkZP1 protein (anti-p95; [26]) was purified by protein A-Sepharose chromatography (Amersham Biosciences Corp., Piscataway, NJ). The IgG was dialyzed against 10 mM sodium phosphate buffer, 50 mM NaCl, pH 7.4, and then coupled to CNBR-activated Sepharose (Amersham). The resulting slurry was resuspended in 25 mM Tris-HCl, 0.01% EDTA, 0.02% NaN3, pH 8.0, and stored at 4°C.
Laying hen serum was ultracentrifuged for 24 h and the lipid layer on top of the suspension as well as the dark red bottom phase including the pellet were discarded. Sepharose-coupled anti-p95 IgG (see above) was equilibrated in phosphate-buffered saline (PBS; 80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, pH 7.5), and the remaining serum fraction was added and rotated overnight at 4°C. The suspension was then transferred to a column and washed with 5–10 column volumes of PBS. Elution of the protein was initiated by incubating the column material in elution buffer (1 M CH3COOH, 0.1 M glycine, pH 2.5) for 15 min and then collecting the eluate by washing with another column volume of elution buffer and one volume of PBS. The eluted fractions were immediately neutralized with 1 M K2HPO4 (200 µl/ml eluted protein), pooled, and dialyzed against PBS, or, if subsequently used for lyophilization, against 50 mM NH4CO3. Protein concentration was measured using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA).
Purification of chkZPC
For purification of chkZPC, the largest follicles of freshly killed laying hens were punctured, the yolk was drained, and the granulosa sheets were isolated as described [27], washed in PBS, and incubated in 1 ml of Dulbecco modified Eagle medium (DMEM) supplemented with 5% glutamine overnight at 39°C. The supernatant containing secreted chkZPC was aspirated the next day, added to anti-p32 antibody (directed against the entire chkZPC protein [27]) that had been previously coupled to CNBr-activated Sepharose as described above, and rotated at 4°C overnight. Washing and elution steps were performed as described for purification of chkZP1.
Immunofluorescent Sperm-Binding Studies
Sperm were collected and resuspended in prewarmed (40°C) DMEM supplemented with 10 mM HEPES and 25 µg/ml kanamycin. An equal amount (v/v; typically 50 µl) of purified chkZP1 (52 ng/µl) or chkZPC (41 ng/µl), in PBS, was added to the sperm suspension and incubated at 40°C for 15 min. Two microliters of this suspension was spotted onto a PAP-Pen-circled area on a glass slide, air dried, and fixed in methanol for 10 min at 4°C. After evaporation of the methanol, the respective antibodies (anti-p95 and anti-p32, respectively) diluted 1:100 in Tris-buffered saline (TBS; 25 mM Tris, 2.6 mM KCl, 75 mM NaCl, pH 8.0) were added to the circled area, and the slides were incubated in humid chambers at room temperature for 1 h. After subsequent washes with TBS, a fluorescent-labeled secondary antibody (Alexa Fluor 488; Molecular Probes, Eugene, OR), diluted 1:500 in water, together with 4',6'-diamidino-2-phenylindole (DAPI; final concentration 300 nmol/L; Molecular Probes) was added, and the slide was incubated in a humid chamber in the dark for 1 h. After final washing steps, the glass slide was covered with a cover slip and inspected under a fluorescent-light microscope (Zeiss, Carl Zeiss, Oberkochen, Germany). More than 95% of the examined spermatozoa were showing fluorescence after incubation with the purified ZP proteins.
Western Blotting
Protein extracts and sera were separated by one-dimensional SDS-PAGE (12% polyacrylamide-bisacrylamide) and transferred to nitrocellulose (Hybond-ECL, Amersham) for immunoblotting. Transfers were performed in 25 mM Tris, 192 mM glycine, 20% methanol for 1 h at 17 V at room temperature, or overnight at 6 V and 4°C. The membranes were blocked with PBS, 0.1% Tween, 5% nonfat dry milk for 1 h, followed by incubation with antiserum (1:20 000) in PBS-t (PBS, 0.1% Tween). After three washes in PBS-t, the membrane was incubated with protein A-horseradish peroxidase (1:5000) for 1 h. Bands were visualized by the enhanced chemoluminescence procedure as suggested by the manufacturer (Amersham). The positions of migration of molecular weight standards (Bio-Rad) were determined by staining with PonceauS (0.5% in 1% acetic acid).
Sperm Ligand Blot
Sperm were collected from Derco Brown roosters, resuspended in sample buffer, and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and nonreducing conditions. After transfer, the membrane was blocked in 5% milk powder in TBS, washed (all washing steps were done with TBS), and incubated with 52 µg of purified chkZP1 or 41 µg of purified chkZPC in 1 ml PBS, respectively, diluted 1:10 in DMEM (Gibco-Invitrogen, Carlsbad, CA), 10 mM HEPES (pH 7.8), overnight at 4°C. Fifty micrograms of conalbumin (Sigma-Aldrich, St. Louis, MO) in 1 ml PBS, diluted 1:10 in DMEM/HEPES, was used as a control ligand. After subsequent washes, the membranes were treated with the respective antibodies and secondary antibodies in TBS and detected by enhanced chemoluminescence (ECL) (Amersham). For detection of conalbumin, the antiserum WS7/8 (gift of Dr. M. Hermann) was used.
ZP1 Degradation
Sperm collected from two Derco Brown roosters were pooled and resuspended in DMEM supplemented with 10 mM HEPES (final pH 7.0). PVM isolated from laid eggs as described earlier [26] was incubated with sperm suspension for 30 min at 40°C. After a 2-min centrifugation step at 720 x g, the supernatant was separated by SDS-PAGE and detected by immunoblotting as described earlier. In addition to the antisera anti-p95 and anti-p32, we also employed an antiserum designated anti-ZP1, which is directed against a peptide of the N-terminus of the mature chkZP1 protein, LLQYHYDCRDFGMQLLAYP [26]. PVM incubated with DMEM/HEPES without sperm was subsequently solubilized in 1x TBS (75 mM NaCl, 2.6 mM KCl, 25 mM Tris, pH 8), 2% SDS, 50 mM dithiothreitol at 42°C overnight and separated by SDS-PAGE.
Sperm-Binding Assay
Analysis of sperm binding was essentially performed as described by Steele et al. [29]. Briefly, PVM from the largest follicle (F1) was isolated as described earlier [26] and washed and suspended in 0.15 M NaCl with 20 mM Tris-HCl, pH 7.5 (NaCl-Tris). Approximately 1 cm2 of PVM was incubated in NaCl-Tris supplemented with 1:100 dilutions in PBS of anti-p32 and anti-p95 antiserum, for 1 h at room temperature. Rooster sperm were collected as described earlier, pooled, and resuspended in DMEM buffered with 10 mM HEPES. The PVM fragments were rinsed in NaCl-Tris and incubated with 100 µl sperm suspension, containing between 1 x 105 and 1 x 106 spermatozoa for 5 min at 40°C, after which time the PVM was removed and rinsed again. The fragments were then fixed in 4% paraformaldehyde in PBS for 1 h, rinsed in NaCl-Tris, and stained with anti-p95 antiserum, diluted in NaCl-Tris, for 1 h. Although the PVM has, in some cases, already been incubated with anti-p95 before, this step was not omitted to guarantee equal treatment of all samples. After a further rinse, the antibody binding was detected with a fluorescent-labeled secondary antibody diluted in NaCl-Tris for 1 h in the dark, rinsed again, spread onto a microscope slide, and mounted with a cover slip. PVM structure and hole formation were monitored by fluorescent microscopy (Zeiss) by counting the holes in three noncontiguous fields at 1000-fold magnification. The number of holes in untreated control samples was taken as 100%.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We next examined the electrophoretic behavior of the purified chkZP1 and chkZPC proteins under reducing and nonreducing conditions. The silver-stained polyacrylamide gel shown in Figure 2A not only demonstrates that the proteins are essentially free of contaminants, but also reveals that chkZPC can self-associate. Thus, under reducing conditions, chkZPC migrates as expected as an approximately 45-kDa protein (lane 1). However, under nonreducing conditions, the signal shifts to an apparent molecular mass of greater than 200 kDa (lane 2). This protein complex contains chkZPC as shown in an immunoblot (Fig. 2B). In contrast, under these conditions, a large percentage of chkZP1 remains monomeric (see Fig. 2C, lane 1), but some self-assembles to form larger aggregates (data not shown).
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To test whether the aggregation of chkZPC can be altered by the presence of chkZP1, we coincubated both proteins at equal concentrations. Analysis by SDS-PAGE under nonreducing conditions and immunoblotting with the respective antisera (Fig. 2C) demonstrates that the large protein complex does not contain chkZP1, but only chkZPC, and that the chkZP1 signal remains unchanged by the presence of chkZPC.
Next, we checked whether the purified chicken ZP proteins possess the capacity to interact with sperm, and if so, which protein can serve as a sperm ligand. Taking advantage of the availability of native affinity-purified ZP proteins, sperm were incubated with either chkZP1 or chkZPC in vitro to allow protein binding and then fixed onto a slide with methanol. After fixation, the respective ZP protein was detected with a specific antibody, followed by a second fluorescent-labeled antibody, whereas the sperm nucleus was counterstained with DAPI. Sperm was then viewed by fluorescence microscopy (see Materials and Methods).
Figure 3 shows that both proteins, chkZP1 and chkZPC, bind at the most apical region of the sperm head. The bright blue staining represents the DAPI-stained sperm nucleus, which also identifies the position of the sperm head. The midpiece region would be at the very bottom of the panels, and the tail, which is not stained either, would leave the picture at the bottom edge.
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Thus, the triangular staining of the chkZP1- and chkZPC-specific antibody, respectively, highlights the very tip of the sperm head, a region identifying the acrosome. Interestingly, both ZP proteins bind to acrosome-intact sperm. The acrosomal status of the sperm was ascertained by binding of the lectin peanut agglutinin (data not shown). Therefore, both chkZP1 and chkZPC can bind to the acrosomal region of the sperm head, but the binding event does not seem to trigger the acrosome reaction. When sperm were simultaneously incubated with both ZP proteins, binding at the same location on the sperm head was observed (data not shown), indistinguishable from incubations with either one alone.
To further investigate the potential involvement of chkZP1 in the binding of sperm to the PVM, we employed an in vitro sperm-egg interaction assay as described by Steele et al. [29] (see Materials and Methods). This assay measures proteolytic lesions in the perivitelline membrane after exposure to rooster sperm. Figure 4 shows that, surprisingly, preincubation with anti-p95 antiserum dramatically reduced the number of bound sperm, indicating that chkZP1 is an important part of the actual PVM-binding reaction that takes place. In contrast, preincubation with an antiserum directed against chkZPC did not display any inhibitory activity. It is not clear if this is due to chkZPC not being necessary for sperm binding, or to the inability of the employed antiserum to block the ZPC-sperm interactions.
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To ascertain the fate of the ZP proteins after sperm binding has occurred, the experiment shown in Figure 5 was performed. Perivitelline membrane was incubated with fresh rooster sperm at 40°C for 30 min. Macroscopically, the PVM disintegrates upon this treatment. We then analyzed the soluble reaction products found in the supernatant by Western blotting using the appropriate antisera. The left panel of Figure 5 shows that there is no detectable change in electrophoretic mobility of chkZPC after exposure to intact sperm. The (–) lane represents ZPC from intact PVM and is included here as a size reference. In contrast, it is obvious from the rightmost panel that chkZP1 is extensively degraded by rooster sperm. The anti-p95 antiserum, which is directed against the entire chkZP1 protein, detects a variety of proteolytic degradation products, whereas a peptide antibody directed against the extreme N-terminus of the protein [26] (shown in the middle panel) only reacts with a fragment of an apparent molecular mass of approximately 22 kDa, which therefore represents the major or only fragment in this digest containing the N-terminus. The fact that the anti-p95 antiserum does not recognize this band indicates that this IgG is not directed against this epitope. Taken together, this experiment demonstrates that sperm degrades chkZP1 but leaves chkZPC unaffected.
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The results described above demonstrate that, in solution and therefore under native conditions, both ZP proteins, either alone or in combination, would bind to sperm. To directly investigate the binding properties of both chkZP1 and chkZPC to sperm proteins (or protein complexes), ligand-binding blots were performed. Freshly collected sperm was separated by SDS-PAGE under reducing and nonreducing conditions, transferred onto a nitrocellulose membrane, and then incubated with a solution of purified chkZP1 or chkZPC, which were then detected with specific antibodies. The results, summarized in Figure 6, show that, under nonreducing conditions, native chkZPC binds specifically to a band of about 180 kDa present in rooster sperm (–ßME). It is of interest that chkZPC, when allowed to interact with a quail sperm extract, also recognizes a band of comparable size, although binding appears significantly weaker (data not shown). In chicken sperm extracts, the 180-kDa band is also bound by chkZP1 under nonreducing conditions (–ßME) but shows much lower affinity when compared with chkZPC, as indicated by a weaker signal. Under reducing conditions, chkZP1 binding cannot be detected. However, chkZPC still recognizes a 
60-kDa band, presumably a component of the 180-kDa complex. In the control lane, the sperm extracts were blotted with conalbumin and probed with an anticonalbumin antiserum, indicating that binding is specific. Furthermore, when ligand is omitted in these experiments, no signal was detected (data not shown). Binding to the 180-kDa complex requires nonreduced sperm proteins but allows denaturation by SDS, indicating a very strong binding reaction, which would be expected for an event essential for reproduction. The chkZP1 also binds to this sperm protein complex, but to a lesser extent.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have used antibody affinity chromatography to isolate milligram amounts of native chkZP1 from the serum of laying hens. Similarly, we have used the supernatant of cultures of avian ovarian granulosa cells to isolate native chkZPC by an analogous chromatographic approach. The products of this one-step purification are electrophoretically indistinguishable from the proteins in the perivitelline membrane. Interestingly, in the absence of reducing agents, chkZPC migrates as an approximately 200-kDa complex, which could possibly correspond to a tetrameric protein. This is in agreement with recent reports that ZP domain-containing proteins have a propensity to polymerize [3]. Under the conditions employed here, the other PVM component, chkZP1, appears as a single monomeric band on SDS polyacrylamide gels. However, we have previously reported that, in detergent-solubilized PVM preparations, chkZP1 is present as a mixture of monomeric and dimeric forms [26], and Takeuchi et al. [25] have communicated a similar finding. Depending mainly on concentration, we also have observed the formation of larger structures from purified native chkZP1 (data not shown). The nature of these associations and the requirements for their formation are currently being investigated. The fact that, in a mixture of chkZP1 and chkZPC, higher order structures contain only chkZPC might indicate that other ovarian polypeptides play a role in PVM assembly or that there are no heteromultimeric units in the chicken PVM structure.
Depending on the species, different functions have been attributed to the individual ZP proteins. In the mouse, ZP3 acts as the primary sperm receptor and inducer of the acrosome reaction [30]. In the pig, a heterodimer between the porcine ZP1 and ZP3 homologues is responsible for sperm binding [10], as is the case in Xenopus laevis [8], where possibly another ZP protein, ZPD, is also involved [31]. It has recently been suggested that not one protein alone, but the supramolecular structure of the ZP is responsible for sperm binding in mammals [32]. In the chicken, both major PVM components chkZP1 and chkZPC bind in vitro individually to rooster sperm and specifically to the acrosomal region of the sperm head. Coincubation of sperm with both proteins shows that their binding sites are coincident. Whether in vivo they bind individually at the same location or form a three-dimensional structure remains to be determined. By ligand blotting, binding of chkZPC to an approximately 180-kDa sperm protein complex can be observed. The chkZP1 also binds this band, albeit with a significantly lower affinity. In sperm extracts separated under reducing conditions, chkZPC also binds a 60-kDa protein, whereas we cannot detect any binding to reduced sperm proteins by chkZP1. If this is due to the loss of a three-dimensional multiprotein structure upon exposure to reducing agents or to the unfolding of a single polypeptide remains to be established.
Sperm binding to the PVM leads to a proteolytic attack that only affects chkZP1, degrading it into discrete fragments, while chkZPC remains intact. In contrast, another group has reported that sperm degrades both components of the chicken PVM [25]. Under the conditions used here, however, degradation of chkZPC is minimal. The reasons for this difference remain unclear. In the previous report, it was demonstrated that an antibody directed against the chicken ZP1 homologue inhibited degradation of both the ZP3 and the ZP1 family members, so that possibly dissolution of the PVM was caused by destruction of a ZP1-containing scaffold structure. This is also supported by the apparent anisotropic composition of the chicken PVM (unpublished observation; also see [25]).
Thus, it appears that binding of rooster sperm to perivitelline membrane is mediated by both chkZP1 and chkZPC. Sperm-specific hydrolysis results in the degradation of chkZP1, possibly enabling individual spermatozoa to penetrate the perivitelline membrane. When probed with an antiserum specific for the N-terminus of the mature ZP1 protein, a 22-kDa fragment is recognized as a product of the hydrolytic digestion. This places the cleavage site closest to the N-terminus right at the beginning of a stretch of repeats rich in Q, G, L, and P of unknown function that are a specific feature of chicken ZP1. Evidence provided by sperm-binding assays that monitored the formation of hydrolytic lesions in the PVM [33, 34] showed that N-linked carbohydrates on the ZP proteins of the chicken are essential for sperm binding. We have preliminary evidence that chkZP1 carries not only N-linked but also O-linked sugars (H. H. Ruckenbauer et al., unpublished), which supports a pivotal role of chkZP1 in the process. Because of the drastic affinity difference of chkZPC and chkZP1 for sperm-derived proteins in the ligand blot shown in Figure 6, it appears at least possible that initial binding occurs through chkZPC, which then enables hydrolysis of chkZP1 by a sperm-associated protease. Alternatively, it is conceivable that higher order structures of ZP proteins such as multimers are required for binding to the 180-kDa protein and that chkZPC is more readily driven into such a form than chkZP1. It is, however, entirely possible that in vivo chkZP1 and chkZPC need to be both present to exhibit significant sperm-binding activity, as the experiments described here were performed under conditions of excess sperm concentrations, or that additional factors are involved.
A further argument for the crucial role of chkZP1 in the fertilization process in chickens comes from the competitive sperm-binding experiments shown in Figure 4. An antiserum directed against chkZP1 (anti-p95) was able to significantly and reproducibly reduce the number of holes in the PVM caused by sperm binding. This would suggest that chkZP1 is not only participating in, but is necessary for, sperm-egg interactions. Antisera directed against chkZPC did not show any inhibitory effect, which can either mean that chkZPC is not necessary for this initial step in sperm recognition and binding, or more likely, that the antiserum is not neutralizing the binding reaction.
The availability of native properly folded and correctly glycosylated ZP proteins provides now the opportunity to study aspects of the mechanisms of both perivitelline membrane assembly and sperm-PVM interactions in vitro.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: FAX: 43 1 4277 9618; franz.wohlrab{at}univie.ac.at
Received: 17 February 2004.
First decision: 2 March 2004.
Accepted: 9 April 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  M Lemoine, I Grasseau, J P Brillard, and E Blesbois A reappraisal of the factors involved in in vitro initiation of the acrosome reaction in chicken spermatozoa Reproduction, October 1, 2008; 136(4): 391 - 399. [Abstract] [Full Text] [PDF]
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T. Sasanami, T. Murata, M. Ohtsuki, K. Matsushima, G. Hiyama, N. Kansaku, and M. Mori Induction of sperm acrosome reaction by perivitelline membrane glycoprotein ZP1 in Japanese quail (Coturnix japonica) Reproduction, January 1, 2007; 133(1): 41 - 49. [Abstract] [Full Text] [PDF]
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