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Regular article |
a Instituto de Biología y Medicina Experimental, Buenos Aires, Argentina
ABSTRACT
Rat epididymal protein DE associates with the sperm surface during epididymal maturation and is a candidate molecule for mediating gamete membrane fusion in the rat. Here, we provide evidence supporting a role for DE in mouse sperm-egg fusion. Western blot studies indicated that the antibody against rat protein DE can recognize the mouse homologue in both epididymal tissue and sperm extracts. Indirect immunofluorescence studies using this antibody localized the protein on the dorsal region of the acrosome. Experiments in which zona-free mouse eggs were coincubated with mouse capacitated sperm in the presence of DE showed a significant and concentration-dependent inhibition in the percentage of penetrated eggs, with no effect on either the percentage of oocytes with bound sperm or the number of sperm bound per egg. Immunofluorescence experiments revealed specific DE-binding sites on the fusogenic region of mouse eggs. Because mouse sperm can penetrate zona-free rat eggs, the participation of DE in this interaction was also investigated. The presence of the protein during gamete coincubation produced a significant reduction in the percentage of penetrated eggs, without affecting the binding of sperm to the oolemma. These observations support the involvement of DE in an event subsequent to sperm-egg binding and leading to fusion in both homologous (mouse-mouse) and heterologous (mouse-rat) sperm-egg interaction. The lack of disintegrin domains in DE indicates that the protein interacts with its egg-binding sites through a novel mechanism that does not involve the reported disintegrin-integrin interaction.
epididymis, fertilization, ovum, sperm
INTRODUCTION
The fertilization process comprises a first phase of close approximation between gametes and a second phase of membrane fusion. Although the visible structural and physiological aspects of gamete fusion have been carefully studied, progress toward identifying the molecules involved in this process has been made only during the last years [1, 2].
The epididymal sperm protein DE is a candidate molecule to mediate gamete membrane fusion in the rat. First described by our laboratory [3], DE (37 kDa) is synthesized in an androgen-dependent manner by the proximal segments of the epididymis and associates with the sperm surface during epididymal transit [4, 5]. Originally localized on the dorsal region of the acrosome, DE migrates to the equatorial segment concomitantly with the occurrence of the acrosome reaction [6]. Relocation of DE to the equatorial segment, the region through which the sperm fuses with the egg [7, 8], together with the results of experiments showing that the polyclonal anti-DE antibody significantly inhibits the percentage of penetrated zona-free rat eggs, supported a role for this protein in sperm-egg fusion [9]. Subsequent studies in which the exposure of zona-free eggs to purified protein DE significantly reduced the percentage of penetrated eggs without affecting sperm-egg binding have provided further evidence for the participation of this protein in sperm-egg fusion, and indicated the existence of DE-complementary sites on the egg surface as well [10, 11]. Indirect immunofluorescence (IIF) studies showed that these DE-binding sites are localized as patches over the entire egg surface, with the exception of the area overlying the meiotic spindle, a region through which fusion rarely occurs [12, 13]. Thus, DE is localized on the fusogenic region of the sperm head, and DE-binding components are localized on the fusogenic area of the egg surface.
The recent sequencing of both an internal peptide of DE and a positive clone detected by immunoscreening of a rat epididymal library [14] indicate that DE presents 100% homology with the mature rat epididymal secretory glycoprotein previously cloned by Brooks et al. [15] and with the rat acidic epididymal glycoprotein (AEG) sequenced by Charest et al. [16]. Analysis of the complete DE sequence showed that it belongs to the CRISP (cysteine-rich secretory proteins) family and exhibits significant homology with other epididymal proteins: mouse protein AEG-1/CRISP-1 (70%) [17, 18] and human protein ARP/CRISP-1 (40%) [19, 20]. However, so far, the role of these DE homologues in gamete fusion has not been investigated.
In the present study, we explored the participation of DE in mouse sperm-egg fusion. In addition, because mouse sperm can fuse with zona-free rat eggs, we examined the participation of DE in this interaction to gain insights regarding the mechanisms involved in sperm-egg fusion between different species.
MATERIALS AND METHODS
Animals
Adult (age, 60–120 days) male and immature (age, 25–30 days) female (C57BL/6JxCBA)F1 mice as well as adult (age, 90–120 days) male and immature (age, 25–30 days) female Sprague-Dawley rats were maintained at 23°C with a 12L:12D cycle. Experiments were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction.
Preparation of Tissue Cytosols and Sperm Extracts
Mouse and rat epididymides and testes were homogenized in four volumes of ice-cold, 20 mM Tris-HCl buffer (pH 7.4) containing 1 mM EDTA, 1 mM dithiothreitol (DTT), 0.2% sodium azide, and 2 mM PMSF using an Ultra-Turrax (IKA-WERK; Jauke & Hunkel, Staufen, Germany). The homogenates were then centrifuged for 20 min at 10 000 x g and 4°C, the supernatants centrifuged at 105 000 x g for 1 h, and the cytosol fractions stored at -20°C until use.
For sperm protein extraction, mouse and rat cauda epididymal sperm were allowed to disperse in PBS at 37°C, washed three times in PBS containing 0.2 mM PMSF (PBS-PMSF), and then incubated for 30 min at room temperature in 1% Triton X-100 in PBS-PMSF. The suspension was centrifuged for 10 min at 13 000 x g, and the supernatant was precipitated at -20°C with 10 volumes of acetone and stored until use.
SDS-PAGE and Western Blot Analysis
Samples were separated by 10% SDS-PAGE under nonreducing conditions according to the method described by Laemmli [21], and proteins were electrotransferred to nitrocellulose [22]. Membranes were blocked for 1 h with powdered skim milk (2% w/v in PBS) and incubated for 2 h with the antibody against rat epididymal DE (anti-DE; 1:200 in blocking solution). The characteristics of polyclonal anti-DE antibody have been reported elsewhere [23].
Membranes were washed thoroughly before incubation for 1 h with biotin-conjugated antirabbit immunoglobulin (Ig) G (1:500 in blocking solution; Sigma, St. Louis, MO). After extensive washing, membranes were incubated for 1 h with ExtrAvidin-horseradish-peroxidase (1:1000; Sigma), and reactive bands were visualized with 3,3'-diaminobenzidine (40 µg/ml in 0.1 M Tris [pH 7.5] and 0.01% v/v H2O2). All incubations were performed at room temperature.
Recovery and Treatment of Oocytes
Immature mouse or rat females were superovulated by an injection (i.p.) of eCG (mouse, 5 IU; rat, 20 IU; Sigma), followed by the administration (s.c.) of hCG (mouse, 5 IU; rat, 25 IU; Sigma) 48 h later. Eggs were obtained from the oviducts of superovulated animals 12–15 h after hCG administration. Cumulus cells were removed by incubating the oocyte-cumulus complexes for 3 min in rat [24] or mouse [25] capacitating medium containing 0.1% w/v hyaluronidase (type IV; Sigma). After washing in fresh medium, the zonae pellucidae were dissolved by treating the oocytes for 10–20 sec with acid Tyrode's solution (pH 2.5) [26]. Finally, zona-free oocytes were thoroughly washed in capacitation medium and distributed among treatment groups.
For experiments involving the effect of DE on gamete fusion, zona-free oocytes were incubated for 30 min before insemination in 100 µl drops (under oil) of capacitation medium alone or medium containing either rat purified protein DE (20–200 µg/ml) [27, 28] or ovalbumin (OA; 200 µg/ml; Sigma) as a control. For IIF experiments, zona-free eggs were incubated for 30 min in 200 µg/ml of purified rat DE or control proteins as described earlier.
Indirect Immunofluorescence
Labeling on unfixed sperm in suspension Cauda epididymal mouse sperm were allowed to disperse in 2 ml of PBS containing 4 mg/ml BSA (PBS-BSA4), and 50-µl aliquots were incubated with either anti-DE (1:10) or normal rabbit serum (NRS; 1:10) for 30 min at 37°C. After washing with 10 volumes of PBS-BSA4, sperm were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (1:50 in PBS-BSA4; Sigma) for 30 min at 37°C. After washing, motile sperm were observed under an epifluorescence microscope.
Labeling on fixed sperm Cauda epididymal mouse and rat sperm were fixed for 10 min in 2% w/v paraformaldehyde in PBS at room temperature. After extensive washing with PBS, sperm were air-dried on poly-L-lysine (0.01 mg/ml)-coated slides, incubated with normal goat serum (NGS; 5% v/v in PBS) for 30 min at 37°C, and then exposed to anti-DE or NRS in 1% v/v NGS in PBS (mouse, 1:50; rat, 1:100) overnight at 4°C. After washing three times in PBS, sperm were incubated for 30 min at 37°C with FITC-conjugated goat anti-rabbit IgG (1:100 in PBS), washed, and mounted in 90% v/v glycerol in PBS. More than 200 cells were examined for each preparation.
Egg labeling The DE-protein-incubated and control zona-free mouse eggs were fixed for 1 h in 1 ml of 2% paraformaldehyde in PBS at room temperature, and then washed several times with PBS containing 10 mg/ml of BSA (PBS-BSA10). Eggs were then incubated for 30 min at 37°C in 100 µl of 5% v/v NGS in PBS-BSA10 and then exposed to anti-DE or NRS (1:50 in 1% NGS in PBS-BSA10) for 2 h at 37°C. After washing in PBS-BSA10 containing 0.02% v/v Tween 20, eggs were incubated for 30 min at 37°C in FITC-conjugated goat anti-rabbit IgG (1:50 in PBS-BSA10), washed, and finally mounted in 90% glycerol in PBS.
Both sperm and eggs were examined with a Nikon Optiphot microscope (Nikon, Tokyo, Japan) equipped with epifluorescence optics.
In Vitro Sperm Capacitation
Mouse sperm Mouse sperm were recovered by incising the cauda epididymal tubules in 0.2 ml of capacitation medium under paraffin oil (saybolt viscosity 125/135; Fisher Scientific Co., Pittsburgh, PA). Aliquots of the original suspension were diluted in fresh capacitation medium, and the final sperm concentration was adjusted to 1 x 106 cells/ml. Culture dishes (Falcon Plastics, Los Angeles, CA) containing 200-µl drops of the sperm suspension were incubated for 90 min under paraffin oil at 37°C in an atmosphere of 5% CO2 in air. For motility assessment, 10 µl of sperm suspension were placed on prewarmed slides, and the percentage of motility was then determined subjectively under the light microscope.
Rat sperm A dense mass of cauda epididymal sperm was placed in a conical tube, covered with 2 ml of capacitation medium, and allowed to swim up at 37°C. After 15 min, the upper sperm layer of the suspension was diluted in capacitation medium, and the final concentration was adjusted to 0.5–1 x 106 cells/ml. Aliquots (500 µl) of this suspension were placed in tissue culture wells (16 mm; Costar, Cambridge, MA) and incubated for 5 h under paraffin oil at 37°C in an atmosphere of 5% CO2 in air.
Evaluation of Sperm-Egg Fusion
For mouse-mouse or mouse-rat fusion assays, capacitated mouse sperm were added to zona-free mouse or rat eggs at a final concentration of 0.5–1.5 x 105 cells/ml. Gametes were incubated for 3–4 h under paraffin oil at 37°C in an atmosphere of 5% CO2 in air. For rat-rat gamete fusion experiments, capacitated rat sperm were added to zona-free rat eggs at a final concentration of 0.5–2.0 x 105 cells/ml, and gametes were coincubated for 2–3 h at 37°C and in an atmosphere of 5% CO2 in air. In all cases, oocytes were then washed in fresh medium to remove loosely adherent sperm, mounted on slides, and examined under a phase-contrast microscope (x400) to evaluate the presence of decondensing sperm heads or pronuclei in the egg cytoplasm.
Alternatively, sperm-egg fusion was evaluated by incubating mouse eggs with Hoechst 33342 (1 µg/ml) before insemination and observing the transfer of DNA-specific dye to the nucleus of the fused sperm as described elsewhere [29]. Because both methods gave identical results, the former procedure was preferable for a large number of samples.
Sperm-Egg Binding Assay
Capacitated mouse sperm were added to zona-free mouse or rat eggs at a final concentration of 0.5–1.5 x 105 cells/ml. Thirty minutes after sperm addition, eggs were removed and washed by transfer through several drops of fresh capacitation medium to remove sperm loosely associated with the egg surface. All washing procedures were done by the same person and using the same pipette. Eggs were then transferred to a drop of 2% v/v glutaraldehyde in PBS, fixed for 20 min at room temperature, washed, and mounted on slides. The number of sperm bound per egg was scored under phase-contrast microscopy at 400x magnification.
Statistical Analysis
Results are expressed as mean values ± SEM for each series of experiments. Statistical significance of the data was analyzed using a one-way ANOVA for the number of bound sperm per egg and the 
2 test for the percentage of oocytes with bound or fused sperm, with significant differences being defined by a P value of less than 0.05.
RESULTS
Localization of Mouse Protein DE on Mouse Epididymal Sperm
To study the presence and localization of DE on mouse epididymal sperm, the ability of the antibody against rat epididymal DE (anti-DE) to recognize the mouse epididymal DE homologue was first examined. For this purpose, mouse testicular and epididymal tissues as well as mouse sperm extracts were analyzed by Western blotting using anti-DE as primary antibody. Results indicated that whereas no bands were observed in mouse testicular cytosol, a band coincident with DE was detected in both mouse epididymal cytosol and a detergent extract from mouse epididymal sperm (Fig. 1).
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Having confirmed the ability of anti-DE to recognize the mouse protein, the localization of DE on mouse sperm was studied by IIF using this antibody. Sperm exposed to NRS were used as a control. Mouse sperm presented a clear fluorescent labeling on the dorsal region of the acrosome (Fig. 2, a and b) similar to that observed for rat spermatozoa (Fig. 2, e and f). The same fluorescent pattern was observed when labeling was performed on unfixed sperm cells. No labeling was observed on unfixed testicular sperm exposed to anti-DE or on either fixed or unfixed epididymal sperm incubated with NRS (Fig. 2, c and d).
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Participation of Mouse DE in Gamete Fusion
To study the participation of DE in mouse sperm-egg fusion, zona-free mouse eggs were incubated for 30 min in medium containing different concentrations of purified rat DE (20–200 µg/ml) and then inseminated with capacitated mouse spermatozoa. Four hours later, the oocytes were recovered and examined for evidence of penetration. Zona-free mouse oocytes incubated in the absence of DE or in the presence of OA were used as controls. As shown in Figure 3, the presence of DE during gamete coincubation produced a concentration-dependent decrease in the percentage of penetrated eggs, with a significant effect being observed at 50 µg/ml and maximum inhibition at 200 µg/ml (80% inhibition, P < 0.001). This inhibition was not observed when a control protein (i.e., OA) was tested at the highest concentration. As previously observed for the rat, inhibition did not result from an effect of the protein on sperm motility or egg penetrability, because no differences were observed in the percentages of motile spermatozoa compared with controls and because normal levels of sperm penetration were observed when DE-incubated eggs were subjected to washing before insemination (data not shown).
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To examine whether the inhibition of egg penetration resulted from an inhibitory effect of DE on the first step of sperm binding to the oolemma, zona-free mouse eggs were inseminated in the presence of 200 µg/ml of DE or OA recovered 30 min after the initiation of gamete coincubation, and examined for evidence of sperm-egg binding. Neither the percentage of eggs with bound sperm nor the number of sperm bound per egg differed significantly from the corresponding controls (% binding: 97% ± 3% for medium alone, 100% for OA and DE; sperm bound per egg: 13 ± 2 for medium alone, 13 ± 1 for OA, and 12 ± 3 for DE). Taken together, these results support the participation of DE in mouse gamete fusion through complementary sites for DE on the mouse egg surface. To study the localization of these DE-binding sites, zona-free mouse eggs were incubated with purified protein DE for 30 min, fixed, and exposed to anti-DE. Zona-free eggs subjected to three different treatments were used as controls: 1) mouse oocytes incubated with DE and then exposed to NRS, 2) mouse oocytes incubated with OA and anti-DE, and 3) mouse oocytes incubated with maltose-binding protein (MBP) and anti-MBP. Results indicated that whereas no egg from the control groups presented evidence of fluorescence (Fig. 4a), all mouse eggs incubated with DE and anti-DE exhibited a patchy fluorescent labeling localized over the entire egg surface, with the exception of a negative area (Fig. 4b).
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Participation of DE in Mouse-Rat Sperm-Egg Fusion
Mouse eggs can only fuse with mouse sperm, but mouse sperm can also fuse with zona-free rat eggs [30]. To examine the participation of protein DE in the fusion process between these two species, zona-free rat eggs were inseminated with mouse sperm in the presence of 200 µg/ml of DE or OA. In a parallel control, zona-free rat eggs were inseminated with rat sperm. The presence of DE during gamete coincubation produced a significant inhibition (80%; P < 0.001) in the percentage of zona-free rat eggs penetrated by mouse sperm, which was similar to that observed for the rat homologous system (90%; Fig. 5). This inhibition did not result from an effect of DE on the first step of sperm-egg binding, because neither the percentage of rat eggs with bound mouse sperm nor the number of mouse sperm bound per rat egg differed significantly (P > 0.05) from those observed in controls (% binding: 97% ± 3% for OA, 93% ± 5% for DE; sperm bound per egg: 14 ± 3 for OA, 10 ± 2 for DE).
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DISCUSSION
The present study provides evidence that the mouse homologue of rat epididymal protein DE plays a role in both the homologous (i.e., mouse-mouse) and the heterologous (i.e., mouse-rat) sperm-egg fusion process.
Western blot experiments using the antibody against rat epididymal DE confirmed the ability of this antibody to recognize the mouse epididymal protein. Subsequent IIF experiments using this antibody revealed that as in the rat, the mouse protein is localized over the dorsal region of the acrosome. The existence of the mouse epididymal homologue has been previously reported [17, 18, 31], but to our knowledge, its localization has not.
Exploration of the involvement of DE in mouse gamete fusion indicated that its presence significantly reduces the percentage of penetrated eggs in a concentration-dependent manner. While significant inhibition was observed with 20 µg/ml of DE in the rat system [10], but the concentration required to significantly inhibit sperm-egg fusion in the mouse was 50 µg/ml. The observation that DE did not affect the first step of sperm-egg binding supports the idea that DE participates in an event subsequent to gamete binding, leading to membrane fusion.
The inhibition of egg penetrability observed in the presence of DE also indicates the existence of complementary sites for the protein on the egg surface. The IIF studies confirmed the binding of protein DE to the mouse egg. This binding was specific for DE-anti-DE as judged by the lack of labeling observed on those oocytes incubated with either another protein (i.e., OA), another antibody (i.e., NRS), or a combination of a protein and its corresponding antibody (i.e., MBP-anti-MBP). The DE-binding sites were localized over the entire egg surface, with the exception of a negative area coincident with a distention of the egg cytoplasm exhibited by some mouse oocytes after removal of the zona pellucida. In both the rat [10] and the mouse [32], this distention corresponded to the region of plasma membrane overlying the meiotic spindle, a region through which fusion rarely occurs. Thus, DE-binding sites are localized on the fusogenic region of the mouse egg surface.
Sperm proteins involved in sperm-egg fusion should be localized on the equatorial segment of acrosome-reacted sperm, because the sperm fuses with the oolemma through this region [7, 8]. In the rat, DE that originally localized on the dorsal region of the acrosome relocalizes to the equatorial segment during the acrosome reaction [6]. The IIF results on mouse capacitated sperm, however, revealed that these were not labeled on the equatorial segment (data not shown). The use of an antibody raised against the mouse epididymal protein might help to clarify this point.
The fact that cross-fertilization occurs after the removal of the zona in many cases suggests that gamete interactions at the membrane level are less specific than those at the zona. However, even at this stage, correct reciprocal relations between gametes are important, because a cross that works in one direction may not work in the other. In this regard, zona-free rat eggs are fertilized by mouse sperm, but not vice versa [30, 33–35]. Considering both the presence of DE on mouse sperm and the existence of DE-binding sites on the rat egg surface, we explored the participation of DE in this cross-species fusion process. The inhibition of egg penetration in the presence of DE was comparable with that observed for homologous rat-rat sperm-egg fusion and, as in the latter, did not involve the first stage of sperm-egg binding. Taken together, these results support the participation of DE and its egg-binding sites in mouse-rat sperm-egg fusion, indicating that the mechanism of fusion between these heterologous gametes involves a molecule shared by both species. That rat sperm do not fuse with mouse eggs [33, 34], in spite of the presence of both DE protein on the rat sperm and DE-binding sites on the mouse egg surface, suggests that other molecules involved in gamete fusion are critical for the interaction between these gametes.
The failure of proteins other than DE to either bind to mouse and rat zona-free eggs or to inhibit mouse and rat sperm-egg fusion [10], together with the results of recent experiments showing that two other members of the CRISP family (i.e., ARP and helothermine [40% and 47% homology with DE, respectively]) [36] do not inhibit rat sperm-egg fusion (unpublished observations), support the idea of a specific participation of DE in both homologous (i.e., mouse-mouse and rat-rat) and heterologous (i.e., mouse-rat) sperm-egg interaction.
Several sperm surface proteins have been implicated in mammalian gamete fusion [1, 2, 37]. One of the best-characterized candidates is fertilin, a testicular heterodimer of 
and ß subunits [38], both of which belong to the ADAM (i.e., a disintegrin and a metalloprotease protein) family of membrane proteins [39]. Because DE and fertilin have both been studied in mouse gamete interaction, a sequence of molecular events involved in gamete fusion can be delineated in this animal model. Strong evidence suggests that fertilin mediates the first step of sperm binding to the oolemma through the interaction of its disintegrin domain with an integrin on the egg surface (Fig. 6A) [40, 41]. The results of this study as well as those previously reported for the rat indicate that DE and its egg-binding sites are not involved in the first stage of sperm-egg binding but in a subsequent event leading to fusion (Fig. 6B). This was clearly shown in the rat by the fact that those sperm bound to the egg surface in the presence of DE became competent to fuse with the oolemma only after the removal of the protein from the incubation media [10]. The lack of disintegrin domains in the DE amino acid sequence also suggests that the protein must interact with its egg-binding sites through a mechanism not involving the disintegrin-integrin interaction. Taken together, these results indicate that the epididymal protein not only participates in a step other than that described for fertilin but also through a novel mechanism. The different conditions (i.e., temperature, pH, and ionic composition of the medium) required for sperm-egg binding and fusion [30] support the idea of distinct mechanisms operating in these two processes.
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Fertilin might act as a fusogenic molecule through a fusion peptide present in the subunit [42], but recent evidence does not favor this possibility [43, 44]. Regarding DE, the lack of hydrophobic domains in its amino acid sequence does not support a direct insertion of this protein into the egg plasma membrane [45]. Thus, other molecules probably participate in the stage of sperm-egg membrane fusion itself (Fig. 6C).
The present results provide, to our knowledge, the first evidence for the coexistence of at least two different mechanisms operating in sperm-egg fusion within the same animal model, supporting the view that mammalian gamete fusion is a complex, multistep process involving different molecules as well as different mechanisms. The molecular basis underlying the interaction between DE and its egg-binding sites is currently being investigated.
ACKNOWLEDGMENTS
The authors thank Dr. Lucrecia Calvo for constructive comments on the manuscript.
FOOTNOTES
First decision: 11 February 2000.
1 Partially supported by WHO grant H9/181/R429 to P.S.C. Both D.J.C. and D.A.E. are Research Fellowship recipients from the National Research Council of Argentina (CONICET). P.S.C. is a Research Career Award recipient from CONICET. 
2 Correspondence: Débora J. Cohen, Instituto de Biología y Medicina Experimental, Vuelta de Obligado 2490, (1428) Buenos Aires, Argentina. FAX: 54 11 4786 2564; dcohen{at}dna.uba.ar
Accepted: March 20, 2000.
Received: January 5, 2000.
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K. P. Roberts, K. M. Ensrud, and D. W. Hamilton A Comparative Analysis of Expression and Processing of the Rat Epididymal Fluid and Sperm-Bound Forms of Proteins D and E Biol Reprod, August 1, 2002; 67(2): 525 - 533. [Abstract] [Full Text] [PDF]
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C. E. Tubbs, J. C. Hall, R. O. Scott, V. P. Clark, T. L. Hermon, and C. Bazemore-Walker Binding of Protein D/E to the Surface of Rat Epididymal Sperm Before Ejaculation and After Deposition in the Female Reproductive Tract J Androl, July 1, 2002; 23(4): 512 - 521. [Abstract] [Full Text] [PDF]
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D. J. Cohen, D. A. Ellerman, D. Busso, M. M. Morgenfeld, A. D. Piazza, M. Hayashi, E. T. Young, M. Kasahara, and P. S. Cuasnicu Evidence That Human Epididymal Protein ARP Plays a Role in Gamete Fusion Through Complementary Sites on the Surface of the Human Egg Biol Reprod, October 1, 2001; 65(4): 1000 - 1005. [Abstract] [Full Text] [PDF]
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 J. H. Olson, X. Xiang, T. Ziegert, A. Kittelson, A. Rawls, A. L. Bieber, and D. E. Chandler Allurin, a 21-kDa sperm chemoattractant from Xenopus egg jelly, is related to mammalian sperm-binding proteins PNAS, September 13, 2001; (2001) 211316798. [Abstract] [Full Text] [PDF]
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J. H. Olson, X. Xiang, T. Ziegert, A. Kittelson, A. Rawls, A. L. Bieber, and D. E. Chandler Allurin, a 21-kDa sperm chemoattractant from Xenopus egg jelly, is related to mammalian sperm-binding proteins PNAS, September 25, 2001; 98(20): 11205 - 11210. [Abstract] [Full Text] [PDF]
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