Journal of Reproduction and Development, Vol. 55, No. 2, 2009, 20134 —Full Paper— Importance of the Porcine ADAM3 Domain in -Egg Interaction Ekyune KIM1)#, Ki-Eun PARK2)#, Ji-Su KIM1), Dong Chul BAEK1,3), Jae-Woong LEE1), Sang-Rae LEE1), Myeong-Su KIM1), Sang-Hyun KIM1), Chan-Shick KIM3), Deog-Bon KOO4), Han-Seok KANG5), Zae-Young RYOO6) and Kyu-Tae CHANG1) 1)National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology, Chung-buk 363-883, Korea, 2)Department of Animal Sciences, Purdue University, West Lafayette, IN 47907, USA, 3)Faculty of Biotechnology, Cheju National University, Jeju 690-756, 4)Department of Biotechnology, Daegu University, Kyungsan 712-714, 5)College of Natural Resources and Life Science, Pusan National University, Gyeongnam 627-706 and 6)School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 702-701, Korea Abstract. In the mouse, ADAM3, a well-characterized testis-specific of the A disintegrin and metalloprotease (ADAM) family, has a crucial role in fertilization by mediating sperm binding to the egg . However, little is known about ADAM3 in other species, such as domestic pigs. We have identified porcine ADAM3 and analyzed the protein. RT-PCR and trypsinization of sperm surface revealed that porcine ADAM3 is expressed at high levels in the testis and on the sperm surface. Furthermore, an IVF inhibition assay with a recombinant porcine ADAM3 disintegrin domain showed that treatment of the disintegrin domain effectively prevented pig sperm-egg interactions. In the present study, we demonstrated the presence of ADAM3a and ADAM3b molecules in the pig and examined their roles in fertilization. Key words: A disintegrin and metalloprotease (ADAM), Disintegrin domain, Porcine, Sperm-egg interaction (J. Reprod. Dev. 55: 156–162, 2009)

he A disintegrin and metalloprotease (ADAM) localized within the endoplasmic reticulum of testicular germ cells, has important roles in various biological processes, such as whereas epididymal sperm contain only ADAM1b on the plasma fertilization, neurogenesis, myogenesis and inflammation [1]. membrane. The ADAM1b-deficient mouse is normal, but the loss Most ADAM proteins have a unique organization, containing an N- of ADAM1a results in male infertility because of the severely terminal signal sequence followed by a pro-domain and metallo- impaired ability of sperm to migrate from the into the ovi- , disintegrin, Cys-rich, epidermal growth factor (EGF)- duct through the uterotubal junction and bind to the egg zona like, transmembrane and cytoplasmic tail domains [2]. The metal- pellucida (ZP) [9, 10]. loprotease domain possesses sheddase activity toward the Cyritestin, known also as ADAM3, is a member of the ADAM ectodomain of membranous precursor proteins [3], whereas the family, and is expressed specifically in male germ cells [8]. It has cytoplasmic domain is related to interaction with Src family protein been suggested that cyritestin is able to bind to the ZP through the tyrosine kinases [4]. It is known that the disintegrin domain is disintegrin domain because pretreatment of eggs with peptides of involved in cell-to-cell adhesion [5, 6]. To date, more then 40 the ADAM3 disintegrin domain inhibited IVF [16, 17]. Moreover, ADAM family genes have been identified in a variety of species the sperm of the cyritestin null male mouse is incapable of binding (http://people.virginia.edu/~jw7g/), and over half of the ADAM to the ZP. Interestingly, several researchers have reported that family is expressed exclusively or predominantly in the mouse tes- there are two human cyritestin genes, CYRN1 and CYRN2, local- tis. Although the roles of several testis-specific ADAMs have been ized on chromosomes 8p12–21 and 16q12, respectively [18]. identified in knockout (KO) mice [7–10], these ADAMs are Evidence suggests that human cyritestin genes are non-functional, expressed in a species-specific manner. Thus, the specific roles of as judged by Western blot analysis using rabbit antisera against testis-specific ADAM molecules in sperm-egg interaction remain human and macaque CYRN1 and the presence of a variety of dele- to be determined. tions, insertions and premature termination in humans [19]. Fertilin, a well-characterized testis-specific ADAM, is a het- However, little is known about ADAM3 in other species, such as erodimeric complex consisting of α (ADAM1) and β (ADAM2) pigs. subunits [11–13]. Two different isoforms of ADAM1 (fertilin We have identified and characterized porcine ADAM3 in an alpha), ADAM1a and ADAM1b, are synthesized in the rodent tes- attempt to elucidate its role(s) in interaction with the ZP. In the tis [14, 15]. Previously, we showed that both ADAM1 isoforms are present study, we found two isoforms of porcine ADAM3, ADAM3a and ADAM3b, on the surface of the sperm that are Accepted for publication: November 17, 2008 Published online in J-STAGE: December 24, 2008 involved in interaction with the ZP. Theses results indicate that Correspondence: KT Chang (e-mail: [email protected]) both ADAM3a and ADAM3b could have important roles in # E Kim and KE Park contributed equally to this study fertilization. THE ROLE OF PORCINE ADAM3 DISINTEGRIN DOMAIN IN FERTILIZATION 157

Materials and Methods 3’ (sense) and 5’-CTCGAGACATTCAAATTCAGGCCG-3’ (antisense) for Probe1 and Probe2, 5’-GAATTCTGC- Total RNA extraction and reverse transcriptase-polymerase AATAATAAGACTACCTATTG-3’ (sense) and 5’-CTC- chain reaction (RT-PCR) GAGATCGGCATTGTCCATTATCAC-3’ (antisense) for Probe3 Total RNAs were obtained from the brain, heart, lung, liver, 5’-GAATTCATGCAATAATAAGACTGCCT-3’ (sense) and 5’- spleen, kidney, testis, uterus, and ovary using Isogen (Nippon CTCGAGTTGTACGTAGTCACTGATAT-3’ (antisense) for Gene, Toyama, Japan) as described previously [20]. A 5 μg sample Probe4. of total RNA was reverse-transcribed to cDNA with superscript III reverse transcriptase using the SuperScript III First-Strand Synthe- Southern blot hybridization sis System (Invitrogen, Carlsbad, CA, USA). DNA fragments of A 10 μg portion of miniature pig genomic DNA was digested by porcine ADAM3a and ADAM3b (GenBank accession Nos NM- BglII, EcoRI and PstI at 37 C overnight. The digested DNA frag- 001097513 and NM-001098595, respectively) were amplified by ments were separated by electrophoresis in 0.8% agarose gel and PCR using first strand complementary DNA from the miniature pig transferred onto Hybond-N+ membranes (Amersham-Pharmacia testis as a template and two sets of primers. The primers for Biotech) as described previously [14]. The blots were hybridized ADAM3a were 5’-TTCTCTGGAGCTCTGGTCAG-3’ and 5’- at 60 C in 5 × SSPE (SSPE is 15 mM sodium phosphate, pH 7.7, TATGAATGCAGTTCTCCACGG-3’, and the primers for 0.18 M NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA) con- ADAM3b were 5’-TTCTCTGGAGCTCTGGTCAG-3’ and 5’- taining 0.02% Ficoll 400, 0.02% polyvinylpyrrolidone and 0.02% TATGACTACATCTCTCTGCAT-3’. The primers for porcine sodium dodecyl sulfate (SDS) and then at 60 C overnight with vacuolar protein sorting 29 (Vps 29), used as a control were 5’- probes prepared as follows. Four DNA probes, Probe1, Probe2, ATGACCCACCAAACACATGC-3’ (sense) and 5’-TTAC- Probe3 and Probe4, were labeled with [α-32P]dCTP (3000 Ci/ CGAAACTGATTGATTGGA-3’ (antisense). mmol, Amersham Pharmacia Biotech) by the random priming pro- PCR was performed in a 50 μl volume containing 200 μM each cedure. The blotted membranes were washed once in 2 × SSC dNTP, 10 μM primer, template DNA and 1 unit of EX Taq DNA (SSC is 15 mM sodium citrate, pH 7.0, 0.15 M NaCl) at room tem- polymerase (Takara, Otsu, Japan). The reaction program was set perature for 10 min, then washed again in 2 × SSC containing 0.1% for initial heat denaturation at 94 C for 180 sec, followed by 35 SDS for 10 min, and then washed a final time in 2 × SSC at room cycles of 94 C for 60 sec, 62 C for 60 sec, and 72 C for 30 sec. The temperature for 10 min. The membranes were analyzed with a PCR products were sequenced after purification by electrophoresis BAS 2000 Bio-Image Analyzer (Fuji Film, Tokyo, Japan) [14]. in 1.5% agarose gel. Preparation of protein extracts Antibodies Fresh epididymal porcine sperm were collected in PBS and Anti-ADAM3a and anti-ADAM3b polyclonal antibodies were washed by centrifugation at 1,000 g for 10 min, and then the pro- raised in rabbits by using cloned cysteine-rich/EGF-like domains teins were extracted in lysis buffer (20 mM Tris-HCl, pH 7.4, 1% corresponding to residues 515–620 of ADAM3a and residues 515– Triton X-100, 0.15 M NaCl, 1% Protease Inhibitor Cocktail 635 of ADAM3b, respectively. In order to clone of these gene [Sigma-Aldrich]) on ice for 6 h. The extract was centrifuged at fragments, each PCR-amplified DNA was ligated into pET-23d 12,000 g for 10 min, and the concentration of protein was deter- (Novagen, Madison, WI, USA), and the construct was expressed in mined by the Bradford method [15]. Escherichia coli BL21 (DE3). To obtain hyperimmune sera, the His-tagged recombinant forms of porcine ADAM3a and ADAM3b Western blot analysis were emulsified with Freund’s complete adjuvant (Sigma-Aldrich, Proteins were denatured by boiling for 3 min in the presence of St. Louis, MO, USA) and injected intradermally into female New 1% SDS and 1% β-mercaptoethanol (β-ME) and separated by Zealand white rabbits (Damool Science, Deajeon, Korea). The SDS-PAGE before transfer onto Immobilon-P membranes (Milli- antibodies raised against porcine ADAM3a and ADAM3b were pore, Bedford, MA, USA). After blocking with 2% skim milk, the purified by fractionation with ammonium sulfate (0–40% satura- blots were incubated with primary antibodies for 2 h and then with tion) followed by immunoaffinity chromatography on a column of horseradish peroxidase-conjugated secondary antibodies for 1 h. Sepharose 4B coupled with the above recombinant protein as Immunoreactive proteins were detected with an ECL Western blot- described [20]. ting detection kit (Amersham Biosciences, Buckinghamshire, UK).

Polymerase chain reaction (PCR) for generation of probes Trypsinization of sperm surface proteins The DNA fragments used as probes were prepared by several Trypsinization of porcine sperm was performed to determine rounds of PCR. Probes, named Probe1 and Probe2, were designed whether the ADAM3 proteins are localized on the sperm surface. for common detection of both ADAM3a and ADAM3b Probe3 and Ejaculated fresh semen was washed three times with PBS and kept Probe4 were designed for specific detection of ADAM3a and on ice for 0, 10, 20, 30 and 40 min in PBS containing 500 μg/ml ADAM3b (329, 329, 378 and 406 bps in lengths, respectively). trypsin (Sigma). Trypsinized sperm samples were washed five PCR was conducted by using cDNA clones encoding porcine times with PBS containing 1% Protease Inhibitor Cocktail (Sigma) ADAM3a and ADAM3b as a template with the following oligonu- and lysed as described above. cleotides as primers: 5’-GAATTCATGATATGGCATACTTGT- 158 KIM et al.

Fig. 1. Alignment of the amino acid sequences of porcine ADAM3a and ADAM3b, and murine ADAM3. Dashes represent gaps introduced to optimize the alignment. Arrows indicate putative starting points of the pro-domain and metalloprotease (MP), disintegrin, Cys-rich, epidermal growth factor (EGF)-like and cytoplasmic domains. Possible binding sequences (QCD) in the disintegrin domain are indicated with an asterisk (*). Repeated sequences in the cytoplasmic tail domain are underlined.

Recombinant proteins follicles 3 to 6 mm in diameter. After washing three times with To express glutathione S- (GST) fusion proteins, each Hepes-buffered Tyrode’s lactate medium (TL-HEPES) [21], disintegrin, Cys-rich and EGF-like domain of porcine ADAM3a approximately 50 oocytes were matured in 500 μl of in vitro matu- and ADAM3b was cloned into pGEX4T1 (Amersham Pharmacia) ration medium in a four-well multidish (Nunc, Roskilde, Denmark) and transformed into Escherichia coli BL21 (DE3). GST fusion at 38.5 C in an atmosphere of 5% CO2 in air. The medium used for proteins expressed in E. coli cells were lysed by sonication in PBS oocyte maturation was NCSU-23 medium [22] supplemented with containing 0.3% Triton X-100, 1 mM dithiothreitol, and 1% Pro- 10% follicular fluid, 0.57 mM cysteine, 10 ng/ml β-ME, 10 ng/ml tein Inhibitor Cocktail. After centrifugation at 16,000 g for 10 min, EGF, 10 IU/ml PMSG, and 10 IU/ml hCG. At this step, dibutyryl the recombinant domains fused to GST remained in the supernatant cAMP (dbcAMP) was added to a final concentration of 1 mM to and were purified on a Sepharose 4B column [20], and the eluates the maturation medium for the experimental samples. After 22 h of were dialyzed at 4 C against PBS. culture, the oocytes were washed three times and then cultured in maturation medium without hormone or dbcAMP for an additional In vitro maturation (IVM) 22 h. After completion of IVM, the oocytes were used for in vitro Pig ovaries were collected from a local slaughterhouse and fertilization (IVF), as described previously [23]. transported to the laboratory at 25–30 C in 0.9% saline supple- mented with 75 μg/ml potassium penicillin G and 50 mg/ml In vitro sperm-egg binding assay streptomycin sulfate. Cumulus-oocyte complexes were aspirated The IVF medium (modified Tris-buffered medium; mTBM) through an 18-gauge needle into a disposable 10-ml syringe from consisted of 113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl2 · 2H2O, 20 THE ROLE OF PORCINE ADAM3 DISINTEGRIN DOMAIN IN FERTILIZATION 159

Table 1. ADAM3 amino acid sequence homology between the pig and mouse MP Dis Cys EGF Cyto Total mADAM3/pADAM3a 61.8 59.6 51.2 50.0 nl 54.2 mADAM3/pADAM3b 62.8 69.7 52.8 57.4 nl 56.8 pADAM3a/pADAM3b 93.2 60.6 53.2 68.5 74.4 75.3 mADAM3, mouse ADAM3; pADAM3a, porcine ADAM3a; pADAM3b, porcine ADAM3b; MP, Metalloprotease domain; Dis, Disintegrin domain; Cys, cysteine-rich domain; EGF, EGF-like domain; Cyto, Cytoplasmic domain; nl, negligibly low.

mM Tris (crystallized free base; Fisher Scientific, Fair Lawn, NJ, and ADAM3b are similar to murine ADAM3, except for the fact USA), 11 mM glucose and 5 mM sodium pyruvate. Fresh ejacu- that they have a much less well-conserved cytoplasmic domain. lated semen was washed three times by centrifugation with Although the metalloprotease domain showed 93% homology Dulbecco’s phosphate-buffered saline (DPBS; Gibco-BRL, Grand overall between porcine ADAM3a and ADAM3b, ADAM3a dif- Island, NY, USA) supplemented with 1 mg/ml BSA (Fraction V; fered from ADAM3b in the C-terminal half region encoding the Sigma), 100 μg/ml penicillin G and 75 μg/ml streptomycin sulfate. disintegrin, Cys-rich, EGF-like and cytoplasmic domains (Table 1 After washing, the spermatozoa were suspended in mTBM (pH and Fig. 3B). As shown in Table 1, the homologies for the domains 7.8). The oocytes were washed three times in mTBM with 2.5 mM of ADAM3a and ADAM3b were 52–69% when the porcine and caffeine/sodium benzoate and 4 mg/ml BSA (fatty acid-free) and murine ADAM3 proteins were compared, although the homologies placed into 50 μl of mTBM under paraffin oil. They were then, for the cytoplasmic domains of both ADAM3a and ADAM3b were treated with the above-mentioned recombinant GST fusion proteins negligibly low. (20 μg/ml) for 30 min at 38.5 C, respectively. Diluted spermatozoa (2 μl) were added to 50 μl of mTBM containing 15–25 oocytes to Gene copy number determination for ADAM3a and ADAM3b give a final concentration of 1.5 × 105 sperm/ml. The oocytes were in the porcine genome incubated with the spermatozoa for 6 h at 38.5 C in an atmosphere To examine whether or not the porcine ADAM3a and ADAM3b of 5% CO2 in air. The eggs were denuded by gentle pipetting in genes existed as single-copy genes, Southern blot analysis of the medium mTBM containing 4% formaldehyde at 4 C, washed with porcine genomic DNA was carried out by using DNA probes spe- medium PVA-PBS and mounted on microscope slides. The sam- cific for ADAM3a (Probe3, Fig. 2B and 3B) and ADAM3b ples were then fixed for 10 min in acetic acid. The number of (Probe4, Fig. 2C and 3B), and with common probes for these genes sperm bound per egg was enumerated using a Leica microscope at (Probe1 and Probe2, Fig. 2A and 3B). The probes detected the spe- 200 × magnification. cific bands of genomic DNA fragments (Figs. 2B and C). These results demonstrate that porcine ADAM3a and ADAM3b are present Statistical analysis as single copy genes. Variations in the rates of sperm binding to the surfaces eggs were analyzed by one-way ANOVA and Duncan’s multiple range Presence of porcine ADAM3a and ADAM3b on the sperm tests. A P value of less than 0.05 (P<0.05) was considered to be surface significant. To examine whether porcine ADAM3a and ADAM3b are present in germ cells, RT-PCR analysis was carried out using spe- Results cific primers against porcine ADAM3a and ADAM3b. The results indicated that porcine ADAM3a and ADAM3b were both Isolation and characterization of porcine ADAM3a and expressed predominantly in the testis among the porcine tissues ADAM3b tested (Fig. 3A). We then performed Western blot analysis for spe- ADAM3 is a single copy gene on chromosome 8 in the mouse, cific detection of the ADAM3a and ADAM3b proteins using but there are two subtypes in humans. A database search of porcine polyclonal antibodies raised against each domain for the cysteine- ADAM3 raised the possibility that there may be more than one gene rich and the EGF-like domains. To verify whether these antibodies for ADAM3 in the pig. We used a PCR-based cloning protocol to were recognized specifically against porcine ADAM3a and identify the presence of putative porcine ADAM3 genes and found ADAM3b (Fig. 3C), we performed an immunoblot analysis of the two homologues of mouse ADAM3 in the pig, which we cloned by porcine sperm extracts. The antibodies recognized 50-kDa proteins 3’-RACE from porcine testis cDNA. The two ADAM3 homo- (Fig. 3C), respectively. We then trypsinized pig to deter- logues identified in the pig are designated ADAM3a and ADAM3b. mine whether the porcine ADAM3a and ADAM3b proteins were The ORFs of porcine ADAM3a and ADAM3b consist of 744 and localized at the sperm surface. Trypsin digestion of the sperm sur- 750 amino acid residues, respectively (Fig. 1). The multi-domain face proteins was maximal at 20 min trypsinization. Our data mouse ADAM3 structure, based on the characterized ADAM struc- showed that both ADAM3a and ADAM3b were localized on the ture, contains pro-domain, metalloprotease, disintegrin, Cys-rich, cell surface of sperm (Fig. 3D). EGF-like, and cytoplasmic domains. Overall, porcine ADAM3a 160 KIM et al.

Fig. 2. Genomic Southern blot analysis of pig genomic DNA, which was digested by three restriction and subjected to Southern blot analysis using 32P-labeled DNA fragment probes common to both porcine ADAM3a and ADAM3b (A) and probes specific to ADAM3a (B) and ADAM3b (C).

Fig. 3. Expression of porcine ADAM3a and ADAM3b in various porcine tissues and localization of both molecules on the surface of the sperm. (A) Expression pattern of ADAM3a and ADAM3b in various pig tissues obtained by RT-PCR analysis. (B) Diagrams of pig ADAM3a and ADAM3b: Pro, pro-domain; MP, metalloprotease domain; Dis, disintegrin domain; Cys, Cys-rich domain; EGF, EGF-like domain; and CT, cytoplasmic domain. (C) The presence of ADAM3a and ADAM3b in pig sperm. The specificities of the anti-ADAM3a and anti-ADAM3b antibodies were verified by immunoreactivity with recombinant ADAM3a and ADAM3b Cys-rich domains produced in E. coli. Arrows indicate specific bands for ADAM3a and ADAM3b. (D) Trypsinization of pig sperm surface proteins. Proteins on the surface of sperm were trypsinized, separated by SDS- PAGE and then analyzed by immunoblotting using antibodies against ADAM3a and ADAM3b. THE ROLE OF PORCINE ADAM3 DISINTEGRIN DOMAIN IN FERTILIZATION 161

This role of mouse ADAM3 is well characterized by the sterility observed in the ADAM3-deficient male mouse [8]. The functions of ADAM3 have been studied extensively in the mouse, but far less is understood about its significance in other animal species. The present study describes the identification of sperm membrane pro- teins ADAM3a and ADAM3b, which may be involved in sperm- egg binding in the pig. The first important observation made in the present study is the existence of two subtypes of ADAM3 in the pig, namely, ADAM3a and ADAM3b, possessing most of the domains of ADAM mole- cules. ADAM3a and ADAM3b possess metalloprotease, disintegrin, Cys-rich, EGF-like and cytoplasmic domains, and their overall amino acid sequences display 54.2% and 56.8% identity with the mouse ADAM3 respectively. Interestingly, multiple sequence alignment demonstrated that the cytoplasmic domain is not well conserved, and the distinctive repeated sequence of the mouse, MTVPGSFNSYAYHGNTDQNF, does not exist in the pig. The extracellular regions of both porcine ADAM3s, including the metalloprotease domain, share a high degree of sequence identity with murine ADAM3 (Fig. 1), Fig. 4. (A) Expression of porcine GST fusion ADAM3a and ADAM3b disintegrin, Cys-rich and EGF-like domains in E. coli. As The biochemical function(s) of these cytoplasmic domains has described in Materials and Methods, the purified proteins were yet to be defined, and further analysis may be required to determine analyzed by SDS/12.5% PAGE under reducing conditions and whether they are related to signal transduction during sperm-egg stained with Coomassie brilliant blue. (B) Effects of GST fusion ADAM3a and ADAM3b domains on sperm-egg binding. Pig interaction. Both porcine ADAM3a and ADAM3b transcripts cumulus-free eggs were incubated for 30 min with the above- show preferential expression in the testis (Fig. 3A). We raised rab- mentioned GST-fusion proteins at a final concentration of 20 μg/ bit polyclonal sera against the Cys-rich domain of ADAM3a and ml. Sperm-egg binding was assayed as described in Materials ADAM3b, respectively. We found that these antibodies recognize and Methods. The ADAM3a disintegrin domain was revealed to be critically important for sperm-egg binding. Data are expressed each molecule specifically and that both ADAM3a and ADAM3b as the mean ± SD of assays performed in triplicate. are localized on the porcine sperm surface. Thus, porcine ADAM3s, like murine ADAM3, may be necessary for fertilization. Our second important observation was that ADAM3a and Effects of recombinant proteins of porcine ADAM3a and ADAM3b are involved in fertilization directly via their disintegrin ADAM3b on sperm-egg binding domains. Many studies have used in vitro assays focused on the In earlier studies of the mechanism of fertilization in mammals, peptides and antibodies comprising the in sperm-egg it has been shown that murine ADAM3 could be a key element in adhesion. The amino acid sequences of the QCD-containing loop fertilization involved directly in association of sperm with the ZP. in murine ADAM3 have been reported to function as the motifs ADAM3 is known to be essential for interaction with the ZP in required for binding the ZP [11, 16]. Even though the disintegrin ADAM1a-, ADAM2-, and ADAM3-deficient mice [8]. Further- homologies of porcine ADAM3a and ADAM3b are less than those more, Kim et al. demonstrated that a murine ADAM3 could of the other domains, the active site forms that regulate sperm-egg interact with the ZP [24]. We used an IVF inhibition assay with interaction during fertilization are highly conserved. To determine recombinant ADAM domains prepared as GST-fusion proteins to whether the disintegrin domain is involved in sperm-egg interac- investigate the roles of porcine ADAM3a and ADAM3b. As tion, we carried out an in vitro assay using recombinant protein shown in Fig. 4, GST-fusion proteins from ADAM3a and (Fig. 4A). Since the recombinant disintegrin domains of both ADAM3b were expressed in E. coli (Fig. 4A). We subsequently ADAM3a and ADAM3b prevent sperm binding to the ZP, our tested whether these domains inhibited sperm-egg binding in the study indicates that both pig ADAM3a and ADAM3b have impor- pig. Dose-dependent binding of both recombinant disintegrin tant roles in sperm-egg interaction. domains inhibited sperm-egg interaction (data not shown). Inhibi- The functional basis of ADAM3a and ADAM3b provides infor- tion of sperm-egg binding was maximal at a protein concentration mation about the structure-function relationship of these two of 25 μg/ml (P<0.05). As seen in Fig. 4B, both recombinant disin- molecules with the porcine ZP, and may prevent polyspermy dur- tegrin domains inhibited sperm-egg bindings. ing IVF in the pig. The presence of the porcine recombinant ADAM3 disintegrin domain may inhibit polyspermy. Further- Discussion more, application of this mechanism may contribute greatly to the development of efficient contraceptives. These in vitro approaches ADAM3 is a key molecule responsible for sperm-egg binding in will be useful in elucidating the roles of ADAM3 in mammalian the mouse, and is necessary for physiologic function of the sperm. fertilization. 162 KIM et al.

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