Journal of Reproduction and Development, Vol. 55, No. 2, 2009, 20134 —Full Paper— Importance of the Porcine ADAM3 Disintegrin Domain in Sperm-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 protein of the A disintegrin and metalloprotease (ADAM) family, has a crucial role in fertilization by mediating sperm binding to the egg zona pellucida. 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 proteins 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) protein family 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 uterus into the ovi- protease, 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].