Possible Function of the ADAM1a/ADAM2 Fertilin Complex in the Appearance of ADAM3 on the Sperm Surface

In mouse, two different isoforms of ADAM1 (fertilin α), ADAM1a and ADAM1b, are produced in the testis. ADAM1a is localized within the endoplasmic reticulum of testicular germ cells, whereas epididymal sperm contain only ADAM1b on the plasma membrane. In this study, we show that the loss of ADAM1a results in the male infertility because of the severely impaired ability of sperm to migrate from the uterus into the oviduct through the uterotubal junction. However, epididymal sperm of ADAM1a-deficient mice were capable of fertilizing cumulus-intact, zona pellucida-intact eggs in vitro despite the delayed dispersal of cumulus cells and the reduced adhesion/binding to the zona pellucida. Among testis (sperm)-specific proteins examined, only the level of ADAM3 (cyritestin) was strongly reduced in ADAM1a-deficient mouse sperm. Moreover, the appearance of ADAM3 on the sperm surface was dependent on the formation of a fertilin protein complex between ADAM1a and ADAM2 (fertilin β) in testicular germ cells, although no direct interaction between the fertilin complex and ADAM3 was found. These results suggest that ADAM1a/ADAM2 fertilin may be implicated in the selective transport of specific sperm proteins including ADAM3 from the endoplasmic reticulum of testicular germ cells onto the cell surface. These proteins then can participate in sperm migration into the oviduct, the dispersal of cumulus cells, and sperm binding to the zona pellucida. In mouse, two different isoforms of ADAM1 (fertilin α), ADAM1a and ADAM1b, are produced in the testis. ADAM1a is localized within the endoplasmic reticulum of testicular germ cells, whereas epididymal sperm contain only ADAM1b on the plasma membrane. In this study, we show that the loss of ADAM1a results in the male infertility because of the severely impaired ability of sperm to migrate from the uterus into the oviduct through the uterotubal junction. However, epididymal sperm of ADAM1a-deficient mice were capable of fertilizing cumulus-intact, zona pellucida-intact eggs in vitro despite the delayed dispersal of cumulus cells and the reduced adhesion/binding to the zona pellucida. Among testis (sperm)-specific proteins examined, only the level of ADAM3 (cyritestin) was strongly reduced in ADAM1a-deficient mouse sperm. Moreover, the appearance of ADAM3 on the sperm surface was dependent on the formation of a fertilin protein complex between ADAM1a and ADAM2 (fertilin β) in testicular germ cells, although no direct interaction between the fertilin complex and ADAM3 was found. These results suggest that ADAM1a/ADAM2 fertilin may be implicated in the selective transport of specific sperm proteins including ADAM3 from the endoplasmic reticulum of testicular germ cells onto the cell surface. These proteins then can participate in sperm migration into the oviduct, the dispersal of cumulus cells, and sperm binding to the zona pellucida. A family of ADAM (adisintegrin and metalloprotease) transmembranous proteins belonging to the zinc protease superfamily is implicated in various biological processes such as fertilization, neurogenesis, myogenesis, cancer, and inflammation (1Schlöndorff J. Blobel C.P. J. Cell Sci. 1999; 112: 3603-3617Crossref PubMed Google Scholar, 2Primakoff P. Myles D.G. Trends Genet. 2000; 16: 83-87Abstract Full Text Full Text PDF PubMed Scopus (518) Google Scholar, 3Seals D.F. Courtneidge S.A. Genes Dev. 2003; 17: 7-30Crossref PubMed Scopus (904) Google Scholar). ADAMs have a unique organization containing an N-terminal signal peptide domain followed by pro-, metalloprotease, disintegrin, cysteine-rich, epidermal growth factor-like, transmembrane, and cytoplasmic tail domains, each of which has been believed to fulfill distinct function(s). The metalloprotease domain exhibits a “sheddase” activity toward the ectodomain of membranous precursor proteins (4Hooper N.M. Karran E.H. Turner A.J. Biochem. J. 1997; 321: 265-279Crossref PubMed Scopus (562) Google Scholar), whereas cell-to-cell adhesion is mediated by the disintegrin domain (5Yuan R. Primakoff P. Myles D.G. J. Cell Biol. 1997; 137: 105-112Crossref PubMed Scopus (205) Google Scholar, 6Linder B. Heinlein U.A.O. Dev. Growth Differ. 1997; 39: 243-247Crossref PubMed Scopus (47) Google Scholar, 7Chen H. Sampson N.S. Chem. Biol. 1999; 6: 1-10Abstract Full Text PDF PubMed Scopus (109) Google Scholar). Although many members of the ADAM family are exclusively or predominantly expressed in the testis, their roles in spermatogenesis and fertilization have not yet been completely elucidated. Mammalian fertilization is a successive, multistep process of events including the adhesion and binding of sperm to the zona pellucida (ZP), 1The abbreviations used are: ZP, zona pellucida; tACE, a testis-specific isoform of angiotensin-converting enzyme; TGC, testicular germ cells; ER, endoplasmic reticulum; UTJ, uterotubal junction. an extracellular glycoprotein matrix surrounding the egg, and the membrane fusion between sperm and egg (8Yanagimachi R. Knobil E. Neill J.D. The Physiology of Reproduction. Raven Press, New York1994: 189-317Google Scholar, 9Wassarman P.M. Jovine L. Litscher E.S. Nat. Cell Biol. 2001; 3: E59-E64Crossref PubMed Scopus (345) Google Scholar). Male mice lacking calmegin (10Ikawa M. Wada I. Kominami K. Watanabe D. Toshimori K. Nishimune Y. Okabe M. Nature. 1997; 387: 607-611Crossref PubMed Scopus (248) Google Scholar, 11Ikawa M. Nakanishi T. Yamada S. Wada I. Kominami K. Tanaka H. Nozaki M. Nishimune Y. Okabe M. Dev. Biol. 2001; 240: 254-261Crossref PubMed Scopus (120) Google Scholar), a testis-specific isoform of angiotensin-converting enzyme (tACE) (12Hagaman J.R. Moyer J.S. Bachman E.S. Sibony M. Magyar P.L. Welch J.E. Smithies O. Krege J.H. O'Brien D.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2552-2557Crossref PubMed Scopus (307) Google Scholar, 13Kessler S.P. Rowe T.M. Gomos J.B. Kessler P.M. Sen G.C. J. Biol. Chem. 2000; 275: 26259-26264Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), and ADAM2 (14Cho C. Bunch D.O. Faure J.-E. Goulding E.H. Eddy E.M. Primakoff P. Myles D.G. Science. 1998; 281: 1857-1859Crossref PubMed Scopus (452) Google Scholar) are all sterile because of the sperm defects in binding to the ZP and in migrating from the uterus into oviduct. Sperm from ADAM3-deficient mice are also defective in ZP binding but can ascend into the oviduct normally (15Shamsadin R. Adham I.M. Nayernia K. Heinlein U.A.O. Oberwinkler H. Engel W. Biol. Reprod. 1999; 61: 1445-1451Crossref PubMed Scopus (197) Google Scholar, 16Nishimura H. Cho C. Branciforte D.R. Myles D.G. Primakoff P. Dev. Biol. 2001; 233: 204-213Crossref PubMed Scopus (220) Google Scholar). An intriguing finding is that the disruption of functional ADAM2 results in the loss or severe reduction of ADAM1 and ADAM3 in testicular germ cells (TGC) and epididymal sperm (14Cho C. Bunch D.O. Faure J.-E. Goulding E.H. Eddy E.M. Primakoff P. Myles D.G. Science. 1998; 281: 1857-1859Crossref PubMed Scopus (452) Google Scholar, 16Nishimura H. Cho C. Branciforte D.R. Myles D.G. Primakoff P. Dev. Biol. 2001; 233: 204-213Crossref PubMed Scopus (220) Google Scholar). Thus, it is reasonable to postulate that ADAM1, ADAM2, and ADAM3 interact with one another to make sperm fertilization-competent. Fertilin is a heterodimeric protein complex present on the sperm surface consisting of ADAM1 and ADAM2 (17Blobel C.P. Wolfsberg T.G. Turck C.W. Myles D.G. Primakoff P. White J.M. Nature. 1992; 356: 248-252Crossref PubMed Scopus (631) Google Scholar, 18Lum L. Blobel C.P. Dev. Biol. 1997; 191: 131-145Crossref PubMed Scopus (89) Google Scholar, 19Waters S.I. White J.M. Biol. Reprod. 1997; 56: 1245-1254Crossref PubMed Scopus (66) Google Scholar). In mouse, two different genes encoding ADAM1a and ADAM1b corresponding to guinea pig ADAM1 are both localized on chromosome 5 (20Nishimura H. Kim E. Fujimori T. Kashiwabara S. Kuroiwa A. Matsuda Y. Baba T. Gene (Amst.). 2002; 291: 67-76Crossref PubMed Scopus (29) Google Scholar). ADAM1a is present within the endoplasmic reticulum (ER) of TGC, whereas epididymal sperm contain only ADAM1b on the cell surface (21Kim E. Nishimura H. Baba T. Biochem. Biophys. Res. Commun. 2003; 304: 313-319Crossref PubMed Scopus (46) Google Scholar). Moreover, although either ADAM is capable of forming the fertilin protein complex with ADAM2 in the ER of TGC, only the ADAM1b/ADAM2 fertilin complex is present on the sperm surface (21Kim E. Nishimura H. Baba T. Biochem. Biophys. Res. Commun. 2003; 304: 313-319Crossref PubMed Scopus (46) Google Scholar). These data imply that ADAM1a and ADAM1b have critical, distinct roles in spermatogenesis and fertilization, respectively. To elucidate the role(s) of ADAM1a in TGC, we produced male mice carrying a disruptive mutation in ADAM1a by homologous recombination. The ADAM1a-deficient male mice are infertile because sperm have a severely impaired ability to migrate from the uterus into the oviduct through the uterotubal junction (UTJ). ADAM3, but not other testis (sperm)-specific proteins, is strongly reduced on ADAM1a-deficient mouse sperm. Comparative experiments using TGC and epididymal sperm of ADAM1a-, ADAM2-, and ADAM3-deficient mice suggest that the ADAM1a/ADAM2 fertilin complex may be implicated in the transport of sperm proteins, including ADAM3, from the ER of TGC onto the cell surface. Generation of Mutant Mice Lacking ADAM1a—A mouse genomic clone, mFAG10 (20Nishimura H. Kim E. Fujimori T. Kashiwabara S. Kuroiwa A. Matsuda Y. Baba T. Gene (Amst.). 2002; 291: 67-76Crossref PubMed Scopus (29) Google Scholar), encoding ADAM1a was used for construction of a targeting vector containing an expression cassette of the neomycin-resistance gene (neo) flanked by a 1.5-kbp genomic region of ADAM1a (see Fig. 1A). The MC1 promoter-driven herpes simplex virus thymidine kinase gene (tk) was also included in the targeting vector for negative selection. Following electroporation of the targeting vector, which had been linearized by digestion with KpnI, into mouse D3 embryonic stem cells, homologous recombinants were selected by using G418 and gancyclovir, as described previously (22Kashiwabara S. Noguchi J. Zhuang T. Ohmura K. Honda A. Sugiura S. Miyamoto K. Takahashi S. Inoue K. Ogura A. Baba T. Science. 2002; 298: 1999-2002Crossref PubMed Scopus (106) Google Scholar). Seven embryonic stem cell clones carrying the targeted mutation were identified from 450 clones resistant to G418 and gancyclovir and then injected into C57BL/6 mouse blastocysts. Chimeric male mice were crossed to ICR female mice (Japan SLC Inc., Shizuoka, Japan) to establish heterozygous mutant lines. Homozygous mice were obtained by mating of heterozygous males and females. All animal experiments were carried out according to the Guide for the Care and Use of Laboratory Animals in the University of Tsukuba. Blot Hybridization—Genomic DNA was prepared from the mouse tail, digested by EcoRI, separated by agarose gel electrophoresis, and transferred onto Hybond-N+ nylon membranes (Amersham Biosciences), as described previously (23Honda A. Yamagata K. Sugiura S. Watanabe K. Baba T. J. Biol. Chem. 2002; 277: 16976-16984Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Total cellular RNA was prepared from testicular tissues using ISOGEN (Nippon Gene, Toyama, Japan) (24Kashiwabara S. Zhuang T. Yamagata K. Noguchi J. Fukamizu A. Baba T. Dev. Biol. 2000; 228: 106-115Crossref PubMed Scopus (82) Google Scholar). The RNA samples were glyoxylated, separated by agarose gel electrophoresis, and transferred onto the nylon membranes. The blots were probed by 32P-labeled DNA fragments and analyzed by a BAS2000 Bio-Image Analyzer (Fuji Photo Film, Tokyo, Japan) (24Kashiwabara S. Zhuang T. Yamagata K. Noguchi J. Fukamizu A. Baba T. Dev. Biol. 2000; 228: 106-115Crossref PubMed Scopus (82) Google Scholar). Preparation of Protein Extracts—TGC and cauda epididymal sperm were obtained typically from 3-month-old mice (25Phelps B.M. Koppel D.E. Primakoff P. Myles D.G. J. Cell Biol. 1990; 111: 1839-1847Crossref PubMed Scopus (146) Google Scholar, 26Yamagata K. Murayama K. Okabe M. Toshimori K. Nakanishi T. Kashiwabara S. Baba T. J. Biol. Chem. 1998; 273: 10470-10474Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). were in a consisting of and protease on for and for The was to in the of and then as described Protein was using a protein ADAM1b, and were prepared as described previously (21Kim E. Nishimura H. Baba T. Biochem. Biophys. Res. Commun. 2003; 304: 313-319Crossref PubMed Scopus (46) Google Scholar, D. Kashiwabara S. Honda A. Yamagata K. M. Okabe M. Baba T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, T. Y. Y. Kashiwabara S. K. M. Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar). was produced in female New White by of protein containing the and epidermal growth of ADAM3 between and The was as described (21Kim E. Nishimura H. Baba T. Biochem. Biophys. Res. Commun. 2003; 304: 313-319Crossref PubMed Scopus (46) Google Scholar, D. Kashiwabara S. Honda A. Yamagata K. M. Okabe M. Baba T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). ADAM2 and ADAM3 were from mouse and were from M. Nakanishi T. Yamada S. Wada I. Kominami K. Tanaka H. Nozaki M. Nishimune Y. Okabe M. Dev. Biol. 2001; 240: 254-261Crossref PubMed Scopus (120) Google Scholar), M. Nakanishi T. Yamada S. Wada I. Kominami K. Tanaka H. Nozaki M. Nishimune Y. Okabe M. Dev. Biol. 2001; 240: 254-261Crossref PubMed Scopus (120) Google Scholar), and enzyme Y. Biol. Reprod. PubMed Scopus Google Scholar, M. J.M. P. PubMed Google Scholar, P. PubMed Scopus Google Scholar) were of M. Blot were by for 5 in the of and separated by and transferred onto membranes with the blots were with for and then with for proteins were by an (Amersham complex formation between two ADAMs was examined, protein samples were in for separated by and transferred onto membranes. To the proteins on the membranes were for in containing and with to and proteins were as described of Sperm into the of sperm from the uterus into oviduct was as described previously M. Nakanishi T. Yamada S. Wada I. Kominami K. Tanaka H. Nozaki M. Nishimune Y. Okabe M. Dev. Biol. 2001; 240: 254-261Crossref PubMed Scopus (120) Google Scholar). female mice were by of Tokyo, Japan) followed by The female mice were with male mice the and the formation of was The were with a of the uterus the in containing with and in Tokyo, were with and then an of the of by with cumulus cells, were from the of ICR mice and in a of a containing and Y. M. T. J. Reprod. 16: Google Scholar) with cauda epididymal sperm from 3-month-old mice were by for in a of in An of the sperm was with the eggs in The eggs and sperm were for in The of the cumulus was according to the described previously D. Kashiwabara S. Honda A. Yamagata K. M. Okabe M. Baba T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). of eggs were with testicular for to cumulus cells, and in a of with An of sperm was to the and the was for in The of sperm to the egg ZP was using an with (Fuji Photo Film, Tokyo, Japan) K. Murayama K. Okabe M. Toshimori K. Nakanishi T. Kashiwabara S. Baba T. J. Biol. Chem. 1998; 273: 10470-10474Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). of in eggs were by sperm in a of for in the eggs were with for to cumulus cells and then The female and male in the eggs were with for and then an as described (14Cho C. Bunch D.O. Faure J.-E. Goulding E.H. Eddy E.M. Primakoff P. Myles D.G. Science. 1998; 281: 1857-1859Crossref PubMed Scopus (452) Google Scholar, 16Nishimura H. Cho C. Branciforte D.R. Myles D.G. Primakoff P. Dev. Biol. 2001; 233: 204-213Crossref PubMed Scopus (220) Google Scholar). of eggs were with for and then The eggs were with and then with sperm for in the eggs were the as described (14Cho C. Bunch D.O. Faure J.-E. Goulding E.H. Eddy E.M. Primakoff P. Myles D.G. Science. 1998; 281: 1857-1859Crossref PubMed Scopus (452) Google Scholar, 16Nishimura H. Cho C. Branciforte D.R. Myles D.G. Primakoff P. Dev. Biol. 2001; 233: 204-213Crossref PubMed Scopus (220) Google Scholar). were from TGC in a consisting of and protease as described by with of protein on for and for 5 The was with Protein and the was on for with the was with the in a containing and for 5 and then analyzed by and were used as To the of ADAM1a in we produced mutant mice lacking ADAM1a by homologous in embryonic stem The targeting was to the region containing the pro-, metalloprotease, disintegrin, and in ADAM1a with 1A). The of heterozygous and mice for mutation of ADAM1a were identified by of genomic DNA the of ADAM1a in testis Moreover, protein of TGC completely the precursor of ADAM1a These data the loss of ADAM1a in male of male and female mice the of mice mice were in and formation of in the of males was severely only a was and only two were males were with for two the male mice are male and female mice and the were also and and in and of the testis was between and mice despite the of cells in the of mice were epididymal sperm of mice not be from of mice by or of not In the of sperm in the cauda and uterus in mice were to and in Thus, is no in sperm and in the male the migration of sperm from the uterus into the oviduct through in Fig. of of the the of sperm in the uterus and a of the oviduct that into the S. Biol. Reprod. PubMed Scopus Google Scholar). However, sperm were not in the and all of in the The of sperm is with of sperm lacking calmegin (10Ikawa M. Wada I. Kominami K. Watanabe D. Toshimori K. Nishimune Y. Okabe M. Nature. 1997; 387: 607-611Crossref PubMed Scopus (248) Google Scholar, 11Ikawa M. Nakanishi T. Yamada S. Wada I. Kominami K. Tanaka H. Nozaki M. Nishimune Y. Okabe M. Dev. Biol. 2001; 240: 254-261Crossref PubMed Scopus (120) Google Scholar), (12Hagaman J.R. Moyer J.S. Bachman E.S. Sibony M. Magyar P.L. Welch J.E. Smithies O. Krege J.H. O'Brien D.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2552-2557Crossref PubMed Scopus (307) Google Scholar), and ADAM2 (14Cho C. Bunch D.O. Faure J.-E. Goulding E.H. Eddy E.M. Primakoff P. Myles D.G. Science. 1998; 281: 1857-1859Crossref PubMed Scopus (452) Google Scholar). Thus, the loss of the ADAM1a is with the ability of sperm to ascend into the oviduct through the To the of ADAM1a in the ER of TGC in vitro fertilization were carried out using cauda epididymal sperm. sperm a in the dispersal of cumulus cells from the cumulus surrounding the eggs The delayed dispersal of cumulus cells may the impaired ability of sperm to in the extracellular matrix of cumulus However, sperm D. Kashiwabara S. Honda A. Yamagata K. M. Okabe M. Baba T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, P. H. Myles D.G. J. Cell Biol. PubMed Scopus Google Scholar) was normally present on the sperm surface of as described eggs were sperm binding to the ZP was severely reduced by the loss of ADAM1a sperm were capable of fertilizing cumulus-intact, eggs normally despite the delayed dispersal of cumulus cells and the impaired ability to the ZP no in the fusion of sperm with eggs was between and mice we that the infertility of male mice is by the of sperm to ascend into the oviduct. ADAM1 and ADAM2 are both as in TGC and into the the of testicular sperm into the in the (21Kim E. Nishimura H. Baba T. Biochem. Biophys. Res. Commun. 2003; 304: 313-319Crossref PubMed Scopus (46) Google Scholar, B.M. Koppel D.E. Primakoff P. Myles D.G. J. Cell Biol. 1990; 111: 1839-1847Crossref PubMed Scopus (146) Google Scholar, C.P. Myles D.G. Primakoff P. White J.M. J. Cell Biol. 1990; 111: PubMed Scopus Google Scholar). The of ADAM2 has been to in the reduction and loss of ADAM1 in TGC and epididymal (14Cho C. Bunch D.O. Faure J.-E. Goulding E.H. Eddy E.M. Primakoff P. Myles D.G. Science. 1998; 281: 1857-1859Crossref PubMed Scopus (452) Google Scholar, 16Nishimura H. Cho C. Branciforte D.R. Myles D.G. Primakoff P. Dev. Biol. 2001; 233: 204-213Crossref PubMed Scopus (220) Google Scholar), the of forming the fertilin protein complex between two ADAMs a of the is present on the ER membrane of TGC and is for the between ADAM1 and ADAM2 M. Nakanishi T. Yamada S. Wada I. Kominami K. Tanaka H. Nozaki M. Nishimune Y. Okabe M. Dev. Biol. 2001; 240: 254-261Crossref PubMed Scopus (120) Google Scholar). two of ADAM1, ADAM1a and ADAM1b, in mouse TGC (20Nishimura H. Kim E. Fujimori T. Kashiwabara S. Kuroiwa A. Matsuda Y. Baba T. Gene (Amst.). 2002; 291: 67-76Crossref PubMed Scopus (29) Google Scholar, E. Nishimura H. Baba T. Biochem. Biophys. Res. Commun. 2003; 304: 313-319Crossref PubMed Scopus (46) Google Scholar), we the loss of ADAM1a the formation of the fertilin protein in TGC and the appearance of proteins for fertilization on the sperm membrane. that testis (sperm)-specific proteins, including ADAM3, tACE, and are normally present in TGC of although only ADAM3 was severely reduced in sperm The of ADAM1b, ADAM2, and were and sperm were the formation of heterodimeric protein between the of ADAM1a or ADAM1b and ADAM2 in TGC was examined, ADAM1a/ADAM2 fertilin was the only complex in mice In we that a precursor of ADAM3 is in TGC, into the a sperm in the and localized on the surface of cauda epididymal sperm not the ADAM3 precursor is normally produced in TGC the reduced level of ADAM3 in sperm is to the transport of the ADAM3 precursor from the ER to the cell surface Thus, the formation of the fertilin complex between ADAM1a and ADAM2 in TGC may be in the appearance of ADAM3 on the sperm surface. Moreover, results the that ADAM3 is one of the in the binding of sperm to the egg ZP ADAM1a-, ADAM2-, and ADAM3-deficient mouse which all show a in binding to the ZP, also all have in that ADAM3 (14Cho C. Bunch D.O. Faure J.-E. Goulding E.H. Eddy E.M. Primakoff P. Myles D.G. Science. 1998; 281: 1857-1859Crossref PubMed Scopus (452) Google Scholar, R. Adham I.M. Nayernia K. Heinlein U.A.O. Oberwinkler H. Engel W. Biol. Reprod. 1999; 61: 1445-1451Crossref PubMed Scopus (197) Google Scholar, 16Nishimura H. Cho C. Branciforte D.R. Myles D.G. Primakoff P. Dev. Biol. 2001; 233: 204-213Crossref PubMed Scopus (220) Google Scholar). To the functional roles of fertilin in TGC, protein from TGC and epididymal sperm of mice lacking ADAM2 or ADAM3 were analyzed by with the level of ADAM1b was reduced in TGC of mice despite the of ADAM3, and fertilin complex between ADAM1a and ADAM2 between ADAM and ADAM2 was in TGC to the loss of ADAM2 sperm completely ADAM1b and ADAM3 as described previously H. Cho C. Branciforte D.R. Myles D.G. Primakoff P. Dev. Biol. 2001; 233: 204-213Crossref PubMed Scopus (220) Google Scholar). In TGC and epididymal sperm of only ADAM3 was whereas the of other proteins were between and mice The ADAM1a/ADAM2 and ADAM1b/ADAM2 fertilin were in TGC These data suggest that the appearance of ADAM3 on the sperm surface may the of the ADAM1a/ADAM2 fertilin complex in Moreover, the formation of the heterodimeric protein complex between the ADAM1b and ADAM2 in TGC may be for the of the of two ADAMs on the sperm surface. The severely reduced of ADAM3 in and sperm H. Cho C. Branciforte D.R. Myles D.G. Primakoff P. Dev. Biol. 2001; 233: 204-213Crossref PubMed Scopus (220) Google Scholar) to postulate a direct of ADAM3 with ADAM1b, or ADAM2 and with ADAM1a/ADAM2 or ADAM1b/ADAM2 fertilin complex in To this we carried out of protein from TGC of and mutant mice using ADAM1b, ADAM2, and ADAM3 In TGC of the and ADAM2 was with both ADAM1a and ADAM1b, whereas the with ADAM1a and ADAM1b in to ADAM1b and ADAM2 were from TGC with or These data are with the of ADAM1a/ADAM2 and ADAM1b/ADAM2 fertilin in both and TGC and with the of only ADAM1a/ADAM2 fertilin and both in and TGC, and However, no ADAM3 was with ADAM1b, or ADAM2 that ADAM3 is of forming a protein complex with ADAM1b, and ADAM2 in Although many ADAM members exclusively expressed in the testis have been is of their roles in TGC show a between the loss of ADAM1a and the transport of ADAM3 onto the cell surface. finding may a for the of the of sperm proteins by fertilin in ADAM1a/ADAM2 fertilin may be implicated in the selective transport of specific sperm proteins, including ADAM3, that in the sperm into the oviduct in the sperm through the of cumulus cells experiments that ADAM1a and ADAM3 are localized in of TGC not The ADAM1a/ADAM2 fertilin complex may the membrane as a in which the selected sperm proteins such as ADAM3 are although this fertilin complex is of with ADAM3 in TGC ADAM1a/ADAM2 fertilin may be also with an the transport of ER the of protein transport by ADAM1a/ADAM2 fertilin in the ER of TGC to be elucidated. D. P. and K. of for protein from TGC and sperm of and ADAM3-deficient M. Okabe and Y. for in the of ADAM1a-deficient mice and ADAM2, and angiotensin-converting S. Takahashi of for of testicular and S. B. of for