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Impaired sperm aggregation in Adam2 and Adam3 null mice

2010; Elsevier BV; Volume: 93; Issue: 8 Linguagem: Inglês

10.1016/j.fertnstert.2010.03.013

1556-5653

Cecil Han, Juntae Kwon, Inju Park, Boyeon Lee, Sora Jin, Heejin Choi, Chunghee Cho,

Genetic and Clinical Aspects of Sex Determination and Chromosomal Abnormalities

Previous studies have shown that sperm from Adam2 and Adam3 knockout mice have defective migration in the female reproductive tracts and cannot bind to the egg's zona pellucida (ZP), which leads to infertility. Here, we report that Adam2 and Adam3 knockout sperm have severely impaired sperm aggregation and that this defect is not restored over time during in vitro cultivation, suggesting the requirement of ADAM2 and ADAM3 in normal sperm association. Previous studies have shown that sperm from Adam2 and Adam3 knockout mice have defective migration in the female reproductive tracts and cannot bind to the egg's zona pellucida (ZP), which leads to infertility. Here, we report that Adam2 and Adam3 knockout sperm have severely impaired sperm aggregation and that this defect is not restored over time during in vitro cultivation, suggesting the requirement of ADAM2 and ADAM3 in normal sperm association. A disintegrin and metalloproteases (ADAMs) are transmembrane glycoproteins that share a conserved multidomain structure. A number of ADAM family members are exclusively or predominantly transcribed in spermatogenic cells (1Cho C. Mammalian ADAMs with testis-specific or -predominant expression.in: Hooper N.M. Lendeckel U. The ADAM family of proteases. Springer, Dordrecht, The Netherlands2005: 239-259Crossref Google Scholar). Male mice lacking some of these ADAMs, such as ADAM1a, ADAM2, or ADAM3, are infertile because the knockout sperm are unable to migrate from the uterus to the oviduct and cannot bind to the egg's zona pellucida (ZP) (2Cho C. Bunch D.O. Faure J.E. Goulding E.H. Eddy E.M. Primakoff P. et al.Fertilization defects in sperm from mice lacking fertilin beta.Science. 1998; 281: 1857-1859Crossref PubMed Scopus (437) Google Scholar, 3Shamsadin R. Adham I.M. Nayernia K. Heinlein U.A. Oberwinkler H. Engel W. Male mice deficient for germ-cell cyritestin are infertile.Biol Reprod. 1999; 61: 1445-1451Crossref PubMed Scopus (192) Google Scholar, 4Nishimura H. Cho C. Branciforte D.R. Myles D.G. Primakoff P. Analysis of loss of adhesive function in sperm lacking cyritestin or fertilin beta.Dev Biol. 2001; 233: 204-213Crossref PubMed Scopus (212) Google Scholar, 5Nishimura H. Kim E. Nakanishi T. Baba T. Possible function of the ADAM1a/ADAM2 Fertilin complex in the appearance of ADAM3 on the sperm surface.J Biol Chem. 2004; 279: 34957-34962Crossref PubMed Scopus (152) Google Scholar, 6Yamaguchi R. Muro Y. Isotani A. Tokuhiro K. Takumi K. Adham I. et al.Disruption of ADAM3 impairs the migration of sperm into oviduct in mouse.Biol Reprod. 2009; 81: 142-146Crossref PubMed Scopus (108) Google Scholar). These ADAMs form complexes with each other and also have a relationship with other reproductive ADAMs, such as ADAM4, ADAM5, ADAM6, and ADAM7, during sperm development (5Nishimura H. Kim E. Nakanishi T. Baba T. Possible function of the ADAM1a/ADAM2 Fertilin complex in the appearance of ADAM3 on the sperm surface.J Biol Chem. 2004; 279: 34957-34962Crossref PubMed Scopus (152) Google Scholar, 7Nishimura H. Myles D.G. Primakoff P. Identification of an ADAM2–ADAM3 complex on the surface of mouse testicular germ cells and cauda epididymal sperm.J Biol Chem. 2007; 282: 17900-17907Crossref PubMed Scopus (43) Google Scholar, 8Kim T. Oh J. Woo J.M. Choi E. Im S.H. Yoo Y.J. et al.Expression and relationship of male reproductive ADAMs in mouse.Biol Reprod. 2006; 74: 744-750Crossref PubMed Scopus (68) Google Scholar, 9Han C. Choi E. Park I. Lee B. Jin S. Kim D.H. et al.Comprehensive analysis of reproductive ADAMs: relationship of ADAM4 and ADAM6 with an ADAM complex required for fertilization in mice.Biol Reprod. 2009; 80: 1001-1008Crossref PubMed Scopus (52) Google Scholar). In the course of analyzing ADAM knockout mice, we found altered behavior of Adam2-/- and Adam3-/- sperm. Here we report a new phenotype in the knockout sperm, a defect in sperm–sperm adhesion.Sperm were obtained from 8-week-old wild-type (WT), Adam2, and Adam3 knockout male mice (2Cho C. Bunch D.O. Faure J.E. Goulding E.H. Eddy E.M. Primakoff P. et al.Fertilization defects in sperm from mice lacking fertilin beta.Science. 1998; 281: 1857-1859Crossref PubMed Scopus (437) Google Scholar, 4Nishimura H. Cho C. Branciforte D.R. Myles D.G. Primakoff P. Analysis of loss of adhesive function in sperm lacking cyritestin or fertilin beta.Dev Biol. 2001; 233: 204-213Crossref PubMed Scopus (212) Google Scholar). Sperm were directly released from the caudal epididymis and vas deferens into prewarmed M16 media containing 0.5% bovine serum albumin (BSA) and then incubated at 37°C/5% CO2. The sperm concentration was adjusted to 2–4 × 106 cells/mL. Sperm aggregation/grouping and motility were examined using a light microscope (DMLB; Leica Microsystems, Wetzlar, Germany) after incubation for 0, 10, 30, and 60 minutes. The number of aggregated sperm groups (at least >10 sperm per group) was counted in each microscopic field in three independent experiments. In addition, live sperm behavior was recorded after 10–20 minutes of incubation using an inverted microscope (DMIL; Leica Microsystems, Wetzlar, Germany) equipped with a digital camera. To validate the genotypes of sperm observed for aggregation, the sperm were subjected to Western blot analysis using anti-ADAM2 and anti-ADAM3 antibodies (9Han C. Choi E. Park I. Lee B. Jin S. Kim D.H. et al.Comprehensive analysis of reproductive ADAMs: relationship of ADAM4 and ADAM6 with an ADAM complex required for fertilization in mice.Biol Reprod. 2009; 80: 1001-1008Crossref PubMed Scopus (52) Google Scholar).We found that most WT sperm formed motile clumps of 15–20 sperm, attaching to each other mostly at the head region within the first 10–60 minutes of incubation (Fig. 1A and Supplementary Movie 1). In contrast, we were unable to identify any distinct group formation in Adam2 knockout sperm (Fig. 1A and Supplementary Movie 2). Most Adam3 knockout sperm also did not form aggregates and only a limited number of sperm groups was observed (Fig. 1A). To make an accurate estimate of the sperm grouping phenotype, we compared the number of sperm aggregates and the number of sperm with motility in each microscopic field between WT and knockout sperm. The degrees of sperm aggregation in Adam2 and Adam3 null sperm were 1% and 27% of the WT, respectively (Fig. 1B). There was no difference in sperm motility between WT and knockout sperm, indicating that defective aggregation is not due to altered motility (Fig. 1B). These results indicate that Adam2 and Adam3 knockout sperm have abnormal behavior in aggregation or grouping.To better understand the nature of sperm aggregation, we determined whether there was a time-dependent change in sperm–sperm association and whether this grouping behavior was reversible. We found that both WT and knockout sperm were rapidly dispersed into single cell suspensions without aggregation when released from the tissues (0 minutes of incubation time) (Fig. 1C). However, WT sperm, but not Adam2 and Adam3 null sperm, exhibited a dramatic increase in aggregation after 10 minutes of incubation and the level of the sperm grouping stayed the same during the period of additional incubation (10–60 minutes) (Fig. 1C). After 60 minutes of incubation, we gently disentangled the aggregated sperm by repeated pipetting and then monitored their behavior. The WT sperm dissociated by pipetting rapidly formed new aggregates, reaching about 90% of the original level of aggregation after 10 minutes of additional incubation (Fig. 1C). Although a lower level of aggregation was observed in Adam3 null sperm, they also showed a pattern similar to that of WT sperm during sperm dissociation analysis by pipetting. Adam2 null sperm did not exhibit any change in aggregation before and after pipetting (Fig. 1C). Taken together, these results suggest that sperm–sperm adhesion is an inherent and reversible phenomenon and that the defect of Adam2 and Adam3 null sperm in this characteristic is not restored over time.Finally, the absence of the ADAM proteins in mutant sperm used for the aggregation analysis was confirmed by Western blot analysis (data not shown). Consistent with previous studies, the level of ADAM2 was weakly reduced in Adam3 null sperm and ADAM3 was completely lost in Adam2 null sperm (data not shown). Previously, the levels of other ADAM proteins, such as ADAM4, ADAM5, ADAM6, and ADAM7, were found to be also affected in Adam2 and Adam3 knockout sperm (8Kim T. Oh J. Woo J.M. Choi E. Im S.H. Yoo Y.J. et al.Expression and relationship of male reproductive ADAMs in mouse.Biol Reprod. 2006; 74: 744-750Crossref PubMed Scopus (68) Google Scholar, 9Han C. Choi E. Park I. Lee B. Jin S. Kim D.H. et al.Comprehensive analysis of reproductive ADAMs: relationship of ADAM4 and ADAM6 with an ADAM complex required for fertilization in mice.Biol Reprod. 2009; 80: 1001-1008Crossref PubMed Scopus (52) Google Scholar). It should be noted that the loss of these ADAM proteins is more severe in sperm from Adam2 knockout mice than Adam3 knockout mice. Similarly, Adam2 null sperm exhibited an almost complete defect in aggregation, but this defect was less severe in Adam3 null sperm (Fig. 1A–C), suggesting that ADAM2 is essential for normal sperm aggregation.Sperm–sperm association has been shown to occur during epididymal maturation after sperm leave the testis and persist after ejaculation in the uterus and oviducts of mated females (10Monclus M.A. Cesari A. Cabrillana M.E. Borelli P.V. Vincenti A.E. Burgos M.H. et al.Mouse sperm rosette: assembling during epididymal transit, in vitro disassemble, and oligosaccharide participation in the linkage material.Anat Rec (Hoboken). 2007; 290: 814-824Crossref PubMed Scopus (10) Google Scholar, 11Yanagimachi R. Mahi C.A. The sperm acrosome reaction and fertilization in the guinea-pig: a study in vivo.J Reprod Fertil. 1976; 46: 49-54Crossref PubMed Scopus (57) Google Scholar). Sperm grouping was recently considered to be an altruistic behavior that provides advantages to fertilization (12Moore H. Dvorakova K. Jenkins N. Breed W. Exceptional sperm cooperation in the wood mouse.Nature. 2002; 418: 174-177Crossref PubMed Scopus (194) Google Scholar, 13Pizzari T. Foster K.R. Sperm sociality: cooperation, altruism, and spite.PLoS Biol. 2008; 6: e130Crossref PubMed Scopus (62) Google Scholar). With regard to a relationship between sperm aggregation and the ADAM-dependent fertilization processes, it is noteworthy that the ADAM1a/ADAM2 complex formed in testicular germ cells during spermatogenesis has been suggested to regulate the integrity of sperm proteins including ADAM3 (5Nishimura H. Kim E. Nakanishi T. Baba T. Possible function of the ADAM1a/ADAM2 Fertilin complex in the appearance of ADAM3 on the sperm surface.J Biol Chem. 2004; 279: 34957-34962Crossref PubMed Scopus (152) Google Scholar, 8Kim T. Oh J. Woo J.M. Choi E. Im S.H. Yoo Y.J. et al.Expression and relationship of male reproductive ADAMs in mouse.Biol Reprod. 2006; 74: 744-750Crossref PubMed Scopus (68) Google Scholar, 9Han C. Choi E. Park I. Lee B. Jin S. Kim D.H. et al.Comprehensive analysis of reproductive ADAMs: relationship of ADAM4 and ADAM6 with an ADAM complex required for fertilization in mice.Biol Reprod. 2009; 80: 1001-1008Crossref PubMed Scopus (52) Google Scholar), implying an indirect role of ADAM2 in sperm functions. Indeed, Adam1b null sperm contain a severely reduced level of ADAM2, but they are normal in progression into the oviduct and binding to the ZP (14Kim E. Yamashita M. Nakanishi T. Park K.E. Kimura M. Kashiwabara S. et al.Mouse sperm lacking ADAM1b/ADAM2 fertilin can fuse with the egg plasma membrane and effect fertilization.J Biol Chem. 2006; 281: 5634-5639Crossref PubMed Scopus (71) Google Scholar). Therefore, it is now considered that the infertile phenotype of Adam2 null sperm is attributable to the lack of ADAM3. Because the degree of a defect in sperm aggregation is not the same between Adam2 and Adam3 null sperm, the molecular mechanism of sperm aggregation could differ from that of the fertilization processes. Alternatively, it is possible that ADAM3 functions in sperm aggregation as well as in the fertilization processes. The additive impairment of sperm aggregation in Adam2 null sperm could be due to the greater loss of other sperm proteins, such as ADAM4, ADAM5, ADAM6, and ADAM7 (8Kim T. Oh J. Woo J.M. Choi E. Im S.H. Yoo Y.J. et al.Expression and relationship of male reproductive ADAMs in mouse.Biol Reprod. 2006; 74: 744-750Crossref PubMed Scopus (68) Google Scholar, 9Han C. Choi E. Park I. Lee B. Jin S. Kim D.H. et al.Comprehensive analysis of reproductive ADAMs: relationship of ADAM4 and ADAM6 with an ADAM complex required for fertilization in mice.Biol Reprod. 2009; 80: 1001-1008Crossref PubMed Scopus (52) Google Scholar), which may also act in sperm aggregation.In conclusion, we discovered that sperm–sperm association or aggregation is impaired in Adam2 and Adam3 knockout sperm. These mutant sperm were previously found to be defective in migration in the female reproductive tracts and to lack the ability to bind to the egg ZP. Our finding provides novel information regarding the role of the ADAM proteins in sperm behavior and a model to investigate a relationship between sperm aggregation and the ADAM-dependent fertilization processes. A disintegrin and metalloproteases (ADAMs) are transmembrane glycoproteins that share a conserved multidomain structure. A number of ADAM family members are exclusively or predominantly transcribed in spermatogenic cells (1Cho C. Mammalian ADAMs with testis-specific or -predominant expression.in: Hooper N.M. Lendeckel U. The ADAM family of proteases. Springer, Dordrecht, The Netherlands2005: 239-259Crossref Google Scholar). Male mice lacking some of these ADAMs, such as ADAM1a, ADAM2, or ADAM3, are infertile because the knockout sperm are unable to migrate from the uterus to the oviduct and cannot bind to the egg's zona pellucida (ZP) (2Cho C. Bunch D.O. Faure J.E. Goulding E.H. Eddy E.M. Primakoff P. et al.Fertilization defects in sperm from mice lacking fertilin beta.Science. 1998; 281: 1857-1859Crossref PubMed Scopus (437) Google Scholar, 3Shamsadin R. Adham I.M. Nayernia K. Heinlein U.A. Oberwinkler H. Engel W. Male mice deficient for germ-cell cyritestin are infertile.Biol Reprod. 1999; 61: 1445-1451Crossref PubMed Scopus (192) Google Scholar, 4Nishimura H. Cho C. Branciforte D.R. Myles D.G. Primakoff P. Analysis of loss of adhesive function in sperm lacking cyritestin or fertilin beta.Dev Biol. 2001; 233: 204-213Crossref PubMed Scopus (212) Google Scholar, 5Nishimura H. Kim E. Nakanishi T. Baba T. Possible function of the ADAM1a/ADAM2 Fertilin complex in the appearance of ADAM3 on the sperm surface.J Biol Chem. 2004; 279: 34957-34962Crossref PubMed Scopus (152) Google Scholar, 6Yamaguchi R. Muro Y. Isotani A. Tokuhiro K. Takumi K. Adham I. et al.Disruption of ADAM3 impairs the migration of sperm into oviduct in mouse.Biol Reprod. 2009; 81: 142-146Crossref PubMed Scopus (108) Google Scholar). These ADAMs form complexes with each other and also have a relationship with other reproductive ADAMs, such as ADAM4, ADAM5, ADAM6, and ADAM7, during sperm development (5Nishimura H. Kim E. Nakanishi T. Baba T. Possible function of the ADAM1a/ADAM2 Fertilin complex in the appearance of ADAM3 on the sperm surface.J Biol Chem. 2004; 279: 34957-34962Crossref PubMed Scopus (152) Google Scholar, 7Nishimura H. Myles D.G. Primakoff P. Identification of an ADAM2–ADAM3 complex on the surface of mouse testicular germ cells and cauda epididymal sperm.J Biol Chem. 2007; 282: 17900-17907Crossref PubMed Scopus (43) Google Scholar, 8Kim T. Oh J. Woo J.M. Choi E. Im S.H. Yoo Y.J. et al.Expression and relationship of male reproductive ADAMs in mouse.Biol Reprod. 2006; 74: 744-750Crossref PubMed Scopus (68) Google Scholar, 9Han C. Choi E. Park I. Lee B. Jin S. Kim D.H. et al.Comprehensive analysis of reproductive ADAMs: relationship of ADAM4 and ADAM6 with an ADAM complex required for fertilization in mice.Biol Reprod. 2009; 80: 1001-1008Crossref PubMed Scopus (52) Google Scholar). In the course of analyzing ADAM knockout mice, we found altered behavior of Adam2-/- and Adam3-/- sperm. Here we report a new phenotype in the knockout sperm, a defect in sperm–sperm adhesion. Sperm were obtained from 8-week-old wild-type (WT), Adam2, and Adam3 knockout male mice (2Cho C. Bunch D.O. Faure J.E. Goulding E.H. Eddy E.M. Primakoff P. et al.Fertilization defects in sperm from mice lacking fertilin beta.Science. 1998; 281: 1857-1859Crossref PubMed Scopus (437) Google Scholar, 4Nishimura H. Cho C. Branciforte D.R. Myles D.G. Primakoff P. Analysis of loss of adhesive function in sperm lacking cyritestin or fertilin beta.Dev Biol. 2001; 233: 204-213Crossref PubMed Scopus (212) Google Scholar). Sperm were directly released from the caudal epididymis and vas deferens into prewarmed M16 media containing 0.5% bovine serum albumin (BSA) and then incubated at 37°C/5% CO2. The sperm concentration was adjusted to 2–4 × 106 cells/mL. Sperm aggregation/grouping and motility were examined using a light microscope (DMLB; Leica Microsystems, Wetzlar, Germany) after incubation for 0, 10, 30, and 60 minutes. The number of aggregated sperm groups (at least >10 sperm per group) was counted in each microscopic field in three independent experiments. In addition, live sperm behavior was recorded after 10–20 minutes of incubation using an inverted microscope (DMIL; Leica Microsystems, Wetzlar, Germany) equipped with a digital camera. To validate the genotypes of sperm observed for aggregation, the sperm were subjected to Western blot analysis using anti-ADAM2 and anti-ADAM3 antibodies (9Han C. Choi E. Park I. Lee B. Jin S. Kim D.H. et al.Comprehensive analysis of reproductive ADAMs: relationship of ADAM4 and ADAM6 with an ADAM complex required for fertilization in mice.Biol Reprod. 2009; 80: 1001-1008Crossref PubMed Scopus (52) Google Scholar). We found that most WT sperm formed motile clumps of 15–20 sperm, attaching to each other mostly at the head region within the first 10–60 minutes of incubation (Fig. 1A and Supplementary Movie 1). In contrast, we were unable to identify any distinct group formation in Adam2 knockout sperm (Fig. 1A and Supplementary Movie 2). Most Adam3 knockout sperm also did not form aggregates and only a limited number of sperm groups was observed (Fig. 1A). To make an accurate estimate of the sperm grouping phenotype, we compared the number of sperm aggregates and the number of sperm with motility in each microscopic field between WT and knockout sperm. The degrees of sperm aggregation in Adam2 and Adam3 null sperm were 1% and 27% of the WT, respectively (Fig. 1B). There was no difference in sperm motility between WT and knockout sperm, indicating that defective aggregation is not due to altered motility (Fig. 1B). These results indicate that Adam2 and Adam3 knockout sperm have abnormal behavior in aggregation or grouping. To better understand the nature of sperm aggregation, we determined whether there was a time-dependent change in sperm–sperm association and whether this grouping behavior was reversible. We found that both WT and knockout sperm were rapidly dispersed into single cell suspensions without aggregation when released from the tissues (0 minutes of incubation time) (Fig. 1C). However, WT sperm, but not Adam2 and Adam3 null sperm, exhibited a dramatic increase in aggregation after 10 minutes of incubation and the level of the sperm grouping stayed the same during the period of additional incubation (10–60 minutes) (Fig. 1C). After 60 minutes of incubation, we gently disentangled the aggregated sperm by repeated pipetting and then monitored their behavior. The WT sperm dissociated by pipetting rapidly formed new aggregates, reaching about 90% of the original level of aggregation after 10 minutes of additional incubation (Fig. 1C). Although a lower level of aggregation was observed in Adam3 null sperm, they also showed a pattern similar to that of WT sperm during sperm dissociation analysis by pipetting. Adam2 null sperm did not exhibit any change in aggregation before and after pipetting (Fig. 1C). Taken together, these results suggest that sperm–sperm adhesion is an inherent and reversible phenomenon and that the defect of Adam2 and Adam3 null sperm in this characteristic is not restored over time. Finally, the absence of the ADAM proteins in mutant sperm used for the aggregation analysis was confirmed by Western blot analysis (data not shown). Consistent with previous studies, the level of ADAM2 was weakly reduced in Adam3 null sperm and ADAM3 was completely lost in Adam2 null sperm (data not shown). Previously, the levels of other ADAM proteins, such as ADAM4, ADAM5, ADAM6, and ADAM7, were found to be also affected in Adam2 and Adam3 knockout sperm (8Kim T. Oh J. Woo J.M. Choi E. Im S.H. Yoo Y.J. et al.Expression and relationship of male reproductive ADAMs in mouse.Biol Reprod. 2006; 74: 744-750Crossref PubMed Scopus (68) Google Scholar, 9Han C. Choi E. Park I. Lee B. Jin S. Kim D.H. et al.Comprehensive analysis of reproductive ADAMs: relationship of ADAM4 and ADAM6 with an ADAM complex required for fertilization in mice.Biol Reprod. 2009; 80: 1001-1008Crossref PubMed Scopus (52) Google Scholar). It should be noted that the loss of these ADAM proteins is more severe in sperm from Adam2 knockout mice than Adam3 knockout mice. Similarly, Adam2 null sperm exhibited an almost complete defect in aggregation, but this defect was less severe in Adam3 null sperm (Fig. 1A–C), suggesting that ADAM2 is essential for normal sperm aggregation. Sperm–sperm association has been shown to occur during epididymal maturation after sperm leave the testis and persist after ejaculation in the uterus and oviducts of mated females (10Monclus M.A. Cesari A. Cabrillana M.E. Borelli P.V. Vincenti A.E. Burgos M.H. et al.Mouse sperm rosette: assembling during epididymal transit, in vitro disassemble, and oligosaccharide participation in the linkage material.Anat Rec (Hoboken). 2007; 290: 814-824Crossref PubMed Scopus (10) Google Scholar, 11Yanagimachi R. Mahi C.A. The sperm acrosome reaction and fertilization in the guinea-pig: a study in vivo.J Reprod Fertil. 1976; 46: 49-54Crossref PubMed Scopus (57) Google Scholar). Sperm grouping was recently considered to be an altruistic behavior that provides advantages to fertilization (12Moore H. Dvorakova K. Jenkins N. Breed W. Exceptional sperm cooperation in the wood mouse.Nature. 2002; 418: 174-177Crossref PubMed Scopus (194) Google Scholar, 13Pizzari T. Foster K.R. Sperm sociality: cooperation, altruism, and spite.PLoS Biol. 2008; 6: e130Crossref PubMed Scopus (62) Google Scholar). With regard to a relationship between sperm aggregation and the ADAM-dependent fertilization processes, it is noteworthy that the ADAM1a/ADAM2 complex formed in testicular germ cells during spermatogenesis has been suggested to regulate the integrity of sperm proteins including ADAM3 (5Nishimura H. Kim E. Nakanishi T. Baba T. Possible function of the ADAM1a/ADAM2 Fertilin complex in the appearance of ADAM3 on the sperm surface.J Biol Chem. 2004; 279: 34957-34962Crossref PubMed Scopus (152) Google Scholar, 8Kim T. Oh J. Woo J.M. Choi E. Im S.H. Yoo Y.J. et al.Expression and relationship of male reproductive ADAMs in mouse.Biol Reprod. 2006; 74: 744-750Crossref PubMed Scopus (68) Google Scholar, 9Han C. Choi E. Park I. Lee B. Jin S. Kim D.H. et al.Comprehensive analysis of reproductive ADAMs: relationship of ADAM4 and ADAM6 with an ADAM complex required for fertilization in mice.Biol Reprod. 2009; 80: 1001-1008Crossref PubMed Scopus (52) Google Scholar), implying an indirect role of ADAM2 in sperm functions. Indeed, Adam1b null sperm contain a severely reduced level of ADAM2, but they are normal in progression into the oviduct and binding to the ZP (14Kim E. Yamashita M. Nakanishi T. Park K.E. Kimura M. Kashiwabara S. et al.Mouse sperm lacking ADAM1b/ADAM2 fertilin can fuse with the egg plasma membrane and effect fertilization.J Biol Chem. 2006; 281: 5634-5639Crossref PubMed Scopus (71) Google Scholar). Therefore, it is now considered that the infertile phenotype of Adam2 null sperm is attributable to the lack of ADAM3. Because the degree of a defect in sperm aggregation is not the same between Adam2 and Adam3 null sperm, the molecular mechanism of sperm aggregation could differ from that of the fertilization processes. Alternatively, it is possible that ADAM3 functions in sperm aggregation as well as in the fertilization processes. The additive impairment of sperm aggregation in Adam2 null sperm could be due to the greater loss of other sperm proteins, such as ADAM4, ADAM5, ADAM6, and ADAM7 (8Kim T. Oh J. Woo J.M. Choi E. Im S.H. Yoo Y.J. et al.Expression and relationship of male reproductive ADAMs in mouse.Biol Reprod. 2006; 74: 744-750Crossref PubMed Scopus (68) Google Scholar, 9Han C. Choi E. Park I. Lee B. Jin S. Kim D.H. et al.Comprehensive analysis of reproductive ADAMs: relationship of ADAM4 and ADAM6 with an ADAM complex required for fertilization in mice.Biol Reprod. 2009; 80: 1001-1008Crossref PubMed Scopus (52) Google Scholar), which may also act in sperm aggregation. In conclusion, we discovered that sperm–sperm association or aggregation is impaired in Adam2 and Adam3 knockout sperm. These mutant sperm were previously found to be defective in migration in the female reproductive tracts and to lack the ability to bind to the egg ZP. Our finding provides novel information regarding the role of the ADAM proteins in sperm behavior and a model to investigate a relationship between sperm aggregation and the ADAM-dependent fertilization processes. The authors thank Drs. Paul Primakoff and Diana Myles (University of California, Davis) for Adam2 and Adam3 knockout mice. Supplemental data Download .mpg (6.96 MB) Help with mpg files Supplementary Movie 1 Download .mpg (8.52 MB) Help with mpg files Supplementary Movie 2 Download .mpg (6.96 MB) Help with mpg files Supplementary Movie 1 Download .mpg (8.52 MB) Help with mpg files Supplementary Movie 2