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Neuronal cadherin: localization in human sperm and assessment of its participation in gamete interaction. Clara I. Marín-Briggilera, Ezequiel M. Lentza, María F. Veigaa, Fernanda González-Echeverríab, Mónica H. Vazquez-Levina.Clara I. Marín-Briggilera, Ezequiel M. Lentza, María F. Veigaa, Fernanda González-Echeverríab, Mónica H. Vazquez-Levina. aInstituto de Biología y Medicina Experimental-CONICET-UBA, bFertilab, Buenos Aires, Argentina. E-mail: cmarin@dna.uba.ar Cadherins belong to a superfamily of membrane glycoproteins involved in cell-to-cell adhesion and signalling1. In particular, neuronal cadherin (N-cad) was first described in the brain and was also found in numerous tissues, including the testis, where mediates inter-Sertoli and Sertoli-germ cells contacts2-4. Preliminary evidence suggests the presence of this protein on human sperm5 and of N-cad transcripts in the male gamete6-7. The aim of this study was to confirm N-cad expression in human sperm, to determine its cellular localization and to assess its participation in the fertilization process. The mature form of N-cad (130 KDa) was detected in human sperm and testis protein extracts by Western immunoblotting using a polyclonal antibody directed towards an extracellular domain of N-cad (Santa Cruz Biotech.). Additionally, a lower Mr form of 113 KDa was found in sperm protein extracts and in seminal plasma.N-cad was mainly immunolocalized in the equatorial segment and in the acrosomal region of ejaculated (62±7 %; 17±10 %, respectively; mean±SEM) as well as 18-h capacitated sperm (51±8 %; 30±8 %, respectively) (n=5). Similar N-cad localization was observed in human testicular sperm. Colocalization studies using anti N-cad and Pisum sativum agglutinin showed that in acrosome-reacted sperm the protein was mainly restricted to the equatorial segment (84±2 % of the cells; n=3). In all cases, a specific N-cad signal was also detected in the sperm flagellum. To assess the involvement of N-cad in the fertilization process, the Hemizona and Hamster Egg Sperm Penetration Assays were done in the presence or absence of the specific antibody. Human sperm preincubation with anti N-cad (100 µg/ml) did not alter binding to homologous zona pellucida (ZP) (Anti N-cad=29±11, Control=33±12 bound sperm/hemizona; n=6). Preliminary results showed that when hamster ZP-free eggs were preincubated with the antibody (20 µg/ml) a reduction in the percentage of penetrated eggs was observed in comparison to control conditions (Anti N-cad=40 %, Control=100 %; n=2).In summary, the present study confirms the presence of N-cad in human sperm protein extracts, describes its localization in testicular, ejaculated, capacitated and acrosome reacted cells and suggests participation of this adhesion molecule in sperm-oolemma interaction.Instituto de Biología y Medicina Experimental-CONICET-UBA, bFertilab, Buenos Aires, Argentina. E-mail: cmarin@dna.uba.ar Cadherins belong to a superfamily of membrane glycoproteins involved in cell-to-cell adhesion and signalling1. In particular, neuronal cadherin (N-cad) was first described in the brain and was also found in numerous tissues, including the testis, where mediates inter-Sertoli and Sertoli-germ cells contacts2-4. Preliminary evidence suggests the presence of this protein on human sperm5 and of N-cad transcripts in the male gamete6-7. The aim of this study was to confirm N-cad expression in human sperm, to determine its cellular localization and to assess its participation in the fertilization process. The mature form of N-cad (130 KDa) was detected in human sperm and testis protein extracts by Western immunoblotting using a polyclonal antibody directed towards an extracellular domain of N-cad (Santa Cruz Biotech.). Additionally, a lower Mr form of 113 KDa was found in sperm protein extracts and in seminal plasma.N-cad was mainly immunolocalized in the equatorial segment and in the acrosomal region of ejaculated (62±7 %; 17±10 %, respectively; mean±SEM) as well as 18-h capacitated sperm (51±8 %; 30±8 %, respectively) (n=5). Similar N-cad localization was observed in human testicular sperm. Colocalization studies using anti N-cad and Pisum sativum agglutinin showed that in acrosome-reacted sperm the protein was mainly restricted to the equatorial segment (84±2 % of the cells; n=3). In all cases, a specific N-cad signal was also detected in the sperm flagellum. To assess the involvement of N-cad in the fertilization process, the Hemizona and Hamster Egg Sperm Penetration Assays were done in the presence or absence of the specific antibody. Human sperm preincubation with anti N-cad (100 µg/ml) did not alter binding to homologous zona pellucida (ZP) (Anti N-cad=29±11, Control=33±12 bound sperm/hemizona; n=6). Preliminary results showed that when hamster ZP-free eggs were preincubated with the antibody (20 µg/ml) a reduction in the percentage of penetrated eggs was observed in comparison to control conditions (Anti N-cad=40 %, Control=100 %; n=2).In summary, the present study confirms the presence of N-cad in human sperm protein extracts, describes its localization in testicular, ejaculated, capacitated and acrosome reacted cells and suggests participation of this adhesion molecule in sperm-oolemma interaction.1. In particular, neuronal cadherin (N-cad) was first described in the brain and was also found in numerous tissues, including the testis, where mediates inter-Sertoli and Sertoli-germ cells contacts2-4. Preliminary evidence suggests the presence of this protein on human sperm5 and of N-cad transcripts in the male gamete6-7. The aim of this study was to confirm N-cad expression in human sperm, to determine its cellular localization and to assess its participation in the fertilization process. The mature form of N-cad (130 KDa) was detected in human sperm and testis protein extracts by Western immunoblotting using a polyclonal antibody directed towards an extracellular domain of N-cad (Santa Cruz Biotech.). Additionally, a lower Mr form of 113 KDa was found in sperm protein extracts and in seminal plasma.N-cad was mainly immunolocalized in the equatorial segment and in the acrosomal region of ejaculated (62±7 %; 17±10 %, respectively; mean±SEM) as well as 18-h capacitated sperm (51±8 %; 30±8 %, respectively) (n=5). Similar N-cad localization was observed in human testicular sperm. Colocalization studies using anti N-cad and Pisum sativum agglutinin showed that in acrosome-reacted sperm the protein was mainly restricted to the equatorial segment (84±2 % of the cells; n=3). In all cases, a specific N-cad signal was also detected in the sperm flagellum. To assess the involvement of N-cad in the fertilization process, the Hemizona and Hamster Egg Sperm Penetration Assays were done in the presence or absence of the specific antibody. Human sperm preincubation with anti N-cad (100 µg/ml) did not alter binding to homologous zona pellucida (ZP) (Anti N-cad=29±11, Control=33±12 bound sperm/hemizona; n=6). Preliminary results showed that when hamster ZP-free eggs were preincubated with the antibody (20 µg/ml) a reduction in the percentage of penetrated eggs was observed in comparison to control conditions (Anti N-cad=40 %, Control=100 %; n=2).In summary, the present study confirms the presence of N-cad in human sperm protein extracts, describes its localization in testicular, ejaculated, capacitated and acrosome reacted cells and suggests participation of this adhesion molecule in sperm-oolemma interaction. 1. Angst et al, 2001; 2. Andersson et al, 1994; 3. Munro and Blaschuk, 1996; 4. Johnson and Boekelheide, 2002; 5. Rufas et al, 2000; 6. Goodwin et al, 2000; 7. Dadoune et al, 2005.

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