Evidence on the participation of epithelial cadherin in sperm-egg interaction, partial characterization of human sperm protein forms, and identification of a novel variant epididymal RNA.

VAZQUEZ-LEVIN MH Y COL

Andover, New Hampshire, EEUU

Congreso; Gordon Research Conference in "Cell Contact and Adhesion".; 2005

Gordon Research Conferences

Anque el resumen no es publicado debe ser enviado a los organizadores para considerar la aceptacion a la reunion cientifica muy limitada en asistentes (150-200 de todo el mundo). Evidence on the involvement of epithelial cadherin in sperm-egg interaction, partial characterization of human sperm protein forms, and identification of a novel variant epididymal mRNA.    Lentz1*, EM; Marín-Briggiler1*, CI; Veiga*, MF, Rivero*, CW; Furlong*, LI; Cameo", M; Vazquez-Levin*, MH. *Instituto de Biología y Medicina Experimental, CONICET. "BR Lab. Buenos Aires, Argentina. mhvaz@dna.uba.ar ( 1both authors equally contributed to the work)   Previous reports have shown evidence on the presence of the adhesion molecule Epithelial cadherin (Ecad) in human1,2 and rat3 sperm and oocytes. Immunocytochemical analyses done by our group confirmed these findings for both human gametes4, and extended them to the murine5 and bovine6 models. In all species studied, Ecad was immunolocalized in sperm regions described to participate in gamete interaction during fertilization. The aim of this study was to evaluate the involvement of Ecad in gamete interaction and to perform biochemical and molecular studies towards the characterization of the human sperm protein form(s). To test Ecad participation in sperm-egg interaction, two in vitro assays were performed in the presence or absence of anti Ecad antibodies: a)the Hemizona-Assay (HZA), which evaluates sperm interaction with the egg´s extracellular matrix (Zona Pellucida; ZP), and b)the Hamster Egg Sperm Penetration Assay (HPA), that assesses the ability of human sperm to bind and fuse to heterologous eggs devoid of ZP. In the HZA, sperm adhesion to the ZP was impaired in sperm preincubated with the anti Ecad antibody (anti Ecad=17+5, Control=30+8 sperm bound/HZ; P<0.02). Similarly, sperm binding and fusion in the HPA was significantly diminished when eggs were preincubated with the anti Ecad antibody (anti Ecad=57±11; Control=91±12% penetrated eggs/total inseminated eggs; P<0.05). In accordance with these findings, studies with the murine model revealed a significant inhibition of fertilization when sperm were preincubated with anti Ecad antibody (anti Ecad: 49±8 % fertilized oocytes relative to control; P<0.001). Four high Mr Ecad forms (Ecad-122, -105, -97 and -86) were detected in sperm, in agreement with reports in cell lines, tissues and fluids7,8, describing the presence of several Ecad protein isoforms. Ecad-86 could be removed from sperm by extraction with a high salt buffer, and by treatment with PI-PLC. Western immunoblot analysis using antibodies towards different Ecad regions indicated that Ecad-86 and Ecad-97 would be truncated in its C-terminal and Ecad-105 in its N-terminal protein ends. Moreover, studies suggested that different Ecad forms would be restricted to specific sperm regions. To investigate whether some of the sperm Ecad forms could be of epididymal origin, screening of a human epididymis expression library was carried out, identifying two clone families: while one group encoded the characterized protein, the other presented a 34 bp deletion, which could result from alternative splicing. As a consequence of the frame shift, the putative protein would lack the transmembrane domain, and may correspond to the sperm Ecad-86 isoform. In summary, the studies: a) have shown evidence on the participation of Ecad in sperm-egg interaction, b) have described several Ecad sperm forms that may be involved in the interaction of sperm with both the ZP and the oolema, and c) have identified a novel variant Ecad mRNA in human epididymis that would encode a short protein isoform. Protein expression analysis of the variant Ecad mRNA and functional assays with the sperm protein isoforms will contribute to the understanding of Ecad function(s) in mammalian fertilization.     References: 1. Rufas et al,  2000; 2. Purohit et al,  2004; 3. Ziv et al, 2002;  4. Marín et al, unpublished data; 5. Veiga et al, unpublished data; 6. Caballero et al, unpublished data; 7. Rios-Doria & Day 2004; 8. Kuefer et al, 2003.         Lentz1*, EM; Marín-Briggiler1*, CI; Veiga*, MF, Rivero*, CW; Furlong*, LI; Cameo", M; Vazquez-Levin*, MH. *Instituto de Biología y Medicina Experimental, CONICET. "BR Lab. Buenos Aires, Argentina. mhvaz@dna.uba.ar ( 1both authors equally contributed to the work)   Previous reports have shown evidence on the presence of the adhesion molecule Epithelial cadherin (Ecad) in human1,2 and rat3 sperm and oocytes. Immunocytochemical analyses done by our group confirmed these findings for both human gametes4, and extended them to the murine5 and bovine6 models. In all species studied, Ecad was immunolocalized in sperm regions described to participate in gamete interaction during fertilization. The aim of this study was to evaluate the involvement of Ecad in gamete interaction and to perform biochemical and molecular studies towards the characterization of the human sperm protein form(s). To test Ecad participation in sperm-egg interaction, two in vitro assays were performed in the presence or absence of anti Ecad antibodies: a)the Hemizona-Assay (HZA), which evaluates sperm interaction with the egg´s extracellular matrix (Zona Pellucida; ZP), and b)the Hamster Egg Sperm Penetration Assay (HPA), that assesses the ability of human sperm to bind and fuse to heterologous eggs devoid of ZP. In the HZA, sperm adhesion to the ZP was impaired in sperm preincubated with the anti Ecad antibody (anti Ecad=17+5, Control=30+8 sperm bound/HZ; P<0.02). Similarly, sperm binding and fusion in the HPA was significantly diminished when eggs were preincubated with the anti Ecad antibody (anti Ecad=57±11; Control=91±12% penetrated eggs/total inseminated eggs; P<0.05). In accordance with these findings, studies with the murine model revealed a significant inhibition of fertilization when sperm were preincubated with anti Ecad antibody (anti Ecad: 49±8 % fertilized oocytes relative to control; P<0.001). Four high Mr Ecad forms (Ecad-122, -105, -97 and -86) were detected in sperm, in agreement with reports in cell lines, tissues and fluids7,8, describing the presence of several Ecad protein isoforms. Ecad-86 could be removed from sperm by extraction with a high salt buffer, and by treatment with PI-PLC. Western immunoblot analysis using antibodies towards different Ecad regions indicated that Ecad-86 and Ecad-97 would be truncated in its C-terminal and Ecad-105 in its N-terminal protein ends. Moreover, studies suggested that different Ecad forms would be restricted to specific sperm regions. To investigate whether some of the sperm Ecad forms could be of epididymal origin, screening of a human epididymis expression library was carried out, identifying two clone families: while one group encoded the characterized protein, the other presented a 34 bp deletion, which could result from alternative splicing. As a consequence of the frame shift, the putative protein would lack the transmembrane domain, and may correspond to the sperm Ecad-86 isoform. In summary, the studies: a) have shown evidence on the participation of Ecad in sperm-egg interaction, b) have described several Ecad sperm forms that may be involved in the interaction of sperm with both the ZP and the oolema, and c) have identified a novel variant Ecad mRNA in human epididymis that would encode a short protein isoform. Protein expression analysis of the variant Ecad mRNA and functional assays with the sperm protein isoforms will contribute to the understanding of Ecad function(s) in mammalian fertilization.     References: 1. Rufas et al,  2000; 2. Purohit et al,  2004; 3. Ziv et al, 2002;  4. Marín et al, unpublished data; 5. Veiga et al, unpublished data; 6. Caballero et al, unpublished data; 7. Rios-Doria & Day 2004; 8. Kuefer et al, 2003.