Epithelial and Neural Cadherin in Mammalian Fertilization: Studies in the Mouse Model

VERÓN, GUSTAVO LUIS; VEIGA, MARÍA FLORENCIA; CAMEO, MÓNICA; MARÍN-BRIGGILER, CLARA ISABEL; VAZQUEZ-LEVIN, MÓNICA HEBE

Jornada; XLII Reunión Anual de la Sociedad Argentina de Andrología 2022; 2022

Successful mammalian fertilization requires a well-orchestrated sequence of molecularevents leading to gamete fusion. Since this interaction involves Ca2+-dependent adhesion events,the participation of the Ca+2-dependent cell-cell adhesion proteins Epithelial (E-cad) and Neural(N-cad) cadherin is envisaged. We have previously reported the expression of E-cad and N-cadin human gametes and showed evidence of their involvement in sperm-oocyte adhesion eventsleading to fertilization. To overcome ethical limitations associated with the use of human gametesin fertilization-related studies, the mouse has been selected worldwide as the experimental modelfor over 4 decades. Herein, we report a detailed study aimed at characterizing the expression ofE-cad and N-cad in murine gametes and their involvement in murine fertilization using specificantibodies and blocking peptides towards both adhesion proteins. E-cad and N-cad protein forms,as well as other members of the adhesion complex, specifically -catenin and actin, were identifiedin spermatozoa, cumulus cells and oocytes protein extracts by means ofWestern immunoblotting.In addition, subcellular localization of these proteins was determined in whole cells using opticalfluorescent microscopy. Gamete pre-incubation with anti-E-cad (ECCD-1) or N-cad (H-63) antibodiesresulted in decreased (p < 0.05) In Vitro Fertilization (IVF) rates, when using both cumulus-oocytescomplexes and cumulus-free oocytes. Moreover, IVF assays done with denuded oocytes and eitherantibodies or blocking peptides against E-cad and N-cad led to lower (p < 0.05) fertilization rates.When assessing each step, penetration of the cumulus mass was lower (p < 0.05) when spermatozoawere pre-incubated with ECCD-1 or blocking peptides towards E-cad or towards both E- and N-cad.Moreover, sperm-oolemma binding was impaired (p < 0.0005) after sperm pre-incubation with E-cadantibody or blocking peptide towards E-cad, N-cad or both proteins. Finally, sperm-oocyte fusionwas lower (p < 0.05) after sperm pre-incubation with either antibody or blocking peptide againstE-cad or N-cad. Our studies demonstrate the expression of members of the adherent complex in themurine model, and the use of antibodies and specific peptides revealed E-cad and N-cad participationin mammalian fertilization.