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

Gustavo Luis Verón,Mónica Cameo,Clara Isabel Marín-Briggiler,María Florencia Veiga,Mónica Hebe Vazquez-Levin

doi:10.3390/cells11010102

Gustavo Luis Verón, Mónica Cameo + Show 3 more

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https://doi.org/10.3390/cells11010102

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Abstract

Successful mammalian fertilization requires a well-orchestrated sequence of molecular events 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-cad in human gametes and showed evidence of their involvement in sperm-oocyte adhesion events leading to fertilization. To overcome ethical limitations associated with the use of human gametes in fertilization-related studies, the mouse has been selected worldwide as the experimental model for over 4 decades. Herein, we report a detailed study aimed at characterizing the expression of E-cad and N-cad in murine gametes and their involvement in murine fertilization using specific antibodies and blocking peptides towards both adhesion proteins. E-cad and N-cad protein forms, as well as other members of the adhesion complex, specifically β-catenin and actin, were identified in spermatozoa, cumulus cells and oocytes protein extracts by means of Western immunoblotting. In addition, subcellular localization of these proteins was determined in whole cells using optical fluorescent microscopy. Gamete pre-incubation with anti-E-cad (ECCD-1) or N-cad (H-63) antibodies resulted in decreased (p < 0.05) In Vitro Fertilization (IVF) rates, when using both cumulus-oocytes complexes and cumulus-free oocytes. Moreover, IVF assays done with denuded oocytes and either antibodies 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 spermatozoa were 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-cad antibody or blocking peptide towards E-cad, N-cad or both proteins. Finally, sperm-oocyte fusion was lower (p < 0.05) after sperm pre-incubation with either antibody or blocking peptide against E-cad or N-cad. Our studies demonstrate the expression of members of the adherent complex in the murine model, and the use of antibodies and specific peptides revealed E-cad and N-cad participation in mammalian fertilization.

Highlights

Fertilization is an exceptional multistep process that involves two highly differentiated cells: the spermatozoon and the oocyte
These studies led to the identification of specific protein forms of the expected molecular weight, 120 kDa for Epithelial cadherin (E-cad) and 135 kDa for N-cad, in the tissue and gamete extracts, accompanied with protein band of lower MW (E-cad: 110, 100, 60 and 35 kDa; N-cad: 93 and 68 kDa) (Figure 2A)
The studies presented in this report have thoroughly evaluated the localization of E-cad, N-cad, and other members of the adherent complex in mouse gametes, and showed evidence on the involvement of both classical cadherins in gamete interaction events leading to fertilization

Summary

Introduction

Fertilization is an exceptional multistep process that involves two highly differentiated cells: the spermatozoon and the oocyte. Mature spermatozoa are placed into the female reproductive tract, where they undergo profound changes required to fully develop their fertilizing capacity in a process denoted as capacitation [5,6] Once they arrive at the oocyte vicinity, spermatozoa penetrate the cumulus oophorus and undergo the acrosomal exocytosis (AE), a process in which the plasma membrane and outer acrosomal membrane fuse, releasing the acrosomal content and exposing the fusogenic region. While the extracellular domain participates in cell-cell adhesion, the cytoplasmic domain is involved in intracellular cell signaling and links E-cad to filamentous actin (F-actin) through adaptor molecules, among them β-catenin [18] Another member of the classical cadherin family is Neural cadherin (N-cad, CDH2), a 135 kDa transmembrane protein first identified as a neural cells adhesion molecule, later was found to be expressed in several tissues [19]. While its structure resembles that of E-cad, N-cad mediates homotypic binding, during tumor progression it participates in heterotypic adhesion events involving E-cad on the cancer cell membrane and N-cad on the fibroblast membrane [27]

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