Exocytosis of a 60 kDa protein (Calreticulin) from activated hamster oocytes

Abstract

The sp50 protein localized at the acrosomal region of guinea pig sperm was suggested to participate in acrosome exocytosis, the acrosome reaction (AIR). On the other hand, the cortical reaction (CR), also an exocytotic event, occurs during egg activation. The aim of the present work was to identify sp50 and also to define if sp50 is present in hamster eggs, as well as its location before and after CR. Sp50 was identified as calreticulin (CRT), based on: (a) its NH2-terminal amino acid (25 aa) sequence, (b) a cross-recognition of pure sp50 and pure CRT with anti-CRT (from Santa Cruz, anti-CRTsc), and anti-sp50 (anti-sp50/CRT) antibodies, respectively, and (c) that both antibodies revealed a 50 kDa protein in a Brij sperm extract. On the other hand, CRT presence in eggs was positively determined by Western blotting (Wb) using anti-sp50/CRT antibody which recognized a 60 kDa protein in the egg extract, and by indirect immunofluorescence (IIF), CRT was located in the cortical granules (CG). It was defined by a granular pattern and co-localization with mannose, a specific carbohydrate of the CG. Additionally, a decrease in CRT concentration occurred in eggs after their activation and, in parallel, the protein was revealed in the egg's incubation medium. In activated eggs with zona pellucida (ZP), CRT remains as a halo in the perivitelline space and around the polar body. From these results we suggest that: (1) CRT is present in the CG of non-activated hamster eggs, (2) CRT is exocytosed during the CR, in response to egg activation, and (3) CRT might participate in the block to polyspermy, together with other CG components. (C) 2001 Wiley-Liss, Inc.