ER-60 and HSP-70 Proteins Are Candidates to Be Contained in Cortical Granules of In Vitro-Matured Porcine Oocytes. (2008)

Cortical granules (CG) are clue organelles in the oocyte since their content is released under oocyte activation (i.e. fertilization) modifying the zona pellucida (ZP) and thus blocking polyspermy. When oocyte is activated by appropriate stimulus such as sperm contact, chemical or electrical stimulation, the CG exocytosis (cortical reaction) is triggered. Very little is known about the molecules presumably stored in the CG that act directly on the zona pelúcida to induce the block to polyspermy. Research on cortical reaction and putative CG enzymes has progressed slowly because the amount of protein in these organelles is very low. Isolation and identification of CG contents in porcine oocytes would help to understand the molecular mechanism involved in blocking polyspermic fertilization. It has been shown that peanut agglutinin (PNA) lectin binds to porcine CG and we recently have isolated proteins with affinity to PNA by lectin precipitation that might be contained in the CG. The objective of this work was to study PNA precipitated proteins from in vitro matured porcine oocytes by means of proteomic analysis and western blot. Immature porcine cumulus-oocyte complexes (COCs) from Landrace x LargeWhite gilts were in vitro matured for 44h in NCSU-37 medium. After in vitro maturation period, COCs were stripped of cumulus cells, washed in PBS and quickly washed through purified water. Then, oocytes were lysed in a fresh water droplet by gentle pipetting using a narrow-bore glass pipette. Once lysed, zona pellucidae were removed and oocyte cytoplasmic content (lysate) collected. Lysate was incubated with PNA-agarose beads (2h, under shaking) and glycoproteins bound to PNA detached adding 100ml of Laemmli buffer (100°C, 5 min). Then, samples were centrifuged (5,000g, 3 min) and the supernatant containing proteins was subjected to SDS-PAGE electrophoresis under reducing condition. A lectin-blot analysis was performed using the PNA lectin conjugated with horseradish peroxidase (PNA-HRP, 10μg/mL). The lectin blot revealed four bands with an apparent molecular weight of 57, 60, 70 and 80kDa approximately. These bands were cut and processed for proteomic analysis that was carried out on a HPLC/MS system consisting of an Agilent 1100 Series HPLC (Agilent Technologies, Santa Clara, CA, USA). Six different peptides corresponding to ER-60 (Disulfide Isomerase ER-60) were identified in the 57kDa band. Nine different peptides corresponding to HSP-70 (Heat Shock Protein 70) were identified in the 70kDa band. Proteomic results were repeated in two replicates. The 57 and 80kDa bands are still under analysis. The presence of ER-60 was confirmed by western blot with a polyclonal antibody against ER-60 diluted 1:1000 (anti-ER-60, Abcam, cat. No. ab13507, England). Further confirmation of the presence of ER-60 and HSP-70 proteins inside the CG must be done by confocal and electron microscopy. These preliminary results show a new approach to study the cortical granule content and the role of cortical reaction in pigs. Supported by MEC (project code AGL2006-03495/GAN) and Fundación Seneca (04542/GERM/06).