233 STUDY OF CORTICAL GRANULE CONTENT IN IN VITRO-MATURED PORCINE OOCYTES BY MEANS OF PNA LECTIN PRECIPITATION

Abstract

Cortical granules (CG) are clue organelles in the oocyte since their content is released under oocyte activation (i.e. fertilization) modifying the zona pellucida and thus blocking polyspermy. Once released, CG are not renewed. Research on cortical reaction and putative CG enzymes has progressed slowly because mammalian eggs contain only picogram quantities of CG-derived proteins (Moller and Wassarman 1989 Dev. Biol. 132, 103–112; Green 1997 Rev. Reprod. 2, 147–156), so the protein(s) responsible for the physiological changes in ZP after cortical reaction are not well known. The objective of this project was to study porcine CG content in in vitro-matured porcine oocytes by means of lectin precipitation with peanut agglutinin (PNA), since this lectin binds to porcine CG (Yoshida et al. 1993 Mol. Reprod. Dev. 36, 462–468). Immature porcine cumulus–oocytes complexes (COCs) from Landrace � Large White gilts were in vitro-matured for 44 h in NCSU-37 medium. After IVM 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, zonae pellucidae were removed and oocyte cytoplasmic content (lysate) collected. Lysate from 1000 IVM-oocytes was incubated under continuous shaking (2 h, room temperature) with 100 µL PNA-agarose (Sigma, St. Louis, MO, USA) so that proteins bound to PNA could be precipitated by centrifugation. After lectin precipitation, proteins were detached from PNA-agarose beads by boiling in reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (5 min, 100�C) (Laemmli 1970 Nature 227, 689). Samples were then centrifuged (5 min, 7000g), the pellet containing PNA-agarose beads was discarded, and the supernatant containing the proteins was collected and further separated by SDS-PAGE. The silver staining of electrophoresis gels revealed eleven bands from 37 to 180 kDa, so a second gel was electrotransferred to a polyvinylidene fluoride (PVDF) membrane (100V, 1 h) and incubated with PNA-horseradish peroxidase (PNA-HRP, 10 µg mL–1) for 1 h. Visualization was accomplished using the enhanced chemiluminiscence (ECL plus) method and Typhoon 9410 following the manufacturer's instructions (Amersham Biosciences, Freiburg, Germany); only four bands of approximately 57 kDa, 60 kDa, 70 kDa, and 80 kDa were observed. These bands will be cut and processed for proteomic analysis for further studies. Preliminary results show that porcine CG-derived proteins can be studied by PNA lectin precipitation. These results could be employed in the future to develop specific antibodies against porcine CG.