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Chun-Che Yen1 , Trish Berger1 *
Department of Animal Science, University of California, Davis, CA 95616 1
Previously, our laboratory has identified a 56kD POMP which is putatively involved in sperm-oocyte interaction. The objective of these experiments was to determine if POMP is exposed on the porcine oocyte plasma membrane at the time of sperm-oocyte binding. Antibodies were produced in pigs and goats to a synthesized multiple antigenic peptide (MAP) corresponding to amino acids 11-21 of POMP. The porcine POMP antibody does not cross-react with porcine serum albumin by ELISA. Oocytes were incubated with porcine or goat POMP antiserum or porcine POMP antibody (IgG) separately then incubated with fluorescently labeled second antibodies (anti-porcine or anti-goat). Bound antibody was visualized using laser scanning confocal microscopy. Both POMP antisera and the POMP IgG bound to the oocyte plasma membrane of unfixed oocytes indicating exposed antigenic sites. Pre-immune porcine or goat serum and pre-immune porcine IgG did not bind to the oocytes. Fab was prepared from porcine POMP IgG using papain to cleave IgG and Protein A and Sephacryl S200 to isolate the fragments. The majority of oocytes bound the porcine POMP Fab. Since the N-terminal sequence of POMP is homologous to the N-terminal sequence of sheep serum albumin (SSA), an antibody (CSSA) was made to an approximate 42kD fragment of SSA which did not include the N-terminus. This CSSA antibody does not appear to bind fixed or unfixed porcine oocytes suggesting homology of POMP with SSA is limited to the N-terminal portion. In conclusion, POMP is exposed on the porcine oocyte surface in an appropriate place to be involved in sperm-oocyte interaction.
This abstract is being presented on Sunday, August 1 at 8:00 AM to 10:15 AM at CUB 2nd Floor Ballroom.