319 PROTEIN GENE PRODUCT 9.5 REGULATES SPERM PENETRATION DURING PORCINE FERTILIZATION IN VITRO

The protein gene product 9.5 (PGP9.5) belongs to a family of ubiquitin C-terminal hydrolases (UCHs), which regenerate monoubiquitin from ubiquitin–protein complexes or polyubiquitin chains by cleaving the amide linkage next to the C-terminal glycine of ubiquitin. Identified in the acrosome of boar spermatozoa, we hypothesized that PGP9.5 might regulate sperm–zona pellucida interactions during porcine IVF. The cumulus–oocyte complexes isolated from slaughterhouse ovaries were cultured in TCM-199 media for 44 h at 38.5°C, 5% CO2 in air. After completion of in vitro maturation (IVM), cumulus cells were removed by 0.1% hyaluronidase, and metaphase II (MII) oocytes were used for IVF. In Experiment 1, oocytes were co-incubated with different sperm concentrations (1 × 106, 5 × 105, and 1 × 105 sperm mL-1) in TBM medium with or without anti-PGP9.5 antibody (1 : 50 dilution). In Experiment 2, oocytes were inseminated with 1 × 106 sperm mL-1 in TBM medium containing different concentrations of extracted oviductal fluids (0, 0.1, 0.5, 1, 2, and 3 µg mL-1) for 6 h. After IVF, oocytes were transferred into NCSU23 medium containing 0.4% BSA for further culture. The fertilization rates were evaluated by DAPI staining at 13 to 19 h. Data were analyzed by ANOVA and Duncan's multiple range test using the SAS program. Polyspermy was increased by the addition of anti-PGP9.5 antibody to the IVF medium (56.5–60.2% at polyspermy). This PGP9.5-antibody-induced polyspermy increase was sustained even with decreasing sperm concentrations. The polyspermy rates were reduced by the addition of isolated porcine oviductal fluid to IVF medium (50.4, 44.8, 28.0, 31.1, 1.6, and 0.0% at oviductal fluid concentrations of 0, 0.1, 0.5, 1, 2, and 3 µg mL-1, respectively). Biochemical analysis by Western blotting detected the appropriate 24-kDa PGP9.5 band in porcine oviductal fluid used for these experiments. Enzymatic UCH activity comparable to activity of recombinant UCH-L3 was detected in sperm extract, whole spermatozoa, and isolated oviductal fluid by fluorometric assay using fluorogenic UCH-substrate ubiquitin-AMC. This UCH activity was not reduced by the general protease inhibitor phenyl methyl sylfonyl fluoride, but it was reduced in a statistically significant manner ( P < 0.05) by the specific UCH-inhibitor ubiquitin aldehyde. In conclusion, the polyspermy increased with different concentrations of sperm in the anti-PGP9.5 antibody, and PGP9.5 was detected in oviductal fluids, suggesting that PGP9.5 is involved in the sperm–zona pellucida interaction during porcine fertilization. This work was supported by the US Department of Agriculture (USDA-NRI grant #2002-35203-12237 to P.S.), the F21C Program of The University of Missouri–Columbia (P.S.), and the ERC Program of the Korea Science and Engineering Foundation (KOSEF grant no. R11-2002-100-00000-0 to Y.J. Yi and C.S. Park) .