Partial purification and characterization of a protein in porcine follicular fluid which restricts sperm-egg...

An attempt was made to isolate and characterize a component in preovulatory porcine follicular fluid (pFF) which has a restricting effect on sperm-egg interaction in vitro. Using the zona-free hamster ova (eggs) penetration assay as an in vitro test system, it was shown previously that the numbers of porcine spermatozoa attached to or penetrated into each egg and the number of eggs with sperm attached or penetrated decreased significantly as the concentration of pFF was increased in the culture medium. In the present study, the component in pFF having these effects was shown to be a heat stable, nonsteroidal substance which retained its activity after dialysis, lyophilization and gel filtration chromatography. The activity was also found to be present in preovulatory homologous serum. Separation of the material on protein type gel filtration columns with detection at 280 nm, together with the banding seen with Coomassie staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), suggests that it is a protein. Based on high pressure liquid chromatographic separation (HPLC) and SDS-PAGE analyses, the bioactivity could be due to a single protein of 87 kD or to one or more of three smaller proteins, possibly disaggregated products of the 87 kD protein, in the range of 26-28 kD.