Fetal bovine serum (FBS) has been widely used in the culture of cumulus-oocyte complexes, since it is believed that FBS stabilizes the expanding cumulus extracellular matrix. In this study, we have identified a factor in FBS that is responsible for this effect. FBS was first fractionated by stepwise precipitation with ammonium sulfate followed by high performance liquid chromatography (HPLC) on a gel filtration column. Active fractions, as determined by their ability to stabilize the cumulus extracellular matrix in a bioassay system, were further purified by HPLC on DEAE ion-exchange and gel filtration columns. The purified factor was exhibited as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr 150,000 and a single peak on a C-8 reverse-phase HPLC. The sequence of the first 14 NH2-terminal amino acids was determined by Edman degradation, revealing significant sequence identity of the bovine serum factor with the light chain shared by members of the inter-alpha-trypsin inhibitor family. Western blot analysis using anti-human I alpha I IgG shows that the antibody reacts positively with the purified factor, and immunodepletion of FBS with anti-I alpha I antibody agarose eliminated its ability to stabilize the extracellular matrix. This factor is found in mouse follicular fluid collected 6 h following human chorionic gonadotropin injection to stimulate ovulation, but not in unstimulated mice. Anti-I alpha I-positive epitopes were also localized within the cumulus extracellular matrix of mouse preovulatory follicles in immunocytochemical preparations using anti-human I alpha I IgG, supporting the possibility that this molecule or molecules may diffuse into follicular fluid after an ovulatory stimulus to act as structural linkers that ensure normal cumulus expansion, through stabilization of the cumulus extracellular matrix thus supporting the process of ovulation.
Relation:
The Journal of Biological Chemistry 267(17):12380-6.
