Following an ovulatory surge of LH and FSH, cumulus cells surrounding the oocyte disaggregate as they endocytose their cell-cell junctions and then synthesize a hyaluronic acid-rich extracellular matrix (ECM). This ECM entraps the cells and in most mammalian species, the integrity of this mucoid mass of matrix and cells is maintained during ovulation. The expanded cumulus mass is ultimately dissolved within the oviduct. Recently we have purified a fraction from bovine serum which is required for stabilization of the cumulus ECM in vitro. Immunoblot and sequence analyses support the view that the single protein in this fraction is a major blood proteinase, inter-alpha-inhibitor (IalphaI), or a member of the IalphaI family of serine protease's. Further evidence of its identity is derived from experiments involving depletion of bovine serum with an anti-IalphaI IgG antibody-agarose complex thus suppressing matrix stabilizing activity. Immunoblot and immunocytochemical localization techniques reveal the presence of this protein only in preovulatory follicles responding to an ovulatory stimulus and in these follicles, IalphaI is specifically localized in the cumulus extra-cellular matrix and at the follicle basement membrane. Moreover Western blot analysis of follicular fluid shows that IalphaI is present within the antral compartment of responding follicles within minutes following an ovulatory stimulus. These findings support the hypothesis that cumulus ECM stabilization in vivo requires the breakdown of a blood-follicle barrier allowing the influx of IalphaI into the follicular antrum where it becomes incorporated into the cumulus ECM. While IalphaI may stabilize the cumulus mass by inhibiting active proteinases within the ECM, it is also possible that this molecule serves as a linker to bind hyaluronic acid within the ECM. Additional preliminary results support the hypothesis that in addition to follicular wall rupture, normal synthesis and stabilization of the cumulus ECM during the preovulatory period are required for normal ovulation. Therefore, the objectives of the present proposal are to further define temporal, physical, and molecular characteristics of the postulated blood-follicle barrier, the molecular mode of action of IalphaI in stabilization of the cumulus ECM, and the possible role of the blood- barrier and IalphaI in the process of ovulation. Finally, we hope to explore possible roles of blood IalphaI levels in idiopathic human anovulatory syndrome focusing initially on anovulation in diabetes mellitus.
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