Formation of ovarian follicular fluid may be due to the osmotic potential of large glycosaminoglycans and proteoglycans.

During mammalian follicle development, a fluid-filled antrum develops in the avascular centre of the follicle. We investigated the hypothesis that follicular fluid contains osmotically-active molecules, sufficiently large so as to not freely escape the follicular fluid. Such molecules could generate an osmotic differential and thus recruit fluid from the surrounding vascularised stroma into the antrum. Follicular fluid was collected from bovine follicles classified histologically as healthy (n = 4 pools) or atretic (n = 4 pools). Dialysis of the follicular fluid at 300 kDa or 500 kDa resulted in a reduction in colloid osmotic pressure of 35% and 60%, respectively, in fluid from healthy follicles and 29% and 80% from atretic follicles. Digestion of follicular fluid with Streptomyces hyaluronidase, chondroitinase ABC or DNase 1 followed by dialysis resulted in reductions in osmotic pressure of 43%, 53% and 43% respectively for fluids from healthy follicles and 34%, 20% and 31% for atretic follicles. Digestion with collagenase I, proteinase K, heparanase 1 or keratanase had no significant effect on the osmotic pressure of follicular fluid of healthy follicles. Ion exchange and size exclusion, Western blotting and ELISA identified the proteoglycans versican and inter-alpha trypsin inhibitor and the glycosaminoglycan hyaluronan in follicular fluid. We conclude that these molecules or aggregates of them are of sufficient size to contribute to the osmotic potential of follicular fluid and thus recruit fluid into the follicular antrum. DNA may also contribute but it is probably not a component that is regulated for this role.

Rabbit epididymal secretory proteins. II. Immunolocalization and sperm association of REP38.

Polyclonal antibody was used to partially characterize REP38, a major rabbit epididymal secretory protein. Western blot analyses and immunohistochemistry indicated that REP38 is only expressed in regions 5 and 6 of the epididymis (corpus epididy-midis) and is localized in the supranuclear region and microvilli of the principal cells in these regions. It was not expressed in other tissues of the body. In region 8 (cauda epididymidis), REP38 was detected in the luminal border and cytoplasm of scattered principal cells, indicating that it may be reabsorbed in this region. This protein accumulated on the sperm plasma membrane downstream of region 5 and was localized predominantly over the acrosomal and postacrosomal regions of the head and the middle piece. Although tightly bound to epididymal sperm, REP38 migrated to the equatorial segment under conditions in vivo that would promote capacitation. When tested in vitro, anti-REP38 IgG reduced the percentage of ova fertilized in a concentration-dependent manner, apparently by blocking sperm-egg fusion.