332. GYCOMIC ANALYSES OF THE GRANULOSA AND THECA OF BOVINE OVARIAN FOLLICLES

Abstract

Development of ovarian follicles involves changes in cell function and remodelling of the follicular wall. Remodelling necessitates changes in the extracellular matrix, including proteoglycans (PGs). PGs contain glycosaminoglycans (GAGs) covalently bound to a protein core. The length of GAG chains in PGs and the degree and pattern of sulphation differs between cell types and change as cells alter their phenotype. PGs that have been identified in follicles include the chondroitin sulphate (CS) PG, versican and inter-a trypsin inhibitor, and the heparan sulphate (HS) PGs, perlecan and type XVIII collagen. The latter two are found in focimatrix, the follicular and the thecal subendothelial basal laminas. To examine GAGs composition in follicles, bovine antral follicles of various sizes were collected. Follicles were dissected and a biopsy taken for histological classification of health. Theca layers and granulosa cells were collected separately and analysed by fluorophore-assisted carbohydrate (FACE) analysis of GAGs following digestion to disaccharides with chondroitinase ABC, hyaluronidase, heparinase, and heparitinases I and II. Four non GAG sugars and 12 different GAG derived disaccharides were identified and quantitated on a per DNA basis. Healthy versus atretic follicles for each cell type were compared and correlation analyses were also undertaken. Immunohistochemistry using CS specific antibodies was also conducted. There was no effect of size on the GAG content for either granulosa or theca cells. The 4- and 6- sulphated CS sugars were the most abundant following digestion in all tissues. Theca had higher levels than granulosa cells of HS derived disaccharides and also of un- or 4- or 6- N-sulphated CS derived disaccharides. Some sulphated CS moieties localised uniquely to the stroma surrounding blood vessels in the theca externa. Atretic follicles had lower amounts of disaccharides derived from HS in both granulosa and thecal cells, suggesting that heparanase may be activated upon atresia.