Running title: Cumulus expansion-inhibiting factors

Studies suggest that oocyte cumulus expansion is regulated by both cumulus expansion-enabling factors (CEEF) and cumulus expansion-inhibiting factors (CEIF). However, whereas there are many reports on CEEF, CEIF has rarely been studied. By cumulus expansion assays using mouse cumulus-oocyte complexes (COCs) and oocytectomized complexes (OOXs), this study demonstrated that whereas follicular fluid (FF) from medium (2-4 mm) goat follicles contained both CEEF and CEIF activities, FF from abattoir or FSH-stimulated large (5-6 mm) follicles contained neither. FF from hCG-stimulated large follicles showed CEEF but not CEIF activities. Whereas medium conditioned with cumulus or mural granulosa cells from 2-4 mm follicles contained only CEEF, theca cell-conditioned medium (CM) showed both CEEF and CEIF activities. Whereas 0.01 mg/ml heparin efficiently inhibited cumulus expansion of mouse COCs in vitro, FF from large follicles that showed no CEIF activity contained much higher concentrations (0.23-0.25 mg/ml) of heparin. None of the glycosaminoglycans (GAGs) tested inhibited cumulus expansion of goat COCs. Among the follicles observed, only FF from medium follicles contained a linoleic acid (LA) level sufficient to inhibit cumulus expansion of both mouse and goat COCs in vitro. CM contained some amount of GAGs but no LA. Together, the results suggest that (i) the FSH and LH surges before ovulation promote cumulus expansion by down regulating CEIF and up regulating CEEF activities, respectively; (ii) GAGs are not the CEIF in goat follicles; and (iii) LA has CEIF activity but additional factor (s) must be involved because theca cell-CM that showed high CEIF activities contained no LA.