Effect of the Factor inhibiting germinal vesicle breakdown on the disruption of gap junctions and cumulus expansion of pig cumulus-oocyte complexes cultured in vitro

The present study was undertaken to explore the regulatory mechanisms for m eiotic resumption of pig cumulus-oocyte complexes (COCs) by assessing the n uclear status of oocytes, the degree of gap junction cell-to-cell communica tion and cumulus expansion after culture of various numbers of COCs in 10 m ul droplets of medium for 24 h. Cap junction communication was examined by confocal laser scanning microscopy after injection of a fluorescent dye, lu cifer yellow, into the oocytes. When one, three or six COCs were cultured i n a 10 mul droplet, germinal vesicle breakdown was observed in > 70% of ooc ytes; increasing the number of COCs in a droplet further actually decreased the proportion of oocytes undergoing germinal vesicle breakdown (10 COCs: 49%; 20 COCs: 21%; 40 COCs: 13%). When six COCs were cultured in a 10 mul d roplet of conditioned medium (prepared previously by culturing 20 intact an d oocytectomized COCs for 24 h), the proportion of oocytes undergoing germi nal vesicle breakdown was significantly reduced compared with the proportio n in fresh medium. An increase in the proportion of category 1 COCs (all ga p junctions within cumulus cells and between cumulus cells and oocyte are f unctionally maintained) was achieved by increasing the number of COCs cultu red in a 10 mul droplet. The addition of conditioned medium to the fresh me dium at a concentration of 50% significantly inhibited cumulus expansion. F rom these results, it is concluded that the factors secreted by cumulus cel ls regulate the disruption of gap junctions and cumulus expansion, and conc urrently control the incidence of germinal vesicle breakdown in pig COCs.