Secretion of paracrine factors enabling expansion of cumulus cells is developmentally regulated in pig oocytes

To demonstrate secretion of cumulus expansion-enabling factor (CEEF) by por cine oocytes, we used an interspecies testing system. Porcine oocytes were used to condition culture medium, and the presence of CEEF was tested using mouse oocytectomized complexes (OOX), which require CEEF for expansion. Fo llicle-stimulating hormone-stimulated expansion and synthesis of hyaluronic acid (HA) by mouse OOX were assessed after 18 h of culture in media condit ioned by porcine oocytes: 1) at different stages of maturation and 2) in wh ich maturation was inhibited with a specific inhibitor of cdk-kinases, buty rolactone I. Fully grown (GV-germinal vesicle), late-diakinesis (LD), metap hase 1 (MI), and metaphase II (MII) oocytes were prepared by culture of ooc yte-cumulus complexes (OCC) for 0, 22, 27, and 42 h, respectively. To block CV breakdown, porcine oocytes were cultured for 27 h in medium supplemente d with butyrolactone 1 (50 mu M). Medium conditioned by oocytes in GV, LD, and after butyrolactone I block allowed full expansion of >90% of mouse OOX , whereas oocytes in MI and MII caused disintegration of mouse OOX without cumulus mucification. To measure synthesis of HA by cumulus cells, 25 mouse OOX were cultured in the conditioned media in the presence of 2.5 mu Ci of D-[6-H-3]glucosamine hydrochloride. After 18 h, incorporation of the [H-3] glucosamine into HA was determined either in complexes (retained HA) or in medium plus complexes (total HA). Total HA accumulation by mouse OOX was no t different from that of intact OCC. However, oocytes in CV, LD, and after butyrolactone I treatment enabled mouse OOX to retain significantly more HA within the complex than oocytes in MI and MII. The results indicate that s ecretion of factors that promote the retention of HA within the complex is developmentally regulated during oocyte maturation.