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Production of Plasminogen Activators (PAs) in Bovine Cumulus-Oocyte Complexes during Maturation In Vitro: Effects of Epidermal Growth Factor on Production of PAs in Oocytes and Cumulus Cells¹

^a The Graduate School of Natural Science and Technology and ^b Faculty of Agriculture, Okayama University, Okayama 700–8530, Japan

We examined whether plasminogen activators (PAs) are produced by bovine cumulus-oocyte complexes (COCs) during maturation in vitro. The effects of epidermal growth factor (EGF) on production of PAs in oocytes and cumulus cells were also examined. When COCs were cultured for 24 h with 30 ng/ml EGF, three plasminogen-dependent lytic zones (58.5 ± 3.5 kDa, 79.0 ± 3.0 kDa, and 113.5 ± 6.5 kDa) were observed. Addition of amiloride, a competitive inhibitor of urokinase-type PA (uPA), to the zymogram eliminated the activity of the 58.5 ± 3.5-kDa zone, suggesting that this band is a uPA. However, since the activity of the remaining two bands was not eliminated, it was suggested that the 79.0 ± 3.0-kDa band is a tissue-type PA (tPA) and the 113.5 ± 6.5-kDa band is possibly a tPA-PA inhibitor (tPA-PAI) complex. In COCs before culture, however, no activity of PAs was detected. At 6 h of culture, the same level of uPA activity was detected in COCs cultured both in the absence and in the presence of EGF. The uPA activity was increased at 12 h of culture but without further increase at 24 h of culture, with higher activity in the presence than in the absence of EGF. The activity of tPA and tPA-PAI was first detected at 24 h of culture in the absence of EGF. In the presence of EGF, however, some activity of tPA-PAI was detected at 12 h of culture. At 24 h of culture, the activity of all PAs was detected in cumulus cells, but only uPA activity was detected in oocytes, with higher activity in the presence than in the absence of EGF. The uPA activity in oocytes was not detected when they were cultured without cumulus cells in either the presence or absence of EGF, although cumulus expansion was stimulated by EGF, exhibiting a time-course similar to that observed in PA production. These results suggest that uPA, tPA, and tPA-PAI are all produced by bovine COCs, but only uPA by oocytes, during maturation in vitro. However, cumulus cells play an essential role or roles in the production of uPA by oocytes, and EGF enhances the roles of cumulus cells.

¹ This work was supported by Grant-in-Aid for Scientific Research A (no. 08556046) from the Ministry of Education, Science, Sports and Culture of Japan and The Monbusho's Grant-in-Aid for the Japan Society for Promotion of Science (JSPS) Fellows. S.-H.C. was a recipient of the JSPS Fellowship for Foreign Researchers in Japan (no. P96283).

² Correspondence: FAX: 81 86 251 8388; kniwa{at}cc.okayama-u.ac.jp

³ Current address: National Livestock Research Institute, Chonan 330–800, Korea.