396 CHANGES OF PLASMINOGEN ACTIVATOR ACTIVITY IN OVIDUCTAL EPITHELIAL CELLS DURING THE ESTROUS CYCLE IN PIGS

Abstract

Plasminogen activator (PA) activity in rat, porcine, and bovine embryos and in oocytes have been demonstrated, but reports of PA activity in the uterus and oviduct are limited. The present study was undertaken to identify changes of PAs in porcine oviductal epithelial cells (pOECs) during the estrous cycle in pigs. pOECs obtained from pigs in post-ovulatory (Post-Ov), early-to-mid luteal (Early-mid L), and pre-ovulatory (Pre-Ov) stages were washed twice with Hank's balanced salt solution (HBSS); the oviductal lumen was flushed with 10 mL HBSS, and centrifuged at 300g for 10 min at 4°C. The collected pOECs were washed twice with DMEM containing 0.1% BSA and centrifuged at 300g for 10 min at 4°C. To examine the changes in activity of PAs in pOEC, 2.0 × 10⁵ fresh cells were cultured in a 4-well dish with DMEM/Ham's F-12 medium containing inactivated 10% FBS and amphotericin under a humidified atmosphere of 5% CO₂ in air at 38°C for 1, 3, 5, and 7 days. The culture medium was replaced every 48 h with fresh medium during culture for 7 days, after which cells and conditioned medium were stored separately at -20°C until they were used for zymographic analysis. PA activities of pOECs were measured using SDS-PAGE, casein-agar zymography, and densitometry. The urokinase-type PA (uPA) was observed at 5–7 days of culture in pOECs of different stages of the estrous cycle in pigs. On the other hand, uPA, tissue-type (tPA), and tPA-PA inhibitor (tPA-PAI) activities were observed at days 1, 3, 5, and 7 of culture in medium conditioned with pOEC from different stages of the estrous cycle. uPA activity was decreased during prolonged culture of Post-Ov pOECs. At this time, uPA activity was significantly (P < 0.05) higher at 1 day than at 3–7 days of culture in Post-Ov pOEC-conditioned medium. tPA activity was significantly (P < 0.05) higher at 5 days than at 1–3 days or 7 days of culture in Post-Ov pOEC-conditioned medium. tPA-PAI activity was significantly (P < 0.05) higher at 7 days than at 5 days of culture in Pre-Ov pOEC-conditioned medium, and tPA-PAI activity was significantly (P < 0.05) higher at Post-Ov than Pre-Ov or Early-mid L in 7 days of culture in pOEC-conditioned medium of different stages of the estrous cycle. All tPA activities were increased until Day 5 and were decreased at Day 7 of culture in pOEC-conditioned medium. uPA activity was observed in pOEC-conditioned medium throughout the estrous cycle. uPA activity was significantly (P < 0.05) different during culture of Post-Ov in pOECs-conditioned medium. tPA-PAI activity was observed at all stages, and tPA-PAI activity was significantly (P < 0.05) different among different stages of the estrous cycle. These results suggest that uPA, tPA, and tPA-PAI are produced by pOEC, and that variations of PA activity are found at different stages of the estrous cycle in porcine oviductal epithelial cells.

This work was supported by grant No. R01-2003-000-10500-0 from the Basic Research Program of Korea Science & Engineering Foundation.