Graduate Thesis Or Dissertation
 

Identification of the plasminogen activator produced by microdissected day 12 to 14 bovine embryonic tissues

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  • The type of plasminogen activator (PA) secreted by bovine embryos was identified. Day 12 to 14 blastocysts were collected at slaughter from estrous-synchronized, superovulated and naturally mated crossbred beef cows. Embryos were either left as intact embryos (E) or microdissected into component embryonic discs (ED) and trophoblastic vesicles (TV). Intact embryos, ED and TV were pre-cultured for 2 d in a Minimum Essential Medium (MEM) with 10% heat-treated fetal calf serum, washed in serum-free MEMa and cultured individually for 5 d in 50-µl microdrops of MEM with 15 mg/ml bovine serum albumin. At 24-h intervals, E, ED and TV were observed for tissue morphology and transferred to fresh microdrops, and media were recovered and frozen at -20°C. At the end of culture, blastocoelic fluid (BF) and embryonic tissues were recovered and frozen at -20°C. Plasminogen activator concentrations in media, tissues and BF were determined using a caseinolytic assay. Antibodies to urokinase-type PA (anti-uPA) and tissue-type PA (anti-tPA), and the urokinase inhibitor, amiloride (AMR), were used to identify the type of PA produced by bovine embryonic tissues. Intact embryos and TV released more PA (P<.05) than ED, and tissues exhibiting expanded blastocoels released less PA (P<.05) than tissues with collapsed blastocoels. Blastocoelic fluid from TV exhibited more PA (P<.05) activity than from ED. Anti-uPA decreased PA activity (P<.05) in media from TV, ED and E compared to controls with nonspecific immunoglobulins and anti-tPA. Amiloride completely eliminated PA activity (P<.05) in tissues, media and BF from TV, ED and E. These results suggest that the PA secreted by the bovine embryo is the urokinase-type.
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