Berg D A, Menino A R
Department of Animal Science, Oregon State University, Corvallis.
Mol Reprod Dev. 1992 Jan;31(1):14-9. doi: 10.1002/mrd.1080310104.
The type of plasminogen activator (PA) secreted by bovine embryos was identified. Day 12-14 embryos were collected from estrus-synchronized, superovulated, and naturally mated crossbred beef cows. Embryos were left intact (E) or microdissected into component embryonic discs (ED) and trophoblastic vesicles (TV). Intact embryos, ED, and TV were pre-cultured for 2 days in Minimum Essential Medium Alpha (MEM alpha) with 10% heat-inactivated fetal calf serum, washed in serum-free MEM alpha, and cultured individually for 5 days in 50 microliters microdrops of MEM alpha with 15 mg/ml bovine serum albumin. At 24 hr intervals, E, ED, and TV were observed for tissue morphology and transferred to fresh microdrops, and medium was recovered and frozen at -20 degrees C. At the end of culture, blastocoelic fluid (BF) and embryonic tissues were recovered and frozen at -20 degrees C. Plasminogen activator concentrations in medium, tissues, and BF were determined by 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 less than 0.05) than ED, and tissues exhibiting expanded blastocoels released less PA (P less than 0.05) than tissues with collapsed blastocoels. Blastocoelic fluid from TV exhibited more PA (P less than 0.05) activity than from ED. Treatment with anti-uPA decreased PA activity (P less than 0.05) in pooled medium and tissues from E compared to treatment with nonspecific immunoglobulins and anti-tPA.(ABSTRACT TRUNCATED AT 250 WORDS)
确定了牛胚胎分泌的纤溶酶原激活剂（PA）的类型。从发情同步、超排和自然交配的杂交肉牛母牛中采集第12 - 14天的胚胎。胚胎保持完整（E）或显微切割成组成胚胎盘（ED）和滋养层囊泡（TV）。完整胚胎、ED和TV在含有10%热灭活胎牛血清的α - 最低必需培养基（MEMα）中预培养2天，用无血清MEMα洗涤，然后在含有15 mg/ml牛血清白蛋白的50微升MEMα微滴中单独培养5天。每隔24小时观察E、ED和TV的组织形态，并转移到新鲜微滴中，收集培养基并在 - 20℃冷冻。培养结束时，回收囊胚腔液（BF）和胚胎组织并在 - 20℃冷冻。通过酪蛋白溶解测定法测定培养基、组织和BF中的纤溶酶原激活剂浓度。使用抗尿激酶型PA抗体（抗 - uPA）和组织型PA抗体（抗 - tPA）以及尿激酶抑制剂阿米洛利（AMR）来鉴定牛胚胎组织产生的PA类型。完整胚胎和TV释放的PA比ED更多（P < 0.05），具有扩张囊胚腔的组织释放的PA比囊胚腔塌陷的组织更少（P < 0.05）。TV的囊胚腔液显示出比ED更多的PA活性（P < 0.05）。与用非特异性免疫球蛋白和抗 - tPA处理相比，用抗 - uPA处理可降低E的合并培养基和组织中的PA活性（P < 0.05）。（摘要截断于250字）
