Ashworth C J, Bazer F W
Animal Science Department, University of Florida, Gainesville 32611.
Biol Reprod. 1989 Feb;40(2):425-33. doi: 10.1095/biolreprod40.2.425.
The secretory protein profile from conceptuses collected from naturally mated ewes on Days 10, 12, 14, and 16 was characterized by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. The presence of the anti-luteolysin ovine trophoblast protein-1 (oTP-1) in culture medium from Day 10 conceptuses was confirmed by fluorography, Western blotting, and radioimmunoassay (RIA). On each of the days studied, oTP-1 was the dominant secretory protein, and was secreted in increasing quantities as pregnancy progressed. In a second experiment, Day 6 embryos were transferred to either Day 6 (SR) or Day 4 (AR) recipients. Three mated ewes (P) received daily injections of 50 mg progesterone on Days 4-9. Controls consisted of 2 groups of pregnant ewes (D8 and D10). Conceptuses and ipsilateral endometrium were collected 4 days following transfer of conceptuses to SR and AR ewes, on Day 10 in P and D10 ewes, and on Day 8 in D8 ewes. Conceptus volume was estimated upon recovery from the uterus. Tissues were cultured with 35S-methionine, and the medium was analyzed for total and trichloroacetic acid-precipitable radiolabeled proteins. Levels of specific endometrial secretory proteins were determined after protein separation by 2D-PAGE and estimation of the radioactivity associated with discrete radiolabeled proteins on fluorographs. The concentration of oTP-1 in conceptus culture medium was estimated by RIA. Thirty endometrial proteins were investigated. All 30 proteins were present in endometrial cultures from SR, AR, D10, and P ewes, but 13 proteins were absent from D8 ewes. Levels of three proteins were higher in AR compared to D8 (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
通过二维聚丙烯酰胺凝胶电泳(2D-PAGE)和荧光自显影技术,对在第10、12、14和16天从自然交配母羊收集的孕体分泌蛋白谱进行了表征。通过荧光自显影、蛋白质印迹法和放射免疫测定(RIA)证实了第10天孕体培养基中存在抗溶黄体素绵羊滋养层蛋白-1(oTP-1)。在研究的每一天,oTP-1都是主要的分泌蛋白,并且随着妊娠进展分泌量增加。在第二个实验中,将第6天的胚胎移植到第6天(同步受体,SR)或第4天(异步受体,AR)的受体母羊中。三只交配母羊(P)在第4至9天每天注射50毫克孕酮。对照组由两组怀孕母羊(D8和D10)组成。在将孕体移植到SR和AR母羊后的第4天、P和D10母羊的第10天以及D8母羊的第8天收集孕体和同侧子宫内膜。从子宫中回收后估计孕体体积。用35S-甲硫氨酸培养组织,并分析培养基中总放射性标记蛋白和三氯乙酸沉淀的放射性标记蛋白。通过2D-PAGE分离蛋白质并估计荧光自显影片上与离散放射性标记蛋白相关的放射性后,测定特定子宫内膜分泌蛋白的水平。通过RIA估计孕体培养基中oTP-1的浓度。研究了30种子宫内膜蛋白。所有30种蛋白都存在于SR、AR、D10和P母羊的子宫内膜培养物中,但D8母羊中不存在13种蛋白。与D8相比,AR中三种蛋白的水平更高(p小于0.05)。(摘要截短至250字)