Salamonsen L A, Wai S O, Doughton B, Findlay J K
Endocrinology. 1985 Nov;117(5):2148-59. doi: 10.1210/endo-117-5-2148.
Ovariectomized ewes were treated with either nothing or implants of estrogen (E), progesterone (P), or E + P. Epithelial and stromal cells from caruncular and intercaruncular regions of sheep endometrium were dispersed by collagenase digestion and enriched by Ficoll gradient separation. Verification of cell types was by electron microscopy, keratin staining (epithelial cells), cell size, and appearance in culture. Epithelial cells were cultured under optimized conditions with [35S]methionine (S-met) and uptake of label by cells and its incorporation into cellular and secreted protein determined. Protein in the medium and lysed cells was analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cells from E-treated animals had higher S-met uptake and incorporation into proteins (cellular and secreted) than cells from ewes treated with nothing and P-treated animals. E effects were not significantly reduced in the presence of P. When secreted protein was expressed as a percent of total incorporated S-met, P treatment either alone or with E increased the proportion of labeled protein secreted by cells. There were no significant differences between caruncular and intercaruncular. Two-dimensional polyacrylamide-gel electrophoresis of secreted proteins showed one major glycoprotein (mol wt, 46,000, isoelectric point, 5.8-6.5) and four minor proteins induced by E + P greater than E, and five minor proteins inhibited by the steroids. Both induction and inhibition of cellular proteins were also apparent, though of lesser magnitude. Overall, whereas E treatment in vivo influenced the rate of incorporation of S-met into proteins by epithelial cells in vitro, P treatment increased the proportion of newly synthesized protein which was secreted. Steroids caused significant alterations in the individual proteins secreted by ovine endometrium.
对去卵巢母羊分别进行不处理或植入雌激素（E）、孕酮（P）或E + P处理。通过胶原酶消化分散绵羊子宫内膜肉阜区和肉阜间区的上皮细胞和基质细胞，并通过Ficoll梯度分离进行富集。通过电子显微镜、角蛋白染色（上皮细胞）、细胞大小和培养中的外观来验证细胞类型。上皮细胞在优化条件下用[35S]甲硫氨酸（S-met）培养，并测定细胞对标记物的摄取及其掺入细胞和分泌蛋白的情况。通过二维十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析培养基和裂解细胞中的蛋白质。与未处理母羊和P处理母羊的细胞相比，E处理动物的细胞具有更高的S-met摄取量以及更高的掺入蛋白质（细胞内和分泌的）的量。在存在P的情况下，E的作用没有显著降低。当将分泌蛋白表示为总掺入S-met的百分比时，单独使用P或与E一起使用P均可增加细胞分泌的标记蛋白的比例。肉阜区和肉阜间区之间没有显著差异。分泌蛋白的二维聚丙烯酰胺凝胶电泳显示，一种主要糖蛋白（分子量46,000，等电点5.8 - 6.5）以及四种次要蛋白在E + P处理下比E处理时诱导程度更高，还有五种次要蛋白受到类固醇抑制。细胞蛋白的诱导和抑制也很明显，尽管程度较小。总体而言，体内E处理影响体外上皮细胞将S-met掺入蛋白质的速率，而P处理增加了新合成蛋白分泌的比例。类固醇导致绵羊子宫内膜分泌的个别蛋白质发生显著变化。
