Farin C E, Imakawa K, Roberts R M
Department of Animal Sciences, University of Missouri, Columbia 65211.
Mol Endocrinol. 1989 Jul;3(7):1099-107. doi: 10.1210/mend-3-7-1099.
The embryonic interferon, ovine trophoblast protein-1 (oTP-1), is considered to be the major protein signal by which the developing ovine conceptus communicates its presence to the mother in order to provide extension of luteal progesterone secretion critical for the establishment of pregnancy. The objective of the present study was to examine the distribution of mRNA for oTP-1 in developing ovine embryos by using in situ hybridization. A total of 11 ovine embryos were collected on days 11, 13, 15, 17, and 23 of gestation (n = 1, 2, 3, 3, and 2, respectively) and were subjected to either immediate paraformaldehyde fixation or culture for 24 h followed by fixation. Fixed embryos were embedded in paraffin and oTP-1 mRNA levels determined by in situ hybridization with a 35S-labeled cDNA probe specific for the 3'-untranslated region of the oTP-1 mRNA. Controls included parallel hybridizations with a 35S-labeled gamma-actin cDNA to detect actin mRNA (positive control) and with 35S-labeled plasmid cDNA (negative control). Hybridization signals were detected by autoradiography and quantified by computer-assisted video image analysis. Ovine TP-1 mRNA levels in tissue were low but detectable on day 11, rose 6.5-fold to peak concentrations on day 13, and declined in a linear fashion through day 23. A low, detectable signal was present in portions of chorionic tissue on day 23. Messenger RNA was localized solely to the trophectoderm and did not appear in the extraembryonic endoderm, yolk sac, and embryonic disc. The relative hybridization signal for actin mRNA was approximately 12-fold lower than that for oTP-1 mRNA on day 13. However, by day 17 oTP-1 and actin mRNA hybridization signals were similar. In conclusion, oTP-1 mRNA is localized in the trophectoderm of the developing embryo, being produced between days 11 and 23 of gestation with peak amounts produced per cell at approximately day 13 of gestation.
胚胎干扰素——绵羊滋养层蛋白-1（oTP-1），被认为是发育中的绵羊孕体向母体传达其存在的主要蛋白信号，以便延长黄体孕酮分泌，这对妊娠的建立至关重要。本研究的目的是通过原位杂交检测oTP-1 mRNA在发育中的绵羊胚胎中的分布。在妊娠第11、13、15、17和23天共收集了11个绵羊胚胎（分别为n = 1、2、3、3和2），并立即进行多聚甲醛固定，或培养24小时后再固定。固定后的胚胎用石蜡包埋，并用针对oTP-1 mRNA 3'-非翻译区的35S标记cDNA探针通过原位杂交测定oTP-1 mRNA水平。对照包括与35S标记的γ-肌动蛋白cDNA平行杂交以检测肌动蛋白mRNA（阳性对照）和与35S标记的质粒cDNA（阴性对照）。通过放射自显影检测杂交信号，并通过计算机辅助视频图像分析进行定量。组织中的绵羊TP-1 mRNA水平在第11天较低但可检测到，在第13天上升6.5倍达到峰值浓度，并在第23天呈线性下降。在第23天，绒毛膜组织的部分区域存在低水平的可检测信号。信使RNA仅定位于滋养外胚层，未出现在胚外内胚层、卵黄囊和胚盘。在第13天，肌动蛋白mRNA的相对杂交信号比oTP-1 mRNA低约12倍。然而，到第17天，oTP-1和肌动蛋白mRNA杂交信号相似。总之，oTP-1 mRNA定位于发育中胚胎的滋养外胚层，在妊娠第11天至23天产生，在妊娠约第13天每个细胞产生的量达到峰值。
