Whitley J C, Shulkes A, Salamonsen L A, Vogiagis D, Familari M, Giraud A S
University of Melbourne Department of Surgery, Austin and Repatriation Medical Centre, Melbourne, Victoria, Australia.
J Endocrinol. 1998 Apr;157(1):139-48. doi: 10.1677/joe.0.1570139.
Synthesis of both mRNA and peptide for gastrin-releasing peptide (GRP) has been demonstrated in the pregnant endometrium of sheep and women. However, it is not known whether GRP is synthesized in the sheep uterus during the oestrous cycle. Furthermore the cellular site of GRP mRNA synthesis in the uterus has not been determined. Therefore we examined the synthesis of GRP and determined the cellular location of GRP peptide and mRNA in sheep uterus taken at different times during the oestrous cycle (duration 17 days) and pregnancy (duration 145 days). Northern blot analysis of RNA isolated from ovine endometrium revealed low or no GRP mRNA at days 4, 10, 12 and 14 of the oestrous cycle and a 24-fold rise in GRP mRNA (normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA) between days 14 and 16. A similar pattern was observed during early pregnancy, with a 12-fold rise in GRP mRNA:GAPDH mRNA between days 17 and 20 of pregnancy. Levels of GRP peptide were determined by RIA and found to be low in endometrium isolated at days 4, 10, 12 and 14 of the oestrous cycle (1.0-1.6 pmol/g) and 4 to 5-fold higher at day 16. In situ hybridization localized GRP synthesis to the epithelial cells of the uterine glands at day 16 of the oestrous cycle and at days 17, 20, 40 and 50 of pregnancy. At day 140 of pregnancy diffuse hybridization to cells of the myometrium was also observed. Immunohistochemistry localized GRP peptide to the apical cytoplasm of uterine glandular epithelial cells at day 16 of the oestrous cycle. For samples obtained at day 20 of pregnancy, the area surrounding the glands also showed moderately strong staining. Further staining in the glandular lumen and the stromal tissue surrounding the glands was apparent at day 140 of pregnancy. No GRP immunoreactivity could be detected in the peripheral plasma during the oestrous cycle or the first 20 days of pregnancy. Sizing chromatography of GRP immunoreactivity extracted from endometrial tissue taken at day 10 of the oestrous cycle revealed two peaks that co-eluted with GRP(1-27) and GRP(18-27). However, during luteolysis and oestrus the major peak of GRP immunoreactivity extracted from endometrial tissue was larger than GRP(1-27) and similar to that seen previously in the gravid ovine endometrium. These studies demonstrate that a peptide similar to, but larger than, GRP is a major product of the glandular epithelium of the ovine uterus during the luteal regression phase of the oestrous cycle and post-blastocyst implantation in pregnancy and provide further evidence that GRP-related peptides have important regulatory roles in uterine function.
在绵羊和女性的妊娠子宫内膜中已证实存在胃泌素释放肽（GRP）的mRNA和肽的合成。然而，尚不清楚在发情周期中GRP是否在绵羊子宫中合成。此外，子宫中GRP mRNA合成的细胞位点尚未确定。因此，我们检测了GRP的合成，并确定了在发情周期（持续17天）和妊娠（持续145天）不同时间采集的绵羊子宫中GRP肽和mRNA的细胞定位。从绵羊子宫内膜分离的RNA的Northern印迹分析显示，在发情周期的第4、10、12和14天，GRP mRNA水平较低或无，而在第14天至16天之间，GRP mRNA（以甘油醛-3-磷酸脱氢酶（GAPDH）mRNA标准化）增加了24倍。在妊娠早期也观察到类似的模式，在妊娠第17天至20天之间，GRP mRNA:GAPDH mRNA增加了12倍。通过放射免疫分析（RIA）测定GRP肽水平，发现在发情周期的第4、10、12和14天分离的子宫内膜中GRP肽水平较低（1.0 - 1.6 pmol/g），而在第16天则高出4至5倍。原位杂交显示，在发情周期的第16天以及妊娠的第17、20、40和50天，GRP合成定位于子宫腺上皮细胞。在妊娠第140天，还观察到子宫肌层细胞的弥漫性杂交。免疫组织化学显示，在发情周期的第16天，GRP肽定位于子宫腺上皮细胞的顶端细胞质。对于在妊娠第20天获得的样本，腺周围区域也显示出中等强度的染色。在妊娠第140天，腺管腔和腺周围的基质组织中有进一步的染色。在发情周期或妊娠的前20天，外周血浆中未检测到GRP免疫反应性。对从发情周期第10天采集的子宫内膜组织中提取的GRP免疫反应性进行大小排阻色谱分析，发现有两个峰与GRP(1 - 27)和GRP(18 - 27)共洗脱。然而，在黄体溶解和发情期间，从子宫内膜组织中提取的GRP免疫反应性的主要峰大于GRP(1 - 27)，与之前在妊娠绵羊子宫内膜中观察到的相似。这些研究表明，一种与GRP相似但更大的肽是发情周期黄体退化阶段和妊娠胚泡着床后绵羊子宫腺上皮的主要产物，并进一步证明GRP相关肽在子宫功能中具有重要的调节作用。
