Discipline of ICT, University of Tasmania, Hobart, Tasmania, Australia.
College of Animal Life Sciences, Kangwon National University, Chuncheon, Republic of Korea.
Theriogenology. 2020 Mar 1;144:16-26. doi: 10.1016/j.theriogenology.2019.12.014. Epub 2019 Dec 23.
The aim of this study was to determine the change of Ras and its guanosine triphosphatases (GTPases) proteins in the bovine corpus luteum (CL) during estrous cycle and investigate protein-protein interaction between hormone receptors and Ras proteins via angiogenetic and apoptotic factors using bioinformatics database. The bovine CLs at proliferation phase (PP), secretion phase (SP), and regression phase (RP) were dissected from abattoir ovaries (n = 4/stage), whole of the tissue samples was used to analyze two-dimensional electrophoresis (2-DE), mRNA, and protein analysis. The protein-protein interaction between the Ras GTPases proteins and hormone receptors were analyzed using Search Tool for the Retrieval of Interacting Genes (STRING) database. The Ras protein activator like 3 (RASAL3), Ras GTPase activating protein 3 (RASA3), Ras guanine nucleotide exchange factors 1 beta (RasGEF1B) were discovered by the 2-DE and mass spectrometry in bovine CLs, and the protein spots of RASA3 and RASAL3 were significantly increased in the SPCL compared to the PPCL, whereas the RasGEF1B was reduced in the PPCL (P < 0.05). The mRNA and proteins expression of progesterone receptor, estrogen receptor alpha (ERα), vascular endothelial growth factor A (VEGFA), angiopoietin 1 (Ang1), VEGF receptor2 (VEGFR2), and Tie2 were significantly increased, but intrinsic and extrinsic apoptotic factors were decreased in PPCL and SPCL compared to RPCL (P < 0.05). Based on STRING database, we determined that RasGEF1B is activated by ERα via VEGFA and VEGFR2, then RasGEF1B activates H-Ras and R-Ras. In addition, the RasGAP protein was significantly increased, however, the RasGEF, H-Ras and R-Ras proteins were reduced in SPCL compared to PPCL and RPCL (P < 0.05). In summary, the RasGEF and Ras proteins were raised during the development, whereas the RasGAP was increased when development was completed, then the Ras and its GTPases dramatically decreased at the regression in bovine CL. In conclusion, these results suggest that Ras and Ras GTPases could be changed during development and regression, activated by the ERα via angiogenetic signaling during proliferation, and may be important to understanding of the Ras and its GTPases system for estrous cycle in bovine CL.
本研究旨在确定牛黄体（CL）在发情周期中 Ras 及其鸟嘌呤核苷酸三磷酸酶（GTPases）蛋白的变化，并通过血管生成和凋亡因子利用生物信息学数据库研究激素受体与 Ras 蛋白之间的蛋白-蛋白相互作用。从屠宰场卵巢中分离增殖期（PP）、分泌期（SP）和退化期（RP）的牛 CL（n=4/期），使用二维电泳（2-DE）、mRNA 和蛋白质分析对整个组织样本进行分析。使用搜索工具检索基因相互作用（STRING）数据库分析 Ras GTPases 蛋白与激素受体之间的蛋白-蛋白相互作用。通过 2-DE 和质谱在牛 CL 中发现 Ras 蛋白激活样 3（RASAL3）、Ras GTP 酶激活蛋白 3（RASA3）、Ras 鸟嘌呤核苷酸交换因子 1β（RasGEF1B），与 PPCL 相比，SPCL 中 RASA3 和 RASAL3 的蛋白斑点显著增加，而 RasGEF1B 在 PPCL 中减少（P<0.05）。与 RPCL 相比，PPCL 和 SPCL 中孕激素受体、雌激素受体 alpha（ERα）、血管内皮生长因子 A（VEGFA）、血管生成素 1（Ang1）、VEGF 受体 2（VEGFR2）和 Tie2 的 mRNA 和蛋白表达显著增加，而内在和外在凋亡因子减少（P<0.05）。基于 STRING 数据库，我们确定 RasGEF1B 通过 VEGFA 和 VEGFR2 被 ERα 激活，然后 RasGEF1B 激活 H-Ras 和 R-Ras。此外，RasGAP 蛋白在 SPCL 中显著增加，而在 PPCL 和 RPCL 中 RasGEF、H-Ras 和 R-Ras 蛋白减少（P<0.05）。总之，在发育过程中 RasGEF 和 Ras 蛋白升高，而在发育完成时 RasGAP 升高，然后在牛 CL 退化时 Ras 和其 GTPases 急剧减少。总之，这些结果表明，Ras 和 Ras GTPases 在发育和退化过程中可能发生变化，通过增殖过程中的血管生成信号被 ERα 激活，这可能对理解牛黄体中的 Ras 和其 GTPases 系统在发情周期中的作用很重要。
