The Ras family members follow the blood progesterone level during formation and regression in bovine corpus luteum

Ras family members regulate cellular differentiation, proliferation and survival. CL formation and regression are regulated by the blood P4 level. This study investigated the association between changes in Ras family members and the serum P4 level and determined protein interactions among Ras family members, hormone receptors, and angiogenetic and apoptotic factors during formation and regression of the bovine CL. RASAL3 and RASA3 were found using proteomics in CL and were significantly increased in the SPCL compared to the PPCL, whereas RasGEF1B was decreased in the PPCL. Hormone receptors and angiogenetic proteins expression was lower in the PPCL and SPCL than that in the RPCL, but apoptotic proteins were increased in the RPCL. The P4 and estrogen receptors positive correlated with RasGEF1B, R-Ras, and H-Ras through VEGFA, VEGFR2 and Tie2 in STRING database. RasGAP, H-Ras and R-Ras protein expression was increased in the PPCL compared to that in the SPCL, whereas RasGEF expression was decreased. In summary, Ras activation and angiogenesis in the CL were positively correlated with the blood P4 during estrous cylce. These results may increase understanding of Ras biological functions following stimulation of hormones and their receptors during tissue proliferation and degeneration.


Introduction
D Clean-Up kit (GE Healthcare, Piscataway, NJ, USA) according to the manufacturer's instructions, and 700 μg protein 158 was dissolved in 300 μL rehydration solution (GE Healthcare) for 1 hour at RT. Proteins into rehydration buffer were 159 incubated with an 18 cm immobilized pH 3-11 nonlinear gradient dry strip (GE Healthcare) for 16 hours at 20℃. As 160 described previously (19), isoelectric focusing (IEF) was performed for protein separation. IEF was performed at hold 161 500 V for 1 hour, gradient 1,000V 1hour, gradient 8,000 V for 3 hours, hold 8,000 V for 1.5 hours, gradient 10,000 V 162 for 3 hours and hold 10,000 V for 1hour. Strips were then equilibrated for 15 min in 5 mL equilibration buffer (50 mM 163 Tris-HCl, pH 8.8, 6.0 M urea, 30% glycerol (v/v), and 2% sodium dodecyl sulfate (w/v) containing 0.8 g dithiothreitol 164 (Sigma), followed by an additional incubation for 15 min in 5 mL equilibration buffer containing 0.1 g iodoacetamide. 165 Separation in the second dimension was accomplished using an 10% SDS-PAGE in a Protean II xi 2-D Cell (Bio-Rad, 166 Hercules, CA, USA) at 50 mA until the bromophenol blue reached the bottom of the gel. Gels were stained in a solution 167 of 0.1% Coomassie Brilliant Blue R-250 (Sigma) comprised of 45% methanol, 10% acetic acid and 45% water. Gels 168 were then scanned using an image scanner and analyzed with ImageJ software (NCBI, USA). The SPCL and RPCL 169 protein spots intensities were normalized to PPCL protein spots intensities for calculation of relative intensity.  172 MALDI-TOF/MS was performed as described previously (19) and condition was listed in Supplementary Table   173 1. Spots were extracted from the gel and washed in 50% acetonitrile (ACN; Sigma) containing 25 mM NH 4 bicarbonate, 174 then incubated with 50% ACN containing 10 mM NH 4 bicarbonate and 100% CAN. Finally, ACN in samples was 175 removed using a speed vacuum. Then, samples were incubated with cold sequencing-grade modified trypsin (Promega, 176 Madison, WI, USA) at 37 o C for 20 h, followed by 50 min incubation with 50% ACN containing 5% trifluoroacetic acid 177 (TFA) at RT. The supernatants were dried for peptide extraction using a speed vacuum, and then diluted with 50% ACN 178 containing 5% TFA. Samples were desalted using a Zip-Tip C18 (Millipore, Milford, MA, USA). Plating was performed 179 using a 4-hydroxy-α-cyano-cinnamic acid matrix solution (Sigma) on a MALDI-TOF/MS plate. Peptides were analyzed 180 using an Ultraflex-TOF/TOF spectrometer (Bruker Daltonics, Hamburg, Germany) and MS-Fit software 181 (http://prospector.ucsf.edu) and data were searched against UniProt database (http://www.uniprot.org/).

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The Gene Ontology (GO) analysis of identified total CL proteins reveals over the half of all proteins at PPCL and 239 SPCL involved in cellular process, single organism cellular process, biological process, cellular response to stimulus, 240 and cell communication, whereas apoptotic process, negative cellular process, negative biological process, endocytosis, 241 negative developmental process, and muscle contraction were mostly related with RPCL (Fig 2A). Based on 242 classification of the identified differentially expressed protein in molecular function, over the 50 % of PPCL and SPCL 243 proteins were involved in molecular function, heterocyclic compound binding, organic cyclic compound binding and 244 ion binding (Fig 2B). Moreover, cellular components ratio of determined proteins was different during estrous cycle, 245 kinds of cellular component were increased in PPCL (Fig 2C) to SPCL (Fig 2D), whereas, decreased at SPCL to RPCL 246 ( Fig 2E). The 3β-HSD, P4R, PGF2αR and ERα mRNA ( Fig 3A) and protein ( Fig 3B) were decreased in RPCL compared 250 to PPCL and SPCL, ERα and OTR proteins expression were higher at PPCL than SPCL and RPCL. Additionally, P4R 251 was positive correlated with ERα but there were little molecular interaction between hormone receptors and Ras 252 regulators according to in STING protein-protein interaction data (Fig 3C). 253 The VEGFA and VEGFR2 mRNA ( Fig 3D) and protein ( Fig 3E) were increased at PPCL and SPCL compared 254 to RPCL. The Ang1 mRNA and protein expression were higher at SPCL than PPCL and RPCL, and Tie2 mRNA was 255 no significantly difference between PPCL and SPCL, but protein was increased at SPCL compared to PPCL and RPCL. 256 ( Fig 3E). According to STING data, there were highly correlative between angiogenetic factors and Ras regulators, 257 moreover both VEGFA and Tie2 had various molecular action to VEGFR2 and Tie2 (Fig 3F). Furthermore, STRING p53, Bax and Casp3; see the Fig 3G and H) were increased compared to PP and SP, most angiogenetic factors and Ras 298 family members were dramatically reduced in RP (Fig 5K). Generally, P4 increases the endometrium, is involved in pregnancy maintenance and inhibits follicle growth in the 302 female reproductive system (4, 5). In practice, continuous P4 synthesis from LSCs is needed to ensure the interaction 303 between intercellular P4 activity and its nuclear receptor and to active steroidogenic enzymes (11, 21). The main function to 18 after ovulation. Our results showed that the P4 concentration of Korean native cattle at days 12 to 18 after ovulation 316 was 3.0 -4.0 ng/mL and that the P4 at the secretion phase was higher than that at the proliferation and regression phases.

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Although the blood P4 level of Korean native cattle heifers was lower than that of previous reports, we confirmed that  The story of small G proteins started more than three decades age and was followed by the discovery of the Ras  Additionally, Ras is controlled by a molecular switch by cycling between the inactive Ras-GDP and active Ras-GTP 336 conformations, and GDP/GTP binding is regulated by RasGEF and RasGAP(30). As such, Ras has been widely studied 337 in ovarian cancer, but studies of Ras family members during formation and regression of the CL induced by P4 produced 338 from the ovary have not been reported to date. has not been reported, this study may contribute to understanding of the roles of the Ras pathway following hormone 359 changes during tissue formation and regression.

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Studies of hormones and their receptors have been widely conducted because formation and regression of the CL 361 into the ovary are repeated in response to various hormone reactions, which control the reproductive cycle (4). Based 362 on the bioinformatics analysis, P4R not only reacted to but its transcription was also regulated by ERα, and the 363 interaction between P4R and ERα was closer than those of PGF2αR, ERα, and OTR with P4 in the PPCL. Similar to P4 364 and E2, the physiological interaction may be more activated in the PP and SP during cellular proliferation and 365 maintenance. However, no association was found between the hormone receptors and the Ras regulators (RASAL3, 366 RASA3, and RasGEF1B). The hormone receptor and Ras regulator aspect of the in silico assay showed that the hormone 367 receptors were not directly regulated by the Ras regulators, we know that there were few study on its receptors and Ras 368 regulators as evidence assay.
plays an important role for proliferation and maintenance in the CL lifespan (34). Furthermore, our study showed that increases endothelial nitric oxide synthase in the periphery of the bovine corpus luteum: the possible regulation 519 of blood flow at an early stage of luteolysis. Reproduction 2008;135: 527-539.