#METABOLOMICS WORKBENCH kborkows_20190729_154556 DATATRACK_ID:1786 STUDY_ID:ST001245 ANALYSIS_ID:AN002068 PROJECT_ID:PR000832 VERSION 1 CREATED_ON August 26, 2019, 1:53 pm #PROJECT PR:PROJECT_TITLE Luteal lipids regulate progesterone production and may modulate immune cell PR:PROJECT_TITLE function during the estrous cycle and pregnancy PR:PROJECT_SUMMARY Despite data indicating an important functional role for bioactive lipids in PR:PROJECT_SUMMARY luteal function, little is known about the patterns of abundance of these lipids PR:PROJECT_SUMMARY in corpus luteum (CL) during luteal development, maintenance, and rescue, in any PR:PROJECT_SUMMARY species. Therefore, the abundance of lipid mediators, including endocannabinoids PR:PROJECT_SUMMARY and oxylipins from cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 PR:PROJECT_SUMMARY (CYP)-dependent metabolism were profiled in the CL on days 4, 11, and 18 of the PR:PROJECT_SUMMARY estrous cycle and on day 18 of pregnancy. The objectives of this study were to PR:PROJECT_SUMMARY identify lipid mediators that regulate luteal function during these transitions, PR:PROJECT_SUMMARY to integrate the lipid profile with a previously published mRNA profile of CL PR:PROJECT_SUMMARY during maternal recognition of pregnancy, and to determine the effect of a PR:PROJECT_SUMMARY subset of lipids on in vitro progesterone production. PR:INSTITUTE University of California, Davis PR:DEPARTMENT Genome and Biomedical Sciences Facility PR:LABORATORY WCMC Metabolomics Core PR:LAST_NAME Fiehn PR:FIRST_NAME Oliver PR:ADDRESS 1315 Genome and Biomedical Sciences Facility, 451 Health Sciences Drive, Davis, PR:ADDRESS CA 95616 PR:EMAIL ofiehn@ucdavis.edu PR:PHONE (530) 754-8258 PR:FUNDING_SOURCE NIH U24DK097154 #STUDY ST:STUDY_TITLE Luteal lipids regulate progesterone production and may modulate immune cell ST:STUDY_TITLE function during the estrous cycle and pregnancy ST:STUDY_SUMMARY Despite data indicating an important functional role for bioactive lipids in ST:STUDY_SUMMARY luteal function, little is known about the patterns of abundance of these lipids ST:STUDY_SUMMARY in corpus luteum (CL) during luteal development, maintenance, and rescue, in any ST:STUDY_SUMMARY species. Therefore, the abundance of lipid mediators, including endocannabinoids ST:STUDY_SUMMARY and oxylipins from cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 ST:STUDY_SUMMARY (CYP)-dependent metabolism were profiled in the CL on days 4, 11, and 18 of the ST:STUDY_SUMMARY estrous cycle and on day 18 of pregnancy. The objectives of this study were to ST:STUDY_SUMMARY identify lipid mediators that regulate luteal function during these transitions, ST:STUDY_SUMMARY to integrate the lipid profile with a previously published mRNA profile of CL ST:STUDY_SUMMARY during maternal recognition of pregnancy, and to determine the effect of a ST:STUDY_SUMMARY subset of lipids on in vitro progesterone production. ST:INSTITUTE University of California, Davis ST:DEPARTMENT Genome and Biomedical Sciences Facility ST:LABORATORY WCMC Metabolomics Core ST:LAST_NAME Fiehn ST:FIRST_NAME Oliver ST:ADDRESS 1315 Genome and Biomedical Sciences Facility, 451 Health Sciences Drive, Davis, ST:ADDRESS CA 95616 ST:EMAIL ofiehn@ucdavis.edu ST:PHONE (530) 754-8258 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Bos taurus SU:TAXONOMY_ID 9913 SU:GENDER Female #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - 575 Treatment:Regressing 4 hour SUBJECT_SAMPLE_FACTORS - 600 Treatment:Regressing 1 hour SUBJECT_SAMPLE_FACTORS - 604 Treatment:Regressing 1 hour SUBJECT_SAMPLE_FACTORS - 616 Treatment:Regressing 4 hour SUBJECT_SAMPLE_FACTORS - 857 Treatment:Regressing 4 hour SUBJECT_SAMPLE_FACTORS - 858 Treatment:Regressing 1 hour SUBJECT_SAMPLE_FACTORS - 862 Treatment:Regressing 4 hour SUBJECT_SAMPLE_FACTORS - 876 Treatment:Regressing 12 hour SUBJECT_SAMPLE_FACTORS - 905 Treatment:Regressing 12 hour SUBJECT_SAMPLE_FACTORS - 909 Treatment:Regressing 12 hour SUBJECT_SAMPLE_FACTORS - 912 Treatment:Regressing 12 hour SUBJECT_SAMPLE_FACTORS - 936 Treatment:Midcycle SUBJECT_SAMPLE_FACTORS - 956 Treatment:Midcycle SUBJECT_SAMPLE_FACTORS - 957 Treatment:Midcycle SUBJECT_SAMPLE_FACTORS - 971 Treatment:D18 cyclic SUBJECT_SAMPLE_FACTORS - 1005 Treatment:Early SUBJECT_SAMPLE_FACTORS - 1006 Treatment:Early SUBJECT_SAMPLE_FACTORS - 1007 Treatment:Early SUBJECT_SAMPLE_FACTORS - 1008 Treatment:D18 cyclic SUBJECT_SAMPLE_FACTORS - 1011 Treatment:D18 pregnant SUBJECT_SAMPLE_FACTORS - 1012 Treatment:D18 cyclic SUBJECT_SAMPLE_FACTORS - 1020 Treatment:D18 pregnant SUBJECT_SAMPLE_FACTORS - 1021 Treatment:D18 pregnant SUBJECT_SAMPLE_FACTORS - 1022 Treatment:D18 cyclic SUBJECT_SAMPLE_FACTORS - 1025 Treatment:D18 pregnant SUBJECT_SAMPLE_FACTORS - 1106 Treatment:Early SUBJECT_SAMPLE_FACTORS - 1129 Treatment:Midcycle SUBJECT_SAMPLE_FACTORS - 1351 Treatment:Regressing 8 hour SUBJECT_SAMPLE_FACTORS - 1352 Treatment:Regressing 8 hour SUBJECT_SAMPLE_FACTORS - 1353 Treatment:Regressing 8 hour SUBJECT_SAMPLE_FACTORS - 1378 Treatment:Regressing 24 hour SUBJECT_SAMPLE_FACTORS - 1379 Treatment:Regressing 24 hour SUBJECT_SAMPLE_FACTORS - 1380 Treatment:Regressing 24 hour SUBJECT_SAMPLE_FACTORS - 1381 Treatment:Regressing 24 hour SUBJECT_SAMPLE_FACTORS - 1342 Treatment:Regressing 8 hour #COLLECTION CO:COLLECTION_SUMMARY For cows assigned to the day 4 group, upon observation of estrus and a dominant CO:COLLECTION_SUMMARY follicle by ultrasound, cows were given an injection of GnRH (Factrel, 100 µg; CO:COLLECTION_SUMMARY Zoetis) in order to precisely time ovulation relative to time of collection for CO:COLLECTION_SUMMARY these early CL. Cows were slaughtered on day 4 following estrus. For samples CO:COLLECTION_SUMMARY collected later than day 4, precise synchrony of ovulation relative to CL CO:COLLECTION_SUMMARY collection was not necessary, so no GnRH was given, and CL were collected via CO:COLLECTION_SUMMARY colpotomy. For CL of pregnancy, cows were bred by artificial insemination and a CO:COLLECTION_SUMMARY uterine flush was performed immediately following CL collection and was examined CO:COLLECTION_SUMMARY for embryo fragments to confirm the presence of a viable pregnancy. For all CO:COLLECTION_SUMMARY samples, tissue was snap frozen in liquid nitrogen immediately following tissue CO:COLLECTION_SUMMARY collection and stored at -80 degrees Celsius thereafter. For in vitro CO:COLLECTION_SUMMARY experiments, three to five dairy cows were used in each group, CL were collected CO:COLLECTION_SUMMARY on day 10-12 of the estrous cycle, and each treatment was applied to cells from CO:COLLECTION_SUMMARY each cow CO:SAMPLE_TYPE Corpus Luteum #TREATMENT TR:TREATMENT_SUMMARY For cows assigned to the day 4 group, upon observation of estrus and a dominant TR:TREATMENT_SUMMARY follicle by ultrasound, cows were given an injection of GnRH (Factrel, 100 µg; TR:TREATMENT_SUMMARY Zoetis) in order to precisely time ovulation relative to time of collection for TR:TREATMENT_SUMMARY these early CL. Cows were slaughtered on day 4 following estrus. For samples TR:TREATMENT_SUMMARY collected later than day 4, precise synchrony of ovulation relative to CL TR:TREATMENT_SUMMARY collection was not necessary, so no GnRH was given, and CL were collected via TR:TREATMENT_SUMMARY colpotomy. For CL of pregnancy, cows were bred by artificial insemination and a TR:TREATMENT_SUMMARY uterine flush was performed immediately following CL collection and was examined TR:TREATMENT_SUMMARY for embryo fragments to confirm the presence of a viable pregnancy. For all TR:TREATMENT_SUMMARY samples, tissue was snap frozen in liquid nitrogen immediately following tissue TR:TREATMENT_SUMMARY collection and stored at -80 degrees Celsius thereafter. For in vitro TR:TREATMENT_SUMMARY experiments, three to five dairy cows were used in each group, CL were collected TR:TREATMENT_SUMMARY on day 10-12 of the estrous cycle, and each treatment was applied to cells from TR:TREATMENT_SUMMARY each cow #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Oxylipins and endocannabinoids were isolated using a Waters Ostro™ Sample SP:SAMPLEPREP_SUMMARY Preparation Plate. Luteal samples were homogenized and 40 ± 8 mg were added to SP:SAMPLEPREP_SUMMARY 2 mL polypropylene tubes spiked with a 5 µL antioxidant solution (0.2 mg/ml SP:SAMPLEPREP_SUMMARY solution BHT/EDTA in 1:1 MeOH:water) and 10 μL 1000 nM analytical deuterated SP:SAMPLEPREP_SUMMARY surrogates as previously described (Agrawal et al., 2017; La Frano et al., SP:SAMPLEPREP_SUMMARY 2017). Samples were then mixed with 35 µL methanol, 550 µL isopropanol w/ 10 SP:SAMPLEPREP_SUMMARY mM ammonium formate, 1% formic acid and 100 µL water, and the tube was placed SP:SAMPLEPREP_SUMMARY in a Geno/Grinder 2010 (SPEX SamplePrep) for 30 sec and centrifuged at 10,000 x SP:SAMPLEPREP_SUMMARY g for 5 min at room temperature. Supernatants were transferred into the Ostro SP:SAMPLEPREP_SUMMARY plate wells and captured in glass inserts containing 10 μL of 20% glycerol in SP:SAMPLEPREP_SUMMARY methanol by applying 15 mmHg of vacuum for 10 min. The eluent was dried under SP:SAMPLEPREP_SUMMARY vacuum and reconstituted with 100 µL, 1:1 MeOH/ACN (v/v) containing 100 nM of SP:SAMPLEPREP_SUMMARY 1-cyclohexyl ureido, 3 dodecanoic acid and 1-phenyl ureido, 3-hexanoic acid urea SP:SAMPLEPREP_SUMMARY used as internal standards (gifts from Dr. B.D. Hammock, University of SP:SAMPLEPREP_SUMMARY California, Davis). The samples were then vortexed and filtered at 0.1µm SP:SAMPLEPREP_SUMMARY through PVDF membranes (Millipore) by centrifugation < 4500 x g (rcf) for 3 min SP:SAMPLEPREP_SUMMARY at 6 ºC. The filtrate was transferred to inserts in amber glass and stored at SP:SAMPLEPREP_SUMMARY -20 ºC for less than 48 hours before analysis by UPLC-MS/MS. Analytes in 5 μL SP:SAMPLEPREP_SUMMARY extract aliquot were separated on a 2.1 mm x 150 mm, 1.7 µm Acquity BEH column SP:SAMPLEPREP_SUMMARY (Waters) using published protocols for oxylipins and endocannabinoids (Agrawal SP:SAMPLEPREP_SUMMARY et al., 2017; Pedersen and Newman, 2018). Samples were held at 10ºC. Separated SP:SAMPLEPREP_SUMMARY residues were detected by negative mode electrospray ionization for oxylipins SP:SAMPLEPREP_SUMMARY and positive mode electrospray ionization for endocannabinoids using multiple SP:SAMPLEPREP_SUMMARY reaction monitoring on an API 6500 QTRAP (AB Sciex). Analytes were quantified SP:SAMPLEPREP_SUMMARY using internal standard methods and 5- to 7-point calibration curves (r2 ≥ SP:SAMPLEPREP_SUMMARY 0.997). Calibrants and internal standards were either synthesized [10,11-DHN, SP:SAMPLEPREP_SUMMARY 10,11-DHHep, 10(11)-EpHep] or purchased from Cayman Chemical, Avanti Polar SP:SAMPLEPREP_SUMMARY Lipids Inc., or Larodan Fine Lipids. Data was processed with AB Sciex MultiQuant SP:SAMPLEPREP_SUMMARY version 3.0.2. The internal standards were used to quantify recovery of SP:SAMPLEPREP_SUMMARY surrogate standards #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Shimadzu Nexera X2 CH:COLUMN_NAME Waters Acquity BEH C18 (150 x 2mm, 1.7um) CH:FLOW_RATE 0.5 mL/min CH:COLUMN_TEMPERATURE 60 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 6500 QTrap MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Multiquant #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS nM MS_METABOLITE_DATA_START Samples 575 600 604 616 857 858 862 876 905 909 912 936 956 957 971 1005 1006 1007 1008 1011 1012 1020 1021 1022 1025 1106 1129 1351 1352 1353 1378 1379 1380 1381 1342 Factors Treatment:Regressing 4 hour Treatment:Regressing 1 hour Treatment:Regressing 1 hour Treatment:Regressing 4 hour Treatment:Regressing 4 hour Treatment:Regressing 1 hour Treatment:Regressing 4 hour Treatment:Regressing 12 hour Treatment:Regressing 12 hour Treatment:Regressing 12 hour Treatment:Regressing 12 hour Treatment:Midcycle Treatment:Midcycle Treatment:Midcycle Treatment:D18 cyclic Treatment:Early Treatment:Early Treatment:Early Treatment:D18 cyclic Treatment:D18 pregnant Treatment:D18 cyclic Treatment:D18 pregnant Treatment:D18 pregnant Treatment:D18 cyclic Treatment:D18 pregnant Treatment:Early Treatment:Midcycle Treatment:Regressing 8 hour Treatment:Regressing 8 hour Treatment:Regressing 8 hour Treatment:Regressing 24 hour Treatment:Regressing 24 hour Treatment:Regressing 24 hour Treatment:Regressing 24 hour Treatment:Regressing 8 hour 1-AG 892 3080 2060 1110 1280 3220 2420 1020 2770 2970 4730 2770 1240 921 20500 281 710 707 717 949 1300 664 1920 917 1460 776 3510 740 618 1160 1690 755 1430 720 3650 1-LG 3010 5280 6480 3540 3790 11100 5850 2930 12600 11200 24800 6710 1290 2820 47200 515 1170 1070 1600 4310 5060 1680 4290 4330 4800 2170 9590 979 886 3890 4710 2000 3330 1500 15900 1-OG 2460 9370 4180 1430 1440 3860 2100 2980 5330 11500 12200 8190 4500 2420 43000 688 2100 3570 1250 2150 10200 1040 2980 2330 4450 2330 7660 1320 756 2210 1970 1380 3310 1130 12500 2-AG 5940 17400 10700 9650 4550 7160 11500 6470 7420 21600 26200 8420 8790 3060 143000 1800 3650 2330 5760 3410 6390 3370 12300 6910 9900 3460 11200 3690 4970 3790 11300 3970 6280 3840 12300 2-LG 12900 17900 20000 13400 13800 21500 18800 23900 30800 42600 71300 19700 7020 7940 154000 3020 4020 3090 9460 16900 26000 6980 13500 21000 25600 10500 25500 4820 3310 9850 10900 5230 9040 5570 36500 2-OG 7850 22200 8810 5870 4780 9980 9540 6150 9310 21800 32500 11400 11900 5610 101000 4230 8030 8340 3530 3660 16400 2900 7440 8200 10700 6360 23600 3640 5140 5870 5720 3860 6120 3290 34600 A-EA 37.4 79.9 114 36.9 41.8 81 73.6 53.2 47.7 78.4 129 19.8 35.7 20.9 309 13.5 10.6 7.27 22.3 27 60.3 26.2 77.9 49.4 36.5 33.8 38 27.6 31.3 42.2 39.1 23.6 39.3 34.3 66.8 aL-EA 2.02 6.84 2.75 6.31 0.925 1.35 1.68 3.98 6.61 35.6 18.1 5.08 3.84 4.56 101 1.56 1.02 0.576 2.26 1.76 3.44 1.93 2.05 3.2 3.08 4.58 0.879 1.71 1.51 2.1 1.27 1.13 1.01 1.16 3.94 D-EA 39.3 49.4 39.3 23.7 27 30.8 63.2 36.5 24.4 125 63.1 21.5 21.8 12.2 480 4.28 6.6 12.1 15.2 17 36.9 24.5 83.2 32.6 34.9 7.64 46.3 20.9 23.8 34.6 32 28.6 36.4 21.6 134 DGL-EA 8.02 11.1 15.8 9.58 8.42 7.59 14.4 10.5 9.9 37.9 26.6 3.81 6.06 5.66 153 2.89 2.64 1.17 8.31 4.46 8.86 7.27 27.9 17.6 13.2 5.27 8.41 6.78 7.38 6.76 8.56 7.12 8.51 5.95 11.1 DH-EA 1.42 1.95 1.07 0.653 0.471 0.584 0.849 1.19 1.85 2.83 2.91 1.07 3.6 1.04 6.7 1.41 1.18 1.05 0.384 0.444 0.98 1.05 2.53 1.23 0.909 1.49 2.47 0.547 1.61 1.06 1.49 0.668 1.47 1.38 3.02 L-EA 214 190 214 1240 45.3 89 85 60.7 83.6 265 225 34.8 26.5 37.5 628 18.9 17.5 21.5 33.9 38.2 79.7 33.7 69.7 54.2 50.5 70.7 61.3 40.1 30 50.4 43.6 31.4 34.8 39.6 97.5 O-EA 816 563 407 5930 19.3 34.8 44.6 38 29.5 89.4 73.5 18.1 61.4 45.3 306 26.5 28.4 12.8 50.8 16.8 66.7 40.5 67.1 39.6 56.4 66.4 45 22.3 34.5 17 26 25.7 30 23.9 36.4 P-EA 2510 1660 896 8330 1190 715 1180 342 1180 674 719 504 911 790 625 849 169 98.7 327 406 806 1030 1300 1520 601 285 954 748 1270 988 249 660 1270 346 942 S-EA 1040 671 356 1470 650 384 678 278 695 506 514 356 1050 386 572 382 151 85.2 286 265 1280 567 1340 1090 559 154 815 450 739 658 143 449 796 153 1290 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Report Order LOD LOQ Ionisation mede Observed InChIKey PubChem ID Chemical class Enzyme Parent FA Units quantitated m/z Q3 Retention times 1-AG 109 0.438 1.46 Positive Yes DCPCOKIYJYGMDN-HUDVFFLJSA-N 16019980 MAG Lipase AA nM 379.302 287.2 11.25043394 1-LG 107 4.57 15.2 Positive Yes WECGLUPZRHILCT-GSNKCQISSA-N 6436630 MAG Lipase LA nM 355.302 263.2 11.34366667 1-OG 111 7.51 25 Positive Yes RZRNAYUHWVFMIP-QJRAZLAKSA-N 12178130 MAG Lipase OA nM 357.302 265.2 12.21410716 2-AG 110 0.657 2.19 Positive Yes RCRCTBLIHCHWDZ-DOFZRALJSA-N 5282280 MAG Lipase AA nM 379.302 287.2 11.25043394 2-LG 108 1.25 4.18 Positive Yes IEPGNWMPIFDNSD-HZJYTTRNSA-N 5365676 MAG Lipase LG nM 355.302 263.2 11.34366667 2-OG 112 2.97 9.91 Positive Yes UPWGQKDVAURUGE-KTKRTIGZSA-N 5319879 MAG Lipase OA nM 357.302 265.2 12.21410716 A-EA 100 0.0623 0.208 Positive Yes LGEQQWMQCRIYKG-DOFZRALJSA-N 5281969 Acyl-EA PLD AA nM 348.3 62.1 10.6254871 aL-EA 98 0.129 0.431 Positive Yes HBJXRRXWHSHZPU-PDBXOOCHSA-N 5283449 Acyl-EA PLD aLA nM 322.2 62.1 9.512628019 D-EA 101 0.0799 0.266 Positive Yes FMVHVRYFQIXOAF-DOFZRALJSA-N 5282273 Acyl-EA PLD Adrenic acid nM 376.3 62.1 11.60083512 DGL-EA 99 0.127 0.425 Positive Yes ULQWKETUACYZLI-QNEBEIHSSA-N 5282272 Acyl-EA PLD DGLA nM 350.3 62.1 11.17465724 DH-EA 102 0.0562 0.187 Positive Yes CXWASNUDKUTFPQ-KUBAVDMBSA-N 53245830 Acyl-EA PLD DHA nM 372.3 62.1 10.51195296 L-EA 97 0.142 0.473 Positive Yes KQXDGUVSAAQARU-HZJYTTRNSA-N 5283446 Acyl-EA PLD LA nM 324.2 62.1 10.62321288 O-EA 96 0.242 0.806 Positive Yes BOWVQLFMWHZBEF-KTKRTIGZSA-N 5283454 Acyl-EA PLD OA nM 326.2 62.1 11.56286658 P-EA 95 5.06 16.9 Positive No HXYVTAGFYLMHSO-UHFFFAOYSA-N 4671 Acyl-EA PLD PA nM 300.2 62.1 11.26566447 S-EA 94 68.1 227 Positive No OTGQIQQTPXJQRG-UHFFFAOYSA-N 27902 Acyl-EA PLD SA nM 328.2 62.1 12.5729662 METABOLITES_END #END