Summary of Study ST001245

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000832. The data can be accessed directly via it's Project DOI: 10.21228/M84Q24 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST001245
Study Title	Luteal lipids regulate progesterone production and may modulate immune cell function during the estrous cycle and pregnancy
Study Summary	Despite data indicating an important functional role for bioactive lipids in luteal function, little is known about the patterns of abundance of these lipids in corpus luteum (CL) during luteal development, maintenance, and rescue, in any species. Therefore, the abundance of lipid mediators, including endocannabinoids and oxylipins from cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP)-dependent metabolism were profiled in the CL on days 4, 11, and 18 of the estrous cycle and on day 18 of pregnancy. The objectives of this study were to identify lipid mediators that regulate luteal function during these transitions, to integrate the lipid profile with a previously published mRNA profile of CL during maternal recognition of pregnancy, and to determine the effect of a subset of lipids on in vitro progesterone production.
Institute	University of California, Davis
Department	Genome and Biomedical Sciences Facility
Laboratory	WCMC Metabolomics Core
Last Name	Fiehn
First Name	Oliver
Address	1315 Genome and Biomedical Sciences Facility, 451 Health Sciences Drive, Davis, CA 95616
Email	ofiehn@ucdavis.edu
Phone	(530) 754-8258
Submit Date	2019-07-29
Publications	Hughes, C.H.K., R. Bosviel, J.W. Newman, J.L. Pate. Luteal lipids regulate progesterone production and may modulate immune cell function during the estrous cycle and pregnancy. Front. Endocrinol. Jun 27, 2019
Raw Data Available	Yes
Raw Data File Type(s)	wiff
Analysis Type Detail	LC-MS
Release Date	2019-09-10
Release Version	1

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Project:

Project ID:	PR000832
Project DOI:	doi: 10.21228/M84Q24
Project Title:	Luteal lipids regulate progesterone production and may modulate immune cell function during the estrous cycle and pregnancy
Project Summary:	Despite data indicating an important functional role for bioactive lipids in luteal function, little is known about the patterns of abundance of these lipids in corpus luteum (CL) during luteal development, maintenance, and rescue, in any species. Therefore, the abundance of lipid mediators, including endocannabinoids and oxylipins from cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP)-dependent metabolism were profiled in the CL on days 4, 11, and 18 of the estrous cycle and on day 18 of pregnancy. The objectives of this study were to identify lipid mediators that regulate luteal function during these transitions, to integrate the lipid profile with a previously published mRNA profile of CL during maternal recognition of pregnancy, and to determine the effect of a subset of lipids on in vitro progesterone production.
Institute:	University of California, Davis
Department:	Genome and Biomedical Sciences Facility
Laboratory:	WCMC Metabolomics Core
Last Name:	Fiehn
First Name:	Oliver
Address:	1315 Genome and Biomedical Sciences Facility, 451 Health Sciences Drive, Davis, CA 95616
Email:	ofiehn@ucdavis.edu
Phone:	(530) 754-8258
Funding Source:	NIH U24DK097154

Subject:

Subject ID:	SU001313
Subject Type:	Mammal
Subject Species:	Bos taurus
Taxonomy ID:	9913
Gender:	Female

Factors:

Subject type: Mammal; Subject species: Bos taurus (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment
SA090787	971	D18 cyclic
SA090788	1022	D18 cyclic
SA090789	1008	D18 cyclic
SA090790	1012	D18 cyclic
SA090791	1011	D18 pregnant
SA090792	1021	D18 pregnant
SA090793	1020	D18 pregnant
SA090794	1025	D18 pregnant
SA090795	1005	Early
SA090796	1106	Early
SA090797	1006	Early
SA090798	1007	Early
SA090799	957	Midcycle
SA090800	936	Midcycle
SA090801	1129	Midcycle
SA090802	956	Midcycle
SA090806	876	Regressing 12 hour
SA090807	912	Regressing 12 hour
SA090808	909	Regressing 12 hour
SA090809	905	Regressing 12 hour
SA090803	604	Regressing 1 hour
SA090804	600	Regressing 1 hour
SA090805	858	Regressing 1 hour
SA090810	1379	Regressing 24 hour
SA090811	1378	Regressing 24 hour
SA090812	1380	Regressing 24 hour
SA090813	1381	Regressing 24 hour
SA090814	616	Regressing 4 hour
SA090815	575	Regressing 4 hour
SA090816	862	Regressing 4 hour
SA090817	857	Regressing 4 hour
SA090818	1352	Regressing 8 hour
SA090819	1342	Regressing 8 hour
SA090820	1351	Regressing 8 hour
SA090821	1353	Regressing 8 hour

Showing results 1 to 35 of 35

Showing results 1 to 35 of 35

Collection:

Collection ID:	CO001307
Collection Summary:	For cows assigned to the day 4 group, upon observation of estrus and a dominant follicle by ultrasound, cows were given an injection of GnRH (Factrel, 100 µg; Zoetis) in order to precisely time ovulation relative to time of collection for these early CL. Cows were slaughtered on day 4 following estrus. For samples collected later than day 4, precise synchrony of ovulation relative to CL collection was not necessary, so no GnRH was given, and CL were collected via colpotomy. For CL of pregnancy, cows were bred by artificial insemination and a uterine flush was performed immediately following CL collection and was examined for embryo fragments to confirm the presence of a viable pregnancy. For all samples, tissue was snap frozen in liquid nitrogen immediately following tissue collection and stored at -80 degrees Celsius thereafter. For in vitro experiments, three to five dairy cows were used in each group, CL were collected on day 10-12 of the estrous cycle, and each treatment was applied to cells from each cow
Sample Type:	Corpus Luteum

Treatment:

Treatment ID:	TR001328
Treatment Summary:	For cows assigned to the day 4 group, upon observation of estrus and a dominant follicle by ultrasound, cows were given an injection of GnRH (Factrel, 100 µg; Zoetis) in order to precisely time ovulation relative to time of collection for these early CL. Cows were slaughtered on day 4 following estrus. For samples collected later than day 4, precise synchrony of ovulation relative to CL collection was not necessary, so no GnRH was given, and CL were collected via colpotomy. For CL of pregnancy, cows were bred by artificial insemination and a uterine flush was performed immediately following CL collection and was examined for embryo fragments to confirm the presence of a viable pregnancy. For all samples, tissue was snap frozen in liquid nitrogen immediately following tissue collection and stored at -80 degrees Celsius thereafter. For in vitro experiments, three to five dairy cows were used in each group, CL were collected on day 10-12 of the estrous cycle, and each treatment was applied to cells from each cow

Treatment ID:

TR001328

Treatment Summary:

For cows assigned to the day 4 group, upon observation of estrus and a dominant follicle by ultrasound, cows were given an injection of GnRH (Factrel, 100 µg; Zoetis) in order to precisely time ovulation relative to time of collection for these early CL. Cows were slaughtered on day 4 following estrus. For samples collected later than day 4, precise synchrony of ovulation relative to CL collection was not necessary, so no GnRH was given, and CL were collected via colpotomy. For CL of pregnancy, cows were bred by artificial insemination and a uterine flush was performed immediately following CL collection and was examined for embryo fragments to confirm the presence of a viable pregnancy. For all samples, tissue was snap frozen in liquid nitrogen immediately following tissue collection and stored at -80 degrees Celsius thereafter. For in vitro experiments, three to five dairy cows were used in each group, CL were collected on day 10-12 of the estrous cycle, and each treatment was applied to cells from each cow

Sample Preparation:

Sampleprep ID:	SP001321
Sampleprep Summary:	Oxylipins and endocannabinoids were isolated using a Waters Ostro™ Sample Preparation Plate. Luteal samples were homogenized and 40 ± 8 mg were added to 2 mL polypropylene tubes spiked with a 5 µL antioxidant solution (0.2 mg/ml solution BHT/EDTA in 1:1 MeOH:water) and 10 μL 1000 nM analytical deuterated surrogates as previously described (Agrawal et al., 2017; La Frano et al., 2017). Samples were then mixed with 35 µL methanol, 550 µL isopropanol w/ 10 mM ammonium formate, 1% formic acid and 100 µL water, and the tube was placed in a Geno/Grinder 2010 (SPEX SamplePrep) for 30 sec and centrifuged at 10,000 x g for 5 min at room temperature. Supernatants were transferred into the Ostro plate wells and captured in glass inserts containing 10 μL of 20% glycerol in methanol by applying 15 mmHg of vacuum for 10 min. The eluent was dried under vacuum and reconstituted with 100 µL, 1:1 MeOH/ACN (v/v) containing 100 nM of 1-cyclohexyl ureido, 3 dodecanoic acid and 1-phenyl ureido, 3-hexanoic acid urea used as internal standards (gifts from Dr. B.D. Hammock, University of California, Davis). The samples were then vortexed and filtered at 0.1µm through PVDF membranes (Millipore) by centrifugation < 4500 x g (rcf) for 3 min at 6 ºC. The filtrate was transferred to inserts in amber glass and stored at -20 ºC for less than 48 hours before analysis by UPLC-MS/MS. Analytes in 5 μL extract aliquot were separated on a 2.1 mm x 150 mm, 1.7 µm Acquity BEH column (Waters) using published protocols for oxylipins and endocannabinoids (Agrawal et al., 2017; Pedersen and Newman, 2018). Samples were held at 10ºC. Separated residues were detected by negative mode electrospray ionization for oxylipins and positive mode electrospray ionization for endocannabinoids using multiple reaction monitoring on an API 6500 QTRAP (AB Sciex). Analytes were quantified using internal standard methods and 5- to 7-point calibration curves (r2 ≥ 0.997). Calibrants and internal standards were either synthesized [10,11-DHN, 10,11-DHHep, 10(11)-EpHep] or purchased from Cayman Chemical, Avanti Polar Lipids Inc., or Larodan Fine Lipids. Data was processed with AB Sciex MultiQuant version 3.0.2. The internal standards were used to quantify recovery of surrogate standards

Sampleprep ID:

SP001321

Sampleprep Summary:

Oxylipins and endocannabinoids were isolated using a Waters Ostro™ Sample Preparation Plate. Luteal samples were homogenized and 40 ± 8 mg were added to 2 mL polypropylene tubes spiked with a 5 µL antioxidant solution (0.2 mg/ml solution BHT/EDTA in 1:1 MeOH:water) and 10 μL 1000 nM analytical deuterated surrogates as previously described (Agrawal et al., 2017; La Frano et al., 2017). Samples were then mixed with 35 µL methanol, 550 µL isopropanol w/ 10 mM ammonium formate, 1% formic acid and 100 µL water, and the tube was placed in a Geno/Grinder 2010 (SPEX SamplePrep) for 30 sec and centrifuged at 10,000 x g for 5 min at room temperature. Supernatants were transferred into the Ostro plate wells and captured in glass inserts containing 10 μL of 20% glycerol in methanol by applying 15 mmHg of vacuum for 10 min. The eluent was dried under vacuum and reconstituted with 100 µL, 1:1 MeOH/ACN (v/v) containing 100 nM of 1-cyclohexyl ureido, 3 dodecanoic acid and 1-phenyl ureido, 3-hexanoic acid urea used as internal standards (gifts from Dr. B.D. Hammock, University of California, Davis). The samples were then vortexed and filtered at 0.1µm through PVDF membranes (Millipore) by centrifugation < 4500 x g (rcf) for 3 min at 6 ºC. The filtrate was transferred to inserts in amber glass and stored at -20 ºC for less than 48 hours before analysis by UPLC-MS/MS. Analytes in 5 μL extract aliquot were separated on a 2.1 mm x 150 mm, 1.7 µm Acquity BEH column (Waters) using published protocols for oxylipins and endocannabinoids (Agrawal et al., 2017; Pedersen and Newman, 2018). Samples were held at 10ºC. Separated residues were detected by negative mode electrospray ionization for oxylipins and positive mode electrospray ionization for endocannabinoids using multiple reaction monitoring on an API 6500 QTRAP (AB Sciex). Analytes were quantified using internal standard methods and 5- to 7-point calibration curves (r2 ≥ 0.997). Calibrants and internal standards were either synthesized [10,11-DHN, 10,11-DHHep, 10(11)-EpHep] or purchased from Cayman Chemical, Avanti Polar Lipids Inc., or Larodan Fine Lipids. Data was processed with AB Sciex MultiQuant version 3.0.2. The internal standards were used to quantify recovery of surrogate standards

Combined analysis:

Analysis ID	AN002068	AN002069
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Shimadzu Nexera X2	Shimadzu Nexera X2
Column	Waters Acquity BEH C18 (150 x 2mm,1.7um)	Waters Acquity BEH C18 (150 x 2mm,1.7um)
MS Type	ESI	ESI
MS instrument type	Triple quadrupole	Triple quadrupole
MS instrument name	ABI Sciex 6500 QTrap	ABI Sciex 6500 QTrap
Ion Mode	POSITIVE	NEGATIVE
Units	nM	nM

Chromatography:

Chromatography ID:	CH001506
Instrument Name:	Shimadzu Nexera X2
Column Name:	Waters Acquity BEH C18 (150 x 2mm,1.7um)
Column Temperature:	60
Flow Rate:	0.5 mL/min
Chromatography Type:	Reversed phase

MS:

MS ID:	MS001919
Analysis ID:	AN002068
Instrument Name:	ABI Sciex 6500 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	Multiquant
Ion Mode:	POSITIVE

MS ID:	MS001920
Analysis ID:	AN002069
Instrument Name:	ABI Sciex 6500 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	Multiquant
Ion Mode:	NEGATIVE