Source: UNIVERSITY OF NORTHERN COLORADO submitted to
REGULATION OF PROSTAGLANDIN SYNTHESIS IN BOVINE LUTEAL CELLS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0214144
Grant No.
2008-35203-19099
Project No.
COLR-2008-00684
Proposal No.
2008-00684
Multistate No.
(N/A)
Program Code
41.0
Project Start Date
Jul 1, 2008
Project End Date
Jun 30, 2011
Grant Year
2008
Project Director
Burns, P. D.
Recipient Organization
UNIVERSITY OF NORTHERN COLORADO
501 20TH STREET
GREELEY,CO 80639
Performing Department
(N/A)
Non Technical Summary
Prostaglandin (PG) F2alpha is a hormone secreted by the uterus and corpus luteum late in the heat cycle to cause regression of the corpus luteum (CL) in the non-pregnant cow and initiate a new heat cycle. The embryo must block prostaglandin (PG) F2alpha secretion from the uterus and CL for successful pregnancy. It is highly possible that the embryo fails to control luteal PGF2alpha secretion after mating, which can lead to regression of the CL and loss of the pregnancy. Omega-3 fatty acids have been shown to disrupt PG synthesis in a number of tissues and it is hypothesized that luteal tissue obtained from cows receiving diets supplemented with fish meal (i.e. luteal cells enriched with omega-3 fatty acids) will have reduced PGF2alpha secretion leading to improved pregnancy rates. The objectives are 1) to examine the effects of omega-3 fatty acids on PGF2alpha-induced cycooxygenase-2 gene expression and 2) to investigate the effects of these fatty acids on key intracellular signaling proteins that may lead to PGF2alpha secretion. It is anticipated that luteal tissue obtained from cows fed a ration supplemented with fish meal will have reduced PGF2alpha secretion. Results for these experiments will provide new knowledge in the cellular and molecular regulation of luteal PGF2aplha synthesis and the influence of omega-3 fatty acids on PGF2alpha synthesis in this tissue. Ultimately, the outcomes from these studies may allow for the development of new feeding strategies with omega-3 fatty acids to improve reproductive performance in dairy and beef cows.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30133101030100%
Goals / Objectives
Our overall hypothesis states that supplementation of beef cow rations with omega-3 fatty acids, eicosapentaenoate (EPA) and docosahexaenoate (DHA), will alter prostaglandin (PG) F2alpha metabolism in luteal cells. To test this hypothesis, non-lactating beef cows will be fed fish meal, a rich source of EPA and DHA, for approximately 60 days. Luteal cell preparations will be made from day 10-12 corpora lutea and subjected to in vitro studies. We will pursue two objectives in the proposed project. Objective 1 will be to examine the effects of omega-3 fatty acids on PGF2alpha-induced cyclooxygenase-2 gene expression and PGF2alpha synthesis in vitro. Objective 2 will be to investigate the effects of omega-3 fatty acids on mitogen-activated protein kinase signaling cascade, which leads to an increase in PGF2alpha secretion.
Project Methods
Non-lactating beef cows will be housed in individual pens and fed a total mixed ration consisting of corn silage. Rations will be supplemented with either fish meal (5% of DMI; a rich source of omega-3 fatty acids) or corn gluten meal (8.1% of DMI; controls). Cows will be fed for 60 d to enrich luteal tissue with omega-3 fatty acids. Cows will be administered two 25 mg injections of PGF2alpha at 14-d intervals on d 36 and 50 of the supplemental period to synchronize estrous cycles. Ovaries will be surgically removed on d 10-12 post-estrus after the second PGF2alpha injection (approximately d 60 of the supplemental period). The corpus luteum will be dissected from ovarian stroma and enzymatically digested using collagenase. Mixed populations of luteal cells will be washed and subjected to in vitro incubation. In experiment 1, cells ( approximately 500,000 per treatment) will be cultured in serum free medium and immediately exposed to 0, 1, 10, 100, or 1000 nM cloprostenol (a PGF2alpha analog) for 0, 12, or 24 h (n = 3 per treatment per time point). The spent medium will be collected and the concentration of PGF2alpha determined using an enzyme immunoassay. After incubations, cells will be washed 3x with phosphate buffered saline, total RNA extracted, and subjected to real time PCR to determine abundance of cyclooxygenase-2 and beta-actin mRNA. In experiment 2, cells (approximately 500,000 per treatment) will be cultured in serum free medium and subsequently exposed to 100 nM PGF2alpha analog for 0, 5, 15, 30, 60 or 90 min (n = 3 per time point). A second set of cells will be exposed to 0, 0.1, 1, 10, 100 or 1000 nM PGF2alpha analog for 15 min (n = 3 per dose PGF2alpha). At the end of the incubation, cells will be collected and washed 3x with phosphate buffered saline. Cell lysates will be prepared and subjected to western blotting. Proteins will be separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. Membranes will be incubated with anti-extracellular regulatory kinase (ERK) 1/2, anti-c-Jun kinase (JNK), anti-p38 kinase, anti-phospho-ERK1/2, anti-phospho-JNK, or anti-phospho-p38 MAP kinase antisera.

Progress 07/01/08 to 06/30/11

Outputs
OUTPUTS: Data collection and analyses for all proposed experiments have been completed. Inclusion of fish meal, a source of omega-3 fatty acids, to the diet increases blood and luteal omega-3 fatty acid content, mitigates prostaglandin (PG) F2 alpha-induced mitogen activated protein kinase signaling, and attenuates PGF2 alpha-induced cyclooxygenase-2 gene expression. We further examined omega-3 fatty acid composition in polar and neutral lipid fraction of luteal tissue collected from fish meal and corn gluten meal (control) supplemented cows. There was no effect (P > 0.05) of lipid fraction on luteal eicosapentaenoic acid, but luteal content of eicosapentaenoic acid was greater (P < 0.05) in both the neutral and polar lipid fractions of tissue collected from fish meal supplemented cows. Regardless of dietary supplementation, there was an effect (P < 0.05) of lipid fraction on docosahexaenoic acid. The polar lipid fraction contained greater docosahexaenoic acid as compared to the neutral lipid fraction. Furthermore, there was greater (P < 0.05) docosahexaenoic acid in both lipid fractions in tissue collected from fish meal cows as compared to tissue obtained from corn gluten meal supplemented cows. Based on results from the current studies, our present working hypothesis l is that omega-3 fatty acids become incorporated in lipid microdomains and prevent the FP receptor from docking with G-proteins and activation of downstream signaling. We have begun investigating the effects of omega-3 fatty acids on lipid microdomains and lateral mobility of the prostaglandin FP receptor in bovine luteal tissues. We have successfully isolated lipid microdomains using sucrose gradients in luteal cells at this time. We are currently using single particle tracking methodology to monitor lateral mobility of the FP receptor within the plasma membrane of luteal cells. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We currently have one manuscript submitted to the Journal of Animal Science. A second manuscript is in preparation and will be submitted by December 2011.

Publications

  • No publications reported this period


Progress 07/01/09 to 06/30/10

Outputs
OUTPUTS: Data analyses have been completed. Results from these experiments were presented at the Society for Study of Reproduction 43rd annual meeting in Milwaukee WI. We will be submitting manuscripts for publication in January 2010. PARTICIPANTS: Mrs. Nicole White was a graduate student in the School of Biological Sciences and completed her M.S. program. Dr. Patrick Burns (PD) assisted Mrs. White with data analysis and writing of abstracts and manuscripts. TARGET AUDIENCES: Outcomes from these experiments were presented at the 2010 Society for Study of Reproduction and will be published in peer-reviewed journals. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Data analyses for the proposed experiments have been completed. Plasma eicosapentaenoic and docosahexaenoic acid increased within 7 to 14 days of supplementation and remained elevated during the experimental period for those cows receiving fish meal in the diet (P < 0.05). This resulted in greater than 260% increase in eicosapentaenoic and docosahexaenoic acid within luteal tissue collected from fish meal supplemented cows. In experiment 1, PGF2alpha induced phosphorylation of p46 JNK (P < 0.05) and p38 MAP kinase (P < 0.07) in a quadratic manner. Abundance of phosphorylated p46 JNK and p38 MAP kinase in luteal cells increased (P < 0.05) when incubated in the presence of 0.1 to 10 nM PGF2alpha and then decreased (P < 0.05) at higher doses. This response was attenuated in cells obtained from cows supplemented with fish meal (P < 0.05). Prostaglandin F2alpha had no effect on phosphorylation of ERK 1 or 2 (P > 0.10). In experiment 2, there was no effect of dose of PGF2alpha or dietary supplementation on Cox-2 gene expression at 0 h (P > 0.10). Prostaglandin F2alpha stimulated Cox-2 gene expression in a quadratic dose manner at 6 h and 12 h of incubation (P < 0.01), but not a 24 h (P > 0.10). Cycclooxygenase-2 mRNA increased in presence of 0.1 to 10 nM PGF2alpha and declined at higher doses. This response to PGF2alpha was attenuated at 6 h (P < 0.01) in tissue obtained from fish meal supplement cows, but not at 12 h (P > 0.10).

Publications

  • White, NR, PD Burns, J Charumilinda, AD Bryant, ZT Prosser, JE Bruemmer, and TE Engle. Effect of omega-3 fatty acids on prostaglandin F2-induced cyclooxygenase-2 (Cox-2) gene expression in bovine luteal cells in vitro. 2010. 43rd Annual Meeting of the Society for the Study of Reproduction:46.
  • Burns PD, NR White, RD Cheatham, R Romero, JE Bruemmer, and TE Engle. Effect of fish meal supplementation on bovine plasma and luteal omega-3 fatty acid content. 2010. 43rd Annual Meeting of the Society for the Study of Reproduction:46.
  • Cheatham, RD, NR White, PD Burns, ES Chestnut, JS Hickman, JKK Michishima, D Suh, JE Bruemmer, and TE Engle. Effect of omega-3 fatty acids on prostaglandin (PG) F2-induced mitogen-activated protein kinase signaling in bovine luteal cells in vitro. 2010. 43rd Annual Meeting of the Society for the Study of Reproduction:46-47.


Progress 07/01/08 to 06/30/09

Outputs
OUTPUTS: OUTPUTS: Experiments 1 and 2 have been completed during the funding period. We are currently analyzing data in the laboratory and anticipate completion in January 2010. Results will be disseminated at the 2010 Society for Study of Reproduction or National Animal Science meeting. We anticipate manuscripts will be submitted for publication in June or July of 2010. PARTICIPANTS: PARTICIPANTS: Mrs. Nicole White is a graduate student in the School of Biological Sciences. She has devoted 100% of her effort to the project and data from these experiments will be part of her M.S. Thesis. Dr. Patrick Burns (PD) assisted Mrs. White with collection of the data. Dr. Jason Bruemmer, Dr. Thomas Hansen and Ms. Zella Brink at Colorado State University assisted in performing animal surgeries. Drs. Terry Engle and Shawn Archibeque also at Colorado State University provided insight on ration formulation and performed wet chemistry analysis on feedstuffs. Zack Johnson at the Colorado State University Agricultural Research, Development, and Education Center was responsible for care of research animals. TARGET AUDIENCE: Outcomes from these experiments will be presented at the 2010 Society for Study of Reproduction or National Animal Science meetings and published in peer-reviewed journals. TARGET AUDIENCES: TARGET AUDIENCE: Outcomes from these experiments will be presented at the 2010 Society for Study of Reproduction or National Animal Science meetings and published in peer-reviewed journals. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
OUTCOMES: We have completed all experiments proposed during year 1 of the funding period. Body weights increased during the feeding period and did not differ between cows supplemented with corn gluten meal and those with fish meal in either experiment 1 or experiment 2 (P>0.05). In experiment 1, plasma eicosapentaenoic and docosahexaenoic acids increased within 14 days of supplementation and remained elevated during the feeding period for those cows receiving fish meal and were greater when compared to cows supplemented with corn gluten meal (P<0.05). Luteal eicosapentaenoic and docosahexaenoic acids were greater, while arachidonic acid was less (P<0.01) for those cows supplemented with fish meal when compared to luteal tissue obtained from cows supplemented with corn gluten meal. We are in the process of (1) analyzing blood and luteal tissues collected in experiment 2 for fatty acid content and (2) investigating the effects of PGF2alpha on the induction of cyclooxygenase-2 gene expression using real-time PCR. Very preliminary data (one animal per supplementation group) show an increase in COX-2 gene expression in luteal cells obtained from corn gluten meal supplemented cows treated for 24 hr with 1, 10, 100 and 1000 nM PGF2alpha, but was attenuated in luteal cells obtained from fish meal supplemented cows. We have begun looking at MAP kinase phosphorylation using Western blot analysis on luteal cells treated for 15 min with 0, 0.1, 1, 10, 100, or 1000 nM PGF2alpha analog and luteal cells treated with or without 100 nM PGF2alpha analog for 0, 5, 15, 30, 60, or 90 min. IMPACT: The supplementation of fish meal may be a practical way to alter omega-3 fatty acids in reproductive tissues of breeding females. These omega-3 fatty acids (eicosapentaenoate and docosahexaenoate) may attenuate PGF2alpha secretion in pregnant females and prevent regression of the corpus luteum assuring maternal recognition of pregnancy.

Publications

  • No publications reported this period