Source: TEXAS A&M UNIVERSITY submitted to NRP
MECHANISTIC ANALYSIS OF OVINE ENDOMETRIAL ANGIOGENESIS DURING PREGNANCY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
0213301
Grant No.
2008-35203-18830
Cumulative Award Amt.
(N/A)
Proposal No.
2007-01216
Multistate No.
(N/A)
Project Start Date
Mar 1, 2008
Project End Date
Feb 28, 2011
Grant Year
2008
Program Code
[41.0]- Animal Reproduction
Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
VETERINARY INTEGRATIVE BIOSCIENCES
Non Technical Summary
Non-Technical Summary: Embryonic loss in domestic ruminants (e.g. cows and sheep) represents a major constraint to agricultural profitability. The central hypothesis is that pregnancy results in increases of factors essential for development of uterine blood vessel networks capable of sustaining the conceptus. This application integrate two approaches: 1) an in vitro model of new blood vessel development and 2) an in vivo model of pregnancy in which the uterus has been surgically segmented to create two different environments, allowing for comparison of the conceptus containing side (site of robust blood vessel development) and the side that does not have a conceptus (site of only moderate blood vessel development). Use of these model systems will allow for the identification of genes that mediate the growth of new blood vessels both in culture and within the pregnant sheep uterus, as well as the establishment of a correlation between genes associated with blood vessel development in culture and within the uterus during pregnancy. Completion of these aims will significantly advance our understanding of pregnancy through identification of factors that functionally regulate new blood vessel development which is the basis for providing nourishment to the developing conceptus. Knowledge gained from these studies will be useful to design management, biotechnology and genetic applications aimed at improving embryonic survival and enhancing production efficiency in animal agriculture.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30136101020100%
Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3610 - Sheep, live animal;

Field Of Science
1020 - Physiology;
Goals / Objectives
The objective of this application is to identify potential factors that stimulate new blood vessel growth (angiogenesis), localize these factors within pregnant sheep uterine tissues, and establish their involvement in regulating vascular tube assembly and vessel invasion in vitro. The central hypothesis is that pregnancy-specific increases in angiogenic factor production by vascular endothelium, smooth muscle, and/or stromal fibroblasts are essential for the development of vessel networks capable of sustaining the developing conceptus.
Project Methods
This research will integrate two approaches: 1) an in vivo model of pregnancy in which the uterus has been surgically segmented to create two distinct environments with differing levels of blood vessel development within the same ewe; and 2) a three-dimensional in vitro model of endothelial cell outgrowth and tube formation in collagen matrices.

Progress 03/01/08 to 02/28/11

Outputs
OUTPUTS: The objectives of the proposal "Mechanistic Analysis of Ovine Endometrial Angiogenesis During Pregnancy" were to identify and localize factors that stimulate new blood vessel growth (angiogenesis) within the pregnant sheep uterus and to test these factors in a laboratory culture system. The outputs completed during this project are discussed by type. Activities include identification of candidate genes that are either up- or down-regulated in areas of new blood vessel growth within the pregnant uterus and assessment of changes in candidate gene expression levels in a 3-dimensional laboratory culture model of angiogenesis. Changes in gene expression were measured using RT-PCR while localization of genes was determined via in situ hybridization. Changes in protein levels were measured using Western Blots and localization was determined via immunohistochemical and immunoflourescence analyses. These experiments and evaluations were conducted on tissues recovered from pregnant sheep as well as cultured cells. The sheep were a surgically manipulated animal model in which the pregnancy was physically restricted to only one side of the uterus (e.g. the gravid horn). The cells tested were commercially available as well as a primary culture provided by a colleague. Events attended that contributed to the objectives of this project include the Texas Forum for Reproductive Sciences, the annual meeting of the Society for the Study of Reproduction, Gordon Research Conference on Reproductive Tract Biology, and the Texas A&M University Interdisciplinary Reproductive Biology Forum. Services completed include tutoring of 24 undergraduate and 3 graduate students on the topic of placental histology using images generated in these experiments. Valuable products of this project are ongoing collaborations with Dr. Ron Magness at the University of Wisconsin and Dr. Guoyao Wu at Texas A&M University. Both of these collaborations have resulted in additional grant submissions and publications, all of which were fostered by completion of these studies. Dissemination of the results of this project were primarily conducted via platform or poster presentation at scientific meetings and thus provided opportunities to meet with professional colleagues, and potential collaborators. Further outreach activities included generation of manuscripts which allowed for review and comment by peers and potential collaborators prior to publication. Manuscripts were published by journals with electronic viewing platforms, however additional copies have been provided upon individual request. 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
Completion of "Mechanistic Analysis of Ovine Endometrial Angiogenesis During Pregnancy" generated changes in knowledge and action of the PI. Specifically, these studies demonstrated a previously unknown role for members of the sphingosine-1-phosphate signaling pathway during sheep pregnancy. These studies also presented the first localization of mRNAs for sphingosine-1-phosphate receptor 1, ADAMTS1, and DLL4 within the sheep placentome. Another novel finding was localization of immunoreactive sphingosine kinase 1 within the sheep uterus and placenta. The PI also increased skill in evaluation of placental histoarchitecture and use of 3-dimensional in vitro cell culture models of angiogenesis. These findings provided the basis for future research in this area and promoted multiple changes in action which include use of ovine uterine artery endothelial cells (provided by Ron Magness, University of Wisconsin) and carnoy's perfusion for fixation of placental tissues (instruction from Pavel Borowicz, North Dakota State University). Finally, data generated from completion of this project created a working model for the role of sphingosine-1-phosphate in regulating uterine angiogenesis as well as providing the preliminary data for National Research Initiative Competitive Grant no. 2009-35203-05725 from the USDA National Institute of Food and Agriculture, which was awarded to project mentors Kayla Bayless and Greg Johnson.

Publications

  • Dunlap KA, Erikson DW, Burghardt RC, White FJ, Reed KM, Farmer JL, Spencer TE, Magness RR, Bazer FW, Bayless KJ, Johnson GA. (20080 Progesterone and placentation increase secreted phosphoprotein one (SPP1 or osteopontin) in uterine glands and stroma for histotrophic and hematotrophic support of ovine pregnancy. Biol Reprod. 79(5):983-90. Epub 2008 Jul 30.
  • White BG, Dunlap KA, Frank JW, Satterfield MC, Burghardt RC, Bazer FW, Johnson GA, Bayless KJ. (2011). Antagonism of the Sphingosine-1-Phospate Signaling Pathway Affects Placental and Fetal Development. International Federation of Placenta Associations. Geilo, Norway. (Submitted).
  • Dunlap KA, LI X, Satterfield MC, Burghardt RC, Erikson DW, Halverson ML, Wu G, Bazer FW, Spencer TE, Magness RR, Bayless KJ, Johnson GA. (2009) Antagonism of the Sphingosine 1-phosphate (S1P) signaling pathway in pregnant ewes alters placentome architecture, amino acid transport and fetal growth. Society for the Study of Reproduction, Pittsburgh, PA. ( #427).
  • Dunlap KA, LI X, Satterfield MC, Burghardt RC, Erikson DW, Halverson ML, Wu G, Bazer FW, Spencer TE, Magness RR, Bayless KJ, Johnson GA. (2009) Antagonism of the Sphingosine 1-phosphate (S1P) signaling pathway in pregnant ewes alters placentome architecture, amino acid transport and fetal growth. Texas Forum on Reproductive Sciences, Houston, TX. (#14).
  • Dunlap KA, Kwak HI, Su SC, Mendoza EA, Burghardt RC, Bazer FW, Magness RR, Johnson GA, Bayless KJ. (2008). Involvement of the Sphingosine-1-Phosphate Cell Signaling Pathway in Regulating Ovine Endometrial Angiogenesis During Pregnancy. Society for the Study of Reproduction, Kona, HI. (#307.)


Progress 03/01/09 to 02/28/10

Outputs
OUTPUTS: Activities completed include evaluation of the Sphingosine 1-Phosphate (S1P) signaling pathway in the gravid and nongravid uterus of unilaterally pregnant sheep via RT-PCR, immunohistochemical, and in situ hybridization analysis. Additionally, in vitro angiogensis models utilizing either Human Umbillical Vein Endothelial Cells, or Ovine Uterine Artery Cells seeded on three dimensional collagen matrices were used to assess the impact of S1P receptor antagonism on endothelial cell invasion. Events attended include the Texas Forum for Reproductive Sciences, the annual meeting of the Society for the Study of Reproduction, and the Texas A&M University Interdisciplinary Reproductive Biology Forum. Dissemination of the results of this project were conducted via platform or poster presentation at these respective events, thus providing opportunities to meet with professional colleagues, and potential collaborators. 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
This project provided for a change in knowledge as the role of the Sphingosine 1-phospate (S1P) signaling pathway in pregnancy associated angiogenesis was further ellucidated. The enzyme responsible for S1P production, sphingosine kinase, was elevated in the gravid vs nongravid uterine horns of unilaterally pregnant ewes. Likewise, the S1P receptor (S1P1) was also upregulated in the gravid tissues, particularly at sites of robust interdigitation and angiogenesis (i.e. placentomes). In vitro experiments conducted show that S1P1 is critical for endothelial cell invasion as antogonism of the receptor either via siRNA, or the pharmacological compound FTY720 result in decreased endothelial cell invasion. There was a detectable increase in expression of the S1P regulated genes delta-like 4 (DLL4) and A disintegrin and metalloproteinase with thromobospondin-like repeats (ADAMTS1) at the sites of maternal-fetal communication and angiogensis within the pregnant sheep. These findings suggest that S1P signaling may be crucial for proper fetal-placental development.

Publications

  • Dunlap KA, Kwak HI, Burghardt RC, Bazer FW, Magness RR, Johnson GA, Bayless KJ.The sphingosine 1-phosphate (S1P) signaling pathway is regulated during pregnancy in sheep. Biol Reprod. 2010 May;82(5):876-87. Epub 2010 Jan 27.


Progress 03/01/08 to 02/28/09

Outputs
OUTPUTS: Activities completed include evaluation of candidate genes that mediate angiogenic sprouting and invasion of Human Umbilical Vein Endothelial Cells into three dimensional collagen matrices via RT-PCR, Western Blot and immunoflourescence analysis. Genes of interest have been investigated in the in vivo models of the pregnant ewe as well as the gravid and nongravid horns of the unilaterally pregnant ewe via the use of Western Blot, in situ hybridization and immunoflourescence. In vitro experiments to antagonize candidate genes have been conducted and analyzed via histological staining, RT-PCR, and Western Blot analyses. Events attended include the Texas Forum for Reproductive Sciences, the annual meeting for the Society for the Study of Reproduction, the Gordon Research Conference on Reproductive Tract Biology, as well as presentation to the Texas A&M University Interdisciplinary Reproductive Biology Forum. Dissemination of the results of this project were conducted via oral or poster presentation at the previously mentioned events as these were opportuntities to meet with professional colleagues and potential partners and collaborators. 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
This project provided for a change in knowledge as the function of secreted phosphoprotein 1 (SPP1 or Osteopontin)in regards to pregnancy associated uterine angiogesis was further ellucidated. Endometrial levels of SPP1 mRNA increase as pregnancy progresses. SPP1 is localized to the epithelial surface of the uterine lumen, as well as being expressed by the stroma of the gravid endomtrium. In vitro experiments demonstrated that SPP1 promotes ovine trophectoderm attachment and is produced by ovine uterine artery endothelial cells as they invade into three dimensional collagen matrices. In addition to SPP1, these experiements have demonstrated that members of the signalling pathway for the proangiogenic lysosphingolipid Sphingosine one phosphate (S1P) are regulated in response to pregnancy. Specifically the receptors S1P1 and S1P3 increase as pregnancy progresses. The enzyme Sphingosine Kinase 1 also increases at areas of fetal and maternal contact within the placentomes. Conversely, both the phosphatase (Sphingosine one phosphate phosphatase) and lyase (Sphingosine Lyase) that are responsible for the inactivation of S1P are absent from the pregant endometrium. Suggesting that S1P is beneficial to angiogensis during pregnancy. Changes in expression of the genes DLL4 and ADAMTS1 further supports the role of S1P in ovine pregnancy. These findings fostered further investigation into the role of the S1P receptor and have demonstrated that in vivo administration of the drug FTY720 (S1P receptor antagonist) alters placentome formation and amino acid composition of placental fluids.

Publications

  • Dunlap KA, Erikson DW, Burghardt RC, White FJ, Reed KM, Farmer JL, Spencer TE, Magness RR, Bazer FW, Bayless KJ, and Johnson GA. (2008)Progesterone and Placentation Increase Secreted Phosphoprotein One (SPP1 or Osteopontin) in Uterine Glands and Stroma for Histotrophic and Hematotrophic Support of Ovine Pregnancy. Biology of Reproduction, 79, 983-990.