Progress 09/01/09 to 08/31/13
Outputs
Target Audience: Efforts on this project are directed towards reducing the economic costs of infertility in domestic ruminants. The approach taken is to study the biology of early pregnancy; a period of high embryo loss that is a primary contributor to infertility. Because the focus of this work is to describe communication between the early embryo and the uterus, the primary target audience for this work is scientists and educators working the discipline of reproductive biology, including those working in Extension. Evidence that these audiences were reached are reflected by peer reviewed scientific publications, theses, and scientific presentations given to audiences including farmers, Extension Educators, industry personnel, veterinarians, ag teachers and scientists. Changes/Problems: Because of the lack of good in vitro models for the ruminant uterus, we added a project to construct an immortalized cell line derived from the bovine endometrial epithelium. This cell line, once fully characterized, will be made available to anyone who requests it. Our work with transient knockdown of MX1 did not justify completing the in vivo experiment, therefore, we refocused efforts on defining the specific function of MX1 using our in vitro models. What opportunities for training and professional development has the project provided? Two graduate students were trained primarily based on their work on this project. In addition, two other graduate students assisted with the conduct of some of the experiments. Five undergraduate students received research training supported by this grant. How have the results been disseminated to communities of interest? Results have been disseminated via peer review scientific publications, theses, abstracts, seminars, symposia and workshops. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Results from this project revealed that conceptus signals induce expression of the myxovirus 1 (MX1) resistance protein as one of the earliest and strongest maternal responses to the presence of an embryo in the uterus. Experiments under this grant showed that 1) MX1 was produced and secreted into the uterine lumen during early pregnancy; 2) MX1 protein interacts with tubuline beta, a structural element in secretory cells that are thought to create cellular "highways" that transport secretory proteins and vesicles; 3) MX1 protein is secreted as a component of exosomes; 4) MX1 protein is localized inside of secreted exosomes; 5.) MX1 is a potential regulator of secretion of exosomes by acting as a mechanochemical enzyme. This research also lead to the understanding that MX2 is also induced during this period of early pregnancy and that MX2 is localized to the nuclear membrane and may regulate nuclear-cytoplasmic trafficking during early pregnancy.
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2013
Citation:
Development and Characterization of a Bovine Uterine Epithelial Cell Line and The Effects of Pregnancy on Gene Expression in Peripheral Blood Leukocytes.
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Progress 09/01/11 to 08/31/12
Outputs
OUTPUTS: To better model bovine uterine responses to signals from the early embryo, we switched cell culture approaches away from existing ovine uterine cell lines. Because no appropriate bovine cell lines currently exist, experiments were conducted to create immortalized cell lines from bovine endometrial epithelium and stroma. Primary uterine epithelial and stromal cells were purified and transfected with viral E6/E7 genes and with the human telomerase gene to generate immortalized cell lines. These cells were then passaged in selective medium to purify transfectants. Cells surviving immortalization were then screened for epithelial and stromal phenotype and response to embryonic signals. Knowledge of conceptus endometrial interactions and fertility regulation was conveyed by giving scientific presentations at Ag Days (Allegheny College), Penn State Nutrition workshop, Cow Camp and Pre-Vet club. A program to increase knowledge of our food production systems was developed and delivered to Honors undergraduate students. In addition, I served as a consultant for a small animal diagnostic company to aid them in developing technologies to improve animal reproduction and health. PARTICIPANTS: Alyssa Birt, Manasi Kamat, Sreelakshmi Vadasudevan: graduate students participated in the project; Luke Harding and Daniel Hartzel: undergraduate students participated in the project. All were trained in various aspects of cell culture, molecular biology, animal handling and blood sampling. Collaborators included Mike Day at Ohio State University, Dave Townson at the University of New Hampshire, Tom Spencer at Washington State University and Joy Pate at Penn State. TARGET AUDIENCES: Target audiences include scientists in the field of reproductive biology and dairy science, dairy producers and students pursuing undergraduate and graduate training in animal science, reproductive physiology and dairy science. These audiences were reached via efforts in teaching, mentoring of graduate students and publishing research in peer reviewed scientific journals. In addition, a new program was developed and targeted Penn State Honors undergraduate students, particularly those with little knowledge of food production systems. This program helped increase their knowledge of food production systems and the challenge of feeding a growing world population. PROJECT MODIFICATIONS: Switched away from immortalized ovine uterine cell lines because of inconsistent results. Decided to create immortalized bovine uterine cell lines. Once completed and validated, these cell lines will be made available to other researchers who need good in vitro models to study the biology of early pregnancy.
Impacts
Activities related to conveying information about early pregnancy and fertility regulation caused farmers to change management practices to improve fertility and profitability. Activities related to conveying information about our food production systems to Honors undergraduate students help them to better understand how food is produced and the challenge of feeding a growing world population. It also help them better define "sustainable" agriculture. Participants reported that the program helped them make more informed choices about the food they purchase and consume.
Publications
- Bridges, G. A., M. L. Mussard, J. L. Pate, T. L. Ott, T. R. Hansen, and M. L. Day. 2012. Impact of preovulatory estradiol concentrations on conceptus development and uterine gene expression. Animal Reproduction Science 133:16-26.
- Racicot, K., A. Schmitt, and T. L. Ott. 2012. The myxovirus-resistance protein, MX1, is a component of exosomes secreted by uterine epithelial cells. American Journal of Reproductive Immunology. 67(6):498-505.
- Racicot, K. E. and T. L. Ott. 2011. The myxovirus resistance protein, MX1, interacts with tubulin beta in uterine glandular epithelial cells. American Journal of Reproductive Immunology. 65:44-53.
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: Experiments were conducted to examine effects of transient knockdown and overexpression of MX1 on secretory profiles of uterine immune cells. An Enzyme-linked immunosorbent assay for MX1 was developed and validated for use in cell culture medium a cellular lysates. Results from this project were dissemination through invited lectures presented at the University of Florida, Department of Animal Science and through 3 lectures presented as part of a dairy production medicine workshop. Experiments were initiated to create immortalized cell lines from bovine endometrial epithelium and stroma. Outputs include licensing two patents to and service as a consultant for an animal biotechnology company. PARTICIPANTS: Participants: Shannon R. Boone, Alyssa Birt M.S. Students (100% effort). Manasi Kamat, doctoral student (10% effort). Mr. Brian Dawson, B.S. Part time laboratory research support (10 hrs/wk), Ms. Bethany Crouse, part time laboratory research support. Collaborators: Dr. Joy Pate, Penn State University- consults on cell culture and data interpretation. Dr. Thomas Spencer, Washington State University- consults on gene knockdown experiments. Dr. Anthony Schmitt, Penn State University-consults on cell biological techniques. Ms. Alyssa Klein completed honors research project on isolation and quantification of exosomes. Ms. Emily Galli received training in cell culture and preparation of immortalized cell lines. TARGET AUDIENCES: Scientists in the field of reproductive biology. Producers who will benefit from development of diagnostic tools for the early identification of failed inseminations and viral infections. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Experiments are ongoing no outcomes to report.
Publications
- Ott, T. L. and C. A. Gifford. 2010. Effects of early conceptus signals in circulating immune cells: Lessons from domestic ruminants. American Journal of Reproductive Immunology 64:245-254.
- Boone, S. R. 2011. Effect of the Myxovirus Resistance Protein, MX1, on Secretion by a Uterine Cell Line. M.S. Thesis. The Pennsylvania State University, University Park, PA. 170 pp.
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: Outputs include conducting and analyzing the results from experiments designed to determine the effects of over expression of MX1 protein on secretion of proteins by ovine glandular epithelial cells. Results from this project have been disseminated by presentation at national and regional scientific meetings and via publication in peer reviewed manuscripts and book chapters. Presentation of research occurred at Cornell University Department of Animal Science, at the USDA PI meeting in Milwaukee, WI, and via three seminars presented at the Three Rivers Veterinary Symposium in Pittsburgh, PA. Outputs included service as a consultant for two animal biotechnology companies. PARTICIPANTS: Participants: Shannon Rae Boone, M.S. Student, responsible for conducting research on project (100% effort). Brian Dawson, B.S. Student, responsible for support for research project (10 hrs/week). Collaborators: Dr. Anthony Schmitt, Ph.D., Faculty in Veterinary and Biomedical Sciences, Penn State University. Consults on cell biological techniques conducted on project. Training: This project has supported training for one M.S. student (Shannon Boone) and three undergraduate students (Brian Dawson, Brianna Isenberg and Alyssa Klein) who have recieved training in laboratory techniques and conducted activities in support of the research. TARGET AUDIENCES: Target audiences: Scientists in the field of reproductive biology. Producers who will benefit from development of diagnostic tools for the early identification of failed inseminations. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Research has lead to a new understanding of the role of MX1 in the uterus during early pregnancy that have been disseminated via peer reviewed publications, one of which (Toyokawa et al., 2007) was recently cited in a review published in Nature (2009). Results have lead to improved approaches and understanding regarding the utility of using MX1 (and other interferon stimulated proteins) as markers for early pregnancy signaling between the embryo and mother in domestic ruminants. Results have supported efforts to develop and commercialize a diagnostic test for failed inseminations in farm animals that would significantly enhance reproductive management and reduce the days between calving and conception compared to current approaches. Results have supported efforts to patent technology for the determination of pregnancy status in cattle and sheep (U.S. Patent 7,125,728 and patents in 7 other countries)in the European Union.
Publications
- Racicot, K.E. and T.L. Ott. 2010. The myxovirus resistance protein, MX1, interacts with tubulin beta in uterine glandular epithelial cells. American Journal of Reproductive Immunology. http://dx.doi.org/10.1111/j.1600-0897.2010.00885.x.
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