Source: PENNSYLVANIA STATE UNIVERSITY submitted to NRP
ROLE OF UTERINE IMMUNE CELLS IN ESTABLISHMENT OF PREGNANCY IN RUMINANTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1012332
Grant No.
2017-67015-26455
Cumulative Award Amt.
$460,000.00
Proposal No.
2016-10015
Multistate No.
(N/A)
Project Start Date
Apr 1, 2017
Project End Date
Mar 31, 2023
Grant Year
2017
Program Code
[A1211]- Animal Health and Production and Animal Products: Animal Reproduction
Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
408 Old Main
UNIVERSITY PARK,PA 16802-1505
Performing Department
Animal Science
Non Technical Summary
This project addresses infertility in the dairy industry, which costs dairy producers more than $500M annually. Furthermore, these results will be extendable to the beef, sheep and goat industries as the physiology of early pregnnacy in these species is similar to dairy cattle. A majority of embryo loss occurs during the first 4 weeks of pregnancy and a portion of these losses are thought to be immune-mediated. There is relativley little known about how the the early embryo affects the abundance immune cells at the fetal-maternal interface in the uterus. We will address this gap in knowledge in these experiments by determining if the substantial difference in fertility between dairy heifers and mature lactating dairy cows is mediated by differences in immune cell response to conceptus signals. Furthermore, we will deterime the effects of two key signaling molecules released by the embryo on uterine immmune cell function. Both of these molecules have been known to science for over 30 years, but their effects on uterine immune cells is largely unknown. Understanding the effects of these proteins on immune function may lead to strategies to reduce early embryo loss and thereby improve reproductive efficiency and profitability of the dairy industry. By improving efficiency, results of these studies contibute to reducing the environmental impact of the dairy industry.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3013410109060%
3013410102040%
Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3410 - Dairy cattle, live animal;

Field Of Science
1090 - Immunology; 1020 - Physiology;
Goals / Objectives
The goal of the project is to understand and reduce infertility which costs the US dairy industry in excess of $500M annually. Key to this goal is understanding why heifers are more fertile than mature lactating dairy cows even though they have the same genetic value for fertility. To address this we will examine changes in immune cell types and function in the uterus during early pregnancy in dairy heifers and mature, lactating dairy cows. These comparisons will be made during the first 3 weeks of pregnancy; a time when the majority of pregnacies fail. We will test the hypothesis that pregnancies fail due to the inability of embryos from mature cows to reprogram uterine immune cells to promote a tolerant phenotype and to participate in uterine remodeling associated with formation of a functioning placenta. Furthermore, we will address the role of two key molecules produced by the early embryo, interferon tau (IFNT) and pregnancy associated glycoprotein (PAG), on these changes using a novel intrauteine infusion model. We will test the hypothesis that the early embryo reprograms immune cell function via release of these two hormones. Finally, we will address the question of how IFNT and PAG interact to regulate immune function at the fetal-maternal interface during early pregnancy. Both of these hormones were discovered as key signalling molecules from the conceptus to the uterus more than 30 years ago, yet we still do not know how they affect immune cell function to facilitate pregnancy establishment. In particular, we will address the hypothesis that PAG play a key role in modulating immune cell function during pregnancy. The results of this work will be used to develop strategies to reduce infertility in the dairy industry.
Project Methods
The general approach will utilize in vivo animal models to ensure results are extendable to the general population of commercial dairy cows. This is espeicially critical for studying immune function because immune cells change rapidly in response any perterbation in their environment. Tissues will be collected from cyclic and pregnant cows and heifers to allow direct comparisons of difference in immune cell types and functions during early pregnancy; a time when the majority of pregnany loss occurs. Immune cells will be isolated using magnet activated cell sorting and then analyzed using flow cytometry. We will capture immune cells in situ using frozen section immunofluorescent microscopy and we will corroborate result by quantifying mRNA abundance key immune cell type and function genes using quantitative PCR. This three-pronged experimental approach will provide a greater degree of confidence for defining changes in immune cells during early pregnnacy. Furthermore, by using 4 time points of early pregnancy we will be able to define temporal changes in experimental readouts during a key window of signaling between the conceptus and the uterus. Using a unique in utero infusion model, we will be able to address the role of key conceptus proteins in uterine immune cell function. This will be facilitated by our access to native forms of these proteins. Finally, we will define the proteome of uterine immune cells exposed to key conceptus signaling molecules to define their role in reprogramming immune cell function during early pregnnacy. Depending on research outcomes, we will attempt to use this information to institute nutritional or management strategies to promote pregnancy establishment. This information will be conveyed by direct outreach to farmers and through organized Outreach activities including producer conferences and symposia. Beyond conducting the experiments and completing data analysis, the success of the project will be measured by our success in publishing the results in relevant, peer-reviewed scientific journals. Furthermore, novel results that could lead to technological approaches or treatments for improving fertility will be disclosed to our technology transfer office and patent applications associated with technological approaches or treatemtns.

Progress 04/01/17 to 03/31/23

Outputs
Target Audience:The target audience for this mechanistic work on reproductive immunology of early pregnancy in dairy cattle is primarily scientists and allied industry professionals who are consumers of the scientific literature. Students, agricultural workers and lay audiences benefit from the work as it is incorporated into general understanding of the physiology of early pregnancy and in presentations on factors that affect fertility in animal production. For example, peer reviewed manuscripts arising from this work have been assigned and discussed in our undergraduate and graduate curriculum and incorporated into general discussions about the physiology of early pregnancy to a variety of audiences. Changes/Problems:Progress was substantially dalayed by COVID and COVID-related restrictions in access to facilities and personnel hiring. We were also slowed becuase our main laboratory facility was relocated to temporary space for 3 yrs of the funding period requiring two complete lab relocations. Finally, we were delayed by lack of adequate progress by one key student assigned to the project. What opportunities for training and professional development has the project provided?Results were published as part of four M.S. Theses and one Ph.D. Dissertation. During the term of the award 6 undergraduate students assisted with various aspects of the work and thus were exposed to animal science research and were trained in laboratory procedures. How have the results been disseminated to communities of interest?Data were presented the 2018, 2019, 2021, 2022 and 2023 annual meetings of the Society for the Study of Reproduction.Results were published in Biology of Reproduction and are currently in revisions with this journal. Three additional papers are in final stages of submission for publication. Papers and intermediate results from these studies were presented and discussed in senior and graduate level courses to hundreds of students. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The goals of this project were to define the role of the immune system in regulating fertility and infertility in dairy cattle. We focused on early pregnancy becuase this is when the vast majority of pregnancies are lost. We conducted experiments to examine effects of conceptus signaling on uterine and peripheral blood immune cell populations and functions during the cycle and early pregnancy in heifers and cows. We determined what cell populations express interferon stimulated genes in response to the conceptus and completed experiments comparing the proportions of peripheral blood immune cells in dairy cows and heifers to define what mediates reduced fertility in cows. We examined expression of key immune cell types and expression of molecules related to their function in immune cells during the cycle and pregnancy and confirmed our hypothesis that early pregnancy results in baised immunological phenotype towards tolerance. We completed experiments examining the effects of intrauterine infusions of IFNT alone or in combination with pregnancy associated glycoproteins (PAG) on uterine and peripheral blood immune cell populations and functions. The results suggest that PAG have little effect on uterine gene expression, but regulate a large number of genes in peripheral blood immune cells. These results are provide novel information on the role of PAG, which were first described nearly 40 years ago but their function during pregnancy remains unclear. To determine what causes the difference in fertility between heifers and cows, we compared key indicators of immune function between cows and heifers on day 18 of the estrous cycle and early pregnancy. Our results confirmed our working hypothesis that cows exhibit elevated immune activation with greater proportions of neutrophils and cytokines associated with inflammation. To determine the effecs of specific concpetus proteins on formation of the plancenta, we examined the temporal and spatial expression of HIF1 alpha, AHR, and several other mediators of angiogenesis in the endometrium in response to conceptus secretory proteins, during the estrous cycle and during early pregnancy. Contrary to our working hypothesis, is appeared as if IFNT reduced expression of molecules associate with angiogensis in the endometirum. Overall, as a result of this work, we have a better idea of the changes in immune cells and function that occur during a period of high embryo loss in dairy cattle. The fact that cows exhibited reduced fertility compared to heifers also show evidence of elevated inflammation suggests that strategies to reduce inflammation during early pregnancy may result in increased fertililty. Data were presented the 2018, 2019, 2021, 2022 and 2023 annual meetings of the Society for the Study of Reproduction. Results were published as part of four M.S. Theses and one Ph.D. Dissertation. Results were published in Biology of Reproduction and are currently in revisions with this journal.

Publications

  • Type: Theses/Dissertations Status: Published Year Published: 2022 Citation: Effects of the estrous cycle and early pregnancy on proportions and functions of peripheral immune cells in dairy cows and heifers. 2022. Neha Oli. Thesis submitted in Partial Fulfillment of the requirements for the Master of Science degree in Integrative and Biomedical Physiology. The Pennsylvania State University.
  • Type: Theses/Dissertations Status: Published Year Published: 2022 Citation: Effects of interferon tau and pregnancy specific protein B on gene expression during early pregnancy. 2022. Ty Montgomery. Thesis submitted in Partial Fulfillment of the requirements for the Master of Science degree in Animal Science. The Pennsylvania State University.
  • Type: Theses/Dissertations Status: Published Year Published: 2021 Citation: EFFECTS OF AN EXTRUDED FLAXSEED SUPPLEMENT ON PLASMA HAPTOGLOBIN, NON-ESTERIFIED FATTY ACIDS, BETA-HYDROXYBUTYRATE, AND COMPLETE BLOOD COUNT IN TRANSITION DAIRY COWS AND HEIFERS. 2021 Devin Marie Cunningham. Thesis submitted in Partial Fulfillment of the requirements for the Master of Science degree in Animal Science. The Pennsylvania State University.
  • Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: Effects of an Extruded Flaxseed Supplement on Transition Cow Milk Production and Reproductive Function and Effects of Early Pregnancy on Mediators of Immune Tolerance in Dairy Heifers. in Partial Fulfillment of the requirements for the Master of Science degree in Animal Science. The Pennsylvania State University.
  • Type: Theses/Dissertations Status: Accepted Year Published: 2023 Citation: IMMUNOLOGICAL AND ENDOMETRIAL CHANGES OF EARLY PREGNANCY IN DAIRY CATTLE. 2023. Maria Isabel da Silva. A Dissertation in Partial Fulfillment of the requirements for the Doctor of Philosophy degree in Integrative and Biomedical Physiology. The Pennsylvania State University.
  • Type: Journal Articles Status: Submitted Year Published: 2023 Citation: da Silva, MI, Oli, N, Gambonini, FA, Pate, JL, Ott, TL. 2023. Effects of parity and early pregnancy on peripheral blood leukocytes in dairy cattle. Biology of Reproduction (in revision).


Progress 04/01/21 to 03/31/22

Outputs
Target Audience:The target audience for this mechanistic work on reproductive immunology of early pregnancy in dairy cattle is primarily scientists and allied industry professionals who are consumers of the scientific literature. Students, agricultural workers and lay audiences benefit from the work as it is incorporated into general understanding of the physiology of early pregnancy and in presentations on factors that affect fertility in animal production. For example, peer reviewed manuscripts arising from this work have been assigned and discussed in our undergraduate and graduate curriculum and incorporated into general discussions about the physiology of early pregnancy to a variety of audiences. Changes/Problems:None. What opportunities for training and professional development has the project provided?Three students completed (Neha Oli) or are currently conducting research (Ty Montgomery/Isabel da Silva) on this project. The latter two attended a scientific meeting where the data were presented. How have the results been disseminated to communities of interest?Results of experiments were presented at the 2021 annual meeting of the Society for Reproductive Investigation (Ott) and at the 2021 annual meeting of the SSR (da Silva). Results were incorporated into teaching at both the undergraduate and graduate levels. What do you plan to do during the next reporting period to accomplish the goals?Complete the transcriptomic analysis from the infusion experiment and publish one paper on the peripheral blood immune cell experiment and two papers on the infusion experiment. Differentially expressed genes identified in the infusion experiment will be validated and tested mechanistically. Experiments designed to characterize the effects of pregnancy on the AHR/ARNT/HIF system will be conducted.

Impacts
What was accomplished under these goals? We completed the uterine infusion experiments and sent endometrium and peripheral blood leukocytes for transcriptomic analysis. we conducted progesterone and PAG assays in blood. Preliminary reports of these analyses were presented at the 2021 Annual meeting of the Society for the Study of Reproduction. qPCR assasys were developed and validtaed for several of the differentially expressed genes, including genes apparently regulated by intrauterine infusion of pregnancy associated glycoproteins.

Publications


    Progress 04/01/20 to 03/31/21

    Outputs
    Target Audience:The target audience reached this reporting period includes scientists and allied industry professionals that are consumers of the scientific literature. Furthermore, presentations to lay audiences were in the general area of this research focus. Changes/Problems:We were challenged with slowed progress due to COVID-related restrictions on access to animals, obtaining reagents/supplies and staffing levels in the laboratories. One doctoral student who worked on the project coverted to a Master of Science program and successfully defended and left the lab. A new student was recruited to pick up the experiments. Training this student delayed progress. What opportunities for training and professional development has the project provided?One undergraduate student with a disability participated in the project and received training in molecular biology techniques and participated in weekly lab meetings. One of the doctoral students working on the project was appointed to a NIH T32 training grant. How have the results been disseminated to communities of interest?Results in 2019 and 2020 were presented in 2021 following resumption of in-person scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?Complete the intrauterine infusion experiment, analyze and publish. Finish writing up and publish the experiments on the differences in immune cell proportions and function in PBL between heifers and cows.

    Impacts
    What was accomplished under these goals? Completed analysis of experiments compariing proportions of PBL in dairy cows and heifers. Data presented at the 2021 meeting of the SSR. Conducted a follow-on study directly comparing key indictars of immune function between cows and heifers on day 18 of the estrous cycle and early pregnancy. Completed part of the experiment to examine the effects of intrauterine infusion of conceptus secretory proteins on uterine and PBL transcriptome related to immune regulation by the conceptus.

    Publications


      Progress 04/01/19 to 03/31/20

      Outputs
      Target Audience:The target audience reached this reporting period include scientists and allied animal industry professionals that are consumers of scientific literature. Furthermore, presenations to lay audiences were made in the general area of this research focus. Changes/Problems:We were challenged in 2019 with validation of antibodies for detecing interferon stimulated genes in peripheral blood leukocytes. These were custom prepared antibodies. This significantly delayed progress on these experiments. Our results from 2019 were not presented at annual meetings in 2020, but will be presented in 2021. What opportunities for training and professional development has the project provided?3 undergraduate students have participated in this project. Two of these students recently received offers of adminssion to graduate programs in reprodutive biology. One of these students, a Millenium Scholar, is also an underrepresented minority student. How have the results been disseminated to communities of interest?Results from 2019 were prepared for presentation at two meetings of international societies in 2020, both of which were canceled due to the pandemic. What do you plan to do during the next reporting period to accomplish the goals?Will completed the analysis of the effects of parity, day and pregnancy status on the proportions and functions of peripheral blood immune cells in dairy cows and heifers. These data will be reported at international meetings in 2021. Will continue with the experiments examining the effects of intrauterine injection of IFNT and PAG on uterine immmune cell populations and functions.

      Impacts
      What was accomplished under these goals? Completed experiments comparing the proportions of peripheral blood immune cells in dairy cows and heifers. Data analysis is ongoing. Examined expression of key immune cell types and expression of molecules related to their function (SIRP and CD47). Initiated experments examining the effects of intrauterine infusions of IFNT alone or incombination with pregnancy associated glycoproteins on uterine immune cell populations and functions.

      Publications

      • Type: Journal Articles Status: Published Year Published: 2019 Citation: Immunological detection of pregnancy: Evidence for systemic immune modulation during early pregnancy in ruminants. Ott, TL Theriogenology 150, 498-503


      Progress 04/01/18 to 03/31/19

      Outputs
      Target Audience:The target audience reached this reporting period include scientists and allied animal industry professionals that are consumers of scientific literature. Changes/Problems:None. What opportunities for training and professional development has the project provided?Two undergraduate student and 2 graduate students participated in this research. How have the results been disseminated to communities of interest?Results presented at the American Dairy Science Association annual meeting. What do you plan to do during the next reporting period to accomplish the goals?Complete comparison with heifers and cows to determine if lower fertility in cows compared to heifers results from differences in immune cell populations and functions.

      Impacts
      What was accomplished under these goals? Determined the effects of the estrous cycle and early pregnancy on the proportion and function of immune cells in dairy heifers. Determined what cell populations express interferon stimulated genes.

      Publications

      • Type: Book Chapters Status: Published Year Published: 2018 Citation: Ott, T.L. and Ealy, A. 2018. Early Pregnancy and Implantation - Interferons. Encyclopedia of Reproduction (2nd Edition). Ed: Flaws J. et al. Elsevier, UK.
      • Type: Journal Articles Status: Published Year Published: 2019 Citation: Ott, T.L. 2019. Symposium Review: Immunological detection of the ruminant conceptus. Journal of Dairy Science doi: 10.3168/jds.2018-15668. [Epub ahead of print]


      Progress 04/01/17 to 03/31/18

      Outputs
      Target Audience:Scientists working in the discipline, and professionals working in allied industries through presenations at scientific meetings. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Several undergraduate students benefited from participation in aspects of this research. Graduate students participated in leading experiments. This lead to presenations at scientific meetings. How have the results been disseminated to communities of interest?Abstracts at scientific meetings and publications in disciplinary journals. What do you plan to do during the next reporting period to accomplish the goals?Will continue work on characterizing changes in immune cells during the estrous cycle and early pregnancy in dairy heifers and cows and test the hypothesis that reduced fertility in dairy cows (compared to heifers) is a result of altered immune function in cows.

      Impacts
      What was accomplished under these goals? Conducted experiments to examine effects of conceptus signaling on uterine and peripheral blood immune cells. Published results in Biology of Reproduction. Initiated experiments to characterize changes in immune cell populations and functions during the cycle and early pregnancy in heifers and cows.

      Publications

      • Type: Journal Articles Status: Published Year Published: 2017 Citation: Vasudevan, S, Kamat, M, Walusimbi, SS, Pate, JL and Ott, TL. 2017. Effects of early pregnancy on uterine lymphocytes and endometrial expression of immune-regulatory molecules in dairy heifers. Biol. Reprod.;97(1):104-118. doi:10.1093/biolre/iox061