Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907
Performing Department
(N/A)
Non Technical Summary
Early loss of pregnancy is a major cause of poor fertility in beef and dairy cows, leading to fewer calves and lower milk production for farmers. Most of these losses happen in the first month of pregnancy, when the embryo is trying to "talk" to the mother's uterus so it can attach, grow, and form a healthy placenta. In this project, we will study a special group of embryo-made proteins, called trophoblast Kunitz domain proteins (TKDPs), which are found only in cattle and related species and are strong candidates to act as keysignals between the embryo and the uterus. Our overall goal is to find out how TKDPs help the embryo survivein the uterus, how they control the growth and specialization of the cells that will form the placenta, and which molecules in the uterus they interact with. To do this, we will turn TKDP genes off in early embryos, and we will use modern gene-reading methods to see how TKDPs change gene activity in both embryo cells and uterine tissue. We expect this work will identify the main gene pathways and uterine signals that depend on TKDPs during the most vulnerable stage of pregnancy. By filling this major knowledge gap about how the cow's uterus and embryo communicate before implantation, the project will provide a scientific foundation for future tools to improve fertility, such as better embryo culture conditions, tests that predict which pregnancies are likely to succeed, and management strategies that reduce early embryo loss in high-producing dairy and beef herds.
Animal Health Component
15%
Research Effort Categories
Basic
85%
Applied
15%
Developmental
0%
Goals / Objectives
This research addresses reproductive efficiency in beef and dairy cattle by investigating the molecular dialogue between the embryo and uterus, a critical priority in USDA animal reproduction research. A significant proportion of pregnancy failures in cattle occur during the first month, coinciding with the period of embryo elongation and attachment. This early loss is strongly associated with a failure in the reciprocal communication required to modify the uterine microenvironment. While the interferon-tau protein is known to signal pregnancy recognition, it does not act alone; a lineage-specific group of proteins known as Trophoblast Kunitz Domain Proteins (TKDPs) are secreted in massive quantities during this same critical window. Despite their abundance and evolutionary conservation in ruminants, their specific biological function remains unknown. In this project, we investigate the functional necessity of TKDPs to understand how they orchestrate pregnancy success. Aim 1: By utilizing targeted gene modulation in bovine embryos and trophoblast models, we will determine if the loss of TKDP expression compromises embryo elongation, survival, and developmental competence. Aim 2: By mapping the transcriptional regulatory landscape of trophoblast proliferation and differentiation, we will define the genomic networks that control how the embryo responds to developmental cues. Aim 3: By isolating and identifying uterine proteins that physically interact with TKDPs, we will uncover the specific molecular targets within the uterine secretion that facilitate embryo-maternal signaling. This research will advance the efficiency of U.S. agriculture by defining the fundamental mechanisms of pregnancy establishment, providing a scientific foundation for novel interventions to reduce embryo mortality in commercial herds.
Project Methods
The project will be conducted using a multi-disciplinary approach integrating embryology, functional genomics, and proteomics. To determine the functional requirement of Trophoblast Kunitz Domain Proteins (TKDPs), we will employ targeted gene modulation (CRISPR/Cas9 and RNA interference) in both in vitro trophoblast spheroid models and ex vivo bovine blastocysts. A unique aspect of this methodology is the generation of specific "knockout" bovine embryos to observe elongation defects in real-time. Results will be analyzed by quantifying developmental metrics such as blastocyst formation rates, elongation length, and cell survival and subjecting them to statistical evaluation (ANOVA and t-tests) to determine significance compared to controls.To map the regulatory landscapes, we will use high-resolution nascent transcript sequencing. Unlike standard RNA-seq which measures steady-state RNA, this method maps the position of active RNA polymerases, providing an immediate snapshot of transcription. Bioinformatic analysis will involve aligning sequencing reads to the bovine genome to identify active promoters and enhancers, followed by differential expression analysis to define the gene networks controlling trophoblast differentiation. Finally, affinity purification mass spectrometry will be used to identify uterine proteins that bind to TKDPs. Proteomic data will be analyzed using spectral matching algorithms to identify high-confidence interacting partners, filtering for biological relevance within the uterine microenvironment.Efforts: Efforts to deliver science-based knowledge will focus on both formal academic instruction and professional dissemination. (1) Laboratory Instruction and Mentoring: A primary effort will be the experiential learning provided to graduate students and a postdoc. They will receive training in advanced embryology, molecular biology, and bioinformatics, directly contributing to their degree requirements and professional development. (2) Scientific Dissemination: We will actively disseminate findings through presentations at national and international scientific conferences (e.g., Society for the Study of Reproduction). These events serve as informal educational forums where methodologies and results are shared with the broader research community.Evaluation: The success of the project will be evaluated through both scientific milestones and quantitative output indicators. Gene editing success will be measured by the efficient generation of TKDP-null embryos and spheroids, confirmed by PCR and Western blotting. Genomics success will be evaluated by the quality of sequencing libraries (read depth and mapping rates). A key indicator will be the identification of statistically significant differentially regulated gene networks between proliferative and differentiated trophoblasts. Proteomics efficiency will be defined by the isolation of at least one high-confidence uterine interacting protein, validated by co-immunoprecipitation assays.Impact Evaluation: Publications: The project aims to produce at least 2-3 peer-reviewed manuscripts in society journals.Student Development: Evaluation includes tracking the progress of students toward their degrees, measured by the successful defense of theses/dissertations and their placement in subsequent scientific roles.Community Engagement: Impact on the target audience will be quantified by the number of conference presentations given and the reach of the datasets deposited in public repositories.