Performing Department
(N/A)
Non Technical Summary
Reproductive problems remain the number one reason for involuntary culling on dairy farms, creating increased economc costs on farms and increased environmental impacts of dairy farming. The total number of eggs in the ovary is established before birth and is nonrenewing. The goal of this project is to generate an improved understanding of the ovarian developmental events that impactfertility over the lifespan. The global hypothesis is that the immune system interacts with the developing ovary to direct the total number and quality of eggs in the ovary at birth and thus the lifetime fertility potential of the ovary.Aim 1 is to investigate how the immune system regulates the death or survival of eggs in the developing ovary. Aim 2 is to investigate how the immune system regulates when eggs and their supporting cells begin to grow. This project will lead to an improved understanding of how the ovary develops and how this impacts long-term fertility. A role for immune cells in early ovarian developmental events suggests a link between ovarian development and whole-body health-- perhaps cows that experienced disease as young calves or were gestated by dams that experienced disease during pregnancy will have reduced ovarian reserve as compared to healthy calves or those from healthy dams.The long-term goal of this project is to improve understanding of ovarian development in cattle, to improve reproductive efficiency. Ultimately, the knowledge generated by this project will be applied to improve reproductive management on dairies, making dairy farming more sustainable and dairy products more economical.
Animal Health Component
100%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Infertility remains the top reason for involuntary culling on dairy farms, causing substantial financial losses and increasing environmental impacts. The ovarian reserve of follicles is established before birth in cattle, and its size and health are key determinants of lifetime fertility. During ovarian reserve establishment, most oocytes die, and those that remain are enclosed by granulosa cells and become follicles. After establishment, this reserve is nonrenewing and is progressively depleted through the reproductive lifespan, as follicles irreversibly activate, begin to grow, and ultimately ovulate. The processes of follicle assembly and activation are poorly understood. Immune cells are detectable in the developing ovary, but their functional roles are unknown. Given that immune cells are regulators of cell death and tissue remodeling, we hypothesize that they regulate these key processes during ovarian development, thus regulating the formation and depletion of the ovarian reserve. The goal of this project is to determine the role of immune cells in early ovarian developmental events, including oocyte attrition, follicle assembly, and follicle activation. The specific aims are:1. To investigate the effect of ovarian immune cells on oocyte attrition and primordial follicle assembly.2. To investigate the effect of ovarian immune cells on primordial follicle activation and growth.
Project Methods
Histology:Ovaries will be fixed, embedded, sectioned, and stained, and follicles will be quantified [26, 32]. Fetuses will be batched every 10 days (ie, day 50-day 59) beginning on day 50 [33] for statistical analysis, but exact age will be retained in the metadata, allowing precise evaluation of times that appear to be key transition points.Flow cytometry: Flow cytometry will be used to profile the immune cell subtypes in the developing ovary. Ovaries will be dissociatedand CD45+ cells will be sorted on a magnetic bead-based sorter. CD45+ cells will be assessed by flow cytometry, to determine relative proportions of immune cell subtypes. Populations of interest are monocytes (CD14), total macrophages (CD11B), proinflammatory M1 macrophages (NOS2, TNFA, CD80, CD86) and anti-inflammatory M2 macrophages (CD163, IL10)[36, 37], total T cells (CD3), T cell subtypes (CD4, CD8 and TCR delta) and B cells (pan-B cell antibody). These bovine-validated antibodies are available from the WSU antibody center, BioRad, or Thermo. Single cell sequencing:For this experiment, ovaries will be dissociated and CD45+ cells isolated, as above. The CD45+ and CD45- populations will be mixed at a 1:1 ratio to allow sequencing of a standard number of cells (~10,000), while still capturing the relatively rare immune cells in significant numbers. Results will be validated by a combination of qPCR and RNA in-situ hybridization.Immunohistochemistry: In this experiment, ovarian immune cell subtypes will be localized in the ovary, relative to oocytes, follicles, and vasculature, by immunohistochemistry. This will determine if immune cells colocalize preferentially with dying oocytes, nascent follicles, vasculature, or ovarian stroma. Cells undergoing death will be labeled with a TUNEL assay, a standard assay for assessing cellular death. Follicles will be labeled with FOXL2, a well-known granulosa cell marker, oocytes with DDX4, and vasculature with vWF.In vitro assays and Cell culture: Ovarian immune cell function will be assessed in vitro. Phagocytic ability of macrophages will be assessed using a commercially available kit. In addition to this assay, T cell proliferation will be assessed using a kit and cytokine production from macrophages, T cells, and ovarian cortex cultures (culture period: 24 hr) from the four stages of development previously described will be assessed using the Luminex Milliplex bovine cytokine/chemokine array. This assay allows the assessment of fifteen cytokines/chemokines, including IFNγ, IL-1α, IL-1β, IL-4, IL-6, IL-8 (CXCL8), IL-10, IL-17A, IL-36RA (IL-1F5), IP-10 (CXCL10), MCP-1 (CCL2), MIP-1α (CCL3), MIP-1β (CCL4), TNFα, VEGF-A. In addition, immune cells will be cocultured with oocytes or ovarian cortices to determine changes in oocyte/follicle survival in the presence of immune cells.