Performing Department
(N/A)
Non Technical Summary
Cattle production is the most important agricultural industry in the U.S. comprisingthe largest share of cash receipts for agricultural commodities. Beef production has a $12.1 billion impact annually to the state of Nebraska economy including 6.5 billion in direct sales.Infertility, defined as failure for cows to conceive and have a calf each year, is a major problem as cash receipts for offspring fund and sustain cow/calf operations. The cost of infertility to U.S. cattle producers was estimated to be over $1.06 billion annually. A major infertility problem in cattle is anovulation which is the failure to ovulate an egg, or to display behavioral estrus and ovulate at the appropriate time. Often anovulation is due to impaired follicle development which can occur when the environment around the follicle has production of too much steroids like androgen. We have identified five populations of bovine and ovine females with 18-26% of the herd/flock that have naturally occuring excess androgen resulting in follicular arrest and anovulation or inability to ovulate.These naturally occurring androgen excess animals have similar characteristics to women diagnosed with polycystic ovary syndrome (PCOS). The disorder PCOS is strongly familial and the prevalence rate is 21% across many human populations with high heritability. Often 60% of daughters with mothers diagnosed with PCOS also have this disorder. We are working within our herd to determine heritability of this androgen excess in females and if it can be passed from dam to offspring.Experiments within the current grant will develop a better understanding of the ways excess androgen may impair how the cells within the ovary communicate and how this impairsfollicle development and ovulation. Once we understand critical communication pathways that are altered we can start to develop potential therapies to treat and rescue the androgen excess in these cows with the goal of being able to reduce the incidence of anovulation and female infertility in cattle and increase the efficiency and sustanability of producer operations.
Animal Health Component
100%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
Our long-term goals are to understand how alterations in ovarian microenvironment may contribute to granulosa cell function and follicular arrest which results in anovulation in cows. We presume that an androgen excess environment is limiting granulosa cell proliferation and promoting cellular differentiation. Our short-term goals are to better understand how Vascular Endothelial Growth Factor A (VEGFA) generated signals or androgen antagonists control the ovarian microenvironment and thereby may inform cellular processes, cellular fate and, in turn, improve our understanding of follicle progression, the ovulatory processes and female fertility. Thus, we are investigating the potential signal transduction pathways that we have determined to be different in granulosa cells from androgen excess (High A4) cows. We know the granulosa cells in the HIgh A4 cows are arrested in the cell cycle. Part of this cell cycle arrest may be due to altered granulosa cell function, and also altered fibrosis in the stroma, potentially, in fibroblasts.Thus, the following objectives will be elucidated:Objective 1- Elucidate how VEGFA165 and androgen antagonists affect regulation of YAP1 activation and transcription of genes that alter granulosa cell function. Our working hypothesis is that VEGFA165 and androgen inhibition (via 17a hydroxylase inhibitor or androgen receptor inhibition) will turn Hippo OFF to dephosphorylate YAP1 allowing for its translocation to the nucleus of granulosa cells to increase transcription of genes promoting cell cycle progression and proliferation and "normal estrogen production". To test this hypothesis, we will conduct RNA sequencing (RNAseq) on androgen inhibitor, VEGFA165 and PBS treated High A4 and Control granulosa cells to identify differentially expressed genes and determine how transcript changes may contribute to proliferation. Furthermore, primary granulosa cells will be cultured with FSH and different doses of androgens and androgen antagonists (synthesis and receptor inhibitor) to determine how A4 affects YAP1 activity. Furthermore, we will employ small molecule inhibitors and siRNA against YAP1 and proteins in the Hippo/YAP1 pathway to determine their role in granulosa cell proliferation/apoptosis and steroidogenic function.Objective 2- Determine how VEGFA165 or androgen inhibition through YAP1 reduces ECM stiffness and fibrosis in High A4 ovaries. Our working hypothesis is that androgen excess causes increased stiffness and fibrosis in ovarian cortex of High A4 cows either directly or indirectly via turning Hippo ON in ovarian stroma fibroblasts. VEGFA165 or androgen inhibition can turn Hippo OFF to dephosphorylate YAP1 and allow for increased migration of cells, and breakdown of adherens junctions in the extracellular matrix, and between cells, reducing fibrosis. We will conduct single cell sequencing (scRNAseq) on ovarian cortex cultures from Control and High A4 cows treated with PBS, androgen inhibitor and VEGFA165 to determine how different cell types within the cortex culture are changed during androgen excess amelioration by either androgen inhibitor or VEGFA165. We will focus on genes in signal transduction pathways (YAP1) that may be involved in follicle growth and apoptosis/autophagy realizing that the processes of fibrosis, oxidative stress, extracellular matrix fluidity/structure will impact follicular progression and maturation. We will focus on how cells, other than granulosa, change to understand how VEGFA165 and/or androgen inhibition affects them and contributes to normalizing the extracellular matrix surrounding follicles providing an appropriate environment for follicle growth, steroidogenesis, and maturation.
Project Methods
Objective 1- Elucidate how VEGFA165 and androgen antagonists affect regulation of YAP1 activation and transcription of genes that alter granulosa cell function. Experiments 1:1a- How does an androgen excess environment affect YAP1 localization in granulosa cells?Granulosa cells will be cultured and treated with different amounts of A4, androgen synthesis inhibitor or receptor antagonist and see if there is an effect on granulosa cell function. We will also look at where YAP1 is localized- either in cytoplasm and nucleus at different timepoints after treatment by fixing cells and conducting immunofluorescence. We will use image J to image the fluorescence and quantitate differences in different treatments. We will conduct western blots to quantitate YAP1 and phopho-YAP1 after treatment with A4 concentrations. We will use phospho antibodies to LATS1/2 and MST1/2 to determine if Hippo was regulated. Spent media from granulosa cell cultures treated with A4 treatments will be collected and analyzed for estrogen, A4 and Cytokine/Chemokine production.Experiment 1:1b- Does VEGFA165 or 17a hydroxylase inhibitor treatment affect where YAP1 is localized in granulosa cells? Granulosa cells will be cultured and transfected with Controlsand YAP1 (siYAP1), or treated with PP2A inhibitor (LB100; 1 μM,) then 48 hr after transfection, granulosa cells will be treated for 24 hrs with PBS, A4, VEGFA165, 17α hydroxylase inhibitor, AR antagonist, and combinations of VEGFA with FSH, A4, AR antagonist, etc. Cells will be collected 24 hr after treatments and will be analyzed. Most of these treatments should inhibit VEGFA165's actions since YAP1 should be knocked down and not able to bind to TEAD or transcription factors.We will then conduct YAP1 localization through immunohistochemistry to determine if it is nuclear or cytoplasmic, conduct Western blot analysis and steroid and cytokine/chemokine assays and granulosa cell proliferation assays.Experiment 1:2a- What are the differentially expressed mRNA transcripts and predicted signal transduction pathways in granulosa cells from High A4 and Control cows? We will conduct RNA seq on granulosa cells from High A4 and Control cowsin vivo. RNA libraries will be made using Illumina Nextera. 150bp, paired-end sequencing will be performed to a minimum depth of 20 million reads per sample using an Illumina NextSeq. Raw data generated from sequencing will be processed through aBioinformatics pipelineby the UNMC bioinformatics core.Ingenuity Pathways Analysis (IPA) will be utilized to identify canonical pathways and predicted regulators . Sequencing data will be validated by droplet digital RT-PCR (ddPCR). A p value of p<0.05 will be considered significant and a tendency p<0.1. Our Power analysiscalculated that granulosa cells from 8 biological replicates per experimental group (8 Control and 8 High A4 cows) will be required for RNAseq.Experiment 1:2bDo DEGs from High A4 and Control cows affect granulosa cell proliferation and steroidogenesis in granulosa cell cultures from slaughterhouse tissue?The 10 DEGs (initially- 10 upregulated and 10 downregulated) in granulosa cells from Control vs High A4 granulosa cells (Experiment 1:2a) with the greatest fold differences detectedbetween the two groups and involved in either proliferation or steroidogenesis or apoptosis will be evaluated by treating granulosa cell cultures from slaughterhouse tissuewith small molecule inhibitors. We will determine how knocking down these genes affects granulosa cell proliferation, steroidogenesis or apoptosis. Media from granulosa cells will be collected for steroid and cytokine determination. We will utilize three different experiments from 3 slaughterhouse collections with 3 wells of granulosa cells/treatment/replicate. Objective 2- Determine how VEGFA165 and androgen inhibition through YAP1 reduces ECM stiffness and fibrosis in High A4 ovaries.Experiment 2:1a.Can we make an High A4 ovarian cortex by treating with excess A4? Cows will be classified into Control and High A4 groups ovariectomized 36-42 hours after PG. We will utilize ovarian cortex from control cows and culture them with different treatments of A4, VEGFA, inhibitors to androgens, inhibitors to androgen receptors and all of this alone or with VEGFA165 for 7 days. Spent media collected from daily media changes will be pooled to determine steroid, and cytokine/chemokine concentrations secreted from ovarian cortex from each treatment group. Cortex cultures from 8 Control cows and 8 High A4 cows will be utilized for this experiment. To determine fibrosis and oxidative stress in treatment groups, ovarian cortex cultures will be fixed, embedded in paraffin, sectioned and stained for Pico Sirius Red to visualize fibrosis. Immunohistochemistry for oxidative stress will be conducted To analyze both fibrosis (PSR staining) and oxidative stress (IF for 4HNE) we will utilize Image J. Follicle staging on sections will occur to determine effect on follicle progression. ddPCR will be conducted for validation of gene expression. Experiment 2:1b-Do inhibitors to YAP1, PP2A activity increase fibrosis, collagen deposition, A4 production, oxidative stress resulting in follicular arrest? Cows will be classified into Control and High A4 groups, estrous cycles synchronized and cows ovariectomized 36-42 hours after PG. We will treat ovarian cortex cultures from Control cows with PBS, inhibitors of YAP1 activation, or LB100 (1 mM) to inhibit PP2A activity, which results in increased YAP1 phosphorylation and localization in the cytoplasm. Ovarian cortex culture pieces will be collected, media changed, and analyzed for fibrosis, oxidative stress and follicle staging of hematoxylin and eosin-stained sections before and after culture. We will utilize 8 Control cows and 8 High A4 cows for this experiment.Experiment 2:2a- To determine expressed transcripts in different cell types within the ovarian cortex in Control and High A4 ovarian cortex cultures and how VEGFA165 alter the transcripts will be determined. Control and High A4 cows will be ovariectomized 36-42 hours after PG to collect ovarian cortex pieces. These will be treated with PBS or VEGFA165 or 17α hydroxylase inhibitor in culture. Cells will be dispersed to develop a cell suspension and then delivered to the UNMC Genomics core to be prepared for scRNAseq using the 10XGenomics X System. For scRNAseq library preparation 10XGenomic will be used followed by sequencing on a NovaSeq 6000. Outputfiles will be converted and aligned and transferred to the UNMC Bioinformatics core to conduct the analysis of cells via R. A computer program will filter the genes expressed by cells and several different criteria must be met for cells and genes to remain in the analysis. Marker genes discriminating different cell cluster will be selected among highly expressed genes (p< 0.01; and fold change greater than 0.25) using Wilcoxon Rank Sum test. The robustness of cell clustering can be confirmed by identifying clusters from the UMAP using density-based clustering methods HDBSCAN. Because of the expense of the scRNAseq we will use 3 cows per treatment with 5 treatment groups for a total of 15 cows.Experiment 2:2b. We will utilize the ovarian cortex cultures that were fixed for histology in Experiment 2:2a to validate the scRNAseq data using antibodies and mRNA probes for FISH which will identify the localization of proteins and mRNAs, respectively in the cortex cells. The specific transcripts and proteins will be selected based on the bioinformatics data described in Experiment 2:2a. Efforts- we will present these data at scientific meetings, through seminars, and in lab meetings or informal lab tours and through peer reviewed publications.Evaluation- we will evalute data through statistical analysis and evaluate progress through production of abstracts and published manuscripts and graduate student and postdoctoral fellow research progress.