Performing Department
(N/A)
Non Technical Summary
Anovulation with or without the presence of cystic ovarian follicles is a significant cause of reproductive dysfunction in cattle. Environmental factors such as heat stress, feed quality and hormonal imbalances all contribute to reduced reproductive efficiency. Follicular development, ovulation and luteinization are controlled by many hormonal signals and by an array of complex intrafollicular mechanisms that regulate both somatic and germ cell development. Interactions among ovarian cells includes both direct cell-cell contacts, indirect cell-ECM contacts and signaling through secreted products. Androgens are among the most abundant signals produced in the bovine ovary. Exposure to high androgen levels causes follicular cysts, anovulation, and reduced fertility in several mammalian species. Cattle with very high follicular androgen levels are less fertile and have altered ovarian function. However, we do not fully understand how excessive androgen stimulation leads to ovarian dysfunction. We hypothesize that overactivation of the androgen membrane receptor ZIP9 will lead to ovarian fibrosis, altered gene expression and impaired ovarian function. This hypothesis will be tested with a combination of in vitro culture of ovarian cells, intact follicles and ovarian explants treated with various androgenic compounds. The results will uncover the consequences of excessive androgen stimulation on ovarian function and could lead to better ways to mitigate these effects to improve reproductive efficiency in cattle.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
Anovulation with or without the presence of cystic ovarian follicles is a significant cause of reproductive dysfunction in cattle. Environmental factors such as heat stress, feed quality and hormonal imbalances all contribute to reduced reproductive efficiency. Our long-term objectives are to increase reproductive efficiency by first understanding the ovarian causes of anovulation and cyst formation and then developing strategies to mitigate these effects to improve ovarian function and reproductive success. Follicular development, ovulation and luteinization are controlled by many hormonal signals and by an array of complex intrafollicular mechanisms that regulate both somatic (theca, granulosa, stromal, luteal, endothelial, and immune) and germ (oocyte) cell development. Interactions among ovarian cells includes both direct cell-cell or cell-ECM contacts and signaling through secreted products. Androgens are among the most abundant signals produced in the bovine ovary with follicular fluid concentrations in the nM range or higher. Exposure to high androgen levels causes follicular cysts, anovulation and reduced fertility in several mammalian species. Cattle with very high follicular androgen levels (mM range) are less fertile, have altered gene expression patterns, fewer large antral follicles and higher deposition of ECM than cattle with lower androgen levels. Thus, excessive androgen stimulation is associated with significant ovarian dysfunction and could be a major cause of anovulation, but the potential mechanism remains obscure. We hypothesize that high levels of androgen activate a novel androgen receptor (ZIP9) leading to impaired granulosa cell differentiation and excessive ECM deposition (fibrosis). We propose a model where androgen binds to ZIP9 protein on follicular cells and promotes intracellular zinc transport, Akt activation and actin polymerization. These intracellular signals, in turn, cause the activation of YAP and SMAD2/3 transcriptional co-activators that when overactivated impair granulosa cell differentiation (steroidogenesis, luteinization) and cause ECM deposition through the induction of the YAP and SMAD2/3 target genes, connective tissue growth factor (CTGF) and Collagen 1. Increased ECM deposition alters the ovarian environment (fibrosis) such that normal follicular development and ovulation are impaired. Excessive ECM deposition could also enhance actin polymerization within ovarian cells leading to further YAP activation and establishment a self-reinforcing loop. The following objectives are proposed to test specific portions of this model:Objective 1. Determine whether testosterone acting through ZIP9 stimulates specific intracellular signaling events in granulosa cells and cultured follicles. Experiments will test whether testosterone binds to ZIP9 to promote activation of Akt and SMAD2/3 pathways, zinc transport and actin polymerization in granulosa cells and cultured follicles.Objective 2. Determine whether testosterone acting through ZIP9 activates YAP and SMAD2/3 pathways leading to impaired granulosa cell differentiation. Experiments in this objective are designed to test whether testosterone through a ZIP9 pathway stimulates YAP nuclear localization and SMAD2/3 phosphorylation and whether this leads to increase cell proliferation and impaired granulosa cell differentiation.Objective 3. Determine whether testosterone acting through ZIP9 induces CTGF, collagen 1 and ECM deposition. These findings will establish a potentially important relationship between testosterone stimulation and changes in the ECM which may contribute to impaired follicular development and ovulation.
Project Methods
The project will be conducted using cells and tissues collected from a local meat processing plant.Cells and tissues from multiple animals will be combined and used in various cell, histological and biochemical assays to examine the consequence of high levels of androgen in cellular, follicular and tissue responses in vitro. In addition, multiple replicates from tissues collected on different days will be analyzed to provide enough statistical power to discriminate differences of 20% or greater between treatment groups. Cellular assays include measures of cell viability, proliferation, morphology. Biochemical assays for protein (western blot, immunostaining, hormone production) and mRNA (qPCR and RNAi knockdown) expression will determine whether intracellular signaling events are altered according to treatment group. Finally, histological analysis of tissue explants will be analyzed for altered follicular development and ECM deposition. Collectively, these approaches will give a more complete picture of the functional consequences of high androgen stimulation in ovarian cells and tissues.