Date on Master's Thesis/Doctoral Dissertation

8-2016

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Biology

Degree Program

Biology, PhD

Committee Chair

Corbitt, Cynthia

Committee Co-Chair (if applicable)

Klinge, Carolyn

Committee Member

Klinge, Carolyn

Committee Member

Perlin, Michael

Committee Member

Eason, Perri

Committee Member

Schultz, David

Author's Keywords

Glyceollin; brain; microarray; rna-seq; qPCR; phytoalexin

Abstract

Glyceollins (Glys), produced by soy plants in response to stress, have anti-estrogenic activity in breast and ovarian cancer cell lines in vitro and in vivo. In addition to known anti-estrogenic effects, Glys exhibit mechanisms of action not involving estrogen receptor (ER) signaling. To date, effects of Glys on brain physiology and function are unknown. The purpose of the experiments summarized in this dissertation was to gain an understanding of the effects of Gly on brain-related functions in the female mouse brain through the observation of changes in gene expression. For our initial studies, we treated ovariectomized Swiss Webster (CFW) mice with 17-β estradiol (E2) or placebo pellets, followed by 11 days of exposure to Glys or vehicle i.p. injections. We then performed microarray (Chapter 2) and RNA-sequencing analyses (Chapter 3) on total RNA extracted from whole brain hemispheres and identified differentially expressed genes (DEGs) between our treatment groups. Our results suggested that Glys, when in combination with E2 (E2+Gly), can oppose the E2 effects on gene expression and vice versa, can regulate genes similarly to E2, and can also have E2-independent effects on gene expression in the female brain. However, the whole brain experiments did not take into account the heterogeneity of the brain. Different brain regions perform unique and distinct functions and can differ markedly in terms of gene expression, so we wanted to determine if Glys had any brain region-specific effects on gene expression. Additionally, as the whole brain studies only included a single time point of exposure to Glys, we evaluated the effects of a single acute dose of Glys (2, 24 and 48 hr) as well as chronic exposure to Glys (multiple doses of Glys for 7 consecutive days) on gene expression in distinct brain regions. Therefore, in Chapter 4, we evaluated the effects of acute vs. chronic doses of Glys alone and in combination with E2 on gene expression in the hypothalamus, hippocampus, and cortex of the female mouse brain. Our results suggest that Glys can rapidly upregulate the expression of genes like growth hormone (Gh) in the hypothalamus, hippocampus and cortex and prolactin (Prl) in the hypothalamus and cortex, 2h or 24h after administration of a single acute dose. Thus Glys may potentially affect neuronal processes like food intake, stress and cognition through its effects on Gh and Prl gene expression in the female mouse brain. As all of the above chapters involve a peripheral administration of Glys (intraperitoneal injections), it was unclear if Glys affect gene expression through direct action at the neuron or through some indirect peripheral effect. To address this issue, in Chapter 5 we screened five immortalized neural cell lines derived from the adult female mouse hypothalamus (mHypoA-50, 51, 55, 59 and 63) for the presence of our genes of interest and E2 responsiveness. Based on consistency of mRNA transcript detection and E2 responsivity, we selected two cell lines (mHypoA-55 and 63) that may be suitable for future experiments to determine the direct effect of Glys on gene expression at the neuron. Together this work provides novel information on the effects of Glys in the brain, which is important in order to develop its use as a dietary supplement and/or therapeutic agent.

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