PROJECT SUMMARY A central role of estrogen is regulating hydration balance in the body. Decreases in body (systemic) hydration, cause systemic vocal fold dehydration. Systemic vocal fold dehydration alters the biology of the tissue. Nevertheless, the importance of estrogens in regulating these biological changes of systemic vocal fold dehydration remain unknown. We address this important clinical and theoretical issue by investigating the transcriptome, proteome, tissue morphology, and ultrasonic vocalization outcomes in a study design that carefully manipulates hormonal state and hydration state. We will also examine the effects of estrogen on the vascular geometry and blood flow to the larynx following the interaction of systemic dehydration with altered hormonal states. We use the rat animal model for these studies since this model allows for robust and rigorous control of hydration and hormonal levels. Because estrogen is important irrespective of biological sex, both male and female rats will be included. To examine the effects of hormonal loss and replacement, we will measure outcomes when reproductive organs have been surgically removed (ovariectomized and orchiectomized rats) and when estrogen replacement with estradiol is delivered. Systemic dehydration will be induced by a translational paradigm of water restriction for 5 days. Animals with free access to water will serve as controls for dehydration. Across 16 animal groups, our first goal is to quantify the effects of hormonal loss on the molecular and histologic signatures of vocal fold dehydration and the influence of estradiol replacement on these signatures. Because hormone receptors are differentially located across vocal fold mucosa and muscle, and these tissue layers have distinct cellular organization and biomechanical properties, the vocal fold mucosa and muscle are examined separately. Our second goal is to investigate the interaction of hormonal status and dehydration on ultrasonic vocalizations. Our third goal is to examine the influence of hormonal status and dehydration on vascular geometry, blood flow, and the physiologic consequences of these changes. By combining molecular, histological, and functional studies, and mechanistic investigations of hemodynamic modifications, this comprehensive proposal seeks to shed fundamental insight into the effects of estrogens on vocal fold dehydration biology. These data are needed to drive clinical recommendations pertaining to vocal fold physiology under conditions of altered hydration and hormonal states.