ARS/SCA: Effect of endophytie-infected tall fescue seed on equine endometiral gene expression and histology

Endometrial biopsies will be collected from 4 normally cycling mares, with each mare sampled during estrus and at day 14 (diestrus) after ovulation, both in an estrous cycle where mares are fed a control grain ration, and in a subsequent estrus cycle where mares are fed the same control grain ration except with the addition of E+ tall fescue seed, resulting in a total of 16 sample times. Multiple biopsies can be obtained from each mare at each time point to obtain sufficient tissue for the studies. Endometrial tissues will be either snap frozen in liquid nitrogen for RNA extraction and additional samples placed immediately into neutral buffered formalin for histology. RNA extraction and Next Generation Sequencing assays will be performed using a reference sample design. From the array data selective transcripts will be validated using quantitative RT-PCR and proteins of interest localized using immunohistochemistry. This technique provides a systematic approach to the study of endometrial transcriptome changes resulting from alkaloids present in E+ fescue seed, and will lead to target genes which then may be of interest to study for epigenetic changes resulting in up- or down-regulation. Histologically, fixed tissue sections will be differentially stained to highlight different structural changes within the endometrial epithelium and vascular structure. Specific stains to be used are hematoxylin and eosin to examine changes in glandular epithelium, the smooth muscle actin stain which highlights smooth muscle cells in the arterial wall, Masson's stain which highlights collagen and muscle fibers, and Verhoeff's stain which highlights internal elastic laminae.