Effect of Pregnancy on Exosomes in the Uterus and Blood of Dairy Heifers

Open Access
Klein, Alyssa Michelle
Area of Honors:
Animal Sciences
Bachelor of Science
Document Type:
Thesis Supervisors:
  • Troy Ott, Thesis Supervisor
  • Troy Ott, Honors Advisor
  • Joy Lee Pate, Faculty Reader
  • exosomes
  • dairy heifers
  • pregnancy
  • peri-implantation
  • uterine histotrophe
Profitability of animal agriculture is closely linked with fertility, making this a very important field of research. During early pregnancy, the conceptus (embryo and associated extraembryonic membranes) is reliant upon secretions from the reproductive tract, but little is understood about how endometrial secretory processes are regulated. Understanding how the embryo communicates with the uterus will facilitate the development of strategies to reduce embryonic loss and improve profitability. Exosomes, the intraluminal vesicles (ILVs) of multivesicular bodies (MVBs) once they have been secreted into the extracellular milieu, could be one potential contributor to the endometrial secretory milieu necessary for conceptus survival and eventual implantation. The objective of this research was to isolate exosomes from peripheral blood and uterine flushes of both pregnant and non-pregnant dairy heifers, and compare the morphology, relative abundance, and protein composition of these exosomes between samples. Our hypothesis is that pregnancy alters both the abundance and proteomic composition of exosomes in the uterine flush and blood plasma of cows. Others in our lab are simultaneously studying the interaction between exosomes and MX1 (a protein involved in the innate immune response). The knowledge gained by my research will contribute to their working hypothesis that MX1 is involved in the formation of exosomes. Briefly, uterine flush and blood plasma was collected from day 17 cyclic and pregnant heifers. The exosomes were then isolated from the samples via sequential centrifugations followed by an ultra high-speed centrifugation and sucrose floatation gradient. The protein compositions were analyzed by Western blot for Alix, MHC II, HSC70, and CD9; the abundance of exosomes was analyzed by Acetylcholinesterase Assay; and the morphology was analyzed by transmission electron microscopy. Differences in protein composition were unable to be determined; however, the acetylcholinesterase assay and transmission electron microscopy revealed the presence of exosomes in both uterine flush and blood plasma of dairy heifers. Data from the acetylcholinesterase assay suggests an elevated concentration of exosomes in both the uterine flush and plasma of pregnant dairy heifers as compared to cyclic dairy heifers.