Role of the Aryl Hydrocarbon Receptor in Endometrium of Dairy Heifers During the Estrous Cycle and Early Pregnancy

Open Access
Author:
Hartzell, Michelle Christina
Area of Honors:
Animal Sciences
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Troy Ott, Thesis Supervisor
  • Troy Ott, Honors Advisor
  • Joy Lee Pate, Faculty Reader
Keywords:
  • AHR
  • embryonic loss
  • pregnancy
  • endometrium
  • dairy heifers
Abstract:
Early embryonic loss in dairy cattle is a major economic cost to dairy producers. Our overall hypothesis is that a portion of these losses is mediated by aberrant response of the uterine immune system to the embryo. This research seeks to improve our understanding of a cellular mechanism that supports establishment of pregnancy. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that reduces T cell proliferation and is involved in differentiation of T regulatory cells (Treg). We hypothesize that AHR-dependent Treg generation and inhibition of immune cell proliferation confers maternal immunosuppression toward the allogeneic embryo. Holstein dairy heifers (n=22) were estrous synchronized and bred by artificial insemination (Day=0) or remained cyclic. Uterine endometrium was collected on day 17 of the estrous cycle (n=9), and days 17 (n=9) and 20 (n=4) of pregnancy. Total cellular RNA was isolated, converted to cDNA, and amplified by PCR using bovine AHR primers. A cDNA amplicon was visualized by gel electrophoresis, excised, and sequenced to determine its identity. Purified cDNA was used to generate a standard curve with a slope of -3.36, efficiency of 98.44%, and R2 of 0.997. Analysis of mRNA abundance for AHR was conducted using RPL19 as the reference gene. Critical threshold data were adjusted for RPL19 and 2-ΔCt values were analyzed using PROC Mixed and orthogonal contrasts. Although AHR was abundantly expressed in the endometrium, there was no difference in mRNA between pregnant and cyclic heifers nor between day 17 and day 20 pregnant heifers (P > 0.10). The presence of AHR protein in the endometrium was detected in all three groups studied by Western blot although the identity of all bands was not confirmed. Immunofluorescence analysis of uterine sections labelled with AHR antibody indicated AHR protein concentration was greater in the shallow stroma (P = 0.05) and tended to be greater in the myometrium (P = 0.06) in day 17 and 20 pregnant heifers compared to day 17 cyclic heifers. A dose response experiment of peripheral blood mononuclear cells (PBMC) cultured with kynurenine for 72 hours produced no measurable effect on total immune cell proliferation. However, individual immune cell types were not examined. This study confirms the presence of AHR mRNA and protein in the bovine endometrium during the estrous cycle and early pregnancy. Activation of this cellular receptor may initiate tolerogenic mechanisms that support early embryonic growth and development.