DEVELOPMENT AND CHARACTERIZATION OF A BOVINE UTERINE EPITHELIAL CELL LINE AND THE EFFECTS OF PREGNANCY ON GENE EXPRESSION IN PERIPHERAL BLOOD LEUKOCYTES

Open Access
Author:
Birt, Alyssa Nicole
Graduate Program:
Animal Science
Degree:
Master of Science
Document Type:
Master Thesis
Date of Defense:
July 09, 2013
Committee Members:
  • Troy Ott, Thesis Advisor
  • Joy Lee Pate, Thesis Advisor
  • Francisco Javier Diaz, Thesis Advisor
Keywords:
  • Immortalized
  • Bovine
  • Endometrium
  • Uterus
  • Pregnancy
  • ISG
  • Peripheral blood
  • IFNT
Abstract:
The uterine endometrium is vital for the production of a complex array of molecules, which make up uterine histotrophe. Uterine histotrophe is essential to the maintenance of a successful pregnancy. Currently there are no in vitro models available to study bovine uterine epithelial cells. Therefore, the objective of the first study presented here was to develop and characterize an immortalized bovine uterine epithelial cell line to investigate the effects of conceptus signaling on the maternal endometrium. Primary epithelial cells were transfected with human papillomavirus (HPV) genes E6 and E7 and human telomerase (hTERT). Cells were treated with interferon tau to study the effects of conceptus signaling on maternal endometrium. In response to IFNT, MX1 and MX2 transcripts were detected in bEP cells, however, neither MX1 nor ISG15 were detectable by Western blotting in bEP cells when treated with interferon tau (IFNT). Further investigation is needed to understand why mRNA, but not protein, for these interferon-stimulated genes was stimulated by IFNT. Interferon tau is a type I IFN produced by the elongating conceptus and is the signal for maternal recognition of pregnancy in ruminants. IFNT acts to up-regulate the expression of interferon-stimulated genes (ISG), both locally in the uterus and systemically in peripheral blood leukocytes. A study was conducted to examine the effects of recombinant bovine somatotropin (rbST) treatment and pregnancy status on peripheral ISG expression. Several IFN- stimulated genes were quantified in peripheral blood leukocytes (PBL) in pregnant (BPP), bred, non-pregnant (BNP), and pregnant but open on Day 60 (BPL) cows 19 days after insemination. Cows were either treated with a single injection of 325 mg of rbST at AI (Single –rbST; S-rbST) or two sequential injections of rbST at AI and 14 days later (Double-rbST; D-rbST). Steady-state concentrations of mRNA for MX1, MX2, ISG15, OAS and RTP4 were quantified and examined for effects of status and treatment. Status effects for MX1, ISG15, and RTP4 were detected in BPP compared to BNP for control cows not treated with rbST. Also, there was a decrease (P≤0.05) in MX1 expression in BPP cows receiving two injections of rbST compared to other treatment groups. When outliers were removed from the BNP group and parity removed from the model, an increase in ISG15 expression was detected in D-rbST cows compared to other treatment groups. Results indicate that rbST injections caused a differential expression of ISG but did not affect the ability to detect pregnancy status in PBL on Day 19. Therefore, factors that affect pregnancy rate in dairy cows can be detected by assaying ISG transcript expression in PBL as early as 19 days after insemination.