Establishment and maintenance of a successful pregnancy requires signaling from the embryo to the mare, a process known as maternal recognition. Six days after fertilization, the trophectoderm (TE), a placenta precursor is formed. Signals emanating from the TE to the uterine environment are critical to maternal recognition of pregnancy. The identity of factors necessary for this process remain unknown. A novel equine induced trophoblast cell line (iTr) that closely mimics the genotype and phenotype of native equine TE was created. Transcriptome analysis of iTr revealed increased expression of growth factor (GF) receptors for Epidermal GF (EGF), Hepatocyte GF (HGF), Fibroblast GF-2 (FGF-2) and Insulin GF (IGF-1), suggesting these GF may be important targets during TE development in the early embryo. We hypothesized that treatment of iTr cells with these GF would induce changes in cell proliferation and expression of genes likely involved in maternal recognition. The objectives of this experiment were to evaluate the effect of these GFs on iTr mitotic response and regulation of genes involved in steroidogenesis.
Equine iTr cells (n = 3) were cultured with 10 ng/mL EGF, HGF, FGF-2 or IGF-1 for 24 hr, with 5-ethynyl-2'-deoxyuridine (EdU) supplementation during the final 2 hr. Subsequently, cells were fixed and EdU positive and total nuclei were enumerated. A parallel plate of iTr cells was treated in a similar manner and lysed for total RNA isolation. Quantitative PCR using gene-specific primers for CYP11A1, PTGS2, PTGES2, and PTGES3 was performed. Data were analyzed by ANOVA with Tukey's post hoc adjustment using the GLM procedure of SAS. Treatment with EGF, FGF-2, HGF, and IGF-1 increased (P < 0.05) iTr proliferation from control levels of 25.33 ± 1.03% to 38.58 ± 1.61%, 45.50 ± 2.94%, and 38.23 ± 2.01% respectively. The 2-ΔΔCT method was used to calculate the fold change (FC) using GAPDH as the reference gene for normalization. Expression of CYP11A2, PTGES2, and PTGES3 was not affected by GF, as measured by qPCR. By contrast, PTGS2 transcript abundance increased (P < 0.05) following FGF-2 (FC = 3.327 ± 0.8291) and HGF (FC = 11.88 ± 4.572) treatment. These results indicate that FGF-2 and HGF may simultaneously induce proliferation and prostaglandin production by TE cells. The combined results of these experiments will improve our understanding of TE morphogenesis and its response to uterine-derived growth factors. / Master of Science / Establishment and maintenance of a pregnancy requires that the mare uterus recognize the presence of the embryo, a process known as maternal recognition of pregnancy. The trophectoderm (TE) are cells on the outer layer of the embryo formed six days after fertilization, which later give origin to the placenta. The TE sends signals from the embryo to the uterus, that are very important for the mare’s recognition of the embryo’s presence. The specific nature of these signals are still unknown in the horse. A cell line (iTr) very similar in aspect and genes to the horse’s native TE has been created in our laboratory. A set of comparative assays have showed that, during the developmental stage of maternal recognition, both the horse TE and the iTr cells share significant identity, and have receptors for the same set of growth factors (GF), suggesting these GF are important for early embryo development and potentially involved in the signaling process of maternal recognition. We proposed that treatment with these GF would induce iTr cells to proliferate and express signals likely involved in maternal recognition in horses. The objectives of this experiments were to evaluate the effect of EGF, HGF, FGF-2 and IGF-1 on iTr cells by measuring proliferation and cellular mechanisms of maternal recognition already established in in other species. Equine iTr cells were cultured with different GF and right before analysis a fluorescent dye that stain dividing cells was added in order to measure the proliferation. Equivalent cell cultures were used to evaluate if the treatment affected the production of hormones involved in signaling maternal recognition. Treatment with all GF induced higher cell proliferation, but HGF also increased the production of one enzyme that participates in producing a very important hormone (prostaglandin E2). The combined results of these experiments add to our understanding of maternal recognition in horses.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/86784 |
| Date | 18 January 2019 |
| Creators | Bonometti, Susana |
| Contributors | Animal and Poultry Sciences, Johnson, Sally E., Knight, James W., Ealy, Alan D. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Thesis |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
Page generated in 0.0019 seconds