Equine Trophectoderm Cells and Their Role in Fetal-Maternal Recognition

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.