Complex Interactions Between Hypoxia Inducible Factors, Insulin-Like Growth Factor-II and Oxygen in Early Murine Trophoblasts

The human first trimester placenta experiences a low oxygen environment. The hypoxia inducible factors (HIFs) mediate the response to low oxygen, inducing genes such as insulin-like growth factor (IGF)-II. Interestingly, IGF-II has been shown to promote placental growth and function. Currently, the interaction between oxygen, IGF-II and HIFs in the regulation of trophoblast behaviour are unclear. Murine implantation sites from days 5.5-10.5 were collected for immunohistochemical analyses. Use of the hypoxia marker pimonidazole indicated that the early mouse implantation site is exposed to low oxygen levels similar to those seen in the early human placenta. HIF-1α protein immunostaining was also observed in the implantation site. Culturing murine ectoplacental cones in decreasing oxygen concentrations (20%, 5% and 1% O₂), either with or without the addition of IGF-II, induced complex responses by trophoblasts in terms of their migration and differentiation. Following 3 days exposure to low oxygen there was reduced EPC outgrowth, reduced Igf2 and increased Tpbp mRNA levels, suggesting commitment to the spongiotrophoblast lineage. In addition, Hif-1α mRNA levels were decreased, whilst Hif-2α mRNA was unchanged. This decrease in Hif-1α may be due to the observed increase in antisense (as) Hif-1α mRNA levels in 1% cultures. Furthermore, expression of Hif-2α and the HIF target genes: asHif-1α, Vegf and Slc2a1 were reduced under low oxygen with the addition of IGF-II. In conclusion, Hif-1α and Hif-2α are differentially regulated by oxygen and IGF-II in cultured trophoblast cells and asHif-1α may mediate the response to prolonged hypoxia in murine trophoblasts.