The renin-angiotensin system (RAS) has roles in placentation, by regulating trophoblast invasion, uterine spiral artery remodelling, and angiogenesis. We have shown that the (pro)renin receptor ((P)RR), which can activate the RAS cascade, promotes proliferation, migration, and invasion of trophoblast cells in vitro. This study aims to assess the physiological role of (P)RR in placentation in vivo.
GFP-expressing lentiviral packaged gene-constructs were used to specifically knock-down (P)RR expression in the trophectoderm of mouse blastocysts (Chakraborty et.al., Bio-protocol, 2017). Zygotes were collected from super-ovulated C57/BL6/CBA F1 female mice and cultured until they reached the blastocyst stage. Blastocysts were then exposed to Acid-Tyrode’s solution for 30s and washed in G2 media, removing the zona pellucida. Blastocysts were incubated for 6h in either vehicle (no virus), control lentivirus (containing vehicle shRNA and GFP; 1x108VP/ml), or (P)RR knockdown virus (containing (P)RR shRNA and GFP; 1x108VP/ml) before being cultured in modified-RPMI media for 96h to examine GFP and (P)RR expression in blastocyst outgrowths. In separate experiments, blastocysts were transferred into recipient pseudo-pregnant Swiss female mice, after which fetal and placental tissues will be collected at embryonic day-10 and 18.
Treatment with both (P)RR knockdown or control lentivirus [1x108VP/ml] increased GFP expression in blastocysts 96h post-transfection, compared to the vehicle control. No significant GFP expression was observed at earlier timepoints. Treatment at 0.5x108VP/ml, compared to the 1x108VP/ml treatment group exhibited sparse, less uniform GFP expression. Initial assessment of embryos infected with control or knockdown virus and transferred into female recipients are underway. We will collect maternal and fetal tissue to assess trophoblast-specific GFP expression, maternal blood to measure plasma s(P)RR levels and placentae to determine the role of (P)RR in placentation.
Given that (P)RR is involved in various trophoblast cell function, we expect that knocking down placental (P)RR will impair placental development and reduce fetal growth.