Placental deficiency of the (pro)renin receptor ((P)RR) reduces placental development and functional capacity

Abstract

The (pro)renin receptor ((P)RR; also known as ATP6AP2) is a multifunctional receptor. The (P)RR activates the tissue renin-angiotensin system (RAS) and is also involved in regulating integral intracellular pathways such as V-ATPase and Wnt/β-catenin signalling. Given this, the (P)RR may be associated with essential pathways in placentation, however its role within the context of pregnancy remains poorly characterised. The first trimester/extravillous trophoblast cell line, HTR-8/SVneo, underwent an siRNA knockdown where they were incubated for 24 h with a negative control siRNA or siRNA targeting ATP6AP2 mRNA. xCELLigence real-time cell analysis was performed to assess the effect of ATP6AP2 mRNA knockdown on HTR-8/SVneo cell proliferation, migration, and invasion. In subsequent experiments, GFP-encoding lentiviral packaged gene-constructs were used to knockdown (P)RR expression in the trophectoderm of C57/BL6/CBA-F1 mouse blastocysts. Blastocysts were incubated for 6 h with vehicle (no-virus), control virus (non-targeting shRNA and GFP), or (P)RR-knockdown virus ((P)RR shRNA and GFP) before transfer into recipient pseudo-pregnant Swiss CD1 female mice. Fetal and placental tissues were collected and assessed at embryonic age (EA) 10 and 18. (P)RR levels were measured in the labyrinth zone of day 18 placentae and stereological Merz grid analysis was performed to determine the volumetric distribution of trophoblasts, fetal capillaries, and the maternal blood space. We showed that a reduction of ATP6AP2 expression in HTR-8/SVneo cells in vitro, impaired trophoblast proliferation, migration, and invasion. In vivo, decreasing placental labyrinth (P)RR expression adversely effected placental physiology, decreasing placental trophoblast number and total surface area available for exchange, while also increasing maternal blood space. Additionally, decreased (P)RR affected placental efficacy evident by the reduced fetal-placental weight ratio. Our study shows that the (P)RR is necessary for appropriate placental development and function.

Keywords: blastocyst; placenta; placental development; prorenin receptor; trophoblast (TC).

FIGURE 1

Transfected embryo mouse model timeline. C57BL/6. CBA (F1) mice were super ovulated with Folligon and Chorulon. F1 mice were mated, and presumptive zygotes were collected at embryonic age (EA) day 1. Embryos were then cultured until the blastocyst stage (EA 2–4) when they underwent zona pellucida removal and lentiviral transfection (EA 5). Blastocysts were then transferred into pseudopregnant recipient Swiss females. Pregnant mice were then weighed until EA 9 or EA 17, with fetal and maternal endpoint collections on EA 10 or EA 18. Excess embryos on EA 5 were cultured and assessed for GFP expression and outgrowth. Created with Biorender.com.

FIGURE 2

ATP6AP2 siRNA transfection in HTR-8/SVneo cells. HTR-8/SVneo cells were treated with 5 nM ATP6AP2 siRNA and cultured for 48 h. ATP6AP2 siRNA successfully decreased both (A) ATP6AP2 mRNA expression; and (B) (P)RR protein levels compared with negative control siRNA treated cells. (C) ATP6AP2 siRNA significantly decreased proliferation, migration, and invasion of HTR-8/SVneo cells when compared with the negative control siRNA. ***: p < 0.0005, ****: p < 0.0001 indicate significant differences between ATP6AP2 and negative control siRNA treatment groups. β-actin (ACTB) was used as a loading control in the representative immunoblot. Data were analysed by Mann-Whitney test and are presented mean +/- SEM. N = 3 experiments conducted in technical triplicates.

FIGURE 3

The effect of lentiviral APT6AP2 transfection on mouse blastocyst outgrowth in vitro. (A) Blastocysts were outgrown for 120 h and visual assessment of GFP was undertaken. GFP expression in blastocysts confirmed that there was successful infection of the lentivirus in the control lentivirus (CV) and the ATP6AP2 siRNA lentivirus (KV) groups. (B) Blastocysts were cultured for 96 or 120 h and outgrowth area was measured in cm². Blastocyst outgrowth area was significantly reduced in the KV compared to the CV group. Outgrowth area was also significantly reduced in the CV group compared to the no lentiviral group (NV). BF: Bright field, GFP: Green fluorescent protein. **: p < 0.005, denotes significant differences between treatments. Individual blastocysts within each image and their outgrowth are outlined in white. Images were acquired at 2.5x magnification. Scale bar: 1000 μm. Data were analysed by Dunnett’s multiple comparisons test and are presented as mean +/- SEM. 96 hrs N = 15–24 blastocysts, 120 hrs N = 9 blastocysts. Biological replicates (96 hrs n = 9–13, 120 hrs n = 6 mice), total number of blastocysts analysed (96 hrs n = 15–24, 120 hrs n = 9).

FIGURE 4

Placental (pro)renin receptor ((P)RR/ATP6AP2) expression in a mouse model of early placental (P)RR deficiency. (A) Green fluorescent protein (GFP) immunofluorescent staining of embryonic age (EA) 10 mouse placenta where Pl: placenta and Fe: mouse fetus. EA 18 transfected mouse placentae were separated into the labyrinth and junctional zones. (B) ATP6AP2 mRNA expression and (C) (P)RR protein levels were significantly reduced in the labyrinth zone of ATP6AP2 lentivirus infected placentae (KV) when compared with the control lentivirus infected placentae (CV). A representative immunoblot of (P)RR protein levels in mouse placenta is shown, ponceau was used as a loading control. NV: No lentivirus, CV: Control lentivirus, KV: ATP6AP2 lentivirus. *: p < 0.05 **: p < 0.005, denotes significant differences between treatments. Data are presented as mean +/- SEM. NV: n = 9 litters with 1–4 fetuses and placentae per litter (21 pups). CV: n = 6 litters with 1–5 fetuses and placentae per litter (13 pups). KV: n = 6 litters with 1–2 fetuses and placentae per litter (8 pups). Scale bar (white line) = 200 μm.

FIGURE 5

Placental localisation of the (pro)renin receptor ((P)RR). (P)RR immunohistochemical staining of embryonic age 18 mouse placental labyrinth regions. (P)RR antibody staining (brown) in (A) no lentivirus infected placentae; (B) control lentivirus infected placentae; (C) ATP6AP2 lentiviral infected placentae; and (D), no primary antibody control. Inset top right: Whole placenta pictures with red boxes highlighting regions of interest. Scale bar: 50 μM at 25x magnification.