*2.1. Auxology*

Animal data are displayed in Table 1. Maternal protein restriction led to a significant decrease in fetal weight (*p* = 0.03) and a significant increase in placental/fetal ratio (*p* = 0.03) at E18.5 in rats. This had no significant influence on placental weight (*p* = 0.11). In our eNOS−/− mice, fetal and placental weights were examined at E15 and E18.5. The animals showed a significant decrease of fetal weight at both time points compared to wildtype controls (*p* < 0.001). Mouse placental weights were significantly decreased at E18.5 (*p* = 0.006), with a similar trend at E15 (*p* = 0.08). The placental-to-fetal weight ratio was una ffected by eNOS deficiency (Table 1).


**Table 1.** Animal auxology. NP: normal protein diet; LP: low protein diet.

\* Mann-Whitney U-Test. † For rats, each group consisted of n = 4 dams each with n = 6 NP/LP pups/damn, respectively. ‡ For mice, groups consisted of n = 6 eNOS−/− vs. n = 5 C57BL/6 dams at both time points with n = 2 pups/dam. Legend: bold values denote statistical significance.

### *2.2. Localization of Rarres1 and 2*

Representative images of Rarres1 and 2 immunohistochemical (IHC) stains are given in Figure 1A,D and Figure 2A,D, respectively. Both proteins shared similar localization in functional placental compartments. In contrast to Rarres1 (cytoplasmic stain, Figure 1), Rarres2 (Figure 2) additionally showed nuclear staining. IHC did not reveal species differences between rat (Figures 1A and 2A) and mouse (Figures 1D and 2D) placentas regarding Rarres1 and 2 localization. Positive staining was mostly present in the cytoplasm of trophoblast giant cells (GC) and spongiotrophoblasts (ST) of rat (Figure 1A) and mouse (Figure 1D) placentas at E18.5. In mice, we did not note differences in Rarres1 and 2 staining in comparison to E15 (data not shown). We additionally found positive staining for both proteins in the yolk sac, decidual stroma, and the umbilical cord lining membrane (data not shown). Glycogen cells and the labyrinth zone (LZ) stained negative for Rarres1 and 2.

**Figure 1.** Rarres1 expression in rat and mouse placenta. (**A**–**C**) Rat placenta, (**D**–**F**) mouse placenta. (**A**,**D**) Immunohistochemical (IHC) stains of methyl Carnoy-fixed placental paraffin sections. Abbreviations: GC = giant cell, BZ = basal zone, ST = spongiotrophoblast, LZ = labyrinth zone, star = glycogen cells. The bar equals 100 μm. (**B**) Maternal protein restriction rat model: placental *Rarres1* mRNA expression on E18.5 (\**p* = 0.03, Mann-Whitney *U*-Test, *n* = 4 NP/LP dams with 6 pups each). E) eNOS−/− mouse model: placental *Rarres1* mRNA expression on E15 and E18.5 (\* *p* = 0.03, \*\* *p* = 0.008 for C57BL/6 and *p* = 0.002 for eNOS−/<sup>−</sup>, ns: *p* = 0.66, Mann-Whitney *U*-Test, WT: *n* = 5 dams, eNOS−/<sup>−</sup>: *n* = 6 dams with 2 pups each). C + F) Analysis of Rarres1 protein expression versus β-Tubulin housekeeper by Western blotting (WB, Rat: ns: *p* = 0.057, Mann-Whitney *U*-Test, *n* = 4 NP/LP dams with *n* = 2 pups each, E18.5; Mouse: ns: *p* = 0.20, Mann-Whitney *U*-Test, *n* = 4 C57B6 and eNOS−/− dams per group with *n* = 1 pup each, E18.5). Abbreviations: LP = low protein diet, NP = normal protein diet in the rat IUGR model with m = male fetus, f = female fetus; C57B6 = C57BL/6 wild type (WT) control strain, eNOS−/− = preeclampsia (PE)/intrauterine growth restriction (IUGR) model eNOS knockout mouse, ns = not significant. RARRES = retinoic acid receptor responders.

**Figure 2.** Rarres2 expression in rat and mouse placenta. (**A**–**C**) Rat placenta, (**D**–**F**) mouse placenta. (**A**,**D**) Immunohistochemical (IHC) stains of methyl Carnoy-fixed placental paraffin sections. Abbreviations: GC = giant cell, BZ = basal zone, ST = spongiotrophoblast, LZ = labyrinth zone, star = glycogen cells. The bar equals 100 μm. (**B**) Maternal protein restriction rat model: placental *Rarres2* mRNA expression on E18.5 (ns: *p* = 0.89, Mann-Whitney *U*-Test, *n* = 4 NP/LP dams with 6 pups each). E) eNOS−/− mouse model: placental *Rarres2* mRNA expression on E15 and E18.5 (E15: ns: *p* = 0.31, E19: ns: *p* = 0.66, Mann-Whitney *U*-test, WT: *n* = 5 dams, eNOS−/<sup>−</sup>: *n* = 6 dams with 2 pups each). C + F) Analysis of Rarres1 protein expression versus β-Tubulin housekeeper by Western blotting (WB, Rat: ns: *p* = 0.10, Mann-Whitney U-Test, *n* =4 NP/LP dams with *n* = 2 pups each, E18.5; Mouse: ns: *p* = 0.49, Mann-Whitney *U*-Test, *n* = 4 C57B6 and eNOS−/− dams per group with *n* = 1 pup each, E18.5). Abbreviations: LP = low protein diet, NP = normal protein diet in the rat IUGR model with m = male fetus, f = female fetus; C57B6 = C57BL/6 wild type (WT) control strain, eNOS−/− = PE/IUGR model eNOS knockout mouse, ns = not significant.

#### *2.3. Expression Analyses of Rarres1*/*2, CmklR1 Receptor, and IL-11*

We detected a small but significant decrease of placental *Rarres1* mRNA expression in our maternal protein restriction rat model at E18.5 (*p* = 0.03, Figure 1B). Sex did not show any significant influence on the expression of *Rarres1*, *2* and *CmklR1* mRNA expression levels (Table 2). In contrast to the rat, we could determine significant di fferences in *Rarres1* mRNA expression between eNOS−/− mice and C57BL/6 wildtype controls at E15 (*p* = 0.03) but not on E18.5 (*p* = 0.66) (Figure 1E). However, a 3.6-fold (eNOS−/−) and 6.5-fold (C57BL/6) temporal increase of placental *Rarres1* mRNA expression was detected from E15 to E18.5 (*p* = 0.008 for C57BL6 mice and *p* = 0.002 for eNOS−/− mice, Figure 1E). Western blot analysis did not reveal significant di fferences in placental Rarres1 protein expression E18.5 in both animal models (rat: *p* = 0.057, mouse: *p* = 0.20, Figure 1C,F).

Placental *Rarres2* mRNA (Figure 2B,E) and protein expression (Figure 2C,F) was neither affected by maternal protein restriction in the rat (PCR: *p* = 0.89, WB: *p* = 0.10, Figure 2B,C), nor eNOS−/− in the mice (PCR: E15: *p* = 0.31, E19: *p* = 0.66, WB: *p* = 0.49, Figure 2E,F). Also, the expression of *Rarres2* remained unchanged from E15 to E18.5 in the mouse (*p* = 0.31 for C57BL6 mice and *p* = 0.13 for eNOS−/− mice, Figure 2E).

Placental *CmklR1* expression was unchanged by maternal protein restriction in the rat and eNOS−/− in the mouse. Gestational age seemed to have no significant influence on *CmklR1* expression in the mouse (Table 3). However, a significant increase of placental interleukin 11 (*IL-11*) mRNA expression at E15 (2.3-fold, *p* = 0.004, Figure 3) was observed in eNOS−/− mice compared to controls. No such change in *IL-11* expression was noted at E18.5 (*p* = 0.99).


**Table 2.** Sex di fferences in mRNA expression (fold-change).

\* Mann-Whitney U-Test; For Rarres1 and 2, groups consisted of n = 4 NP/LP dams with n = 3 female/male pups, respectively. For CmklR1, groups consisted n = 3 NP/LP dams with n = 2 female/male pups, respectively. Abbreviations: LP = low protein diet, NP = normal protein diet in the rat IUGR model; m = male fetus, f = female fetus.


**Table 3.** Placental *CmklR1* mRNA expression (fold-change).

\* Mann-Whitney U-Test. † For rats, groups consisted of n = 3 dams per group (NP/LP) with n = 5 pups each; ‡ For mice, groups consisted of n = 6 eNOS−/− vs. n = 5 C57BL/6 dams at both time-points with n = 2 pups/dam. Abbreviations: LP = low protein diet, NP = normal protein diet in the rat IUGR model; C57BL/6 = Wild type (WT) control, eNOS−/− = PE/IUGR model knockout mouse.

**Figure 3.** Placental *IL-11* mRNA expression in eNOS−/− mice on E15. (\*\* *p* = 0.004, Mann-Whitney *U*-test, WT: *n* = 5 dams, eNOS−/<sup>−</sup>: *n* = 6 dams with 2 pups each) Abbreviations: C57B6 = C57BL/6 wild type (WT) control strain, eNOS−/− = PE/IUGR model knockout mouse.
