Complex patterns of cell growth in the placenta in normal pregnancy and as adaptations to maternal diet restriction

Abstract

The major milestones in mouse placental development are well described, but our understanding is limited to how the placenta can adapt to damage or changes in the environment. By using stereology and expression of cell cycle markers, we found that the placenta grows under normal conditions not just by hyperplasia of trophoblast cells but also through extensive polyploidy and cell hypertrophy. In response to feeding a low protein diet to mothers prior to and during pregnancy, to mimic chronic malnutrition, we found that this normal program was altered and that it was influenced by the sex of the conceptus. Male fetuses showed intrauterine growth restriction (IUGR) by embryonic day (E) 18.5, just before term, whereas female fetuses showed IUGR as early as E16.5. This difference was correlated with differences in the size of the labyrinth layer of the placenta, the site of nutrient and gas exchange. Functional changes were implied based on up-regulation of nutrient transporter genes. The junctional zone was also affected, with a reduction in both glycogen trophoblast and spongiotrophoblast cells. These changes were associated with increased expression of Phlda2 and reduced expression of Egfr. Polyploidy, which results from endoreduplication, is a normal feature of trophoblast giant cells (TGC) but also spongiotrophoblast cells. Ploidy was increased in sinusoidal-TGCs and spongiotrophoblast cells, but not parietal-TGCs, in low protein placentas. These results indicate that the placenta undergoes a range of changes in development and function in response to poor maternal diet, many of which we interpret are aimed at mitigating the impacts on fetal and maternal health.

Fig 1. Number of trophoblast nuclei in each zone of the mouse placenta as determined using stereology.

(A) Absolute number of trophoblast cell nuclei and total placenta volume. (B) Distribution of trophoblast cell nuclei among different layers of the placenta. (C) Number of trophoblast nuclei within the labyrinth layer (blue bars), as well as labyrinth volume (purple line) and trophoblast cell volume (orange line) from E12.5 to E18.5. (D) Trophoblast cell volume calculated as total trophoblast cell volume in the labyrinth (from C) divided by the number of trophoblast nuclei from E12.5 to E18.5. Different letter labels indicate significant differences between days (p<0.05) and are valid for both volume and total trophoblast number (n = 3).

Fig 2. Ki67 and Phospho-histone H3 immunofluorescence staining in P-TGCs, junctional zone and labyrinth.

Histological sections were dual stained for Ki67/K18 (red/green respectively) or phospho-histone H3/K18 (red/green respectively) and nuclei are counterstained with DAPI (blue). Phospho-histone H3 was detectable at E12.5 in sinusoidal-TGCs within the labyrinth (white arrow; x400 magnification in the inset) but not in SynT cells (red arrow). MBS–maternal blood space; FBS–fetal blood space.

Fig 3. Weights of fetuses and placentas, and fetal/placental weight ratios, in control and dietary protein restricted pregnancies.

(A) Fetal weights separated by sex and embryonic day (E). (B) Placental weights separated by sex and embryonic day. (C) Fetal to placental weight ratio (F:P) separated by sex and embryonic day. Explanatory variables–diet, sex, litter; Co-variates–maternal weight, litter size. Letter labels denote statistically significant main effects of diet within days (p<0.05). Main effects of sex (if present) are denoted by symbols at respective days (p<0.05). No diet by sex interactions were present. Number of conceptuses in each group (from left to right): E13.5–28, 32, 41, 39; E16.5–24, 37, 26, 24; E18.5–23, 42, 19, 32.

Fig 4. Labyrinth growth in low protein pregnancies is mediated by trophoblast hypertrophy and nutrient transport function.

(A) Total labyrinth area separated by diet and sex (n = 4–5). (B) Glucose and amino acid transporter expression in male and female placentas. Fold change in protein restricted placentas (bars) relative to control diet placental expression (dotted line). * p<0.05; ** p < 0.01 (n = 3).

Fig 5. RNA sequencing analysis of Con and LP placentas at E16.5.

(A) Principal component analysis with different points representing different litter (number) and sex within litter (male versus female). (B) Heat map of differentially expressed genes. (C) Gene network diagrams produced by GeneMania program, showing relationships between genes that are up- (red) or down-regulated (blue) in LP placentas (n = 5).

Fig 6. Spongiotrophoblast (SpT) cells contribute to the reduction in junctional zone size due to changes in cell size and number.

(A) Junctional zone area. (B) Total SpT cell area was measured in Image J using automated thresholding, distinguishing SpT cells by expression of Prl8a8 mRNA. (C) Spongiotrophoblast cell number (n = 4–5).

Fig 7. Sinusoidal trophoblast giant cell (S-TGC) and spongiotrophoblast (SpT) cell ploidy in control and protein-restricted pregnancies.

(A) Frequency of S-TGCs at each ploidy level across gestation. (B) Frequency of SpT cells at each ploidy level across gestation (n = 6).