Mid- to late term hypoxia in the mouse alters placental morphology, glucocorticoid regulatory pathways and nutrient transporters in a sex-specific manner

Abstract

Maternal hypoxia is a common perturbation that can disrupt placental and thus fetal development, contributing to neonatal impairments. Recently, evidence has suggested that physiological outcomes are dependent upon the sex of the fetus, with males more susceptible to hypoxic insults than females. This study investigated the effects of maternal hypoxia during mid- to late gestation on fetal growth and placental development and determined if responses were sex specific. CD1 mice were housed under 21% or 12% oxygen from embryonic day (E) 14.5 until tissue collection at E18.5. Fetuses and placentas were weighed before collection for gene and protein expression and morphological analysis. Hypoxia reduced fetal weight in both sexes at E18.5 by 7% but did not affect placental weight. Hypoxia reduced placental mRNA levels of the mineralocorticoid and glucocorticoid receptors and reduced the gene and protein expression of the glucocorticoid metabolizing enzyme HSD11B2. However, placentas of female fetuses responded differently to maternal hypoxia than did placentas of male fetuses. Notably, morphology was significantly altered in placentas from hypoxic female fetuses, with a reduction in placental labyrinth blood spaces. In addition mRNA expression of Glut1, Igf2 and Igf1r were reduced in placentas of female fetuses only. In summary, maternal hypoxia altered placental formation in a sex specific manner through mechanisms involving placental vascular development, growth factor and nutrient transporter expression and placental glucocorticoid signalling. This study provides insight into how sex differences in offspring disease development may be due to sex specific placental adaptations to maternal insults.

Figure 1. The effects of maternal hypoxia on maternal parameters and fetal glucose

The effect of maternal exposure to hypoxia (filled bars, 12% oxygen) from E14.5 to E18.5 on maternal plasma corticosterone (A), maternal plasma glucose (B), maternal average daily food consumption per gram of body weight (C) and maternal average daily water consumption per gram of body weight at E18.5 (D) compared to controls (open bars). All data are represented as means ± SEM. Control, n = 8; hypoxia, n = 7. *P < 0.05.

Figure 2. The effects of maternal hypoxia on markers of placental and fetal hypoxia

The effect of maternal exposure to hypoxia (12% oxygen) from E14.5 to E18.5 on pimonidazole adducts in placental tissue 60 min after pimonidazole injection (60 mg kg⁻¹ i.p.) (A), and placental immunohistochemical staining of HIF1A (red staining) in the junctional zone of placentas from control female fetuses (B), the junctional zone of placentas from hypoxic female fetuses (C), liver tissue taken from control female fetuses (D) and liver tissue taken from hypoxic female fetuses (E). Insets in B–E show isotype control staining in similar sections.

Figure 3. The effects of maternal hypoxia on placental morphology

A and B, the effect of maternal exposure to hypoxia (filled bars, 12% oxygen) from E14.5 to E18.5 on labyrinth total blood space (A) and labyrinth tissue space in placentas (B) from female fetuses compared to controls (open bars). n = 6 per per treatment group. C and D, representative images of placentas from female fetuses of control (C) and hypoxic dams (D). Images are taken at 400× magnification and scale bars represent 25 μm. Data are represented as means ± SEM. *P < 0.05 for unpaired t testing.

Figure 4. The effects of maternal hypoxia on placental transporter expression

A–D, the effect of maternal exposure to hypoxia (filled bars, 12% oxygen) from E14.5 to E18.5 on the relative mRNA levels of Slc2a1 (A), Slc38a2 (B), Slc2a3 (C) and Slc38a2 (D) in E18.5 placentas of male and female fetuses compared to controls (open bars). E and F, representative sections showing GLUT1 protein levels (GLUT1 encoded by Slc2a1 gene) and tissue distribution in control placentas (E) and hypoxic placentas (F). Insets in E and F show ‘no antibody’ control staining in adjacent sections. Data are represented as means ± SEM, with data being normalised to the mean of the values from the male control samples. *P < 0.05 for Sidak's multiple comparison testing compared to controls of same sex. n = 9–12 per sex from 8 litters per treatment group. Images are taken at 400× magnifiaction and scale bars represent 25 μm.

Figure 5. The effects of maternal hypoxia on placental vasculogenesis factor expression and the insulin like growth factor system

A–F, the effect of maternal exposure to hypoxia (filled bars, 12% oxygen) from E14.5 to E18.5 on the relative mRNA levels of Vegfa (A), Kdr (B), Flt1 (C), Igf2 (D), Igf1r (E) and Igf2r (F) in E18.5 placentas of male and female fetuses compared to controls (open bars). n = 9–12 per sex from 8 litters per treatment group. G and H, representative sections showing IGF1R protein levels and tissue distribution in control placentas (G) and hypoxic placentas (H). Insets in G and H show no antibody control staining in adjacent sections. Data are represented as means ± SEM, with data being normalised to the mean of the values from the male control samples *P < 0.05 for Sidak's multiple comparison testing compared to controls of the same sex. Images are taken at 100× magnifiaction and scale bars represent 100 μm.

Figure 6. The effects of maternal hypoxia on placental glucocorticoid regulatory system

A–D and F, the effect of maternal exposure to hypoxia (filled bars, 12% oxygen) from E14.5 to E18.5 on the relative mRNA levels of Crh (A), Crhr1 (B), Hsd11b2 (C), Nr3c1 (D) and Nr3c2 (F) in E18.5 placentas of male and female fetuses compared to controls (open bars). n = 9–12 per sex from 8 litters per treatment group. E, G and H, the effects of hypoxia on (E) HSD11B2 protein levels with representative Western blots for placentas of (G) male and (H) female fetuses shown below. n = 6 per sex per treatment group. Data are represented as means ± SEM, with data being normalised to the mean of the values from the male control samples. *P < 0.05 for Sidak's multiple comparison testing compared to controls of same sex.