Lack of placental neurosteroid alters cortical development and female somatosensory function

Abstract

Placental endocrine function is essential to fetal brain development. Placental hormones include neurosteroids such as allopregnanolone (ALLO), a regulator of neurodevelopmental processes via positive allosteric modulation of the GABA_A receptor (GABA_A-R). Using a mouse model (plKO) in which the gene encoding the ALLO synthesis enzyme is specifically deleted in trophoblasts, we previously showed that placental ALLO insufficiency alters cerebellar white matter development and leads to male-specific autistic-like behavior. We now demonstrate that the lack of placental ALLO causes female-predominant alterations of cortical development and function. Placental ALLO insufficiency disrupts cell proliferation in the primary somatosensory cortex (S1) in a sex-linked manner. Early changes are seen in plKO embryos of both sexes, but persist primarily in female offspring after birth. Adolescent plKO females show significant reduction in pyramidal neuron density, as well as somatosensory behavioral deficits as compared with plKO males and control littermates. Assessment of layer-specific markers in human postmortem cortices suggests that preterm infants may also have female-biased abnormalities in cortical layer specification as compared with term infants. This study establishes a novel and fundamental link between placental function and sex-linked long-term neurological outcomes, emphasizing the importance of the growing field of neuroplacentology.

Keywords: GABAA receptor (GABAAR); allopregnanolone (3α,5α-THP); neuroplacentology; placenta; postmortem human brain; preterm birth; somatosensory cortex (S1).

Figure 1

Impact of placental ALLO insufficiency on the cerebral cortex transcriptome at P30. (A, B) MA plots for the cortical RNAseq analysis of plKO vs Ctrl mice at P30 in males (A) and females (B). M, log ratio; A, mean average. DEGs are represented as colored dots (blue for males and magenta for females). (C, D) Top 15 significant enriched gene functional groups identified with Ingenuity Pathway Analysis software (Qiagen) in males (C) and females (D). P-values were calculated using right-tailed Fisher’s exact tests.

Figure 2

Diffusion Tensor Imaging (DTI) analysis in the somatosensory cortex of Ctrl and plKO mice at P30. Heatmap representing mean diffusivity (MD) across the different somatosensory areas. MD was significantly decreased in the female plKO somatosensory cortex, as compared with their littermate controls. Data presented in a hierarchical tree. Three-way sex by genotype by region ANOVA: main genotype effect: ★★★★★p<0.0005; sex × genotype interaction: #p<0.05. Tukey’s multiple comparison test: ****p<0.001. n = 11 M Ctrl, 8 F Ctrl, 13 M plKO, 11 F plKO. F, female; M, male.

Figure 3

Long-term effect of placental ALLO insufficiency on cortical lamination. (A) Representative immunofluorescent staining for Cux1 (red) and FoxP2 (cyan) in the female S1 area at P30. (B, C) Quantification of (A) shows that the density of Cux1+ upper layer principal cells is significantly decreased (layers II-IV) in plKO females but that the density of FoxP2+ deeper layer principal cells remained unaltered. n = 8-15 M Ctrl, 5-12 F Ctrl, 6-9 M plKO, 7-14 F plKO. Data presented as mean ± SEM. Two-way sex by genotype ANOVA with Sidak’s post-hoc comparison test: *p < 0.05 identified in magenta for females. Cux1, Cut like homeobox 1; F, female; FoxP2, Forkhead box protein P2; M, male. DL, deep layer; UL, upper layer.

Figure 4

Long-term effect of placental ALLO insufficiency on behavior. (A) Schematic diagram of the novel object recognition test, designed to assess the somatosensory function. (B) Female plKO mice have significant deficits in the novel object recognition task, as measured by the recognition index (RI), which corresponds to the ratio between the time spent exploring the new object relative and the total exploration time. n = 12 M Ctrl, 8 F Ctrl, 10 M plKO, 8 F plKO. Data presented as mean ± SEM. (C) Diagram of open field paradigm. (D) Total distance travelled by Ctrl and plKO mice in open field test. (E) Time spent in center quadrants of the open field. n = 6 M Ctrl, 9 F Ctrl, 8 M plKO, 6 F plKO. (F) Diagram of alternating Y-maze. (G) Alternation score (# alternations/# of possible alternations x 100). n = 9 M Ctrl, 14 F Ctrl, 7 M plKO, 5 F plKO. Data presented as mean ± SEM. Two-way sex by genotype ANOVA with Sidak’s post-hoc comparison test: **p < 0.01 identified in magenta for females. Illustrations in (A, C, F) were prepared using BioRender.com.

Figure 5

Influence of placental ALLO insufficiency on mitosis and cell cycle kinetics in the cerebral cortex during mid-to-late gestation. (A) Immunofluorescent staining of pH3, a mitotic marker, in the cerebral cortex at E15.5. (B) Quantification of (A) reveals no change in the proportion of SVZ cells undergoing active mitosis in the plKO cerebral cortex at E.15.5. n = 8 M Ctrl, 6 F Ctrl, 5 M plKO, 7 F plKO. (C) Overview of experiments to determine cell cycle exit and kinetics at E15.5. (D) The different phases of cell cycle. (E) Immunofluorescent staining of EdU (magenta), marking proliferating cells at E15.5, dual pulsed with BrdU (green), injected 3 hours later, to identify cells in cycle and cells that had exited. (F) Quantification of the cell cycle exit ratio in the SVZ shows a significant loss in cell cycle exit in plKO mice at E15.5. (G) Significant increase in the SVZ cell cycle length (Tc) in plKO females associated with an (H) elongated S-phase (T_S). n = 6 M Ctrl, 5 F Ctrl, 4 M plKO, 5 F plKO. Data presented as mean ± SEM. Two-way sex by genotype ANOVA with Sidak’s post-hoc comparison test: *p < 0.05 identified in magenta for females. F, female; M, male; pH3, phospho-Histone 3; SVZ, subventricular zone.

Figure 6

Effect of placental ALLO insufficiency on cortical neurogenesis. (A) Overview of the experiments: BrdU was injected at E15.5, and embryos were euthanized at E17.5. (B) Determination of the localization of proliferating E15.5 cells (BrdU, green) by co-staining with Satb2 (red) upper layer principal cell marker and Ctip2 (cyan) lower layer principal cell marker. (C) Quantification of (B) shows that while deeper layers were unaltered, (D) fewer E15.5 born cells localized to the upper layers of the cortical plate. n = 5 M Ctrl, 4-5 F Ctrl, 3-4 M plKO, 4-5 F plKO. Data presented as mean ± SEM. Two-way sex by genotype ANOVA with Sidak’s post-hoc comparison test. Main effect of genotype: ★★p < 0.01. Ctip2, COUP-TF-interacting protein 2; F, female; M, male; Satb2, Special AT-rich sequence-binding protein 2. SVZ, sub-ventricular zone.