Intrauterine Growth Restriction Causes Abnormal Embryonic Dentate Gyrus Neurogenesis in Mouse Offspring That Leads to Adult Learning and Memory Deficits

A, Percentage of duration of time spent with the left or right object on training day. Light gray bars denote the left object. Dark gray bars denote the right object. Sham and IUGR offspring spent an equal duration of time exploring left and right objects without sex differences. NS, No significance. B, Discrimination index [(time spent with novel object – time spent with old object)/time spent with novel object plus time spent with old object)] on testing day. +1, More time spent with the novel object; 0, no preference; −1, More time spent with the old object. IUGR females spent more time with the old object, demonstrating a lack of memory to the exposure of the old object from previous day compared with sham females. p = 0.048, IUGR females versus sham females. C, Percentage of time spent freezing in contextual conditioning. IUGR offspring of both sexes spent less time freezing when the audible tone was heard but no footshock followed. p = 0.039, IUGR females compared with sex-matched sham controls; p = 0.049, IUGR males compared with sex-matched sham controls. D, Percentage of time spent freezing in cued test. IUGR offspring of both sexes spent less time freezing when placed in the same chamber in which a previous audible tone gave rise to a footshock. p = 0.035, IUGR females compared with sex-matched sham controls; p = 0.048, IUGR males compared with sex-matched sham controls. Two-way ANOVA with Tukey’s HSD post hoc test was used to examine the effects of treatment (sham or IUGR), sex, or both sex and treatment on each main effect (percentage of time spent exploring left or right object, discrimination index, percentage of time freezing in contextual conditioning or cued test).

A, Representative photomicrographs of Sox2⁺ NSCs in E15.5 developing DG. Blue DAPI stain denoted cell nuclei. i, ii, Magnified views of Sox2⁺ NSCs in the white dashed regions of A. IUGR offspring of both sexes showed decreased percentages of Sox2⁺ NSCs (number of Sox2⁺ cells divided by the number of DAPI⁺ cells in the yellow dashed outlined area) in the VZ and MZ. p = 0.04, IUGR males and females compared with sex-matched shams. F, Fimbria. Yellow dashed outlines delineate the quantified areas that were manually traced in ImageJ beginning at the dentate notch (DN) to the beginning of the fimbria on the ventricular side and across to the pial surface. B, Representative photomicrographs of EdU⁺ proliferative cells in E15.5 developing DG. Blue DAPI stain denoted cell nuclei. IUGR offspring of both sexes showed decreased percentages of EdU⁺ proliferative cells (the number of EdU⁺ cells divided by the number of DAPI⁺ cells in the yellow dashed outlined area; i.e., cells in S phase of cell cycle, in VZ and MZ. p = 0.045, IUGR males and females compared with sex-matched sham controls. Student’s t test was used to compare sham and IUGR offspring for Sox2 and EdU, given that no differences were noted when sex was separated. C, Representative photomicrographs of E15.5 sham and IUGR developing DG with EdU and pHH3 at Ser 10 immunofluorescent costaining. White dashed boxed areas denoted as i and ii are enlarged regions demonstrating the colocalization of red EdU⁺ cells with green pHH3⁺ cells in yellow (arrows). Both IUGR males (p = 0005) and females (p = 0.0004) had a decreased number of EdU⁺/PHH3⁺ colocalized cells compared with sex-matched shams showing that IUGR progenitors had fewer cells completing mitosis of the cell cycle. Two-way ANOVA with Tukey’s HSD post hoc test was used to examine the effects of treatment (sham or IUGR), sex, or both sex and treatment on the number of EdU⁺/pHH3⁺ colocalized cells. D, Representative photomicrographs of Tbr2⁺ in E15.5 developing DG. Blue DAPI stain denoted cell nuclei. IUGR offspring of both sexes showed increased percentages of Tbr2⁺ INPs (the number of Tbr2⁺ cells divided by the number of DAPI⁺ cells) in the VZ, MZ, and FDJ. p = 0.038, IUGR males and females compared with sex-matched sham controls. Student’s t test was used to compare sham and IUGR offspring given no differences were noted when sex was separated.

A, Representative photomicrographs of Sox2⁺ NSCs and EdU⁺ proliferative cells in E19 dentate anlage. Blue DAPI stain denoted cell nuclei. IUGR offspring of both sexes showed decreased percentages of Sox2⁺ NSCs in the VZ and migratory stream (MS). p = 0.022, IUGR males compared with sex-matched sham controls; p = 0.005, IUGR females compared with sex-matched sham controls. By contrast, IUGR offspring of both sexes showed similar percentages of EdU⁺ proliferative cells in VZ and migratory stream. Dotted eclipses delineate the area of dentate anlage manually traced in ImageJ. B, Representative photomicrographs of Tbr2⁺ INPs in E19 dentate anlage. IUGR female offspring showed an increased percentage of Tbr2⁺ INPs in the MS and dentate anlage (p = 0.01, IUGR females compared with sham females). IUGR males had a percentage of Tbr2⁺ INPs similar to that of sham males. Two-way ANOVA with Tukey’s HSD post hoc test was used to examine the effects of treatment (sham or IUGR), sex, or both sex and treatment on the percentages of Sox2⁺, EdU⁺, or Tbr2⁺ cells.

A, Representative photomicrographs of NeuroD⁺ NPs in E19 dentate anlage. IUGR offspring of both sexes had increased percentages of NeuroD⁺ NPs (number of NeuroD⁺ cells divided by number of DAPI⁺ cells) in dentate anlage (p = 0.015, IUGR males and females compared with sex-matched sham controls). B, Representative photomicrographs of Prox1⁺ immature and mature glutamatergic granule neurons in E19 dentate anlage. IUGR offspring of both sexes had increased percentages of Prox1⁺ granule neurons (the number of Prox1⁺ cells divided by the number of DAPI⁺ cells) in dentate plate and granule cell layers of the future suprapyramidal and infrapyramidal blades (p = 0.009 and p = 0.008, respectively in IUGR males and females compared with sex-matched sham controls). Two-way ANOVA with Tukey’s HSD post hoc test was used to examine the effects of treatment (sham or IUGR), sex, or both sex and treatment on the percentages of NeuroD⁺ or Prox1⁺ cells.

P18 and P40 hippocampal volumes (in mm³) including DG and CA regions. A, IUGR decreased P18 hippocampal volumes in both males and females compared with sex-matched sham controls. p = 0.024, IUGR compared with sham controls. B, IUGR also decreased P40 hippocampal volumes in both males and females compared with sex-matched shams. p = 0.0007, IUGR mice compared with sham controls. Sex differences in hippocampal volumes are shown to the right of A and B. Two-way ANOVA with Tukey’s HSD post hoc test was used to examine the effects of treatment (sham or IUGR), sex, or both sex and treatment on hippocampal volumes.

A–D, Representative photomicrographs of P18 (A, B) and P40 (C, D) hippocampal DG showing double-labeling of DCX and Calb1 immunofluorescent staining. DAPI was used as a counterstain for all cell nuclei that have been rendered non-nuclear under the Imaris “DAPI surface reconstruction” mode for quantification. GC, Granule cells; Hy, hilus. Within each DCX fluorescent image in A–D, an enlarged region boxed as region a shows the DCX cell body volume and dendritic branching. Quantification of P18 sham and IUGR hippocampi using DAPI showed similar DG volumes. NS, No significance. Quantification of P40 sham and IUGR hippocampi however showed smaller DG volumes (p = 0.043). Quantification of DG cell counts showed no difference between sham and IUGR hippocampal DG at P18 or P40. Quantification of DCX⁺ cell characteristics showed that IUGR decreased P18 DCX⁺ cell volumes but increased mean dendritic lengths. Last, quantification of Calb1⁺ cell characteristics showed no differences in cell volumes or counts between sham and IUGR at P18 or P40 (NS). Given that these were all nonparametric variables, Mann–Whitney U test was used to compare sham and IUGR groups. No sex differences were noted.