Intrauterine hyperglycemia affects cognition of both F1 and F2 male offspring
We introduced moderate hyperglycemia during pregnancy through injection of STZ. Male F1 adults were then intercrossed to unexposed females to obtain F2 offspring (Fig. 1a). Both F1 and F2 offspring were analyzed in behavioral tests at young adulthood at 3–4 months old.
The spontaneous explorative and locomotor activity was assessed in the open field test. F1-GDM mice showed normal total explorative distance as well as the explorative activity in the central area (Fig. 1b). The spatial working memory was examined using the Y-maze. F1-GDM mice performed worse than control in Y-maze (Fig. 1c), suggesting impaired spatial memory. The similar result was observed in novel object test (NOR). In the NOR test, the amount of time spent with the novel object compared with the total time spent exploring both object represents an index of recognition memory. Compared to F1-control, F1-GDM mice spent less times investigating the novel object despite similar total exploration times, revealing a robust memory deficit (Fig. 1d).
Similar to F1 mice, F2-GDM mice showed normal locomotor activity (Fig. 1e), but displayed a significant deficit in spatial memory in the Y-maze test although there was no obvious difference in NOR test or object-in-place task (OiPT) associative recognition memory (Fig. 1f-h).
Altered transcriptome profiling in adult hippocampus from F1 and F2-GDM
The Gene expression patterns of hippocampus from 4-month-old F1 and F2 offspring (n = 5 per group) were analyzed using microarray to investigate the effect of long-term reprogramming caused by intrauterine hyperglycemia. By using a stringent threshold and significant criteria of adjusted P-value < 0.05 and |log2FoldChange| > 1, a total of 451 DEGs including 218 up-regulated and 234 down-regulated genes were identified in F1-GDM compared to Control, and, 1050 DEGs including 511 up-regulated and 539 down-regulated genes were identified in F2-GDM compared to Control (Fig. 2a, 2c, Table S2, Table S3).
Enrichment curves computed by Gene set enrichment analysis (GSEA) of F1 hippocampi were highly consistent with F2. GSEA of the hippocampi transcriptome in F1 and F2-GDM vs. Control revealed the robust enrichment of curated gene sets for axon guidance and Wnt signaling pathway, indicating that the expression of member genes were decreased in the F1-GDM and F2-GDM group (NES < 0), although the changes of F2-GDM were not as significant as F1-GDM. GSEA also provided insights into changes of activated pathways in F1-GDM and F2-GDM, including cocaine addiction, dopaminergic synapse and neuroactive ligand-receptor interaction (NES > 0) (Fig. 2b, 2d, Table S4,Table S5).
Gene ontology (GO) analysis revealed that DEGs were mainly enriched in ‘learning or memory’, ‘cognition’, ‘neuron to neuron synapse’, ‘postsynaptic specialization’, and ‘cAMP-mediated signaling’. There were several shared GO terms in both F1 and F2 hippocampus (Fig. 2e).
The overlapped DEGs of hippocampus in both F1 and F2 offspring
By overlapping DEGs of hippocampus in F1 and F2 offspring, we found that there were 106 genes up-regulated in both F1-GDM and F2-GDM mice compared to control mice, and 117 genes down-regulated in both F1-GDM and F2-GDM mice. There is no differentially expressed gene showing inconsistent tendency in F1-GDM and F2-GDM mice (Fig. 3a). According to the significance of the difference and gene function, we verified some meaningful candidate genes by qPCR, including Akap7, Atp6ap2, Camk2b, Dlgap1, Drd1, Gpr88, Map1b, Penk, S100b, Tanc1, Tubb2b, Wnt5a, Zeb2 (Fig. 3b). The results of qPCR were consistent with that of the microarray. Enrichment analysis of 223 shared DEGs indicated that GO terms were mainly involved in biological process such as ‘negative regulation of nervous system development’, ‘regulation of dopamine receptor signaling pathway’, ‘forebrain development’, ‘negative regulation of neuron differentiation’, ‘axon development’, ‘regulation of neurotransmitter transport and synapse organization’ and so on (Fig. 3c,Table S6).
The overlapped differentially methylated genes of F1-GDM sperm and differentially expressed genes of F2-GDM hippocampus
The F1-GDM male were exposed to hyperglycemia in utero. Therefore the cognitive impairment could be due to direct disruption of neurodevelopment by hyperglycemia. In comparison, cognition impairment and gene expression chagnes in F2-GDM is most likely caused by epigenetic mechanisms such as DNA methylation because F2-GDM is from F1-GDM males breeding with unexposed naïve females. We therefore used genome methylation sequencing to search for differentially methylated loci of sperm between control and F1-GDM. Intrauterine hyperglycemia resulted in 408 DMLs associated with 345 genes that were then used in enrichment analysis (Table S7). The DMLs were distributed in the upstream 2 k (8.58%), 5′-untranslated region (5′-UTR, 1.47%), coding sequence (CDS, 23.28%), introns (56.37%), 3′-UTR (5.15%), downstream 2 k (5.15%) (Fig. 4a). GO analysis identified a cluster of differentially methylated genes that were strongly related to ‘neuron development’, ‘neuron differentiation’ and ‘organ growth’ (Fig. 4b). By overlapping differentially methylated genes of sperm in F1 offspring and expressed genes of hippocampus in F2 offspring, we found 56 hypermethylated genes in F1-GDM sperm compared to Control. These 56 genes were down-regulated in F2-GDM hippocampus compared to Control (Fig. 4c). GO analysis showed that 56 genes were enriched in ‘neuron to neuron synapse’, ‘postsynaptic density/specialization’, ‘neuron projection development’, ‘structural constituent of synapse/postsynapse’, including Akap7, Camk2b, Dlgap1, Tanc1, Tubb2b, Wnt5a and Zeb2, most of which were down-regulated in hippocampus of both F1-GDM and F2-GDM (Fig. 2b). Additionally, within the ‘nerve system development, GO:0007399’ term in MGI database (http://www.informatics.jax.org/), a network of hypermethylated genes related to axon guidance (Htr7), positive regulation of astrocyte differentiation (Fryl), negative regulation of oligodendrocyte differentiation (Nfix), regulation of postsynaptic density assembly (Hoxb3), had a tendency for low expression in the F2-GDM versus the Control samples (Table 1). Of these genes, hypermethylated CpGs was most frequent in CDS regions, followed closely by intron regions.
Table 1
The crucial genes associated with central nervous system development are hypermethylated in F1-GDM sperm and down-regulated expression in F2-GDM hippocampus.
|
RRBS of Sperm
(F1-GDM vs. Ctrl)
|
Microarray of Hip
(F2-GDM vs. Ctrl)
|
Symbol
|
Start
|
End
|
meanMethy
Ctrl
|
meanMethy
F1-GDM
|
Element
|
logFC
|
adj. P Value
|
Tubb2b
|
34127486
|
34127641
|
16.03%
|
81.88%
|
CDS
|
-3.0865
|
0.001
|
Htr7
|
35969594
|
35969756
|
7.22%
|
73.70%
|
CDS 3'-UTR
|
-1.7638
|
0.249
|
Dlgap1
|
70516618
|
70516771
|
6.80%
|
86.87%
|
CDS
|
-1.2927
|
0.186
|
Tanc1
|
59843298
|
59843455
|
65.81%
|
81.41%
|
CDS
|
-1.1135
|
0.026
|
Zeb2
|
44988693
|
44988893
|
14.49%
|
85.39%
|
CDS
|
-0.7887
|
0.645
|
Wnt5a
|
28518390
|
28522909
|
26.62%
|
76.36%
|
CDS Intron
|
-0.5713
|
0.211
|
Hoxb3
|
96345925
|
96346054
|
7.08%
|
28.65%
|
CDS
|
-0.4266
|
0.644
|
Nfix
|
84784452
|
84784657
|
27.85%
|
45.75%
|
Intron
|
-0.2088
|
0.678
|
Nfix
|
84704134
|
84704384
|
38.83%
|
64.20%
|
Intron
|
-0.2088
|
0.678
|
Akap7
|
25251588
|
25251727
|
25.82%
|
77.69%
|
Intron
|
-0.2014
|
0.529
|
Camk2b
|
6010025
|
6010230
|
49.49%
|
89.20%
|
Intron
|
-0.1977
|
0.253
|
RRBS, reduced representation bisulfite sequencing; Hip, hippocampus; F1-GDM, the first filial of gestational diabetes mellitus; F2-GDM, the second filial of gestational diabetes mellitus; Ctrl, control group. |