PRECONCEPTION GUT DYSBIOSIS AFFECTS ENTERIC NERVOUS SYSTEM DEVELOPMENT AND FUNCTION OF OFFSPRING IN MICE
Preconception health affects not only fertility but also offspring health. Maternal gut dysbiosis may be related to intestinal development in offspring, but there is no evidence of the relationship between the preconception gut microbiota and offspring enteric nervous system (ENS) development. Therefore, we designed an animal study to investigate the connection between preconception antibiotic using and the development and function of the ENS in offspring.
Method:
Female C57BL/6 J mice were divided into 2 groups and given oral nonabsorbable antibiotic gavage (ABX) for one week, or water gavage as a control. Then they were pregnant and provided free access to food and water. Whole gut was collected at E13.5, E18.5, juvenile (3-week-old) and adult (8-week-old) offspring. The development and function of gut and ENS were evaluated by qPCR, H&E staining, immunofluorescence and electron microscopy. Transcriptome showed E18.5 gene expression of embryonic colon. Adult mice were exposed to water avoidance stress (WAS)/sham modeling. Gut motility and visceral sensation were measured. Feces and serum of E18.5 dams, juvenile and adult offspring were collected for metagenomic sequencing and targeted metabolomics analysis. Intervention with Limosilactobacillus reuteri (L.reuteri) during gestation was to rescue effect of preconception ABX.
Result:
Preconception ABX affects gut and ENS development in offspring. ABX mice have shorter intestine, slower ENS migration, lower density and less tuj1+ and sox10+ area in embryonic gut (Fig. 1B-C). Genes related to gut and ENS development are down-regulated in E18.5 colon of ABX embryo (Fig. 1D-E). ABX resulted in shortening of colon crypt and reduction of goblet cell in juvenile mice (Fig. 1F). The count of myenteric plexus neurons was reduced in adult ABX mice (Fig. 1G). Preconception ABX affects offspring's intestinal function and susceptibility to diseases. ABX mice have slower gut motility and weaker visceral sensation. However, the fold change in colonic transit time, ChAT+ neurons percentage, visceral sensitivity and the damage of colon epithelium in ABX-WAS mice was more severe (Fig. 1G, 2A-B). In addition to reveal the vertical transmission of maternal gut microbiota (not disclosed), multi-omics analysis of E18.5 embryonic transcriptome, maternal microbiome and metabolome found L.reuteri is one of the key species related to ENS development (Fig. 2C-E). Intervention with L.reuteri during gestation can partially improve ENS dysplasia caused by ABX (Fig. 2F-H).
Conclusion:
Preconception gut dysbiosis leads to dysplasia of the ENS in offspring, which appeared during embryonic stage, mediated by maternal gut microbiota and ultimately bring about intestinal diseases susceptibility. This study helps to identify preconception risk factors for gut development and provides a reference for preconception health care.
Figure 1: Effect of preconception ABX treatment on embryo, offspring, and offspring after WAS exposure. A: The main experimental schematic diagram of the study. B: Length, ENS migration and density of E13.5 embryonic intestinal (bar=50μm, n=6). C: Tuj1+ nerve fiber and Sox10+cell of E18.5 embryonic colon (bar=50μm, n=6). D: GO enrichment of E18.5 transcriptome. E: E18.5 ENS development-related mRNA expression in embryonic colon (n=7-8). F: Crypt and goblet cell in the colon of juvenile offspring stained by H&E and AB-PAS staining (20x, n=7-9). G: Hu+ and choline acetyltransferase+ (ChAT+) neuron staining in myenteric plexus of adult offspring after WAS/sham exposure (20x, n=8). Mean±SEM, *p<0.05, **p<0.01, ***p<0.001.
Figure 2: Multi-omics analysis intervention identified key species in maternal gut microbiota related to ENS development. A: Bead latency time and CRD-EMG at 20, 40, and 60 mmHg of offspring after WAS/sham exposure (n=6-9). B. Electron microscopy of colon epithelial of offspring after WAS/sham exposure (bar=500nm, n=3).C: Different species of E18.5 maternal gut microbiota. D: different metabolites of E18.5 maternal cecal content. E: Microbiota-metabolome-transcriptome correlation analysis reveals key species and key metabolites in maternal fecal. F: experimental schematic diagram of L.reuteri intervention during gestation. G-H: Length, ENS migration and density of E13.5 embryonic intestinal (bar=50μm, n=6). I: Tuj1+nerve fiber and Sox10+cell of E18.5 embryonic colon (bar=50μm, n=6). Mean±SEM, *p<0.05, **p<0.01, ***p<0.001.
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