Gut Biogeography Accentuates Sex-Related Differences in the Murine Microbiome

Abstract

Recent studies have highlighted the influence of factors such as sex and sex-linked hormones on microbiome composition, raising concerns about the generalizability of findings. Here, we explore whether gut geography, specifically the upper and lower gastrointestinal tract (GI), contributes to sex-linked microbiome differences in mice. We collected microbial samples throughout the length of the GI from male and female C57B6/J mice at 6- and 8-weeks old, and conducted 16S rRNA sequencing. Our findings revealed significant sex-related differences, with Clostridium_sensu_stricto_1 more abundant in the male colon, while females exhibited higher levels of Dubosiella newyorkensis across all organs at 6 weeks. We also observed decreased Shannon alpha diversity in the small intestine compared to the lower GI, and this diversity decreased further at 8 weeks. Interestingly, our results suggest that age mitigates sex-related, but not gut geography-related differences in beta diversity, with implications for experimental outcomes and treatment strategies. This study underscores the dynamic nature of microbial diversity, influenced by sex, age, and GI localization, emphasizing the need for a more comprehensive understanding of microbiome dynamics in experimental research and clinical interventions.

Keywords: gut microbiota; murine microbiome; sex differences; upper gastrointestinal tract.

Figure 1

Sex and Age Differences Vary Between Upper and Lower GI. (A) Shannon alpha diversity throughout all collected GI locations for both sexes at 6 and 8 weeks old. Significance values as follows: ** p < 0.01, *** p < 0.001. Non-significant values are not labeled. (B) Beta dispersion analysis using Canberra method comparing upper and lower GI samples at 6 and 8 weeks old (p-val: 0.029). (C) Genus-level relative abundance plot of proximal small intestine and colon for both sexes at 6 and 8 weeks old. “Other” label refers to the sum of the rest of the features found in each group (beyond the top 23). “Uncultured” is a SILVA database term which refers to samples for which the genus-level taxonomic assignment was resolved, but this level was unassigned/unannotated in the SILVA database itself. Repeated labels correspond to different OTUs. (D) Taxa significantly associated with sex and proximal small intestine or colon calculated with DESeq2 R package (v.1.40.2).

Figure 2

Taxa significantly associated with sex and age. (A–D) Taxa significantly associated with estradiol in females at 8 weeks of age identified using the MaAsLin2 package (v.1.4.0). Plotted relative abundance and Mann-Whitney statistical analysis. Significance values as follows: * p < 0.05 and **** p < 0.0001, ns = non-significant. (E) Female and (F) male upper GI at both 6 and 8 weeks old using the DESeq2 R package (v.1.40.2). Dashed lines indicate a p-adjusted value of 0.05.