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. 2020 Sep 29;15(9):e0239681.
doi: 10.1371/journal.pone.0239681. eCollection 2020.

"VSports" The Znt7-null mutation has sex dependent effects on the gut microbiota and goblet cell population in the mouse colon

Mary E Kable  1   2 , Niknaz Riazati  2 , Catherine P Kirschke  3 , Junli Zhao  4 , Surapun Tepaamorndech  5 , Liping Huang  2   3
Affiliations

The Znt7-null mutation has sex dependent effects on the gut microbiota and goblet cell population in the mouse colon (VSports)

Mary E Kable et al. PLoS One. .

VSports app下载 - Abstract

Cellular homeostasis of zinc, an essential element for living organisms, is tightly regulated by a family of zinc transporters. The zinc transporter 7, ZnT7, is highly expressed on the membrane of the Golgi complex of intestinal epithelial cells and goblet cells. It has previously been shown that Znt7 knockout leads to zinc deficiency and decreased weight gain in C57BL/6 mice on a defined diet. However, effects within the colon are unknown. Given the expression profile of Znt7, we set out to analyze the changes in mucin density and gut microbial composition in the mouse large intestine induced by Znt7 knockout. We fed a semi-purified diet containing 30 mg Zn/kg to Znt7-/- mice with their heterozygous and wild type littermates and found a sex specific effect on colonic mucin density, goblet cell number, and microbiome composition. In male mice Znt7 knockout led to increased goblet cell number and mucin density but had little effect on gut microbiome composition. However, in female mice Znt7 knockout was associated with decreased goblet cell number and mucin density, with increased proportions of the microbial taxa, Allobaculum, relative to wild type. The gut microbial composition was correlated with mucin density in both sexes VSports手机版. These findings suggest that a sex-specific relationship exists between zinc homeostasis, mucin production and the microbial community composition within the colon. .

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Znt7-/- mice show reduced body weight after 4 weeks on a semi-purified diet (30 mg/kg Zn).
Offspring, including Znt7-/-, Znt7+/-, and WT produced from Znt7+/- breeding pairs, were fed a semi-purified rodent diet containing 30 mg Zn/kg ad libitum between 6- and 10-weeks of age. Body weights obtained at 6- and 10-weeks of age are shown. All values are expressed as mean±S.E. (error bars), n = 6-12/group. *, p<0.05 (to the WT control in the same sex group); **, p<0.01 (to the WT control in the same sex group). WT, wild type control mice; Het, heterozygous Znt7 knockout mice; Znt7KO, homozygous Znt7 knockout mice.
Fig 2
Fig 2. Mucin staining and quantification in the mouse colon.
(A) Representative staining from the colon tissue (1 cm distal to the cecum) isolated from (a) female WT, (b) female Znt7-/-, (c) male WT, and (d) male Znt7-/- mice. The sections were stained with Alcian blue/PAS and counterstained with Nuclear Fast Red to visualize cell nuclei. Arrows indicate goblet cells. * indicates the lumen side of the colon and ** indicate the basal side of the colon. ⁂ indicates the epithelium of the villus. Scale bar = 25 μm. (B) Representative images for automated quantification of color shades in the colonic villus using TissueQuant software. (a) An Alcian blue/PAS stained image. The solid and open arrows indicate neutral and acidic mucins stained magenta and bright blue, respectively. The gray arrow denotes the overlapping of neutral and acidic mucins (dark blue/purple). TissueQuant masking for (b) magenta color shade for neutral mucins, (c) blue color shade for acidic mucins, and (d) dark blue/purple color shade for both neutral and acidic mucins.
Fig 3
Fig 3. Sex specific differences in mucin density and goblet cell numbers in the colon.
(A) Quantification of mucin density measured by Alcian blue/PAS staining in the villi of male and female WT and Znt7-/- mice. (B) Goblet cell numbers in the villi of the same mice. (C and D) Correlation of mucin density measurement with goblet cell numbers from females and males, respectively. Total color in pixels represents the sum of magenta, blue, and purple intensities, as described in Fig 2, to represent total mucin content. All values are expressed as mean±S.E. (error bars), n = 15–18 villi from 5–6 mice/sex/genotype. *, p<0.05; **, p<0.01.
Fig 4
Fig 4. mRNA expression of the Znt and Zip genes in the mouse colon.
(A) mRNA expression of the Znt genes 1–10 and (B) mRNA expression of the Zip genes 1–14 in colonic tissue distal to the cecum measured by qRT-PCR. The expression of Actb was used as an internal reference for quantitation of target gene expression using the 2–ΔΔCt method [23]. Values represent the average of technical triplicates for n = 4–5 mice/genotype/sex. The expression of the target genes was compared to the transcription level in male WT by Student’s t-test. *, p<0.05; **, p<0.01. WT, wild type; Znt7KO, Znt7 knockout.
Fig 5
Fig 5. Microbial community structure differs between male and female Znt7+/+(WT) mice.
(A) Principal coordinate analysis of the weighted UniFrac distance measures between gut microbial communities from male and female WT mice. (B) Boxplots of the relative abundances of bacterial taxa that were significantly differentially abundant between male and female mice as determined by DESeq2 analysis. (C and D) Boxplots of the relative abundance of (C) Coriobacteriaceae and (D) AF12 with the y-axes adjusted showed differences between sexes more clearly than in panel B. *, p<0.05; **, p<0.01.
Fig 6
Fig 6. Sex specific effects of Znt7 knockout on overall bacterial community structure.
(A and B) Principal coordinate analysis of the weighted UniFrac distance measures between microbial communities from Znt7+/+ (WT), Znt7+/- (HET) and Znt7-/- (KO) in (A) male and (B) female mice. (C and D) Boxplots of microbial community evenness, represented by Pielou's evenness index and Shannon diversity index respectively. *, p<0.05; **, p<0.01.
Fig 7
Fig 7. Bacterial taxa significantly differed by Znt7 genotype in a sex dependent manner.
(A and B) Barplots showing proportions of bacterial taxa present in mouse feces. The microbial community composition of each mouse is represented by a single bar. The proportions of taxa are shown on the y-axis and the cage that each mouse was housed in during the study is shown on the x-axis. Taxa present at less than 2% relative abundance were grouped into the “Other” category. The most specific taxonomic classification obtained is shown and the displayed taxon level is represented by a single letter code preceding the classification; o = order, f = family, g = genus. (C and D) Boxplots of the relative abundance of Allobaculum and Coriobacteriaceae respectively within WT, Znt7+/- (HET) and Znt7-/- (KO) mice. A log ratio test, followed by pairwise testing performed in DESeq2 showed a significant sex-specific effect of genotype for these bacterial taxa. *, p<0.05; **, p<0.01.
Fig 8
Fig 8. Relationship between mucin density and microbial community in the mouse colon.
(A and B) The y-axes in each graph show mucin density values for a subset of the mice. The x-axis shows (A) the first dimension of principal coordinates from the analysis of weighted UniFrac measurements between the microbial communities or (B) the relative proportion of Allobaculum in each 10-week old mouse. A regression line describing the linear relationship between each of the two variables and mucin density is shown. Spearman rho = 0.53472, p = 0.01142 and Spearman rho = -0.53811, p = 0.01083 for each of the comparisons respectively.
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