Inhibition of inosine metabolism of the gut microbiota decreases testosterone secretion in the testis

Abstract

Growing evidence indicates that gut microbiota is involved in the regulation of the host's sex hormone levels, such as through interfering with the sex hormone metabolism in the intestine. However, if gut microbiota or its metabolites directly influence the sex hormone biosynthesis in the gonad remains largely unknown. Our previous study showed that colistin, as a narrow-spectrum antibiotic, can significantly downregulate the serum testosterone levels and thus enhance the antitumor efficiency of anti-PD-L1 in male mice; however, the underlying mechanism for the regulation of the host's testosterone levels remains uninvestigated. In the present study, we analyzed the impact of colistin on the immune microenvironment of the testis as well as the composition and metabolism of gut microbiota in male mice. Our results showed that colistin has an impact on the immune microenvironment of the testis and can downregulate serum testosterone levels in male mice through inhibition of Akkermansia, leading to destroyed inosine metabolism. Supplement with inosine can restore testosterone secretion probably by prompting the recovery of the intestinal mucus barrier and the serum lipopolysaccharides levels. All these findings reveal a new pathway for the regulation of the host's sex hormone levels by gut microbiota.IMPORTANCEThis study demonstrates that exposure to even narrow-spectrum antibiotics may affect the host's testosterone levels by altering the gut microbiota and its metabolites. Our findings provide evidence that some specific gut bacteria have an impact on the sex hormone biosynthesis in the testis.

Keywords: Akkermansia; antibiotic; colistin; gut microbiota; inosine; testis; testosterone.

Fig 1

The impacts of colistin on serum testosterone levels and testicular immune microenvironment in male mice. After treatment with 1 mg/mL of colistin in sterile drinking water for 1 week, serum testosterone levels were measured by ELISA in non-castrated mice (a) or castrated mice (b). (c) Gating strategy for flow cytometry analysis. PD-L1 expression (d), infiltration of macrophages (e), TNFα expression in macrophages (f), and infiltration of CD8⁺ T cells (g), CD8⁺ IFNγ⁺ T cells (h), CD8⁺ Granzyme B⁺ T cells (i), CD4⁺ T cells (j), CD4⁺IFNγ⁺ Th1 cells (k), CD4⁺IL-4⁺ Th2 cells (l), CD4⁺IL-17⁺ Th17 cells (m), and CD4⁺ Granzyme B⁺ T cells (n) in the testis were analyzed by FACS. All data are presented as mean ± SEM. *P < 0.05, and **P < 0.01. CTR, control; COL, colistin.

Fig 2

Effects of colistin on the gut microbiota composition in male mice. (a) Comparison of alpha diversity indices at the species level. (b) Comparison of the total absolute abundance. Principal component analysis of the absolute (c) and relative (d) abundances of ASVs. The absolute (e) and relative (f) composition of the gut microbiota at the phylum level. (g) Comparison of the absolute abundances of Verrucomicrobia, mean ± SEM, Mann-Whitney test. *P < 0.05.

Fig 3

The alteration of the gut microbiota at different taxonomic levels and changes in the potential functional profiles. (a) Comparison of the gut microbiota composition between the groups by statistical analysis of taxonomic and functional profiles at the genus level. P values were derived from the Wilcoxon rank sum test. (b) LEfSe analysis of the gut microbiota composition. (c) Co-occurrence network graphs of the gut microbiota. (d) Heat map of differentially abundant KEGG pathways based on the PICRUSt2 analysis.

Fig 4

Colistin treatment changes the gut microbiota metabolism in male mice. (a) The Pearson correlation of quality control samples. (b) PCA analysis of the QC and experimental samples in both positive and negative modes. (c) OPLS-DA score plot. (d) Cross-validation plot with a permutation test repeated 200 times. The intercepts of the goodness-of-fit (R²) are greater than the goodness-of-prediction (Q²), and the intercept of Q² is less than 0, indicating no overfitting. (e) Volcanic map of the differential metabolites (red, upregulated; blue, downregulated). Student’s t-tests were performed for the comparison. (f) Heat map of differential metabolites. (g) KEGG pathway enrichment analysis of differential metabolites and the pathway diagram of purine metabolism derived from the KEGG database (red, upregulated; blue, downregulated).

Fig 5

Supplementation of inosine promotes the recovery of the gut barrier and testicular immunosuppressive microenvironment and restores the serum testosterone levels in male mice. (a) Schematic diagram of the experimental design and sample collection. For inosine treatment, mice were given 300 mg/kg inosine by gavage daily for 1 week, with sterile PBS as the vehicle control. (b) Serum testosterone levels were measured by ELISA. (c) Representative images of PAS/Alcian Blue staining of the colon. Scale bars: 20 µm. (d) The thickness of the mucus layer. (e) Serum levels of LPS. PD-L1 expression (f) and infiltration of CD4⁺ Granzyme B⁺ T cells (g) in the testis were analyzed by FACS. All data are presented as mean ± SEM. *P < 0.05, and **P < 0.01. CTR, control; COL, colistin; INO, inosine; CI, colistin plus inosine.

Fig 6

Inhibition of Akkermansia decreases the production of inosine in the gut, leading to the disruption of the colonic mucus layer. The abnormal serum LPS level due to the increased gut permeability may contribute to the inhibition of testosterone secretion by modulating the immune microenvironment within the testis.