Disrupted spermatogenesis in a metabolic syndrome model: the role of vitamin A metabolism in the gut-testis axis

Abstract

Objective: Effects of the diet-induced gut microbiota dysbiosis reach far beyond the gut. We aim to uncover the direct evidence involving the gut-testis axis in the aetiology of impaired spermatogenesis.

Design: An excessive-energy diet-induced metabolic syndrome (MetS) sheep model was established. The testicular samples, host metabolomes and gut microbiome were analysed. Faecal microbiota transplantation (FMT) confirmed the linkage between gut microbiota and spermatogenesis.

Results: We demonstrated that the number of arrested spermatogonia was markedly elevated by using 10× single-cell RNA-seq in the MetS model. Furthermore, through using metabolomics profiling and 16S rDNA-seq, we discovered that the absorption of vitamin A in the gut was abolished due to a notable reduction of bile acid levels, which was significantly associated with reduced abundance of Ruminococcaceae_NK4A214_group. Notably, the abnormal metabolic effects of vitamin A were transferable to the testicular cells through the circulating blood, which contributed to abnormal spermatogenesis, as confirmed by FMT.

Conclusion: These findings define a starting point for linking the testicular function and regulation of gut microbiota via host metabolomes and will be of potential value for the treatment of male infertility in MetS.

Keywords: bile acid metabolism; obesity; vitamins.

Figure 1

High-caloric load induced by excessive energy intake disrupted spermatogenesis in a metabolic syndrome (MetS) model. (A) The representative testicular sections are stained with H&E. Scale bar=100 μm. (B) The percentage of abnormal tubules in testicular samples from each group (n=5). (C) The representative epididymal sections are stained with H&E. (D) Statistical analysis of sperm counts (n=5). (E) UMAP plot of germ cell clusters in testes defined by scRNA-seq analysis. (F) The percentage of each germ cell cluster from three groups. (G) Single-cell trajectories of SPG subsets are shown with cells ordered in pseudotime. Branch point represents distinct germ cell transition states. (H) The SPG trajectory is colored by three cell states. (I) Heatmap represents significant DEGs between each cluster at differentiated SPG and SSC pool stages at branch point. (J) Gene Ontology terms of differentially expressed genes (DEGs) in the SPG cluster from (a) ND versus MED and (b) ND versus HED groups. HED, high-energy diet; MED, medium-energy diet; ND, normal diet; scRNA-seq, single-cell RNA-seq; SPC, spermatocyctes; SPG, spermatogonia; SSC, spermatogonia stem cell; UMAP, uniform manifold approximation and projection.

Figure 2

Absorption of vitamin A was disturbed in a MetS model. (A) Orthogonal projections to latent structures-discriminant analysis (PLS-DA) score plot for discriminating the intestine digesta metabolome from the ND, MED and HED groups (n=5). (B) Disturbed metabolic pathways in the ND versus MED and ND versus HED groups. (C) Heatmaps of the differential metabolites including eight vitamins that were altered through MED and HED feeding compared with ND feeding. (D) Comparison of the relative abundance of (a) vitamin E, (b) retinol, (c) retinene, (d) cholic acid, (e) glycocholic acid, (f) lithocholic acid and (g) tauroursodeoxycholic acid in the indicated groups. HED, high-energy diet; MED, medium-energy diet.

Figure 3

Profiling of the gut microbiota was altered in a MetS model. (A) Bray-Curtis principal coordinate analysis (PCoA) plot of gut microbiota based on the operational taxonomic unit metrics of the samples in the ND, MED and HED groups (n=9). (B) Bacterial taxonomic profiling at the phylum level of intestinal bacteria from different treated groups. (C) (a) Shannon index and (b) Simpson index of gut microbiota in three treated groups. (D) Heatmap of differential abundance from the MED and HED groups compared with the ND group. The color of the spots in the left panel represents the relative abundance at the genus level in each group. HED, high-energy diet; MED, medium-energy diet; MetS, metabolic syndrome.

Figure 4

The alteration of gut microbiota was closely associated with the bile acid levels. (A) Pearson’s correlations of retinol and retinene levels with the levels of (a) CA, (b) LCA and TUDCA. Red indicates a positive correlation, and blue indicates a negative correlation. (B) Spearman’s correlation analysis of differential genus microbiota levels with CA, LCA and TUDCA levels. (C) Spearman’s correlation analysis of Ruminococcaceae_NK4A214_group abundance with (a) CA, (b) LCA and (c) TUDCA levels. CA, cholic acid; LCA, lithocholic acid; TUDCA, tauroursodeoxycholic acid.

Figure 5

The effect of abnormal vitamin A metabolism was transferable to testis. (A) The testicular vitamin A levels of the ND and HED fed groups were measured (n=6). (B) Serum vitamin A levels of the ND, MED and HED-fed groups were examined (n=6). (C) The representative images of RBP4 and RDH10 immunofluorescent (IF) staining in sections of testicular tissue. Scale bar=50 μm. (D) The IF staining for STRA8 and SYCP3 is shown together with MVH in representative sections of testicular tissue. Scale bar=25 μm. (E) Western blot of STRA8, RDH10, MVH, GAPDH and β-ACTIN in each indicated group. (F) Quantification protein levels of (a) STRA8, (b) RDH10 and (c) MVH. HED, high-energy diet; MED, medium-energy diet.

Figure 6

Faecal transplants of HED microbiota exhibited disrupted spermatogenesis induced by a notable reduction in levels of vitamin A and RDH10. (A) Study design diagram of the faecal transplant experiment. (B) Vitamin A level measured using an ELISA kit (n=6 for ND-FMT; n=10 HED-FMT). (C) The qPCR analysis of Ruminococcaceae_NK4A214_group abundance (n=9). (D) H&E staining of ND-FMT and HED-FMT testicular tissue. (E) Percentage of abnormal tubules in testicular tissue from each group (n=7). (F) Representative IF staining images of STRA8 and RDH10 in sections of testicular tissue. Scale bar=50 μm. (G) Western blotting of STRA8, RDH10, MVH and β-ACTIN in each indicated group. (H) Quantification protein levels of (a) STRA8, (b) RDH10 and (c) MVH. FMT, faecal microbiome transplantation; HED, high-energy diet; IF, immunofluorescent.

Figure 7

Graphical abstract. Diagram illustrating the proposed vitamin A metabolism via the gut–testis axis being indispensable for spermatogenesis. The disrupted vitamin A absorption was affected by reduced bile acid levels, which mainly due to the dramatically decreased abundance of Ruminococcaceae_NK4A214_group. Notably, the abnormal vitamin A metabolism were transferable to the testicular cells through the blood circulation, which led to abnormal spermatogenesis in the MetS model. MetS, metabolic syndrome.