Impairment of spermatogenesis and sperm motility by the high-fat diet-induced dysbiosis of gut microbes

Abstract

Objective: High-fat diet (HFD)-induced metabolic disorders can lead to impaired sperm production. We aim to investigate if HFD-induced gut microbiota dysbiosis can functionally influence spermatogenesis and sperm motility.

Design: Faecal microbes derived from the HFD-fed or normal diet (ND)-fed male mice were transplanted to the mice maintained on ND. The gut microbes, sperm count and motility were analysed. Human faecal/semen/blood samples were collected to assess microbiota, sperm quality and endotoxin.

Results: Transplantation of the HFD gut microbes into the ND-maintained (HFD-FMT) mice resulted in a significant decrease in spermatogenesis and sperm motility, whereas similar transplantation with the microbes from the ND-fed mice failed to do so. Analysis of the microbiota showed a profound increase in genus Bacteroides and Prevotella, both of which likely contributed to the metabolic endotoxaemia in the HFD-FMT mice. Interestingly, the gut microbes from clinical subjects revealed a strong negative correlation between the abundance of Bacteroides-Prevotella and sperm motility, and a positive correlation between blood endotoxin and Bacteroides abundance. Transplantation with HFD microbes also led to intestinal infiltration of T cells and macrophages as well as a significant increase of pro-inflammatory cytokines in the epididymis, suggesting that epididymal inflammation have likely contributed to the impairment of sperm motility. RNA-sequencing revealed significant reduction in the expression of those genes involved in gamete meiosis and testicular mitochondrial functions in the HFD-FMT mice.

Conclusion: We revealed an intimate linkage between HFD-induced microbiota dysbiosis and defect in spermatogenesis with elevated endotoxin, dysregulation of testicular gene expression and localised epididymal inflammation as the potential causes.

Trial registration number: NCT03634644.

Keywords: diet; endotoxin; inflammation; intestinal microbiology.

Figure 1

High-fat diet (HFD) altered gut microbiota with impaired sperm production and motility. (A) Principal coordinate analysis (PCoA) plot is generated using operational taxonomic unit metrics based on the Bray-Curtis similarity for the samples in the normal diet (ND) and HFD groups. The centre point coordinate of the ellipse is the mean value of PCO1 and PCO2, respectively, in the corresponding group. The values of PCO1 and PCO2 are shown in bar plots, n=10 for each group. (B) The mean percentages of each community contributed by the indicated phyla, n=10 for each group. (C) Sperm from cauda epididymis of mice in the ND group or the HFD group was counted in a haemocytometer under a light microscope, n=6 for each group. (D) Sperm motility of mice in the ND group and the HFD group was analysed by computer-assisted semen analysis, n=6 for each group. Data are expressed as mean±SEM. ***p<0.001. (E) Histological examination in the HFD-treated testis. The sections were stained with H&E. ns, no significant difference. Scale bar=50 µm.

Figure 2

Faecal microbiota transplanted from the HFD mice reduced sperm quality in recipient mice. (A) Schematic diagram of faecal microbiota transplantation experiment. ND, normal diet; HFD, high-fat diet; ND-FMT, normal diet faecal microbiota transplantation; HFD-FMT, high-fat diet faecal microbiota transplantation; i.g., intragastric gavage. (B) Statistical analysis of sperm counts after FMT. Sperm from cauda epididymis of mice in the ND-FMT group or the HFD-FMT group was counted in a haemocytometer under a light microscope. (C) Motility of sperm from the ND-FMT and the HFD-FMT mice was assessed by computer-assisted semen analysis. (D) Representative H&E staining images showing the impairment of seminiferous tubule in the HFD-FMT testis. Scale bar=50 µm. (E) The expression levels of the genes associated with different testicular cells were determined by real-time PCR. (F) Serum endotoxin levels in the ND-FMT mice and the HFD-FMT mice, n=6 for each group. ns, no significant difference. *p<0.05, **p<0.01, ***p<0.001.

Figure 3

Diet-dependent and FMT-responsive alteration of gut microbiota community. (A) Phylum comparison for the abundance of gut microbiota between the ND-FMT and HFD-FMT treated mice. No significant difference in the microbe composition between the two groups. (B) Abundance of family microbes in HFD-FMT mice was compared with the ND-FMT mice. Two-tailed Wilcoxon rank-sum test was used to determine significance. The average abundance of microbes >0.01% was compared. **p<0.01. (C) Similarity analysis of microbes on the genus level was compared in the ND-FMT and the HFD-FMT treated mice. AU values, approximately unbiased values. (D) Abundance of genera microbes in HFD-FMT mice was compared with the ND-FMT mice, n=5 for each group. The average abundance of microbes >0.01% was compared. *p<0.05, **p<0.01.

Figure 4

Correlation of gut Bacteroides and Prevotella collected from clinical sample with sperm motility and blood endotoxin. (A) The combined abundance of the genus Bacteroides-Prevotella collected from healthy donors and the patients with asthenozoospermia, oligozoospermia and teratozoospermia was negatively correlated with sperm motility, n=60 in total. (B) The abundance of Bacteroides in the gut of healthy donors and the patients with asthenozoospermia, oligozoospermia and teratozoospermia was positively correlated with blood endotoxin level, n=60 in total. LPS, lipopolysaccharide. (C) The correlation between the abundance of Prevotella copri and sperm motility in all 60 faecal samples. The dots in the red circle represent the abundance of P. copri >15%, whereas those in the black circle represent the abundance of P. copri <3%. (D) The correlation between the subpopulation with high abundance of P. copri (>15%) and sperm motility (n=20, p<0.05). (E) The correlation between the subpopulation with low abundance of P. copri (<3%) and sperm motility (n=40).

Figure 5

High-fat diet (HFD)-induced dysbiosis of gut microbiota leads to intestinal CD3⁺ T cell and macrophage aggregation and epididymal inflammation. (A) Histological examination of FMT-treated intestines. The sections were stained with H&E. Scale bar=50 µm. (B) Immunofluorescent analysis of CD3 (green) was performed in the small intestine samples of the microbe-transplanted mice. Nuclei were stained with DAPI (blue). The lower panels were enlarged from the upper panels. Scale bar=20 µm. (C) The mRNA levels of CD3e in the small intestines of the microbe-transplanted mice were determined by real-time PCR assays. (D) Immunohistochemistry determination of macrophage infiltration in the small intestines of microbe-transplanted mice. Scale bar=50 µm. (E) The mRNA levels of a macrophage marker, F4/80, in the small intestines of the microbe-transplanted mice were determined by real-time PCR assays. (F) The mRNA levels of pro-inflammation cytokines and NFκB-p65 in the caput epididymis of the microbe-transplanted mice were determined by real-time PCR assays, n=6 for each group. ns, no significant difference. *p<0.05, **p<0.01. CXCL, C-X-Cmotif chemokine; IL, interleukin; MCP, monocyte chemoattractant protein; NFκB, nuclear factor kappa B; TNF, tumour necrosis factor.

Figure 6

Alterations of gene expression in HFD-FMT treated testis. (A) Volcano plot showing the changes of testis genes (fold change ≥2) in the HFD-FMT mice compared with the ND-FMT mice. Five hundred eighty-three genes were found to be downregulated (green dots) and 133 genes upregulated (red dots). The red lines indicate the log2 fold change at 1 and −1, n=4 for each group. (B) Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of differentially expressed genes, n=4 for each group. (C) Heat map of differentially expressed genes in ND-FMT and HFD-FMT treated testis, n=4 for each group. (D) Real-time PCR confirmations for the mitochondrial genes, n=5 for the ND-FMT group and n=6 for the HFD-FMT group. (E) The mRNA levels of meiosis relative genes in the testis were significantly downregulated in the HFD-FMT mice, detected by real-time PCR, n=6 for each group. *p<0.05, **p<0.01.