Gut microbiome-driven regulation of sex hormone homeostasis: a potential neuroendocrine connection

Abstract

The gut microbiome is known to have a bidirectional relationship with sex hormone homeostasis; however, its role in mediating interactions between the primary regulatory axes of sex hormones and their productions is yet to be fully understood. We utilized both conventionally raised and gnotobiotic mouse models to investigate the regulatory role of the gut microbiome on the hypothalamic-pituitary-gonadal (HPG) axis. Male and female conventionally raised mice underwent surgical modifications as follows: (1) hormonally intact controls; (2) gonadectomized males and females; (3) gonadectomized males and females supplemented with testosterone and estrogen, respectively. Fecal samples from these mice were used to colonize sex-matched, intact, germ-free recipient mice through fecal microbiota transplant (FMT). Serum gonadotropins, gonadal sex hormones, cecal microbiota, and the serum global metabolome were assessed. FMT recipients of gonadectomized-associated microbiota showed lower circulating gonadotropin levels than recipients of intact-associated microbiota, opposite to that of FMT donors. FMT recipients of gonadectomized-associated microbiota also had greater testicular weights compared to recipients of intact-associated microbiota. The gut microbiota composition of recipient mice differed significantly based on the FMT received, with the male microbiota having a more concerted impact in response to changes in the HPG axis. Network analyses showed that multiple metabolically unrelated pathways may be involved in driving differences in serum metabolites due to sex and microbiome received in the recipient mice. In sum, our findings indicate that the gut microbiome responds to the HPG axis and subsequently modulates its feedback mechanisms. A deeper understanding of interactions between the gut microbiota and the neuroendocrine-gonadal system may contribute to the development of therapies for sexually dimorphic diseases.

Keywords: Gut microbiota; endocrine system; estrogen; sex hormones; testosterone.

Figure 1.

Study schema. 8-week-old C57BL/6J fecal microbial transplant (FMT) donor mice had sex hormone status altered through gonadectomy and subcutaneous hormone pellet implantation resulting in six donor groups: (1) intact male controls (INT-M, n = 13); (2) orchiectomized males (ORX-M, n = 13); (3) orchiectomized males with testosterone (12.5 mg/pellet 60-day release) supplementation (ORX+T-M, n = 10); (4) intact female controls (INT-F, n = 7); (5) ovariectomized females (OVX-F, n = 8); (6) ovariectomized female with 17Beta-estradiol (0.1mg/pellet 60-day slow release) supplementation (OVX+E-F, n = 8); Eight weeks after surgery, fecal samples were collected from donor mice and used to colonize 6-week-old germ-free C57BL/6J mice via FMT resulting in six FMT recipient groups: INT-M FMT recipient (n = 8); ORX-M FMT recipient (n = 8); and ORX+T FMT recipient (n = 9); INT-F FMT recipient (n = 7); OVX-F FMT recipient (n = 6); and OVX+E FMT recipient (n = 8). Recipient mice were sex matched to donor mice and were euthanized 4 weeks after colonization.

Figure 2.

Circulating gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), were measured in the serum of male and female microbiota donor mice with surgical alteration of sex hormone status (a–d) and in gnotobiotic hormonally intact recipient mice (Male E&F; Female I&J). Testicular weight was measured in male gnotobiotic recipients (g) and uterine weight female gnotobiotic recipients (k). Intragonadal sex hormone levels were evaluated in gnotobiotic recipients (h&l). Statistics were performed using direct contrasts comparisons of gonadectomized FMT donors and recipients with respective controls: intact-associated microbiota donors and recipients, and gonadectomized donors and recipients supplemented with sex steroids. P-values were corrected using the Sidak methodology and shown directly above the comparison. Effect sizes (d) were calculated using Cohen’s d to quantify the strength of the observed effects.

Figure 3.

Principal coordinate analysis (PCoA) plots of cecal microbiota in gnotobiotic recipients are shown using the Jaccard distance metric (a&b). Co-occurrence networks were created for both male and female cecal microbiota from adjacency matrices (c&d) and further delineated with minimum spanning analysis, which builds a network with the fewest necessary edges, highlighting the most critical relationships within the gut microbiota (e&f). Red lines denote negative relationships between nodes, while green lines denote positive relationships. Branches of minimum spanning tree are highlighted with shadowing. Differential abundance analysis was performed at the genus level in male (g–i) and female fecal microbial transplant (FMT) recipient mice (k–m). Enzyme activity of beta-glucuronidase (GUS) was determined in the cecal content of male (j) and female (n) recipient mice. Differential abundance was assessed by Kruskal Wallis with Dunn’s post hoc test. Effect sizes (d) were calculated as Cohen’s d to quantify the strength of the observed effects.

Figure 4.

Untargeted metabolomics was performed in serum of gnotobiotic recipient mice along with germ-free controls. Principal coordinate analysis (PCA) was used to assess global metabolomic profile, revealing the largest factor driving metabolomic profile to be sex (a). Elastic net feature selection was then used to eliminate for features that vary in relation to sex (b). Metabolomic data from gnotobiotic recipient groups were used to construct a network using minimum spanning tree algorithms (c). Points shown in green represent metabolites most impacted by microbiota received via fecal microbial transplant (FMT). Differentially abundant metabolites of interest were determined in male(d–i) and female (j–o) FMT recipient groups and assessed by direct contrast comparisons of gonadectomized FMT donors and recipients with respective controls: intact-associated microbiota donors and recipients, and gonadectomized donors and recipients supplemented with sex steroids. P-values were corrected using the Sidak method and shown directly above comparisons. Effect sizes (d) were calculated as Cohen’s d to quantify the strength of the observed effects.

Figure 5.

Serum untargeted metabolomics from gnotobiotic recipients of altered-sex hormone associated microbiome. Heatmap of the z scores of the top 100 metabolites determined to be differentially present in females (a) and males (b) with hierarchical clustering based on microbiome received.

Figure 6.

Spearman correlations with FDR corrections between top 100 differentially present serum untargeted metabolomics and gut microbiota at the genus level in male gnotobiotic recipients. Red circle denoted positive associations, and blue circle denotes negative associated with the size of the circle indicating the strength (larger circle represents a stronger association). Statistically significant associations (p< 0.05) are shown with red box.

Figure 7.

Spearman correlations with FDR corrections between top 100 differentially present serum untargeted metabolomics and gut microbiota at the genus level in female gnotobiotic recipients. Red circle denoted positive associations, and blue circle denotes negative associated with the size of the circle indicating the strength (larger circle represents a stronger association). Statistically significant associations (p< 0.05) are shown with red box.