Sex hormones, sex chromosomes, and microbiota: identification of Akkermansia muciniphila as an estrogen-responsive bacterium

Microbiota composition is known to be linked to sex. However, separating sex hormones and sex chromosome roles in gut microbial diversity is yet to be determined. To investigate the sex chromosome role independent of sex hormones, we used the four-core genotype mouse model. In this mouse model, males with testes and females with ovaries have XX or XY sex chromosome complement. In gonadectomized four-core genotype mice, we observed a significant decrease in the levels of estradiol ( P < 0.001) and progesterone ( P < 0.03) in female and testosterone ( P < 0.0001) in male mice plasma samples. Independent of sex chromosome complement, microbial α diversity was increased in gonadectomized female but not male mice compared to sex-matched gonad-intact controls. β diversity analysis showed separation between male ( P < 0.05) but not female XX and XY mice. Importantly, Akkermansia muciniphila was less abundant in gonadectomized compared to gonadal intact female mice ( P < 0.0001). In the presence of β -estradiol, A. muciniphila growth exponentially increased, providing evidence for the identification of a female sex hormone-responsive bacterium ( P < 0.001).


Introduction
Sex-specific determinants of health are increasingly recognized as essential contributors to gender-based medicine (Clayton & Collins 2014, Mauvais-Jarvis et al. 2020).From a genetic perspective, sex chromosomes are determinants of sex, but from a developmental perspective, sex hormones are determinants of both sex and gender (Mauvais-Jarvis et al. 2020).Dissection of the role of sex hormones and sex chromosomes is critical in the development of gender-based medicine, including hypertension and arterial stiffening (Ogola et al. 2018, Reue & Wiese 2022).
Gut microbiota plays a vital role in maintaining the homeostasis of the gastrointestinal tract and the host through nutrient digestion, drug metabolism, e230010 A Sakamuri and others 1:1 and immunomodulation (Wu & Wu 2012, Shimada et al. 2022).Decrease in microbial diversity is linked to many disorders and conditions, including metabolic syndrome (Dabke et al. 2019).Sex differences in human and rodent microbial composition are documented in cardiovascular disease Lactobacillus is shown to be greater in women, providing protective effects than in men (Shastri et al. 2015, Razavi et al. 2019, Sinha et al. 2019, Virwani et al. 2023).Sex difference based on sex steroids estradiol and testosterone has shown distinctive impacts on gut microbes with increased Firmicutes-Bacteroidetes ratio in women than in men (Shastri et al. 2015).However, the impact of sex chromosomes on shaping the composition of gut microbiota remains unexplored.
Experimental designs for testing genetic contributions are performed by comparing female and male subjects.In contrast, sex hormonal effects is assessed by comparing experimental gonadectomized animals with control animals without gonadectomy or by comparing aged with young human subjects (Horstman et al. 2012, Mauvais-Jarvis et al. 2020).In the present study, we exploit the four-core genotype (FCG) mouse model wherein gonadal sex is not determined by the sex chromosomes but by the presence or absence of Sry, a testis-determining gene established during male development (Arnold 2020).Therefore, XX and XY mice with the Sry gene develop testes and are males (XX Sry M and XY −Sry M).XX and XY mice without the Sry gene develop ovaries and are females (XXF and XY -F).These four genotypes allows the use of this model to study the differences in phenotypes caused by sex chromosome complement (XX vs XY), the differential effects of ovarian and testicular hormones, and the interactive effects of sex chromosomes with the sex hormonal effects.
Bilateral gonadectomy impacts gut microbial composition regulated by estrogen and testosterone, indicated by the abundance of Akkermansia and Ruminococcaceae in female and male mice (Org et al. 2016).In a population study, Akkermansia is shown to be more abundant in women than men (Naito et al. 2018).Additionally, Akkermansia is vital in mitigating diet-induced obesity in mice (Everard et al. 2013).Whether Akkermansia is impacted by sex hormones, or the interaction of sex hormones and chromosomes is unknown.Therefore, dissecting the magnitude of genetic and hormonal sex-conferring mechanisms is needed.Here, we examine the microbiota composition of the FCG model.By superimposing gonadectomy, we further investigate the relative effects of genetic vs sex hormones on their abilities to reshape gut microbiota composition.

Animals
Four-core genotype (FCG) mice on the C57BL/6J background were used.Studies were conducted in mice between 15 and 16 weeks old.Male (M) mice XY −Sry acquired from Dr Franck Mauvais-Jarvis (Tulane University) were bred with female (F) breeders (XX) on C57bl/6J (RRID: IMSR_JAX:000664) background purchased from JAX (Bar Harbor, ME, USA).Breeding XY −Sry and XX mice produced the four-core genotypes: XXF, XX -Sry M, XY − F, and XY −Sry M. Mice were genotyped using the following primers to PCR amplify the Sry locus: transgene forward primer: 5′-AGC CCT ACA GCC ACA TGA TA-3′, transgene reverse primer: 5′-GTC TTG CCT GTA TGT GAT GG-3′, forward: 5′-CTG GAG CTC TAC AGT GAT GA-3′, reverse: 5′-CAG TTA CCA ATC AAC ACA TCA C-3′, internal positive control forward: 5′-CAA ATG TTG CTT GTC TGG TG-3′ and internal positive control reverse: 5′-GTC AGT CGA GTG CAC AGT TT-3′.All mice were housed together as either males or females independent of their sex chromosome complement and maintained in a temperature-controlled vivarium under a 12-h darkness 12-h light cycle with free access to standard chow (PicoLab® Rodent Diet 20 #5053) and drinking water.Animal experiments were in accordance with the NIH Guide for the Care and Use of Laboratory Animals approved and monitored by the Augusta University Institutional Animal Care and Use Committee.

Gonadectomy
Mice were ovariectomized or castrated at 8 weeks of age followed by 8 weeks to clear endogenous sex hormones.All procedures followed aseptic techniques.Briefly, mice were placed under a heating pad with 3-4% isoflurane-oxygen mixture, eye ointment was applied, and hair was shaved around the incision site.Alcohol (70%) and betadine were used to clean incision sites.This was followed by administration of buprenorphine 0.1 mg/kg.Ovariectomy was performed by cutting a small incision on both sides of the mouse hip below the rib cage.Ovaries were located above the oviduct on the uterine horn and excised.Castration was performed by locating the scrotum and making a small midline cut above the bladder followed by excision of testicles.Muscle tissue was sutured with absorbable suture and the skin was stapled.Mice were left on heating pads to recover and monitored for pain and stress.Awake and alert mice were placed back in their cages with daily monitoring postsurgical monitoring for wound healing and distress.

Fecal matter collection
All fecal matter were collected in the morning between 2 and 5 h into the darkness cycle (8:00-11:00 h; CST).Mice were placed in an empty clean cage without bedding and allowed time to roam the cage and deposit fecal matter at will.Two pellets were collected and frozen immediately for 16S rRNA gene sequencing.These frozen samples were shipped to the University of Toledo Microbiome Core for further processing.A total of six to eight mice were used for each group analysis.(Invitrogen), 1 µL of each primer (5 µM), and 2.5 µL of 5 ng/µL DNA.All samples were reconstituted in water for a final volume of 25 µL.Thermocycling was performed in a Bio-Rad T100TM thermal cycler and the cycling conditions were as follows: initial denaturation at 95°C for 5 min, followed by 25 cycles of 95°C for 30 s, 58°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 5 min for target PCR.Index PCR was carried out in eight cycles, with an initial denaturation at 95°C for 3 min, followed by 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 5 min.Each PCR amplicon sample was purified in two rounds using AMPure XP beads (Beckman Coulter Inc. Brea, CA, USA).Each concentration of purified index PCR products was measured using the Qubit dsDNA HS Assay kit with Qubit 3.0 fluorometer (Life Technologies).The 4 nM of each amplicon was pooled equally.Pooled library was checked for quality using a 2100 Bioanalyzer (Agilent) before sequencing.Library Denaturing and MiSeq Sample Loading was done according to Illumina User Guide Illumina MiSeq System.The 10 pmol/L denatured and diluted library with 10% PhiX was loaded on an Illumina MiSeq V3 flow cell kit with 2 × 300 cycles.

Quality filtering, ASV picking, and data analysis
Chimeric sequences were identified and filtered using Quantitative Insights in Microbial Ecology (QIIME II) software package (version 2021.11) with custom scripts for analyzing all samples and for filtering groups based on comparisons.All samples were rarified at 30,500 sequencing reads.The reads were denoized, merged, and chimera filtered with Divisive Amplicon Denoising Algorithm 2 resulting in an amplicon sequence variants (ASV) table (Callahan et al. 2016).The taxonomic assignments of ASVs representative sequence were performed with a naive Bayes classifier, which was trained on the SILVA database (version 138.1).Bray-Curtis principal coordinate analysis (PCoA) was performed using the previously reported method (Lu et al. 2023).Sankey diagram was generated using the open resource at OmicStudio (https://www.omicstudio.cn).

Measurement of steroids and statistical analysis
While mice were under isoflurane anesthesia, blood was collected by intracardiac puncture and drawn into a syringe prefilled with 50 µL of 0.5M pH 8.0 EDTA.The samples were centrifuged for 10 min at 1000 g, and plasma supernatant was collected and immediately stored at −80°C.The samples were shipped to Creative Proteomics (Shirley, NY, USA) for steroids analysis using electron spray ionization liquid chromatography-mass spectrometry.Steroids including ∆4, ∆5, 5α-reduced, and conjugated steroids sulfates were assessed.Data were quantitated as ng/mL in plasma.

Bacterial culture
Akkermansia muciniphila BAA-835 was procured from the American Type Culture Collection.This bacterium

Statistical analysis
Data was analyzed using GraphPad Prism version 9.1 (GraphPad Software).Outliers were identified by ROUT method (Q=1%).Two-way ANOVA was used to compute main effect (sex hormone, sex chromosome, and interaction) followed by Sidak's multiple comparisons test to determine the difference between groups (XX vs XY and gonadal intact versus gonadectomy).Each dot on the bar graph represents the number of samples.P < 0.05 was considered significant.

Figure 1
Plasma steroid analysis of gonadal intact and gonadectomized mice.Females: (A) Estradiol was significantly decreased by ovariectomy (P < 0.001) independent of sex chromosome complement.(B) The main effect of ovariectomy did not impact testosterone levels, but an increase was noted in XYF (P = 0.04).(C) There was an overall sex chromosome but not sex hormone effect in Aldosterone levels greater in XXF than XYF (P < 0.01).(D) Castrated male mice indicated no significant difference in estradiol levels (P = 0.5).(E) However, testosterone was significantly decreased in castrated mice (P < 0.0001) independent of the mice genotype.(F) There was no significant effect of castration on aldosterone levels (P = 0.9).Data were analyzed as two-way ANOVA to test for the main effect (sex hormone and chromosome), and Sidak's multiple comparisons test was used for the difference between groups.For each group, the dot plot bar graphs indicate sample size (n = 4-6 samples).Error bars depict standard error mean.

Gut microbiota was not significantly impacted by sex chromosomes in female mice
Next, the gut microbial composition was evaluated in all eight groups of mice (gonadal intact and gonadectomized male and female mice with either XX or XY sex chromosome complement).Bray-Curtis principal coordinate analysis (PCoA), which is a measure of distance in the bacterial communities between groups, was significantly different between intact male (XY) and intact female (XX) groups (Fig. 2A).Interestingly, there was a trending difference between XY and XX males (Fig. 2B).However, no difference was found between XY and XX females (Fig. 2C).The data suggested that sex chromosomes impacted microbiota composition in males while sex hormones affected females.

Gonadectomy increased α-diversity of the gut microbiota in female mice
Next, we evaluated the changes in α-diversity of the gut microbiota in the gonadectomized mice.Remarkably, all four parameters (i.e., richness, evenness, observed taxon, and diversity) were higher in the female gonadectomized mice than in the female intact mice independent of the sex chromosome (Fig. 3A, B, C, and D).This data suggested that female sex hormones impacted the gut microbiota more than sex chromosomes.

Gonadectomy decreased Akkermansia in female mice
We focused on the microbial analysis in the female mice to understand which bacterial taxa were mainly impacted by gonadectomy.Interestingly, we did not observe significant differences in PCoA between intact and gonadectomized female mice with X or Y chromosomes (Supplementary Fig. 3A and B).The Sankey diagram was generated to visualize the significant alterations in bacterial communities at different taxonomic levels.
Major shifts in bacterial composition in Verrucomicrobia were found in the female gonadectomized mice, regardless of sex chromosomes complement (Fig. 4A).Additional phyla alterations are indicated in Sankey diagrams of major gut microbiota, including Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria (Fig. 4B, C, D, and E), respectively.Analysis of bacterial composition at the genus level showed that Akkermansia was significantly reduced in the gonadectomized XX and XY female mice (Table 1).This data suggested that a female sex hormone was positively associated with the abundance of Akkermansia.In male mice, there were no significant differences in PCoA between gonadal intact and castrated mice independent of their sex chromosome complement (Supplementary Fig. 4A and B).However, bacterial composition at the genus level indicated Alloprevotella, uncultured organism, Rikenella, and Blautia enrichment in XYM, while Candidatus Arthromitus, Romboutsia, and Turicibacter were abundant in XXM gonadal intact mice (Supplementary Table 3 and 4).

β-estradiol promoted the growth of A. muciniphila
To evaluate the effect of a female sex hormone on the growth of Akkermansia, we cultured A. muciniphila in vitro under anaerobic conditions in the presence and absence of β-estradiol.A. muciniphila was chosen because it is the most abundant and relevant Akkermansia species in humans (Geerlings et al. 2018).Growth of A. muciniphila was monitored by a linear regression curve to associate bacterial numbers with OD600 values (Fig. 5A).As seen in Fig. 5B, supplementation of β-estradiol, at concentrations of 2.2 µM and 10 µM, had a positive, dosedependent effect on the growth of A. muciniphila over 4 h.

Discussion
The most significant findings of our study are that (1) we demonstrated that sex hormones override sex chromosomes in shaping gut microbial composition and (2) that we identified A. muciniphila as a β-estradiolresponsive bacterium.
Cardiovascular studies focusing on identifying sex differences have been reliant on the primary sex hormones estradiol and testosterone in females and males, respectively (Xue et al. 2005).The differences between females and males are not limited to sex steroids but include sex chromosomes XX in females and XY in males (Mauvais-Jarvis et al. 2020).Previously, it has been Microbial analysis of α-diversity.All groups of samples were analyzed by 16S sequencing.(A) There was an overall effect of gonadectomy (P = 0.01) with a significant impact in Richness that was increased in XXF (P = 0.03) and XYF (P < 0.001).(B) Similarly, evenness was increased in XXF (P = 0.04) and XYF (P = 0.02).(C) The observed taxon followed a similar trend with a significant impact of gonadectomy (P < 0.01) and an increase in XXF (P < 0.01) XYF (P = 0.0003).(D) Shannon diversity indicated a similar trend with an increase in XXF (P = 0.02) and (P = 0.02).Data were as two-way ANOVA to test for the main effect (sex hormone and chromosome), multiple unpaired t-tests were used to test for differences between groups.Each group (n 6-8 samples) indicated by the dot plot bar Error bars depict standard error mean.

e230010
A Sakamuri and others 1:1 challenging to identify sexual dimorphism based on sex chromosomes due to limitations on animal models (Reue & Wiese 2022).In this study, we used the four-core genotype mouse to identify gut microbiome and steroid changes in gonadal intact and gonadectomized male and female mice.Our study is the first to evaluate both hormonal and chromosomal effects on gut microbiota characteristics.Consistent with previous publications, the comparison of gut microbial composition between males and females showed significant differences.However, the chromosomal effect was more dominant in the male mice.In contrast, we barely observed differences between XXF and XYF mice, suggesting a diminished chromosomal effect in females.Following this, we evaluated the role of diminishing female sex hormones in gut microbiota and demonstrated that the genus Akkermansia in phylum Verrucomicrobia was remarkably depleted in the gonadectomized XXF and XYF mice.Importantly, our in vitro culture assay provided direct evidence for a stimulatory effect of the female hormone β-estradiol on the growth of A. muciniphila.
Sex hormones have a role in shaping the gut microbiome.For example, testosterone exposure to nonobese type 1 diabetic mice is shown to protect male mice from pancreatic islet inflammation that is reversed by androgen receptor antagonism (Markle et al. 2013, Yurkovetskiy et al. 2013).Several studies have shown protective effects of estradiol in gut microbiome, including more Bacteroidetes and less Firmicutes phyla known to mitigate hypertension and inflammation (Yang et al. 2015,

Jang et al. 2019). Estrogen-responsive bacteria including
Akkermansia is shown to play a role in alleviating colitis disease in mouse model of estrogen beta receptor deletion (Naito et al. 2018, Ma et al. 2022).Our study shows that decline in circulating testosterone levels in male mice tends to decrease richness and diversity in XXM but not in XYM mice suggesting an X chromosome effect.While one can argue that smaller testicular size in XXM than XYM mice contributes to the differential effect, testosterone levels were similar in both groups as previously reported and in our data (Gatewood et al. 2006).Castrated mice from three different strains (C57BL/6J, C3H/HeJ, and DBA/2J) fed a regular chow diet indicated altered gut microbiome composition (Org et al. 2016).However, we did not observe overt changes in our study.Interestingly, gonadectomy, in addition to a high-fat diet, is shown to impact gut microbiota with increased bile acids in males than females, suggesting protection in female mice (Org et al. 2016).In the case of FCG mice, Sry transgene is located on an autosome (Burgoyne & Arnold 2016).Previous studies on sex differences have not shown whether Sry linked to the Y chromosome versus autosome impacts gut composition (Org et al. 2016).Our data wherein gonadectomized female mice increased alpha diversity independent of sex chromosome are similar to findings in Sprague-Dawley rats, suggesting that richness and phylogenetic diversity are impacted by loss of sex hormones in fecal samples (Kaliannan et al. 2018, Diviccaro et al. 2022).
Given that we collected fecal matter and not mucosa samples our analysis was limited to changes in the stool.Nevertheless, microbiome diversity in the mucosa is indicated to differ by sex as shown by the enrichment of Lactobacillus and Oscillibacter in gonadectomized females than in male rats (Diviccaro et al. 2022).
Our results are consistent with data from C57BL/6J and DBA/2J strains, indicating that A. muciniphila is upregulated in gonadectomized females than male mice (Org et al. 2016).Akkermansia has been suggested to be linked to female sex hormones and provides beneficial effects in metabolic syndrome by mitigating inflammation and improving intestinal barrier integrity (Santos-Marcos et al. 2023).Analysis of 1135 human gut metagenome reported that, after correction for all the variables (i.e., dietary, lifestyle, medication, etc.), A. muciniphila was the only bacterial taxon associated with sex, with a higher abundance in females (Sinha et al. 2019).Postmenopausal women have a decrease in gut diversity associated with a reduction in estradiol (Menni et al. 2018, Peters et al. 2022).Akkermansia muciniphila, Clostridium lactatifermentans, Parabacteroides johnsonii, and Veillonella seminalis are decreased in postmenopausal women  (Peters et al. 2022).Sex hormone changes during aging impact the gut microbiota and can play a pivotal role in identifying probiotics (An et al. 2018).Decreases in estradiol and Akkermansia are associated with metabolic syndrome in postmenopausal women and mice (Org et al. 2016, Peters et al. 2022).These studies suggested that Akkermansia is beneficial in regulating blood pressure, inflammation, and metabolic syndrome (Org et al. 2016, Ottman et al. 2017, Sun et al. 2019).
It is shown that prenatal testosterone exposure causes high blood pressure and gut microbiota dysbiosis (Sherman et al. 2018).We did not determine the growth of Akkermansia in the presence of testosterone because there was no significant difference in gut microbiome composition in castrated versus testes intact male mice.Therefore, we proceeded to evaluate the estradiol effect on the growth of Akkermansia because 17β-estradiol is shown to lower Proteobacteria and increase Akkermansia in ovariectomized mice with metabolic syndrome (Diviccaro et al. 2022).This concurs with our finding of downregulated Akkermansia in ovariectomized XXF and XYF mice, indicating a role for estrogen in regulating gut bacteria.While Akkermansia, Anaerostipes, and Lachnoclostridium were decreased with gonadectomy, multiple genera were increased in abundance in female mice (XXF and XYF), including Eubacterium, Romboutsia, and Turicibacter.Enrichment of Blautia in gonadal intact XYF mice suggested its association with the Y chromosome, given its abundance in control stool of male rats (Diviccaro et al. 2022).There was no overt change in Firmicutes and Bacteroides; however, our study shows a significant phylum change in Verrucomicrobia in female mice.
A decrease in 11-deoxycorticosterone was indicated in female mice independent of their sex chromosome complement.This is consistent with lower progesterone and corticosterone levels indicated in primary follicular depletion in rats (Carolino et al. 2019).In male mice, increased Tetrahydroxy 11-deoxycortisol suggested an underlying inflammatory response to upregulate glucocorticoids, adrenal, and metabolic dysfunction (Araujo-Castro et al. 2023, Suminska et al. 2023).Steroid analysis indicated that gonadectomy mediated dysregulation of mineralocorticoid synthesis and perhaps gut microbial composition.Nonetheless, based on the reported associations of Akkermansia with sex (Liu et al. 2017, Kaliannan et al. 2018, Moore & Pluznick 2023) we chose to focus on Akkermansia and demonstrate for the first time that the growth of A. muciniphila is indeed stimulated by estradiol.The enrichment of Akkermansia in gonadal intact female mice and the bacterium growth in the presence of estrogen points to the hormonebacterium interdependence as demonstrated in our study.Intestinal enzyme β-glucuronidases can reactivate conjugated estrogens, including estrone and estradiol, through glucuronidation (Ervin et al. 2019).The estrogendeconjugating β-glucuronidase enzyme known as estrobolome includes Clostridium perfringens, B. fragilis, and R. gnavus (Ervin et al. 2019).Akkermansia accounts for a more significant portion of the estrobolome phylum, indicating its correlation to the presence of estrogen in gonadal intact mice (Candeliere et al. 2022).
Our study provided a baseline to understand the contribution of sex hormones and sex chromosomes to the gut microbiome.However, there are drawbacks to Pro-growth effect of β-estradiol on A. muciniphila.(A) Linear regression curve of intact cell number and OD600 (R 2 = 0.9).(B) OD600 readings were recorded within the first 4 h.Two-way ANOVA was used followed by Tukey multiple comparisons.* P < 0.05, *** P < 0.001, and **** P < 0.0001.

Figure 3
Figure 3Microbial analysis of α-diversity.All groups of samples were analyzed by 16S sequencing.(A) There was an overall effect of gonadectomy (P = 0.01) with a significant impact in Richness that was increased in XXF (P = 0.03) and XYF (P < 0.001).(B) Similarly, evenness was increased in XXF (P = 0.04) and XYF (P = 0.02).(C) The observed taxon followed a similar trend with a significant impact of gonadectomy (P < 0.01) and an increase in XXF (P < 0.01) XYF (P = 0.0003).(D) Shannon diversity indicated a similar trend with an increase in XXF (P = 0.02) and (P = 0.02).Data were as two-way ANOVA to test for the main effect (sex hormone and chromosome), multiple unpaired t-tests were used to test for differences between groups.Each group (n 6-8 samples) indicated by the dot plot bar Error bars depict standard error mean.* P < 0.05 and *** P < 0.001.