Progesterone Receptor Levels in the Nerve System Controlling the Human Gut

Both the classical nuclear progesterone receptor PR-A/B (Figure 1) and the non-classical progesterone receptor "Progesterone Receptor Membrane Component 1" (PGRMC1; Figure 2) were detected immunohistochemically in all sections of the intestine. A well-defined fluorescence signal was observed in the ganglion cells of the myenteric plexus (Tuj-1 positive; red Figure 2, Figure 1) of the intestine. The expression could be found throughout the myenteric plexus and at its borders, but was not exclusively in this area. It was also expressed between and parallel to muscle fibers. Samples from the jejunum, ileum, and colon showed similar distribution and intensity of PGRMC1 and PR-A/B, with consistent signal localization in ENS ganglion cells across all subjects.

As receptor expression was not restricted to Tuj-1-positive cells, co-expression of PGRMC1 with SOX10 (Figure 3)-positive cells, as a marker for ENS progenitors and enteric glia, as well as GFAP (Figure 4) as a glial cell marker, was also investigated. Figure 3 shows that for SOX10, a partial co-expression with PGRMC1 was observed, whereas no overlap was found for GFAP; therefore, only the ileum is shown as an example in Figure 4.

RT-PCR analysis confirmed, in addition to the immunohistochemical stainings, the expression of PR-A/B and PGRMC1, but also the membrane-bound progesterone receptors, membrane progestin receptors alpha and beta (mPRα, mPRβ), which are normalized to the housekeepers (β-Actin and GAPDH).

Immunofluorescence staining revealed distinct expression patterns of progesterone receptors in the ENS, co expressed with both neuronal and glial lineage cells. The higher expression of PGRMC1 in females suggests a gender-specific regulatory mechanism that may be crucial for maintaining gut homeostasis. The regional differences in mPRα and mPRβ expression between large and small intestines point to specialized functions in different gut segments, potentially reflecting the unique neurochemical environments and physiological demands of these regions.

The partly downregulated receptor levels in females behind the Cannon–Böhm point may reflect the unique neurochemical environment in this transition zone, where parasympathetic innervation shifts from the vagus nerve to the pelvic splanchnic nerves. This anatomical landmark could be particularly relevant for understanding site-specific vulnerability and responsiveness to hormonal signals in the gut.

The age-related decline in PR-A/B and mPRα expression, particularly in female individuals over 60, aligns with known hormonal changes during aging [22]. This decline may contribute to the increased susceptibility to gastrointestinal disorders observed in older populations [23]. The stable expression of PGRMC1 across age groups suggests this receptor may respond to different regulatory mechanisms, potentially involving oxidative stress or inflammatory pathways, as demonstrated by [24].

The presence of progesterone receptors on Beta-III-Tubulin-positive neurons indicates a direct influence on neuronal function. Beta-III-Tubulin is a specific marker for neuronal cells and plays a crucial role in axonal transport and structural integrity [25,26]. The expression of progesterone receptors might exert specific effects on the neurons of the ENS, potentially modulating gut motility, sensory processing, and neuroimmune interactions [27]. As a co-expression of progesterone receptors with Sox-10-positive cells was also found, this might additionally hint at enteric glia cell contribution. This finding is particularly interesting in the context of inflammation, immunological, and degenerative processes, as enteric glia play a crucial role in maintaining gut homeostasis and responding to injury or inflammation. While our study provides evidence for the expression of progesterone receptors in the myenteric plexus, the precise cellular localization of these receptors remains to be fully elucidated. Our double staining experiments revealed a co-expression of PGRMC1 with Sox-10 as well as Tuj-1-positive cells, suggesting that PGRMC1 may be expressed in enteric glia as well as neurons. However, the lack of co-expression with GFAP indicates that not all glial cells express PGRMC1. These findings highlight the complex cellular distribution of progesterone receptors in the ENS and the need for further investigation to fully understand their functional roles.

The elevated expression of PR-A/B and mPRα in the females pre Cannon–Böhm point suggests an adaptive mechanism for hormonal fluctuations, e.g., during the menstrual cycle and pregnancy. Progesterone's role in increasing occludin expression [28] and modulating intestinal muscle contraction [29] highlights its importance in maintaining gut barrier function and motility. These findings suggest that progesterone receptors may play a crucial role in the gender-specific differences observed in gastrointestinal disorders and their response to hormonal changes.

The higher expression of these receptors in females may reflect an evolutionary adaptation to support the increased metabolic demands and immune modulation required during pregnancy [30]. This gender-specific pattern could have important implications for understanding the higher prevalence of certain gastrointestinal disorders in women and the potential protective effects of progesterone in maintaining gut health [31].

Recent studies have shown that sex hormones can modulate the gut microbiome composition, which in turn affects gut barrier function and immune responses [32]. This interaction between sex hormones and the microbiome could provide additional insights into the gender-specific differences we observed in progesterone receptor expression.

Our findings regarding dietary patterns present an intriguing paradox. While previous studies have demonstrated significant dietary influences on gut physiology [33], our study also found a significant impact on progesterone receptor expression, but only in females. This discrepancy may stem from the less extreme dietary modifications in our cohort or the complex interplay between diet, microbiota, and hormonal regulation.

The age-related patterns we observed, particularly the decline in receptor expression after 60, underscore the importance of hormonal changes in gut physiology. This decline may contribute to the increased susceptibility to gastrointestinal disorders observed in older populations. The lack of significant correlation between age and PGRMC1 expression suggests that this receptor may be modulated by different mechanisms, potentially involving oxidative stress or inflammatory processes.

Recent research has highlighted the role of dietary components in modulating gut hormone signaling. For example, polyphenols found in fruits and vegetables have been shown to interact with nuclear receptors, including progesterone receptors, and may influence gut health [34]. These findings suggest that while we did not observe significant effects of diet on receptor expression in our study, more nuanced dietary interventions or specific bioactive compounds may have an impact.

The correlation between BMI and receptor expression underscores the obesity-microbiota-receptor expression paradigm, at least in females. Our findings suggest that BMI alone may not capture the complex biological processes influencing receptor expression. The microbiome's role in hormonal modulation becomes particularly evident when considering age-related changes. The decline in bacterial diversity and increase in pro-inflammatory microbes with aging [35] may contribute to the reduced functionality of progesterone receptors we observed in older individuals. This age-related dysbiosis could impair the gut's ability to respond appropriately to hormonal signals, potentially contributing to the development of gastrointestinal disorders [36].

Recent studies have shown that the gut microbiome can produce neuroactive compounds that influence the ENS and CNS [37,38]. These findings suggest that the microbiome may not only modulate hormone receptor expression but also directly influence neuronal function in the gut [39].

The neuroprotective potential of progesterone in PD is particularly compelling. Our findings suggest that progesterone receptors in the ENS could modulate inflammatory processes and oxidative stress, both of which are key factors in PD pathogenesis [40,41]. The identification of mPRα as a key mediator of neuroprotection opens exciting possibilities for developing targeted therapies [42]. The gender differences in PD prevalence further support the potential of hormone-based therapies, particularly for women experiencing hormonal changes during menopause. The protective effects of progesterone may be particularly relevant for women in this life stage, as decreasing hormone levels correlate with increased susceptibility to neurodegenerative diseases [43].

Recent studies have shown that progesterone can modulate the gut–brain axis, potentially influencing PD progression [27,44]. This interaction between the ENS and CNS highlights the importance of considering the gut in the development of PD therapies. Targeting progesterone receptors in the ENS may offer a novel approach to modulating PD pathology through the gut–brain axis.

While our study provides valuable insights, several questions remain. The clinical relevance of these findings for PD and other neurodegenerative diseases needs to be explored through longitudinal studies. The development of selective progesterone receptor modulators that target specific signaling pathways could offer new therapeutic options. Combination therapies that integrate hormonal modulation with existing PD treatments may prove particularly effective, especially when tailored to individual patient characteristics. These personalized approaches could consider factors such as gender, age, and hormonal status to optimize treatment outcomes.

Recent advances in single-cell sequencing technologies could provide more detailed insights into the cellular distribution of hormone receptors in the ENS [45]. These techniques could help identify specific cell populations that express progesterone receptors and their functional roles in gut physiology.

We acknowledge several limitations in our study. The use of tissue samples from patients with pre-existing bowel diseases may have influenced our findings, though it provided a clinically relevant context. The cross-sectional design prevented us from establishing causality or tracking longitudinal changes. The absence of direct microbiome analysis limited our ability to explore these complex interactions.

While our study provides valuable insights into the expression of progesterone receptors in the myenteric plexus, the potential role of these receptors in the submucosal plexus warrants further investigation. The submucosal plexus is closer to the mucosa and may be more directly involved in interactions with the microbiome. Future studies should aim to include the submucosal plexus to provide an even more detailed understanding of progesterone receptor distribution and its potential roles in the intestine.

Despite these limitations, our study significantly advances the understanding of progesterone receptor expression in the human ENS and provides a robust foundation for future research. The comprehensive dataset we have generated offers new perspectives on potential therapeutic approaches for neurodegenerative diseases and highlights the importance of considering hormonal factors in gut physiology.

All human tissue samples were obtained in accordance with ethical guidelines and regulatory standards. Informed consent was obtained from all participants, and samples were collected during planned surgical procedures. To ensure anonymity, all samples were anonymized prior to analysis. The study was approved by the relevant ethics committee, and all procedures were performed in compliance with the principles of the Declaration of Helsinki. For subsequent analyses, tissue samples were categorized according to patient age, with a cutoff at 60 years, whereby patients older than 60 years were considered postmenopausal.

Human intestinal segments (jejunum, ileum, colon) were either fixed in 4% paraformaldehyde (PFA) for immunofluorescence staining or stored in phosphate-buffered saline (PBS) for RT-PCR and Western blot analysis. For cryosectioning, each intestinal segment was placed in 4% PFA in the operating room and stored at 4 °C for 48 h. After thorough washing with PBS, the tissue was cut transversely into 1–2 cm pieces and incubated in a 30% sucrose solution for another 48 h, followed by freezing in isopentane at −40 °C.

The cryostat (CryoStar NX50, Thermo Scientific, Darmstadt, Germany) was cleaned with NaOH-EDTA (0.1 M NaOH, 1 mM EDTA). The frozen tissue was tempered at −20 °C, with the temperature of the microscope slides maintained between −18 °C and −16 °C for 45 min. Each segment was stabilized with a tissue freezing medium (#2309309815, Tissue-Tek^® O.C.T Compound, Sakura Finetek, CA, USA) to prevent distortion during sectioning. Precise adjustment of the sample holder allowed for even sectioning of the tissue layers, which were cut in the transverse plane at a thickness of 14 μm. Sections were checked for proper orientation using light microscopy (BZ-X800, Keyence Corporation, Osaka, Japan) before being placed on special slides and dried at room temperature for 10 min.

After three 5 min washes with PBS, the tissue was permeabilized with 0.3% Triton-X-100 (#T8532; Sigma-Aldrich, St. Louis, MO, USA) in PBS for 15 min, followed by three 5 min PBS washes. Cryosections were outlined with Pap-Pen (#MKP-2, Kisker Biotech GmbH & Co. KG, Steinfurt, Germany) to limit antibody usage. Non-specific binding sites were blocked with donkey serum (S30-M, Sigma-Aldrich, St. Louis, MO, USA; 1:50 in PBS) for 30 min, followed by three 5 min PBS washes. Sections were then incubated overnight with the primary antibody at 4 °C. To visualize the progesterone receptors, either a goat-derived PGRMC1 antibody (Anti-PGRMC1-antibody, ab48012; 1:100 in PBS) or a mouse-derived PR-A/B antibody (PR-AT 4.14, Thermo Fisher, Darmstadt, Germany, # MA1-410; 1:300 in PBS) was used as the specific primary antibody. After another PBS wash (3 × 5 min), secondary antibodies, either donkey anti-goat (Donkey Anti-Goat, ab150129; Abcam, Camebridge, UK; 1:1000 in PBS) or goat anti-mouse (#A11001, Invitrogen, Fisher Scientific GmbH, Schwerte, Germany; 1:500 in PBS), were applied for 2 h, then removed by three 5 min PBS washes. A neuron-specific beta-III-tubulin antibody (#MAB1195, R&D Systems, Singapore, 1:750 in PBS) was used as a second primary antibody to label the neuronal tissue for 2 h at 4 °C, followed by the same washing procedure. The second secondary antibody, donkey anti-mouse (A32744, Invitrogen, Fisher Scientific GmbH, Schwerte, Germany; 1:750 in PBS), was applied for 2 h, followed by sequential washing steps. Cell nuclei were counterstained with DAPI for 30 min (B-1155, Sigma-Aldrich, St. Louis, MO, USA; 1:1000 in PBS). Intensive PBS washes (3 × 5 min) were performed before embedding the samples in a fluorescence mounting medium (F6937, Sigma-Aldrich, St. Louis, MO, USA).

The photographs were taken using a Keyence fluorescence microscope (BZ-X800, Keyence Corporation, Osaka, Japan) with a 20× objective (Plan Apochromat 20×, BZ-PA20, NA 0.75, WD 0.6 mm), and the same exposure time was used for each coverslip in the different channels (DAPI: 1/35 s, PGRMC1 and PR A/B: 1 s, TUJ1: 1 s).

Human intestinal segments were immediately placed in ice-cold PBS in the operating room. After transport to the laboratory, the mucosa was removed, and the remaining sample was cut into 1–2 cm pieces and frozen in isopentane at −40 °C.

Total RNA was extracted using the NucleoSpin^® RNA Kit (REF 740955.50, Macherey-Nagel, Düren, Germany). First, 30 mg of tissue from the myenteric plexus of each human intestinal sample was cut and homogenized. Then the manufacturer's protocol was followed, with the exception that for the elution of the RNA, 30 μL of nuclease-free water was added directly to the center of the column matrix, followed by centrifugation for 1 min. The isolated RNA (~300 ng/μL) was either immediately processed for further analysis or stored at −80 °C.

For cDNA synthesis, the GoScript™ Reverse Transcription Mix, Oligo(dT) (#A2791, Promega, Walldorf, Germany) was used according to the manufacturer's protocol. The samples were then stored at 4 °C for immediate RT-qPCR or at −20 °C for later use.

The gene expression of various progesterone receptors in the myenteric plexus was analyzed using RT-qPCR. The expression levels of the housekeeping genes GAPDH and Beta-Actin, as well as the expression levels of the genes mPRalpha, mPRbeta, PGRMC1, PRA/B, and beta-III-Tubulin (Table 1), were measured in triplicate and in at least three independent runs. The following components were combined: 10 μL GoTaq qPCR Master Mix (#A6001, Promega, Walldorf, Germany), 1.4 μL of forward primer, 1.4 μL of reverse primer, diluted cDNA (1:10), and ddH₂O to a final volume of 20 μL. The samples were analyzed using the CFX96 Real-Time PCR Detection System (BioRad, Hercules, CA, USA), which involved an initial heating step at 95 °C for 2 min, followed by 40 amplification cycles of 15 s at 95 °C and 60 s at 60 °C. Melt curves were recorded after each cycle to detect individual PCR products.

For statistical analysis, Microsoft Excel (Microsoft Corporation, Redmond, WA, USA) and GraphPad Prism version 7.0a (GraphPad, San Diego, CA, USA) were used. The 2^−Δct method was used to calculate relative gene expression levels, which were then plotted as mean ± standard deviation (SD). Prior to statistical analysis, data were tested for normal distribution using the Shapiro–Wilk test, and statistical outliers were removed using the ROUT method with a Q of 5%. Only groups with a sample size of n ≥ 3 were included in the analysis. All in all, 63 human samples were collected, whereof 38 were from male patients and 25 from female patients. We were able to collect 6 samples from Jejunum, 19 from Ileum, 1 from ascending colon, 5 from transversal colon, 15 from descending colon, 14 from sigmoid colon and 3 from rectum. The samples were used for immunohistochemical or molecular biological analysis, or both. Significant differences between groups were assessed using one-way ANOVA followed by Tukey's post hoc test. Statistical significance is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.