Biochanin a modulates steroidogenesis and cellular metabolism in human granulosa cells through TAS2Rs activation: a spotlight on ovarian function

Abstract

Background

Endocrine-disrupting chemicals (EDCs) interfere with the endocrine system and negatively impact reproductive health. Biochanin A (BCA), an isoflavone with anti-inflammatory and estrogen-like properties, has been identified as one such EDC. This study investigates the effects of BCA on transcription, metabolism, and hormone regulation in primary human granulosa cells (GCs), with a specific focus on the activation of bitter taste receptors (TAS2Rs).

Methods

Primary human GCs from 60 participants were treated with 10 µM BCA, and selective antagonists were used to block TAS2R activation. The study assessed the expression of TAS2R14 and TAS2R43, and analyzed the impact on StAR and CYP17A1 gene expression. Intracellular calcium levels, lipid droplet size, and mitochondrial network complexity were measured to evaluate cellular metabolism and energy dynamics.

Results

BCA treatment significantly upregulated TAS2R14 and TAS2R43 expression, leading to a 70% increase in StAR mRNA levels and a twofold increase in CYP17A1 expression (p < 0.05). These effects were reversed by TAS2R antagonists. Additionally, BCA treatment decreased intracellular Ca²⁺ levels (p < 0.01) and reduced lipid droplet size (p < 0.001), both of which were counteracted by antagonists. Enhanced mitochondrial network complexity (p < 0.001) was also observed, suggesting increased mitochondrial fusion and improved cellular energy dynamics.

Conclusion

The findings indicate that BCA modulates transcriptional and metabolic processes in GCs through the activation of TAS2Rs, highlighting their role in endocrine regulation. The statistically significant results emphasize the relevance of further exploring the effects of EDCs like BCA on reproductive health. Collaborative research efforts are essential to address and mitigate the adverse impacts of EDCs on fertility.

Introduction

Endocrine-disrupting chemicals (EDCs) are environmental contaminants known to interfere with the endocrine system, leading to various adverse effects on reproductive health and development. Recent declines in male and female fertility have been associated with exposure to various EDCs [1]. These compounds can mimic or block hormones, disrupt their production, or interfere with their signalling pathways, thereby impacting crucial physiological processes [2]. In females, EDCs have been implicated in numerous reproductive health issues, including a reduced number of viable oocytes, early onset of puberty, limited reproductive lifespan, complications in embryo implantation, endometriosis, and fibroids [3,4,5]. The growing body of evidence linking EDCs to reproductive disorders highlights the importance of understanding their mechanisms of action at the cellular and molecular levels [6, 7]. Phytoestrogens are the most common natural EDCs, plant-derived compounds found in food and drinks that mimic or interact with estrogenic hormones due to their structural similarity to 17β-estradiol, the primary female sex hormone [8]. Among these, the isoflavone Biochanin A (BCA), known for its anti-inflammatory and estrogen-like properties [9], is also a natural bitter compound that acts as a ligand for taste bitter receptors (TAS2Rs), a specific subset of GPCRs traditionally associated with bitter taste perception [10,11,12].

In previous studies, BCA demonstrated to link and activate TAS2R14 and TAS2R39, as other flavonoid compounds [10]. TAS2Rs are implicated in regulating various physiological functions, including hormone biosynthesis, cellular metabolism, and even immune responses [13, 14]. Upon activation, TAS2Rs can initiate multiple intracellular signaling cascades, including those involving calcium mobilization and cAMP production [15]. Notably, taste receptors are expressed not only in taste buds but also in non-gustatory tissues, including reproductive organs [13, 16, 17]. In the ovary, particularly in granulosa cells, TAS2Rs are crucial for follicle development and steroidogenesis [12, 18, 19]. Still, while their activation’s exact effect on steroidogenesis remains unclear, some data suggest it may inhibit progesterone production, potentially through NO/cGMP and apoptotic signaling [20]. Considering this, the binding of EDCs to TAS2Rs could potentially lead to altered signaling and dysregulation of critical processes like steroidogenesis, highlighting the EDC’s potential to influence reproductive health via TAS2R pathways. Despite the natural disruptor, BCA has been extensively studied concerning cancer and cardiovascular health [21], its possible influence on reproductive health, particularly through its interaction with novel TAS2R-induced signaling pathways, remains underexplored. We recently demonstrated that BCA is a potent agonist of TAS2Rs, as it effectively induces their expression in hGL5 cells—an immortalized cell line that closely mirrors the characteristics of primary follicular granulosa cells [12]. Therefore, to fully understand the physiological impact of BCA through these receptors on ovarian steroidogenesis, blockers of TAS2Rs are propelling to the forefront of investigations in the latest studies. One of the most known TAS2R antagonists p-(dipropylsulfamoyl) benzoic acid (Probenecid). Probenecid inhibits TAS2R16, 38 and 43 through an allosteric reaction mechanism. Probenecid is an FDA-approved inhibitor of the Multidrug Resistance Protein1 (MRP1) transporter, that is clinically used to treat gout in humans or is co-administered with antibiotics and chemotherapeutics to reduce their excretion [22]. It selectively inhibits the binding of bitter ligand β-glucopyranosides including salicin, arbutin, and phenyl-β-d-glucopyranoside [23]. 4-sulfamoyl benzoic acid (SBA) is a probenecid analogue, reported as a TAS2R14 antagonist, so it competes with caffeine, which is one of its agonists [24]. Finally, (R)- citronellal is reported to act as a general allosteric inhibitor of TASR43 and TASR46, especially studied for its ability to fully block caffeine-induced calcium channels in the cells expressing those receptors [25]. Although BCA has been widely researched in the context of cancer and cardiovascular health, its possible impact on reproductive health, especially through its interaction with newly discovered TAS2R-induced signaling pathways, has not been thoroughly investigated. Therefore, this study aims to investigate the effect of the natural endocrine disruptor BCA on ovarian steroidogenesis in primary granulosa cells.

The findings of this study demonstrate that BCA activates TAS2Rs in GCs, leading to a notable upregulation of StAR and CYP17A1 gene expression, increased estrogen secretion, and decreased progesterone levels. Additionally, BCA treatment results in reductions in intracellular Ca²⁺ levels and lipid droplets, while enhancing mitochondrial network complexity. Taken together, these results verified a crucial role for endocrine disruption in granulosa cells, revealing how bitter taste receptors specifically influence endocrine regulation. The findings offer valuable insights into the complex effects of endocrine disruptors, suggesting potential new broader implications of EDCs on reproductive health.

Materials and methods

Study design and human biological sample collection

The goal of this study was to evaluate the effect of BCA as an EDC on steroidogenesis and TAS2R regulation in human primary GCs. The cells were obtained from 60 women who had undergone in vitro fertilization at Siena University Hospital’s Assisted Reproduction Unit. All participants gave written, informed consent for the use of their samples and data. The study complied with the Declaration of Helsinki and was approved by the University of Siena’s ethical committee. (CEAVSE, Protocol number 18370, 2 October 2020). This study was performed following good clinical practice guidelines and written informed consent was obtained from all patients. In all cases, the reason for having IVF was male infertility factor. The laboratory investigations were carried out blindly.

Human primary granulosa cells isolation and culture

After follicular fluid samples were collected, GCs were isolated from follicular fluid according to a previously described procedure [26]. The collected GCs were cultured for 1–2 days before the treatments at 37 °C and (5% CO2), in Dulbecco Modified Eagle Medium (Invitrogen) containing 10% fetal bovine serum, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin and treated/stored according to the analyses.

Cell proliferation assay and treatment protocols for biochanin a and antagonists

The most suitable GC concentration for experimental testing was determined using the Cell Counting Kit-8 (CCK-8, Abcam, Cambridge, UK) according to the manufacturer’s instructions. Eight replicates were measured for each cell concentration, and the average data were used to create the calibration curve. The cytotoxicity of BCA and its antagonists —PRO, SBA, and CITRO— was assessed by measuring the viability of the treated GC using cell counting kit-8 after overnight incubation at 37 °C and (5% CO2). All of them were purchased from Sigma Aldrich (Madrid, Spain). These compounds were dissolved in dimethyl sulfoxide (DMSO) and diluted in the media containing Dulbecco’s Modified Eagle Medium (DMEM, 1X), containing fetal bovine serum (FBS) (10%), L-glutamine (1%), penicillin/streptomycin (1%), non-essential amino acids (1%) not exceeding a final DMSO concentration of 0.1% (v/v) to avoid the toxic effect of the cells. Different concentrations of each compound were tested, considering optimal TAS2R stimulation concentrations reported in the literature. The highest non-toxic doses of these compounds were selected based on the brief 24-h exposure period. The absorbance was measured after 4 h incubation (37 °C, 5% CO2) with CCK-8 (10 µL). Four wells per compound were analyzed, and the average absorbance was calculated. Background absorbance from wells with media but no cells were subtracted from the readings of the test wells. Additionally, wells with cells but no stimulation served as a control group for assessing cytotoxicity. Based on the cytotoxicity results, the treatment conditions were established as follows: Untreated (DMSO only), BCA 10 µM, BCA 10 µM + PRO 1 mM, BCA 10 µM + SBA 30 µM, CITRO 150 µM, for 24 h.

Mitotracker staining and image processing using the mitochondria analyzer

GCs were plated on a coverslip, treated for 24 h treatments and incubated with MitoTracker^® Red CMXRos probes at 37 °C for 15 min. This probe diffuses across the plasma membrane and accumulates in active mitochondria. After incubation, cells were fixed with Paraformaldehyde 4% for 15 min at RT, washed 3 times with PBS 1X and counterstained with DAPI. The coverslips were, mounted and images were captured using a Leica 6500 microscope equipped with LAS software. Images were analyzed using Mitochondria Analyzer, an ImageJ plugin for processing and analyzing fluorescent mitochondria. This plugin requires image thresholding to quantify the morphological features of the identified mitochondrial structures, carefully setting a comprehensive set of parameters to mathematically describe key aspects of mitochondrial morphology. With the 2D approach, mitochondrial size is characterized by area and perimeter, while the shape is defined by form factor (FF) and aspect ratio (AR). We assess the overall connectivity and complexity of the mitochondrial network by analyzing the skeletonized network, measuring the number of branches, branch junctions, and the total length of the branches (Chaudhry, Shi, and Luciani, 2020).

RNA extraction, complementary DNA preparation and qPCR

Total RNA was isolated from GCs with a RNeasy Micro kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. At the end of the protocol, RNA extracted from each sample was diluted in a final volume of 20 µL of water. The purity and the concentration of RNA were evaluated by reading on NanoDrop^® ND-100 UV-vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). 200 ng of the extracted RNA was reverse transcribed into cDNA, using the iScript gDNA Clear TM cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA). We started with a treatment of samples with a master mix of DNase to remove contaminating genomic DNA. To inactivate the effect of DNase, the samples were incubated in a thermal cycler at 25 °C for 5 min and at 75 °C for another 5 min. 4 µL of the iScript reverse Transcription Supermix was added to each sample; they were then incubated in a thermal cycler (5 min at 25 °C, 20 min at 46 °C, 1 min at 95 °C).

Gene expression of TAS2R14, TAS2R43, StAR, CYP17A1 and GADPH was measured by Real-Time qPCR. The PCR mix consisted of Green Master Mix FAST ROX 2X (Genaxxon Bioscience GmbH, Ulm, Germany), primers 1X, cDNA and water. The PCR conditions were 3 min at 95ºC, followed by 40 cycles of denaturation at 95ºC for 10 s, annealing at 60ºC or 58º for 10 s and primer extension at 72ºC for 20 s. To normalize the expression levels of the gene transcripts of GCs a simultaneous mRNA expression profiling of the housekeeping gene GADPH was also performed in all the analyzed samples. All amplification reactions were conducted in triplicate by qRT-PCR on a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories) gene-specific primer sets used in this study are listed in Supplemetary Table 1. Melting curve analysis was also performed to confirm the specificity of the products obtained. Changes in gene expression levels were calculated by the 2^−ΔΔCt method.

Western blot analysis

Western blotting was performed as previously described [27]. Briefly, 50 µg of total proteins were denatured, separated on 10% polyacrylamide gel and then transferred onto a nitrocellulose membrane (GE Healthcare, Chicago, IL, USA). After blocking for 1 h in 5% nonfat dry milk, the membrane was incubated overnight at 4 °C with primary antibodies (see Supplementary Table 2) diluted in 1% nonfat dry milk/TTBS (TBS containing 0.2% Tween 20). After washing in TTBS, the membrane was incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (see Supplemetary Table 2). Membranes were stripped using a harsh stripping buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 100 mM β-mercaptoethanol. Membranes were incubated in the buffer at 37 °C for 15–20 min with gentle agitation, followed by three washes in TBS-T (5 min each). Stripped blot did not have any remaining background staining and therefore could provide a true assessment of other antibodies. After stripping, membranes were blocked in 5% BSA or non-fat milk for 1 h and reprobed using standard WB procedures. Immunostained bands were visualized by chemiluminescence with ImageQuant LAS 4000 (GE Healthcare).

Immunofluorescence analysis

Immunofluorescence was performed as previously described [28]. Antibodies used in this study are listed in Supplementary Table 2.

Oil red O (ORO) staining

The amount of lipids accumulated in GCs was evaluated using the Oil red O staining method according to a previously described protocol [29], with slight modifications. 0.5 g of ORO were resuspended in 100 mL of isopropanol (ORO stock solution). The working solution was prepared with 30 mL of this stock diluted with 20 mL ddH₂O (ORO-saturated solution). Following EDC treatments, cells were washed 3 times with PBS and fixed with 4% paraformaldehyde for 10 min. Cells were then washed twice with PBS and were subsequently stained with Oil red O (Sigma Aldrich) in the dark for 15 min at room temperature. Thereafter, cells were stained with hematoxylin, washed with PBS, and photographed using a phase contrast microscope (Olympus, Tokyo, Japan). For the quantification of the droplets, cells were plated in 96 wells, with treatments conducted in replicates of 8. The protocol was followed as outlined, with a modification in the final step, where isopropanol was added to extract Oil red O. Supernatant was subjected to determination of optical density (OD) value at 540 nm using the automatic enzyme immunoassay analyzer.

Progesterone and estradiol quantification

To assess the production of P4 and (Estradiol) E2, 72-hour preincubation period of granulosa cell monolayers was conducted, with medium replacement occurring after 48-h and again after 24-h (1 mL medium per well). in the culture medium for 24-h with BCA antagonist and the control. The P4 and E2 assays demonstrated sensitivities of 6 and 1 pg/tube, respectively. Serial dilutions of medium samples exhibited a similar pattern to the standard curves for the steroids under investigation. All analyses were conducted in triplicate. The concentration of steroid hormones in the spent culture medium was measured by Immulite 2000 (Siemens).

Intracellular cAMP assessment

Free cAMP production (nM) in the samples was assessed using an ELISA cAMP Direct Immunoassay Detection Kit (Abcam, ab138880) according to the manufacturer’s instructions. ELISA plates were analyzed using a Synergy MX plate reader (BioTek, USA). Only ELISA plates that had R2 > 0.90 in their standard curve were included in the analysis. ELISA assay was performed in duplicate for each sample analyzed.

ROS detection

Carboxy-2′,7′-dichlorodihydrofluorescein diacetate dye (DCFH-DA) (Life Technologies Corp., Carlsbad, CA) was used to detect intracellular H₂O₂. This dye is oxidized by H₂O₂ to the highly fluorescent derivative 2′,7′-dichlorofluorescein (DCF). Detection of intracellular ROS in GCs by DCFH-DA assay was assessed. Briefly, after removing the medium, cells were incubated with DCFH-DA solution (10 µM in PBS) at 37 °C for 15 min. Then they were centrifuged to remove the DCFH-DA solution and resuspended with PBS. Fluorescence was measured using excitation and emission wavelengths of 480 and 520 nm, respectively (Synergy HTX multi-mode reader, BioTek, Winooski, VT, USA), and normalized to the number of live cells. All experiments were repeated three times.

Intracellular calcium measurement

Intracellular Calcium levels were assessed using a Colorimetric Calcium Assay Kit (Assay Genie MAES0091). The cells were collected and washed with PBS (0.01 M, pH 7.4) 1–2 times. The sample was then centrifuged at 1000 g for 10 min. The supernatant was discarded, and the cell sediment was retained. Homogenization medium was added at a ratio of cell number (1 × 10^6) to deionized water (µL) = 1:200. The sample was sonicated in an ice water bath. After this, centrifugation was carried out at 10,000 g for 10 min. The supernatant was taken and preserved on ice for detection. If detection was not performed on the same day, the cell sample (without homogenization) could be stored at −80 °C for up to one month.

Calcium content (mmol/g prot) was calculated using the following formula: (ΔA610 - b) ÷ a × f ÷ Cpr.

Where: a: The slope of standard curve; b: The intercept of standard curve; f: Dilution factor of sample before test; Cpr: Concentration of protein in sample, gprot/L ΔA610: Absolute OD (ODSample – ODBlank). Intracellular Calcium levels were measured in duplicate for each sample analyzed.

Statistical analysis

Statistical analyses were performed with GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). All data obtained from the experiments were analyzed for normality using the Shapiro-Wilk, followed by the parametric or non-parametric tests, ANOVA or Kruskal-Wallis respectively, as appropriate. Densitometric analyses were completed using ImageJ software. Statistical significance was set at p < 0.05.

Results

BCA affects TAS2R14 and TAS2R43 expression and regulates steroidogenesis

To clarify the association between the natural endocrine disruptor BCA and TAS2Rs expression in human GCs, we first tested the cytotoxic effect of this compound on human primary GCs isolated from the follicular fluid of women undergoing IVF. BCA did not affect the viability of GCs. After determining the optimal concentration, we observed that adding BCA to human primary GCs led to the induction of both TAS2R14 and TAS2R43 expression (Fig. 1A-B). This effect was blocked by sulfamoyl-benzoic acid (SBA), a selective antagonist of TAS2R14 [24], and by both probenecid (PRO) and citronelle (CITRO), two selective antagonists of TAS2R43 [25, 30] (Fig. 1A-B). Notably, when administered individually, PRO, SBA, and CITRO did not influence TAS2R expression, confirming that the observed effects are attributable to receptor blockade rather than any direct action of the antagonists themselves (Supplementary Fig. 1).

Fig. 1

Relative mRNA expression levels of TAS2R14 (A), TAS2R43 (B), StAR (C), and CYP171A (D) in GCs (n = 30) were measured under various conditions: untreated (Ctrl), treated with BCA 10 µM for 24-h, and co-treated with BCA 10 µM plus either PRO 1 mM (BCA + PRO), SBA 30 µM (BCA + SBA), or CITRO 150 µM (BCA + CITRO) for 24-h. Antagonists were preincubated for 1 h before the addition of BCA. The means ± SD mRNA levels are expressed as a relative fold change compared to Ctrl after normalizing on the housekeeping gene GADPH. Statistical significance was calculated by Ordinary One-way ANOVA test (multiple comparisons) and indicated with letters. E-F Progesterone (E) and E2 (F) secretion from GCs, expressed as fold change relative to the control (set to 1). Different letters and * indicate statistically significant differences (p ≤ 0.05)

Our data also provide evidence that PRO and CITRO can efficiently inhibit TAS2R14, and that SBA can counteract the effect of BCA in inducing TAS2R43 expression. All antagonists (PRO, SBA and CITRO) significantly decreased TAS2Rs expression, thus confirming this EDC directly affects the TAS2Rs pathway.

Previous research has shown that TAS2R agonist saccharin can modulate factors involved in steroidogenesis [20]. We, therefore, assessed the expression of the steroidogenic acute regulatory protein (StAR), which transports cholesterol to the inner mitochondrial membrane and is a rate-limiting regulator of steroid production (Christenson and Strauss, 2000). When stimulated with BCA, GCs had a 70% increase in StAR mRNA levels compared to unstimulated GCs (p < 0.05). We further found that 17α-hydroxylase (CYP17A1), a critical enzyme in the steroidogenesis pathway, showed a two-fold increase in expression when treated with BCA, as compared to the control. However, when combined with antagonists, the expression of both StAR and CYP17A1 was significantly reduced (p < 0.05).

After 24-h exposure to BCA and TAS2R antagonists, we also measured GC’s steroid hormones estrogen and progesterone levels (Fig. 1E-F). BCA treatment resulted in a significant decrease in progesterone secretion (p < 0.0001), an effect that was notably counteracted by both SBA and CITRO (p < 0.001), while PRO did not show any statistically significant effect (Fig. 1E). In contrast, BCA treatment led to a substantial increase in estrogen secretion (p < 0.0001), which was partially reduced by the antagonist SBA and CITRO (Fig. 1F).

Several studies have demonstrated that ERK1/2 is involved in regulating progesterone production in granulosa cells [31, 32]. To further confirm BCA’s role in modulating steroidogenesis in these cells, we assessed ERK1/2 activation by immunoblotting (Fig. 2A-B-C-D-) using a phospho-specific antibody that detects dually phosphorylated ERK1 and ERK2, a widely accepted method for indirectly measuring ERK activity. As shown in Fig. 2C, the levels of activated ERK (pERK) significantly decreased following BCA treatment (p < 0.05). Additionally, intracellular cAMP levels were measured in the same cells, revealing a decrease in this second messenger in BCA-treated cells (Fig. 2E), partially reversed by TAS2R antagonists. This finding aligns with the known ability of TAS2Rs to activate PDE [33] and confirms that BCA, via TAS2R receptors, can modulate steroidogenesis in human granulosa cells. The same trend was obtained when the levels of intracellular calcium were measured (Fig. 2F).

Fig. 2

A Representative image of western blot analysis of pERK 1/2 and total ERK 1/2 expression in GCs (n = 20) were measured under various conditions: untreated (Ctrl), treated with BCA 10 µM for 24-h, and co-treated with BCA 10 µM plus either PRO 1 mM (BCA + PRO), SBA 30 µM (BCA + SBA), or CITRO 150 µM (BCA + CITRO) for 24-h. Antagonists were preincubated for 1 h before the addition of BCA. Equal protein loading of the preparations was verified using Ponceau staining. The image is representative of three independent experiments. B-D Computer-assisted semi-quantitative analysis of the overall relative intensity of the bands. The intensity was measured (pixel/mm2) and then normalized relative to Ponceau staining. Values are expressed as fold change as respect to the control. Different letters indicate statistically significant differences (p ≤ 0.05). E Intracellular cAMP concentration in GCs. Results (expressed as mean ± SD) are expressed as a percentage relative to untreated cells, which are set at 100%. Experiments were each performed in duplicate. F Intracellular Calcium levels (mM) in GCs according to different treatments, measured in duplicate. Statistical analyses were conducted using GraphPad Prism 9, employing a one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test to assess significant differences. Different letters indicate statistically significant differences (p ≤ 0.05)

BCA affects lipid droplet (LD) homeostasis

OilRed O staining, which selectively targets neutral lipids, revealed an increase in the cytoplasmic accumulation of intracellular lipids in the GCs treated with BCA compared to untreated cells (Fig. 3A). However, this difference doesn’t reach statistical significance (p > 0.05). Administration of PRO and SBA, resulted in a reduction in lipid content, as shown in Fig. 3B. In contrast, CITRO, didn’t counteract the lipid-accumulation effect of BCA. Interestingly, further analysis of lipid droplet morphology in BCA-treated GCs showed smaller, predominantly spherical lipid droplets (p < 0.001) (Fig. 3C). The treatment with TAS2Rs antagonists significantly increased the size of lipid droplets. (p < 0.001).

Fig. 3

Lipid analysis through Oilred O staining (n = 10). A Representative images of Oilred O staining in Ctrl, BCA and antagonists PRO, SBA and CITRO 1-h pretreatment before BCA addition. B Lipid content relative to the Ctrl (C) GC lipid droplets mean diameter measure in µm. Results are presented as means ± standard deviations of at least three experiments. Statistics were performed in GraphPad Prism 9 using one-way analysis of variance (ANOVA) with Tukey’s post hoc test to assess significant differences. Different letters indicate statistically significant differences (p ≤ 0.05)

TAS2R antagonist reverts the effect of BCA on mitochondrial dynamic

Mitochondria are crucial organelles where steroidogenesis begins, making their proper organization essential for accurate hormone production. Evaluating the mitochondrial network is key to understanding mitochondrial dynamics. The Mitochondrial Analyzer software excels in detecting morphological differences and structural features, providing an accurate assessment of mitochondrial organization.

Using this approach, we observed that the number of mitochondria per cell remained unchanged, while BCA treatment significantly increased the mean mitochondrial area (p < 0.001) compared to the control, indicating activation of mitochondrial fusion (Fig. 4A-B). This effect was effectively reversed by TAS2Rs antagonists (p < 0.001), which, counteracted the BCA-induced changes and appeared to inhibit mitochondrial fusion (Fig. 4A-B).

Fig. 4

A Mitochondrial staining (red) of GCs (n = 10) exposed to different compounds. Nuclei were DAPI-stained (blue). On the right part example of the skeletonization of the image required by the Software. Ctrl = untreated cell (control). Magnification: 630×. Staining pictures are representative of five independent experiments. Results from mitochondrial network analysis in GCs using Mitochondria Analyzer. A summary statistic box plots show the median (horizontal lines), first to third quartile (box), and the most extreme values within 1.5 times the interquartile range (vertical lines) for: the Number of mitochondria per cell (B), Mean area of mitochondria (C), N^o branches per mitochondrion (D), total branches length per mitochondrion (E), mean form factor for all conditions (F). Different letters indicate statistically significant differences (p ≤ 0.05)

In addition to changes in mitochondrial number, area, and length, it is essential to investigate alterations in mitochondrial networks, as energy metabolism is closely linked to these network modifications [34]. BCA treatment significantly increased both the number of branches and the total branch length per mitochondrion, indicating enhanced network complexity (Fig. 4C-D; p < 0.001). Conversely, antagonist treatment led to a significant reduction in both branch number (Figure A; p < 0.02) and total branch length (Fig. 4C-D; p < 0.009). Finally, we measured the average form factor to quantify overall mitochondrial network complexity, where higher values denote more intricate networks and lower values indicate simpler ones. As shown in Fig. 4F, the average form factor is significantly higher in BCA-treated GCs, further confirming the increase in network complexity (Fig. 4E; p < 0.001). The antagonists effectively counteracted BCA-induced changes, resulting in a notable decrease in mitochondrial network complexity as well as a higher level of potential energy.

The presence of a robust mitochondrial network resulting from the mitochondrial fusion process contributes to improved energy efficiency as well as to a more efficient oxidative phosphorylation (OXPHOS) [35]. By contrast, fission is related to increased uncoupling, and increased ROS production [36]. To validate the induction of mitochondrial fusion by the TAS2R agonist BCA, we assessed the level of ROS in treated GCs. The figure illustrates that the addition of BCA leads to a reduction in ROS production, partially counteracted by TAS2R selective antagonists, although statistical significance is not reached (Fig. 5A).

Fig. 5

A ROS/cell % over control (n = 12). The experiments were repeated for three times. B Representative image of western blot assay for COX4 in GCs untreated (CTRL), treated with 10µM BCA or with BCA and TAS2R antagonist SBA, CITRO e PRO (n = 5). Band intensity, normalized to Ponceau staining, is reported as fold change relative to the control group. C Immunofluorescence detection of COX4 visualized in green and β-Tubulin (red) in GCs untreated (Ctrl), treated with 10µM BCA or with BCA and TAS2R antagonist SBA, CITRO e PRO (n = 10). DAPI nuclear staining in blue. Scale bars represent 25 𝜇m

Immunoblot analysis of GC demonstrated high levels of COX4 expression, with BCA treatment resulting in a significant increase in its relative abundance (Fig. 5B); this increase was not reversed by antagonist treatment. Immunofluorescent staining revealed that COX4 exhibited tight perinuclear clustering in untreated cells, while BCA treatment led to an expansion of mitochondria throughout the cytoplasm (Fig. 5C).

Discussion

This study elucidates the impact of the natural endocrine disruptor BCA on granulosa cell function, emphasizing the critical mediating role of TAS2R receptors. Using specific TAS2R antagonists, we demonstrate that 10µM BCA effectively activates TAS2Rs in primary GCs, resulting in a significant upregulation of the steroidogenic enzymes StAR and CYP17A1. These enzymes, crucial in the early stages of steroidogenesis, act as specific and sensitive indicators of both estrogen and progesterone synthesis. Notably, they reflect the rate of progesterone synthesis, which occurs independently of other key steroidogenic enzymes like CYP19A1. Meanwhile, CYP17A1 plays an indirect but critical role in estrogen production by providing the androgenic substrates required for aromatization [37]. This approach enabled us to capture both the upstream and downstream effects of steroidogenesis in granulosa cells, providing a comprehensive view of the hormonal dynamics following BCA treatment. Specifically, we observed that BCA-induced upregulation of STAR and CYP17A1 is associated with increased estrogen secretion and decreased progesterone levels. Direct hormone measurement provided a clear assessment of BCA’s effects on granulosa cell steroidogenesis, eliminating potential biases related to post-transcriptional and translational regulation of enzyme expression.

In bovine granulosa cells, Biochanin A exhibits a dose-dependent, biphasic effect on steroidogenesis, stimulating progesterone production by 50% at 185 nmol/L, while inhibiting it at concentrations exceeding 176 nmol/L [38]. Our findings, showing a similar effect on steroidogenesis with BCA at a concentration of 10 µM, reinforce these observations. Importantly, the effects of BCA are reversed by selective TAS2R inhibitors, confirming that BCA exerts its influence through bitter taste receptors.

While the precise impact of ovarian TAS2R activation on steroidogenesis is still unclear, evidence suggests that these receptors play a role in regulating hormone synthesis and balance by influencing steroidogenic pathways across various cell types [2, 7]. For instance, studies have shown that activating bitter taste receptors may inhibit progesterone production, possibly through NO/cGMP and apoptotic signalling pathways [20]. In the murine PCOS model, oral administration of KDT501, a ligand for the TAS2R4 orthologue, effectively alleviated PCOS-related endocrine and metabolic disorders and restored reproductive function, surpassing the effects of the PPARγ agonist; specifically, KDT501 reduced levels of testosterone and androstenedione without significantly affecting LH or FSH, and it also decreased hepatic lipid accumulation and body fat [18].

To elucidate the mechanisms underlying the BCA’s effects, we demonstrate that BCA treatment significantly reduces levels of activated ERK (pERK). This finding is consistent with previous research showing that ERK1/2 plays a crucial role in regulating progesterone production in granulosa cells [39]. Notably, both ERK and PKA are critical mediators of optimal steroidogenesis, with ERK playing a pivotal role in correctly positioning StAR on the mitochondrial membrane [40, 41].

The observed decrease in ERK levels may explain the reduction in progesterone production, even with an increase in StAR transcription. In addition, our study identified a significant reduction in intracellular cAMP levels, a crucial second messenger in LH-induced steroidogenesis. These findings collectively highlight BCA’s role in targeting key signaling pathways essential for optimal steroidogenesis, likely through its interaction with TAS2R receptors.

The reduction in lipid droplet size following BCA treatment aligns with its known ability to inhibit lipid accumulation [42]. This finding also reinforces the role of TAS2Rs in modulating cellular metabolism and energy dynamics, in particular, suggesting that BCA might alter lipid storage or utilization, potentially impacting cellular energy balance and steroidogenesis [12, 43]. In this regard, TAS2R138 has been shown to promote the degradation of lipid droplets in neutrophils [44], further supporting its involvement in lipid metabolism.

We also provide clear evidence that BCA influences mitochondrial activity. Biochanin A has been shown to promote mitochondrial biogenesis and the production of functional mitochondria [45]. In our experimental model, BCA significantly increases COX4 levels, a key mitochondrial function marker [46].The production of ROS is inversely correlated with COX4 levels, a key indicator of mitochondrial function; COX4 regulates BMI1 expression by reducing mitochondrial ROS production [47, 48].

Moreover, by using Mitochondrial Analyzer, we observed that BCA-treated cells exhibited increased mitochondrial network complexity, indicative of enhanced mitochondrial fusion- an essential process for steroid production [40].

It has been reported that cells exhibit higher respiratory activity and greater oxidative capacity when mitochondria are hyperfused [49], while fragmented mitochondria are linked to lower energy demand, reduced respiratory activity and increased cellular stress [49, 50].

A well-developed mitochondrial network, like the one induced by BCA treatment, is associated not only with mitochondrial fusion and increased OXPHOS capacity but also with changes in mitochondrial dynamics—such as density, number, and spatial distribution—that can influence mitochondria-driven ROS propagation [36, 51]. Our results showed that BCA treatment reduces ROS production, an effect that is partially offset by TAS2R selective antagonists, though this difference did not reach statistical significance.

Despite its promising in vitro effects, the relevance of Biochanin A (BCA) to in vivo ovarian function requires careful consideration. Given its low oral absorption and bioavailability, achieving a physiologically relevant concentration such as 10 µM in human serum is unlikely under normal dietary conditions [52, 53]. In fact, the maximum plasma concentration of BCA achievable with a daily oral intake of 5–50 mg/kg body weight in rats is ≤ 1 µM, which aligns with the typical physiologically relevant concentration [54]. Yet, this chronic, low-dose exposure can still yield significant biological effects over time, as seen in other isoflavones like genistein and resveratrol. Indeed, chronic genistein intake in animal models has been shown to modulate hormone levels, reduce oxidative stress, and improve metabolic profiles, despite serum concentrations being in the nanomolar range [55]. Similarly, another phytoestrogen, resveratrol, demonstrates strong anti-inflammatory and antioxidant effects in vivo despite its low bioavailability, owing to its active metabolites and prolonged low-dose exposure [56]. BCA metabolites, including glucuronides and sulfates, are present in the serum and may contribute to its biological effects. While unmetabolized BCA is typically found at low serum concentrations (in the nanomolar range), these metabolites may enhance and prolong its activity. This is facilitated by enterohepatic circulation, a process that recycles metabolites back into the bloodstream, thereby extending their bioactive presence [52]. Thus, while the in vitro responses are promising, further research is needed to understand how BCA could impact ovarian function in a living organism.

Conclusion

In summary, this study offers a comprehensive analysis of how the natural endocrine disruptor BCA impacts primary GCs through TAS2R activation. The observed changes in hormone levels, cellular metabolism, and mitochondrial dynamics reveal the complex interplay between TAS2Rs and endocrine regulation. These findings underscore the importance of further research into the broader implications of EDCs on reproductive health, particularly highlighting how TAS2R can serve as a sentinel in reprotoxic studies. Understanding how TAS2R-mediated pathways might be targeted or modulated could be key to mitigating the adverse effects of these disruptors on fertility.