Bitter Taste Receptors in the Reproductive System: Function and Therapeutic Implications

Abstract

Type 2 taste receptors (TAS2Rs), traditionally known for their role in bitter taste perception, are present in diverse reproductive tissues of both sexes. This review explores our current understanding of TAS2R functions with a particular focus on reproductive health. In males, TAS2Rs are believed to play potential roles in processes such as sperm chemotaxis and male fertility. Genetic insights from mouse models and human polymorphism studies provide some evidence for their contribution to male infertility. In female reproduction, it is speculated that TAS2Rs influence the ovarian milieu, shaping the functions of granulosa and cumulus cells and their interactions with oocytes. In the uterus, TAS2Rs contribute to uterine relaxation and hold potential as therapeutic targets for preventing preterm birth. In the placenta, they are proposed to function as vigilant sentinels, responding to infection and potentially modulating mechanisms of fetal protection. In the cervix and vagina, their analogous functions to those in other extraoral tissues suggest a potential role in infection defense. In addition, TAS2Rs exhibit altered expression patterns that profoundly affect cancer cell proliferation and apoptosis in reproductive cancers. Notably, TAS2R agonists show promise in inducing apoptosis and overcoming chemoresistance in these malignancies. Despite these advances, challenges remain, including a lack of genetic and functional studies. The application of techniques such as single-cell RNA sequencing and CRISPR/Cas9 gene editing could provide deeper insights into TAS2Rs in reproduction, paving the way for novel therapeutic strategies for reproductive disorders.

Graphical Abstract

The potential functions of TAS2Rs in various components of the male and female reproductive systems.

1. Introduction

Taste receptors, long thought to be the guardians of our dietary preferences, have recently captured the interest of scientists for their wide-ranging functions beyond the taste buds and gustatory system. These G protein-coupled receptors (GPCRs), particularly the type 2 taste receptor (TAS2R) that mediate bitter taste, were initially recognized for their role in protecting animals and humans from ingesting potentially harmful substances^1,2. In recent years, however, the expression and function of TAS2Rs (or Tas2rs in other species) have been demonstrated in a variety of non-gustatory systems, ranging from respiratory to immune. This intriguing discovery has prompted further investigation into their roles in physiological and pathophysiological processes.

Despite a plethora of comprehensive reviews elucidating the extraoral functions of TAS2Rs^3-12, their role within the reproductive system, which is crucial for understanding and potentially improving reproductive health, has not yet been systematically addressed. This gap in the literature is striking, given the evidence for the presence of TAS2Rs in various components of the female and male reproductive systems, including the ovary, uterus, placenta, cervix, vagina, testis, and spermatozoa.

The purpose of this review is to shed light on this evolving area of research. We aim to provide a thorough review of the current state of knowledge regarding the role of TAS2Rs in the reproductive system and discuss their therapeutic implications for fertility, obstetrics, gynecology, and reproductive cancers. We hope to provide insight into how these receptors may influence reproductive health and disease, thereby contributing to better therapeutic and preventive strategies in reproductive medicine.

For this purpose, we conducted a comprehensive search using PubMed. Our search included original studies published in English up to September 2023, focusing on the following words or phrases combined with ‘bitter taste receptor’: non-gustatory, extraoral, male or female reproductive system, reproductive cancer, fertility, infertility, ovary, oviduct, uterus, placenta, vagina, cervix, prostate, sperm, testis, and epididymis.

1.1. TAS2Rs in the gustatory system

Bitter taste, one of the five basic tastes that also includes sweet, salty, sour, and umami, is primarily sensed by TAS2Rs, also known as bitter taste receptors. These receptors were first discovered in type 2 taste cells, which are chemosensory cells located in the taste buds^1,13. They play a critical role in initiating protective responses to prevent the ingestion of potentially harmful substances². TAS2Rs belong to a subclass of class A GPCRs with seven transmembrane domains. Bitter taste perception is a highly conserved chemosensory system among vertebrates¹⁴. However, each species is endowed with a unique repertoire of TAS2R/Tas2r genes. For example, humans possess 25 different TAS2Rs, while mice have 35, cats have 12, chickens have 3, and American bullfrogs, in an impressive display of diversity, harbor 180 unique Tas2r genes¹⁵.

The molecular mechanisms underlying bitter taste perception in the gustatory system are driven by a common taste transduction cascade in which TAS2Rs participate alongside type 1 taste receptors (TAS1Rs) that are responsible for sweet and umami tastes^2,14,16-20. This canonical pathway involves a cascade of molecular components, including heterotrimeric G-protein subunits (e.g., α-gustducin or GNAT3, Gβ3, and Gγ13), phospholipase C beta 2 (PLCβ2), inositol 1,4,5-trisphosphate receptor (IP3R), and transient receptor potential cation channel subfamily M member 5 (TRPM5) (Figure 1).

Figure 1. The canonical bitter taste receptor signaling pathway in the taste buds.

Bitter compounds bind to TAS2Rs on type 2 taste cells, causing GNAT3 and β3/γ13 to dissociate. β3/γ13 then activates PLCβ2, which generates IP3 to trigger Ca²⁺ release from the ER. The increase in Ca²⁺ opens TRPM5 channels, leading to Na⁺ influx and membrane depolarization. ATP is then released through CALHM1/3 channels and stimulates purinergic receptors on nerve fibers, translating the bitter taste into an electrical neural signal to the gustatory nucleus in the brainstem. The pathways that relay the signal to the thalamus and gustatory cortex are not labeled and shown.

When activated by a bitter substance, TAS2Rs trigger the dissociation of the GNAT3 from the Gβ3/Gγ13 subunits (Figure 1). This, in turn, activates PLCβ2, which generates inositol-1,4,5-phosphate (IP3). The newly formed IP3 binds to IP3R on the endoplasmic reticulum, inducing the release of Ca²⁺ from internal stores. The increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) induces the opening of the membrane-associated TRPM5 channel, allowing an influx of Na⁺ ions. This ion exchange causes membrane depolarization and the subsequent release of adenosine triphosphate (ATP) through the calcium homeostasis modulator 1 and 3 (CALHM1 and CALHM3) channels^21-23. Finally, this released ATP activates purinergic receptors on afferent nerve fibers, converting the chemical signal from the bitter compound into an electrical signal that is transmitted to the gustatory nucleus in the brainstem. From there, the signal is relayed to the thalamus and ultimately to the gustatory cortex in the cerebral cortex, culminating in the perception of bitter taste.

Understanding the intricate details of TAS2R function and bitter taste perception in the gustatory system is not only a matter of academic interest but also a stepping stone to unraveling their diverse roles in other biological systems, including the reproductive system.

1.2. The extraoral roles of TAS2Rs

While the initial identification of TAS2Rs was in the oral cavity, our understanding of these versatile receptors has expanded tremendously over the years. A plethora of studies have highlighted the expression of bitter taste receptors and their downstream signaling components in numerous cells and tissues beyond the tongue. These include vital systems such as the respiratory and gastrointestinal tracts, the urinary system, the brain, the immune system, and even within tumors^3-11.

The functionality of these extraoral TAS2Rs is as diverse as their locations. They are involved in a wide range of physiological and pathophysiological processes that are tailored to the specific type and location of the cell in which they are expressed.

A striking example is within the respiratory system, where TAS2Rs have been detected in the motile cilia of epithelial cells. Here, bitter compounds stimulate the ciliary beat frequency in a dose-dependent manner by inducing an increase in [Ca²⁺]_i. This response helps clear potentially harmful inhaled substances from the lungs, suggesting a protective role for TAS2Rs in the respiratory system²⁴. In addition, activation of TAS2Rs in airway smooth muscle cells, which leads to airway relaxation, has sparked interest in developing bitter tastants as potential asthma medications^25-27.

In the gastrointestinal tract, TAS2Rs play a critical role in the activation of type 2 innate immunity. Upon detection of a parasitic infection, Tas2r-expressing tuft cells in the intestine release interleukin 25 (IL-25) via the canonical taste transduction pathway. This IL-25 signals intestinal type 2 innate lymphoid cells to secrete interleukin 13 (IL-13), which subsequently induces stem and progenitor cells in the crypt to differentiate into tuft and goblet cells. This chain of events triggers the "weep and sweep" response, which aids in the expulsion of the parasites^28-30.

Similarly, in the urinary system, the expression of Tas2rs has been demonstrated in brush cells, which are cholinergic chemosensory cells located in the urethra. Upon stimulation with bitter tastants or uropathogenic Escherichia coli, these cells release acetylcholine, activating the classical taste transduction cascade. This cascade triggers a reflex contraction of the bladder detrusor muscle, which helps to flush out harmful contents from the urethral lumen³¹.

The discovery of these extraoral roles of TAS2Rs highlights their importance beyond mere taste perception. Understanding their function in various physiological and pathophysiological processes may reveal novel therapeutic strategies for a variety of diseases. As summarized in the Graphic abstract figure above, TAS2Rs are proposed to be involved in a wide range of reproductive functions. Their potential involvement extends to areas of infertility, obstetrics, gynecology, and reproductive cancers. In the following sections, we will review the evidence supporting each of these proposed roles.

2. The roles of TAS2Rs in the male reproductive system

Taste receptors, including TAS2Rs, in the male reproductive system have been reviewed^32-34. Therefore, rather than delving into aspects that have been thoroughly reviewed in the literature, we provide a concise summary of TAS2Rs in the male reproductive system with a focus on their potential roles in sperm chemotaxis, fertility, and infertility.

Ejaculated sperm must undertake a challenging journey to reach the oviduct for successful fertilization. In guiding sperm on this journey, chemotaxis was first found to play a critical role in directing sperm to the egg in sea urchins³⁵. This phenomenon of sperm chemotaxis toward eggs has since been documented in various life forms, from lower plants to mammals^36,37. The discovery of taste receptors outside the oral cavity brought to light the fact that taste is one type of chemical sense. Hence, it is plausible to hypothesize that taste sensing may be involved in the process of sperm chemotaxis.

The first indication of this was the detection of sweet taste receptors (TAS1R3) in mouse testes³⁸. Subsequently, a key component of taste transduction, α-gustducin, was found in mammalian spermatozoa before TAS2Rs were identified³⁹ (Table 1).

Table 1.

TAS2Rs in the mouse reproductive system

Receptor	Tissue or cell type	method	reference
TAS2R105	spermatids	genetic labeling	Li and Zhou, 2012
TAS2R131	testis, epididymis, sperm (12 weeks)	genetic labeling	Voigt et al., 2012
TAS2R131	testis (P3, P9, and P17)	genetic labeling	Voigt et al., 2015
TAS2Rs	testis	qRT-PCR	Xu et al., 2013
TAS2R105, TAS2R108	testis	in situ hybridization	Xu et al., 2013
TAS2R108	ovary	qRT-PCR	Wu et al., 2019
TAS2R126, TAS2R135, TAS2R137, and TAS2R143	uterine smooth muscle from pregnant mice	qRT-PCR	Zheng et al., 2017
TAS2R143/TAS2R135/TAS2R126 Cluster	epithelium of mouse vagina and cervix	genetic labeling	Liu et al., 2017

Using genetically modified mice, Li and Zhou (2012) made an important discovery about TAS2R105 in the male mouse reproductive system⁴⁰. They found that TAS2R105 is expressed in early to mid-stage round spermatids during sperm development, but not in spermatocytes or spermatogonia.

Voigt et al made a related discovery concerning Tas2r131-expressing cells in the male reproductive system. They identified these cells in both testis and epididymis using enhanced green fluorescent protein (EGFP) as a marker. The fluorescence intensity of these cells increased as sperm development progressed^41,42. Tas2r131-expressing cells were present in the seminiferous tubules of the adult testes⁴¹ and in specific structures in the neonatal testis⁴². In the epididymis, these cells were found only in the lumen of the tubules⁴¹. Interestingly, the absence of α-gustducin, a component associated with taste signals in the epididymal epithelial cells⁴³, suggests that the Tas2r131-expressing cells in the epididymis may actually be spermatocytes. The epididymis is a critical role for sperm maturation, where sperm gain motility and the ability to fertilize oocytes. It's possible that during their journey through the epididymis, sperm cells interact with secretory molecules and mature under the influence of TAS2R131. Recent studies have also identified a subset of TAS2Rs and their downstream signaling components in the human testis and sperm⁴⁴ (Table 2).

Table 2.

TAS2Rs in the human reproductive system

Receptor	Tissue or cell type	method	reference
TAS2R3, TAS2R4, TAS2R14, TAS2R19, and TAS2R43	testis and sperm	qRT-PCR, western blot, and immunostaining	Governini et al., 2020
TAS2R3, TAS2R4, TAS2R14, TAS2R19, and TAS2R43	freshly isolated granulosa cells and cumulus cells	qRT-PCR, western blot, immunostaining	Semplici et al., 2021
TAS2R3, TAS2R4, TAS2R14, TAS2R19, and TAS2R43	immortalized primary follicular granulosa cells hGL5	qRT-PCR, western blot, immunostaining	Luongo et al., 2022
TAS2R1, TAS2R4, TAS2R10, TAS2R14, and TAS2R38	fallopian tube tissue, normal uterine tissue, ovarian cystadenocarcinoma tumor tissue, human ovarian cancer cell line (OVCAR4 and OVCAR8), ovarian clear cell carcinoma cell lines SKOV3, prostate cancer cell lines (DU145, LNCaP, and PC3), benign prostatic hyperplasia cells line BPH-1, and endometrial cancer cell line HEC-1a	qRT-PCR, western blot	Martin et al., 2019
TAS2R4, TAS2R5, TAS2R10, TAS2R13, and TAS2R14	fresh human myometrium	qRT-PCR	Zheng et al., 2017
TAS2R3, TAS2R4, TAS2R5, TAS2R7, TAS2R8, TAS2R10, TAS2R13, TAS2R14, TAS2R31, TAS2R39, TAS2R42, TAS2R43, TAS2R45, and TAS2R50	human myometrial cell line hTERT-HM	qRT-PCR	Zheng et al., 2017
TAS2R38	endocervix, placenta, and placenta cell line JEG-3	immunostaining	Wölfle et al., 2016
TAS2R14	placenta, trophoblast cell line HTR-8/Svneo, and trophoblast-derived choriocarcinoma cells JAR, JEG, and BeWo.	PCR, immunostaining	Taher et al., 2019

In 2013, Xu and colleagues demonstrated the expression of all Tas2r members in mouse testes using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and further validated the presence of two members (Tas2r105 and Tas2r108) in testis sections by in situ hybridization⁴⁵. Notably, they found that bitter tastants induced an increase in [Ca²⁺]_i in spermatids, with individual spermatids showing different ligand activation profiles⁴⁵. This suggests that each spermatid may have a unique TAS2Rs profile. As calcium triggers several physiological events in spermatozoa, such as hyperactivation, chemotaxis, capacitation, and the acrosomal reaction, TAS2Rs may play a pivotal role in male fertility. Figure 2 illustrates a potential role for TAS2Rs in sperm chemotaxis. Bitter tastants, including progesterone or chemokines, which can be secreted by the cumulus-oocyte complex, could potentially activate TAS2Rs in sperm. This activation could trigger the release of calcium from internal stores^46,47, resulting in an increase in [Ca²⁺]_i, which in turn could enhance sperm flagellar beating and hyperactivation. Given that CatSper is a dominant Ca²⁺ channel for sperm activation^48,49, it is plausible that the activation of TAS2Rs could functionally couple with CatSper. Although progesterone is known to activate CatSper⁵⁰, it would be interesting to investigate whether its activation of CatSper involves TAS2Rs. This is particularly pertinent considering that progesterone, with a threshold less than its level present in the cumulus⁵¹, is an agonist of TAS2R110, TAS2R114, or TAS2R46⁵². Additionally, it is notable that human sperm generate complex Ca²⁺ oscillations involving Ca²⁺ release from the internal store upon exposure to a progesterone gradient that mimics in vivo fertilization conditions^53,54.

Figure 2. Potential roles of TAS2Rs in regulating sperm chemotaxis.

Bitter tastants, such as progesterone or chemokines, which are secreted by the cumulus-oocyte complex, could potentially activate TAS2Rs on sperm. This activation might enhance sperm flagellar beating and hyperactivation by triggering an increase in [Ca²⁺]_i through two possible pathways. The first pathway involves the release of calcium from internal stores, and the second results from calcium influx through the CatSper channel.

Although various investigations have reported the TAS2R profile in the male reproductive system, only the study by Li and Zhou (2012) provides direct genetic evidence that TAS2Rs play a role in fertility⁴⁰. Interestingly, they found that the deletion of cells expressing Tas2r105 results in smaller testes and male infertility but not the deletion of TAS2R105 itself⁵⁵. Given that individual taste receptor cells can express a subset of Tas2rs and that different TAS2Rs can respond to a single agonist^2,52,56,57, it is plausible that a deletion of TAS2R105 alone could be compensated for by other TAS2Rs. However, the depletion of Tas2r105-expressing cells results in the loss of function of multiple TAS2Rs, thereby leading to male infertility.

In addition, polymorphisms in human TAS2Rs have been associated with male infertility. In 2010, Aston and colleagues published the first report of a nonsynonymous single nucleotide polymorphism (SNP) (located at the 887 bp position in the TAS2R38 coding region), rs10246939, associated with azoospermia⁵⁸. However, this finding was not replicated in two subsequent studies^59,60. The different ethnic groups used in these three studies may explain the discrepancies. In addition to TAS2R38, two other TAS2R SNPs have been reported to be associated with male infertility. Homozygous carriers of the G allele at the 371 bp position of the TAS2R14 coding sequence (rs3741843) showed decreased progressive sperm motility compared to heterozygotes and homozygotes of the A allele. Men with the homozygous T allele at position 1422 bp upstream of TAS2R3 (rs11763979) are more likely to have fewer normal acrosomes than those with the heterozygous genotype and homozygous carriers of the G allele⁶¹. These findings are exciting, but it is crucial to further validate the roles of these TAS2R alleles in sperm dysfunction and understand the underlying mechanisms driving these changes.

3. The roles of TAS2Rs in the female reproductive system

The female reproductive system is a complex network of organs, each of which plays a unique role in reproduction. Critical functions, including the menstrual cycle, ovulation, fertilization, pregnancy, and parturition, are all integral to successful mammalian reproduction. Given the unique characteristics of different cell types in different regions of this system, we will discuss the presence and role of TAS2Rs in each tissue separately.

3.1. TAS2Rs in the ovary

The ovaries play critical roles in hormone production and oocyte development. In particular, ovulation, when the dominant follicle is released from the ovary into the fallopian tube, relies heavily on granulosa and cumulus cells for oocyte maturation and fertilization.

During the antral follicular phase, the granulosa cells differentiate into cumulus cells and mural granulosa cells, resulting in an antrum filled with follicular fluid. This fluid contains essential regulatory molecules such as hormones, steroids, and electrolytes derived from serum ultrafiltration and follicular cell secretions, which are necessary for oocyte maturation. During ovulation, a cumulus-oocyte complex is released, which includes cumulus cells, some follicular fluid, and the oocyte. This complex promotes fertilization by facilitating the interaction between the cumulus oophorus and the sperm in the fallopian tube.

Mural granulosa cells and cumulus cells extend their functions beyond simply nurturing the oocyte. They can act as chemosensors, releasing specific molecules that maintain the quality of developing oocytes and guide sperm for successful fertilization. Cell membrane receptors capable of sensing various molecules in this reproductive microenvironment could be a significant advantage for successful reproduction.

Regarding chemosensory receptors, the expression of Tas2r in the ovary was first reported on day 9 in newborn Tas2r131 GFP reporter mice⁴² (Table 1). Subsequent studies with qRT-PCR, Western blotting, and immunostaining revealed that TAS2R3, TAS2R4, TAS2R14, TAS2R19, TAS2R43, and some of their downstream signaling components, α-gustducin and α-transducin, were expressed in either granulosa cells or cumulus cells⁶² (Table 2). Among them, TAS2R14 was the most highly expressed in both granulosa cells and cumulus cells. Intriguingly, while no study has directly demonstrated the presence of TAS2Rs in oocytes, an examination of publicly available RNA sequencing datasets from the EMBL-EBI Expression Atlas (https://www.ebi.ac.uk/gxa/experiments/E-ERAD-401) revealed the expression of Tas2r108 and Tas2r137 in metaphase II oocytes from various mouse strains⁶³.

In addition, the levels of TAS2R14 and TAS2R43 were higher in young women than in older women. Using a protein network analysis tool, the authors found that TAS2R14 and its various downstream signaling components are central functional hubs⁶². While these findings demonstrate the presence of TAS2Rs in the ovary and suggest potential roles in ovarian function, there has been a lack of functional studies to establish these roles in the ovary. Using saccharin, an agonist for both TAS1R2 and TAS2R31 (known as TAS2R140 in the rat), Jiang et al. suggested that activation of TAS2R140 may reduce the protein expression of steroidogenesis-related factors in the rat, resulting in the inhibition of ovarian progesterone production⁶⁴ (Figure 3A).

Figure 3. Potential functions of TAS2Rs in the ovary.

(A) The activation of TAS2Rs promotes the production of nitric oxide (NO), leading to increased levels of cyclic guanosine 3',5'-monophosphate (cGMP). This, in turn, reduces the protein expression of steroidogenesis-related factors, ultimately inhibiting progesterone production in the corpus luteum. (B) In granulosa cell or cumulus cell, the binding of endocrine disrupting chemical (EDC) to TAS2Rs inhibits lipid droplet production. This results in a decrease in progesterone secretion, thereby regulating follicular maturation before ovulation and contributing to quality control of the oocyte after ovulation.

Luongo et al. recently identified a subset of TAS2Rs (i.e., TAS2R3, TAS2R4, TAS2R14, TAS2R19, and TAS2R43) in an immortalized human granulosa cell line (hGL5)⁶⁵. Their study focused on the effects of endocrine disrupting chemicals (EDCs) - substances that interfere with the synthesis, transport, metabolism, or binding actions of hormones critical for reproductive functions, homeostasis, and developmental processes – on mitochondrial morphology and intracellular lipid content. Since mitochondrial morphology is a proxy for the health of a cell and undergoes dynamic changes, the authors used the Mitochondrial Network Analysis tool to measure several mitochondrial descriptors, such as the number of individuals and networks, mean branch size, mean branch length, and network size, to calculate the mitochondrial footprint.

Among several EDCs analyzed by these authors, biochanin A (BCA) and caffeine, two potent TAS2R agonists, were observed to modulate TAS2R expression in hGL5 cells. BCA increased the expression of TAS2R14 and TAS2R43 and decreased the abundance of TAS2R19. In contrast, caffeine significantly decreased the expression of TAS2R3, TAS2R19 and TAS2R43 but increased the abundance of TAS2R14. Regarding cellular effects, BCA increased both the mitochondrial footprint and median branch length, whereas caffeine decreased the mitochondrial footprint. Furthermore, both BCA and caffeine reduced the intensity of oil red staining in hGL5 cells, indicating a reduction in intracellular lipids. However, only caffeine reduced the amount of lipid deposition.

Given the critical role of mitochondria in steroid hormone biosynthesis in steroidogenic cells and the functional importance of lipid droplets in steroidogenesis⁶⁶, these authors further evaluated the effects of these two EDCs on sex steroid hormone production. They found that BCA induced an increase in estrogen secretion and a decrease in progesterone secretion, whereas caffeine did not affect estrogen levels but appeared to inhibit progesterone secretion. These findings suggest a mechanistic role for TAS2Rs in mediating the effects of EDCs on follicular granulosa cell function⁶⁵ (Figure 3B).

In addition to normal physiological conditions, the therapeutic potential of targeting TAS2Rs in ovarian disorders has also been explored. Polycystic ovary syndrome (PCOS), one of the most common reproductive disorders in women, is a complex condition often associated with obesity. Wu et al. recently discovered that the mouse ovary expresses Tas2r108. Moreover, using a high-fat diet-induced mouse model of PCOS, these authors found that a hop derivative called KDT501, an agonist of TAS2R108, may activate these receptors in the ovary. Their activation could potentially ameliorate PCOS-associated endocrine and metabolic disorders, leading to the restoration of reproductive function⁶⁷.

3.2. TAS2Rs in the uterus

The investigation of TAS2Rs in the human and mouse uterus has been largely focused on the myometrium, an area to which our research group has actively contributed. We were the first to identify a subset of TAS2Rs expressed in the human myometrium, the human myometrial cell line hTERT-HM, and the mouse myometrium⁶⁸ (Table 1 and Table 2). Similarly, Martin et al. reported the expression of several TAS2R mRNAs in normal human uterine tissue ⁶⁹. In addition, we observed that bitter tastants, such as chloroquine, induced relaxation in precontracted uterine smooth muscle strips, a relaxation effect superior to that of currently used tocolytics⁶⁸.

To understand the molecular mechanism behind this, we studied single smooth muscle cells from the mouse myometrium and found that bitter tastants triggered the activation of classical TAS2R downstream signaling components, which in turn reversed uterotonic-induced increases in [Ca²⁺]_i and cell shortening in myometrial cells (Figure 4A). This reversal effect is likely mediated via the inactivation of voltage-dependent Ca²⁺ channels, as has been observed in airway smooth muscle cells²⁶. To validate the role of TAS2Rs in this process, we knocked down TAS2R10 and TAS2R14 (chloroquine receptors) with RNA interference (RNAi) in the human myometrial cell line hTERT-HM. Our results highlighted TAS2R14 as the mediator of chloroquine action in human myometrial cells⁶⁸.

Figure 4. Potential roles of TAS2Rs in the uterus.

(A) Activation of TAS2Rs inhibited the calcium influx induced by uterotonics through voltage-dependent calcium channels (VDCC), thereby maintaining uterine smooth muscle relaxation and preventing preterm labor. (B) In the syncytiotrophoblast, TAS2Rs activation may bolster innate immune defense functions. Sensing of quorum sensing molecules (QSMs) released by infecting microorganisms by TAS2Rs leads to the production of nitric oxide (NO), which is then secreted to combat the invading pathogens. Additionally, TAS2R activation triggers the release of cholecystokinin (CCK) by the TAS2R/Tas2r-expressing cells, which in turn upregulates ATP-binding cassette (ABC) transporters, thereby facilitating the extrusion of harmful compounds from the cell.

Preterm birth, occurring before 37 weeks of gestation, remains a leading cause of infant morbidity and mortality⁷⁰. Despite multiple proposed and varying triggers, spontaneous preterm labor is almost always associated with increased uterine contractility prior to full term^71,72. Given the above findings, we propose that chloroquine's role in uterine relaxation could potentially help prevent preterm birth. We investigated the effects of chloroquine in mouse models of preterm birth induced by the bacterial endotoxin lipopolysaccharide or the progesterone receptor antagonist mifepristone. Our studies showed that chloroquine was more effective than other previously used tocolytics, namely, MgSO₄ or beta-2 agonists, in preventing preterm birth in mice⁶⁸. This protective effect was diminished when the TAS2R signaling component α-gustducin was genetically deleted, further supporting our hypothesis that bitter tastants such as chloroquine can prevent preterm birth through TAS2R signaling (Figure 4A).

This finding points toward an exciting new application of chloroquine as a potential tocolytic for human preterm contraction. The clinical safety and efficacy of chloroquine, along with its analog hydroxychloroquine, are well documented in conditions such as malaria, systemic lupus erythematosus (SLE), and rheumatoid arthritis^73,74. Leroux et al. found that hydroxychloroquine significantly reduced preterm birth and alleviated intrauterine growth restriction, thereby decreasing neonatal morbidity in women with SLE⁷⁵. Similarly, Kroese et al. showed that hydroxychloroquine use in pregnant women was associated with a gestational period longer by 2.4 weeks in the SLE population⁷⁶. In a population-based prospective pregnancy cohort, Berard et al. observed a nonsignificant 13% decrease in the risk of prematurity among those using chloroquine and hydroxychloroquine in the second/third trimester⁷⁷. The question remains: is the extended gestation caused by chloroquine or hydroxychloroquine in humans due to their activation of TAS2R, which leads to the relaxation of the uterine smooth muscle? Our study supports this possibility; however, further investigation is needed, particularly among women who have a tendency toward preterm labor and birth but do not have SLE.

Chloroquine acts as an agonist for multiple TAS2Rs in both animals and humans. Given the broad expression of Tas2rs across different organs, an agonist that interacts with several TAS2Rs could potentially activate diverse biological processes, which might lead to previously unidentified side effects. We found that phenanthroline, a specific TAS2R5 agonist, reversed human term uterine contraction induced by oxytocin and several contractile inflammatory mediators⁷⁸. This suggests that activating TAS2R5 alone might trigger a broad spectrum of uterine relaxation. This discovery is significant because parturition and preterm labor are complex processes involving a range of contractile hormones and inflammatory mediators. Therefore, an effective tocolytic for preterm labor should ideally be capable of broad-spectrum uterine relaxation. Consequently, phenanthroline and its derivatives could potentially pave the way for a new class of uterine relaxants.

3.3. TAS2Rs in the placenta

The placenta plays a unique role in regulating the transfer of oxygen and metabolites between the pregnant individual and the developing fetus. This multifaceted organ, however, is vulnerable to infections and inflammation due to its intricate function and complex structure. These pathologies can significantly impact the health of both the pregnant individual and the fetus. Surprisingly, Wölfle et al. (2016) discovered high TAS2R38 expression in the syncytiotrophoblasts of the human placenta⁷⁹ (Table 2). The placental cell line JEG-3 also showed a robust signal when stained with a TAS2R38 antibody. Exposure to the TAS2R38-specific agonist phenylthiocarbamide induced a calcium influx that could be inhibited by the TAS2R38 inhibitor probenecid. In the human upper airway epithelium, TAS2R38 stimulated calcium-dependent nitric oxide production in response to quorum sensing molecules (QSMs) released by infecting microorganisms, thereby promoting innate defense via increased cilia beating, mucus clearance, and direct antimicrobial effects of nitric oxide⁸⁰. The syncytiotrophoblast, a multinucleated cell layer that forms the barrier between the fetal and maternal circulation, may use TAS2R38 in a similar sentinel role, generating an innate defense upon sensing QSMs during a placental infection in pregnancy (Figure 4B).

In another study, TAS2R14 was found to be coexpressed with cholecystokinin (CCK) in the human placenta, the human trophoblast cell line HTR-8/SVneo, and three other trophoblast-derived choriocarcinoma cell lines, JAR, JEG, and BeWo⁸¹ (Table 2). Both CCK and TAS2R14 agonists induced an increase in [Ca²⁺]_i. However, no functional assay was performed on TAS2R14 in the placenta. Drawing an analogy with the findings of TAS2R38 expressed by enteroendocrine cells in the gut, the authors proposed potential physiological or pathophysiological roles for TAS2R14. In the gut, activation of TAS2R38 by taste compounds leads to the secretion of CCK, which, in turn, upregulates P-glycoprotein (also known as multidrug resistance protein 1 or ABCB1 transporters) in neighboring enteric cells⁸². This results in the extrusion of the noxious compound into the intestinal lumen, thereby limiting intestinal absorption. It is possible that in the placenta, autocrine CCK may exert similar immunomodulatory effects (Figure 4B). As such, these ideas warrant further investigation to fully understand their potential implications for protecting the fetus from potentially harmful chemicals.

3.4. TAS2R in the cervix and vagina

There has been limited research on TAS2Rs in the lower part of the female genital tract. Wölfle and colleagues were the first to report TAS2R38 expression in the mucosal epithelium lining the endocervical crypts⁷⁹ (Table 2). Shortly thereafter, using the Tas2r143 reporter mouse (Tas2r143-CreERT2;Rosa26flox-mT-stop-flox-mG), Liu et al. revealed EGFP-positive cells in the epithelium of the mouse vagina and cervix, suggesting the possible expression of the Tas2r143/Tas2r135/Tas2r126 cluster⁸³ (Table 1).

In the vagina and cervix, the majority of EGFP-positive cells in the Tas2r143 reporter mouse are mitotically active basal epithelial cells (K5-positive), while a small fraction are terminally differentiated epithelial cells (K10-positive)⁸³. These mitotically active basal epithelial cells have the capacity to migrate upward and differentiate into terminally differentiated epithelial cells⁸⁴. Given that the vagina and cervix are open to the external environment and at risk of infection by sexually transmitted pathogens, it is plausible that Tas2r-expressing cells in the epithelium could play a critical role in the initial defense against these pathogens. They might achieve this possibly by promoting cell migration to the superficial layer, proliferation, and differentiation into protective epithelial cells (Figure 5A). However, this hypothesis needs to be confirmed with further research.

Figure 5. Hypothetical roles of TAS2Rs in the vagina and cervix.

(A) In the vagina, invading pathogens activate TAS2Rs in basal epithelial cells. This activation promote the migration, proliferation, and differentiation of these cells into superficial protective epithelial cells, enhancing the defensive barrier. (B) In the cervix, the binding of hormones to TAS2Rs results in an increase cytosolic Ca²⁺, which in turn stimulates the secretion of mucin, contributing to the protective mucosal barrier.

While no functional assays on TAS2R38 were performed in the cervix, it is worth noting that TAS2R38 modulates thyroid-stimulating hormone-dependent intracellular Ca²⁺ signals in human thyroid epithelial cells. This, in turn, regulates the secretion of the thyroid hormones triiodothyronine and thyroxine⁸⁵. Given these observations, it is plausible to hypothesize that TAS2R38 could serve a similar function within the epithelium of endocervical crypts, potentially sensing hormonal signals to regulate mucus secretion (Figure 5B).

4. The role of TAS2Rs in reproductive cancers

Costa et al. recently provided a comprehensive summary of bitter taste signaling in cancer⁸⁶. Considering their extensive review, our focus here will be a brief overview of TAS2Rs in reproductive cancers. Reproductive cancers, including prostate, breast, ovarian, cervical, uterine, vaginal, and vulvar cancers, pose a significant health concern. Each presents unique challenges in terms of diagnosis, treatment, and prognosis, with ovarian cancer often being particularly difficult to detect in its early stages. Therefore, the discovery of novel pathways or targets for reproductive cancers is of great interest to researchers.

Singh et al found that TAS2R4 mRNA and protein levels in breast cancer were lower than those in noncancer cell lines⁸⁷. This decrease was further confirmed in breast cancer and normal breast tissues⁸⁸. Martin et al demonstrated that a subset of TAS2Rs, specifically TAS2R1, TAS2R4, TAS2R10, TAS2R14, and TAS2R38, were expressed at lower levels in endometrial and ovarian cancer cell lines than in normal uterine tissue⁶⁹. The same study also showed that this group of TAS2Rs was detected in four prostate cancer cell lines. Intriguingly, the expression levels in three prostate cancer cell lines (DU145, LNCaP, and PC3) were decreased compared to those in benign prostate hyperplasia cells (BPH-1 cells). These observations support the general pattern that TAS2Rs are downregulated in cancer cells^87-91. However, the biological and pathogenic implications of this alteration in reproductive cancers are not yet fully understood. Interestingly, Singh et al revealed that activation of TAS2R4 and TAS2R14 induced apoptosis and impaired chemotactic migration in breast cancer cells⁸⁸. Furthermore, Seo et al found that overexpression of TAS2R8 or TAS2R10 inhibited self-renewal capacity as well as migration and invasion in the neuroblastoma cell line SK-N-BE(2)C⁸⁹. Strikingly, subcutaneous injection of these overexpressing cells resulted in reduced tumor incidence and growth⁸⁹. Therefore, it is plausible that modulating the level of TAS2Rs may influence cell proliferation and renewal capacity in reproductive cancers (Figure 6A).

Figure 6. Potential roles of TAS2Rs in the development, treatment, and combating chemotherapy resistance in gynecological cancers.

(A) In cancer cells, a relatively low expression level of TAS2Rs is associated with increased proliferation and tumor formation. Therapeutically, enhancing the expression of TAS2Rs can reduce tumor growth. Additionally, the binding of bitter agonists to TAS2Rs promotes cell apoptosis, thereby potentially decreasing tumor occurrence. (B) Bitter agonists can reverse chemotherapy resistance in cancer cell by inhibiting the expulsion function of ATP-binding cassette (ABC) transporter through the activation of the TAS2R signaling pathway.

Several TAS2R agonists have shown potential anticancer activity. In particular, the TAS2R14 agonists noscapine and diphenhydramine have been shown to induce proapoptotic effects on the ovarian cancer cell line SKOV3⁶⁹. Epigallocatechin gallate, a ligand of TAS2R14 and TAS2R39, induced apoptosis specifically in MCF7 and MDA-MB-231 breast cancer cells. It does this by increasing PARP, caspase-3, and caspase-9, which leads to cell cycle arrest ^92-94. Kaempferol, also a ligand of TAS2R14 and TAS2R39, increased apoptosis via the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) and telomerase pathways in human cervical cancer cells or via the death receptors/Fas-associated death domain (FADD)/caspase-8 pathway in ovarian cancer cells^95,96. Resveratrol, another agonist of TAS2R14 and TAS2R39, suppresses cervical cancer cell proliferation and induces apoptosis via the mitochondrial and p53 pathways⁹⁷. Genistein, which also activates TAS2R14 and TAS2R39, decreased the incidence of ovarian cancer by inhibiting nuclear factor-kappa B (NF-kB) and B-cell lymphoma 2 (Bcl-2) while activating nuclear factor erythroid 2-related factor 2 (Nrf2) and Bcl-2-associated X protein (Bax)⁹⁸. In xenograft mouse models of breast cancer, quercetin, an agonist of TAS2R14, delayed and decreased tumor growth and inhibited the metastatic ability of cancer stem cells in proliferating and generating mammospheres^99,100. Therefore, a potential approach in the development of treatments for reproductive cancers involves exploiting agonist-mediated activation and overexpression of TAS2Rs (Figure 6A).

While the potential of TAS2R agonists in cancer treatment is promising, another significant challenge in cancer therapy is resistance to chemotherapeutic agents. This drug resistance often leads to treatment failure, relapse, or death^9,86,101. Interestingly, bitter compounds such as quercetin and resveratrol, which have been extensively studied as coadjuvants in combination with chemotherapeutic drugs in several cancers, show promise⁸⁶. Specifically, in ovarian cancer cells, quercetin treatment in combination with platinum reversed platinum resistance by restoring extracellular signal-regulated kinase (ERK) phosphorylation and inducing cancer cell apoptosis¹⁰². A potential mechanism for reversing chemoresistance involves these bitter-tasting compounds acting as substrates or inhibitors of ABC transporters. Duarte et al. showed that silencing TAS2R14 increased the expression of ABCC4 (a member of the ABC transporter family) compared to mock- and siRNA scramble-transfected cells in a choroid plexus papilloma cell line¹⁰³. Since quercetin activates TAS2R14, it is plausible that its effect on platinum resistance in ovarian cancer cells may be mediated through activation of TAS2R (Figure 6B).

5. Challenges and future directions

Our understanding of TAS2Rs in the reproductive system faces several significant challenges. At present, there is a heavy reliance on qRT-PCR, largely due to a lack of reliable commercial antibodies. While qRT-PCR is a reliable and sensitive technique utilized to quantify the level of a specific section of mRNA in a tissue sample composed of different cell types, it does not provide information about which cell types express TAS2Rs. This limitation curtails our understanding of the unique roles TAS2Rs play within different cells in the reproductive system. To overcome this, in addition to developing robust TAS2R antibodies, we could also use advanced new molecular biology techniques to study TAS2Rs in the reproductive system. Two such techniques are single-cell RNA sequencing¹⁰⁴ and spatial transcriptomics¹⁰⁵. With these approaches, one can accurately identify TAS2R-expressing cells in the reproductive system.

Although TAS2R expression is well documented in various reproductive tissues, comprehensive functional studies are unfortunately still sparse. Existing functional assays primarily rely on immortalized or carcinoma cell lines and employ strategies such as [Ca²⁺]_i measurement with TAS2R agonists and RNAi, a molecular technique to decrease protein translation¹⁰⁶. While these methods are informative, they provide limited insight into TAS2R functions in their native environment, underlining the need for more in vivo or in situ studies. Furthermore, much of the current research has only speculated about the functional implications of TAS2Rs in the reproductive system, often drawing parallels to similar events in other extraoral tissues.

Crucially, we still lack concrete genetic evidence concerning the functions of TAS2Rs in the reproductive system. Only a few Tas2rs, including Tas2r131 and the Tas2r143/Tas2r135/Tas2r126 cluster, have been tagged genetically in mice^42,83. Similarly, only a handful of Tas2r knockout mice involving Tas2r105 and the Tas2r143/Tas2r135/Tas2r126 cluster have been generated in different laboratories^55,107-109. Notably, no cell-specific knockout mouse has been reported yet. Most of the existing genetic evidence from other systems is derived from the deletion of taste signaling downstream components, such as α-gustducin and TRPM5. Given that the signaling cascade is shared by taste receptors, including the 35 TAS2Rs, the sweet taste receptor, and the umami taste receptor, it is critical to generate specific Tas2r knockout mice to uncover direct evidence for the involvement of corresponding TAS2Rs in the proposed functions in the reproductive system. Furthermore, considering that different TAS2Rs can be activated by the same agonist, the presence of compensatory effects cannot be ruled out if not all TAR2Rs with similar functions are deleted. Therefore, the generation of multiple Tas2rs knockouts becomes a necessity.

CRISPR/Cas9 gene editing technology, known for its efficacy in targeting multiple genes simultaneously^110,111, was used by our team and two other groups to generate Tas2r143/Tas2r135/Tas2r126 cluster deletion mice^107-109. With 35 Tas2rs located in multiple clusters on the mouse chromosome, it is feasible to create a Tas2r-less mouse through a series of stepwise applications of CRISPR/Cas9. Such Tas2r deletion mice could provide deeper insights into the role of TAS2Rs in the reproductive system.

In the oral cavity, TAS2Rs function as a defense mechanism, detecting potentially harmful or toxic substances in our food and preventing their ingestion^1,2. Outside the gastrointestinal tract, the likelihood that bitter components of a food will reach the extraoral tissues where TAS2Rs/Tas2rs are expressed is relatively low. However, the female reproductive tract, which is open to the outside, is susceptible to invasion by pathogenic microorganisms. Thus, TAS2Rs, particularly those expressed in the cells of the vagina and cervix, may serve as a defense by detecting potentially harmful invaders and initiating an innate immune response.

A recent study identified a group of chemosensory cells in the porcine endometrium. These cells are immunoreactive for taste transduction components (PLCβ2 and TRPM5), as well as the chemosensory cell markers acetylcholine synthesizing enzyme and choline acetyltransferase¹¹². These chemosensory cells may function as sentinels and respond immunologically to uterine pathogens. Furthermore, since parturition or preterm labor is an inflammatory process^71,113, these uterine chemosensory cells could potentially sense changes in cytokine and chemokine secretion by infiltrating immune cells. Finally, since progesterone, a vital hormone during pregnancy and parturition, is a known ligand of TAS2R110, TAS2R114, and TAS2R46⁵², it would be worthwhile to investigate whether these chemosensory cells express TAS2Rs.

It is worth noting that many scientific discoveries in animals do not translate directly to humans. This caution holds true in the context of investigating the role of TAS2R in reproduction as well. For example, while a mouse has evolved 35 Tas2rs, humans have only 25 TAS2Rs. Some functional human TAS2Rs, such as TAS2R5, have no counterparts in mice¹¹⁴, making it difficult to use mice as animal models to study these TAS2Rs. However, modern technologies can help overcome these challenges. For example, one could perform in vitro tissue culture and genetic manipulation. McCloskey et al. (2014) investigated the function of the potassium channel KIR7.1 by lentiviral-mediated knockdown or overexpression in mouse uterine strips in vitro¹¹⁵. Similarly, in vitro TAS2R knockdown in human uterine strips will allow us to investigate whether the relaxation effect of bitter tastants is mediated by TAS2Rs. Additionally, the recent combination of organoid culture, a method that produces miniaturized and simplified versions of organs, with microfluidic technology has led to the development of a 'human-on-a-chip' platform¹¹⁶. This platform enables high-throughput clinical applications, drug discovery, and toxicology studies. Such advanced methods can accelerate the evaluation of the role of TAS2Rs in the reproductive system. For example, one could identify endogenous ligands for TAS2Rs expressed in granulosa and cumulus cells, thereby revealing the underlying molecular mechanisms by which TAS2Rs affect follicular maturation, ovulation, and fertilization.

Finally, although there has been extensive research on TAS2Rs in the reproductive system, no study has directly investigated potential changes in TAS2Rs or examined the role of these receptors in obstetric and gynecologic diseases. These diseases often involve complex hormonal and cellular changes that may involve TAS2Rs, and understanding the role these receptors may play could provide invaluable insight into disease mechanisms. This could open new avenues for treatment strategies, diagnostics, and even preventive measures. It is therefore crucial that future research efforts on TAS2Rs are directed toward this underexplored area.

6. Conclusions

In summary, the presence and likely roles of TAS2Rs in the reproductive system offer new horizons for understanding reproductive health and disease. Research into this field has revealed the extensive expression of TAS2Rs in reproductive tissues and their potential involvement in key reproductive processes, such as fertility, immune surveillance, steroidogenesis, and uterine contractility.

These findings promise to have a significant impact on the field of reproductive medicine. The potential to develop TAS2R-targeted therapies could provide a novel approach to treating male and female infertility, preventing preterm birth, or addressing reproductive cancers. However, research to understand the full potential of TAS2Rs in reproductive health is just beginning. There remain significant gaps in our knowledge, particularly regarding the role of TAS2Rs in obstetric and gynecologic diseases, which warrant further investigation. As we delve deeper into this evolving field, we can look forward to discoveries that may provide new approaches to reproductive health and disease management.

Abbreviations

TAS2R

type 2 taste receptor or bitter taste receptor

TAS1R

type 1 taste receptor

GPCR

G protein-coupled receptor

ER

endoplasmic reticulum

cGMP

cyclic guanosine monophosphate

EDC

endocrine disrupting chemical

QSM

quorum sensing molecule

NO

nitric oxide

CCK

cholecystokinin

CatSper

cation channel in sperm (a calcium channel of mammalian spermatozoa)

PLCβ2

phospholipase C beta 2

IP3R

inositol 1,4,5- trisphosphate receptor

TRPM5

transient receptor potential cation channel subfamily M member 5

CALHM1/3

calcium homeostasis modulator 1/3 channel

[Ca²⁺]_i

intracellular Ca²⁺ concentration

ATP

adenosine triphosphate

IL-25

interleukin 25

IL-13

interleukin 13

EGFP

enhanced green fluorescent protein

qRT-PCR

quantitative reverse transcription-polymerase chain reaction

SNP

single nucleotide polymorphism

BCA

biochanin A

PCOS

polycystic ovary syndrome

RNAi

RNA interference

hTERT-HM

human telomerase reverse transcriptase-infected human myometrial cell line

SLE

systemic lupus erythematosus

ABCB1

ATP-binding cassette subfamily B member 1 transporter or P-glycoprotein

CRISPR

clustered regularly interspaced palindromic repeats

Cas9

CRISPR-associated endonuclease 9