G-Protein Coupled Hormone Receptors of the Hypothalamic-Pituitary-Gonadal Axis are Targets of Endocrine Disrupting Chemicals

Endocrine-disrupting chemicals have received significant concern, since they ubiquitously persist in the environment and are able to induce adverse effects on health, and more particularly on reproductive function. Most of the studies focused on nuclear hormone receptors as mediators of sex steroid hormones signaling. However, there are increasing evidences that peptides hormones of the Hypothalamo-Pituitary-Gonadal axis are targets of endocrine-disrupting chemicals (as Gonadotropin-Releasing Hormone, Follicle-Stimulating Hormone, Luteinizing Hormone…). The majority of these hormones act on G protein-coupled membrane receptors. This review summarizes the effects of endocrine-disrupting chemicals on homeostasis of peptides hormone of Hypothalamo-Pituitary-Gonadal axis and on their G protein-coupled membrane receptors signaling revealed by experimental, clinical, and epidemiological studies in human.


Introduction
Public concern of endocrine-disrupting chemicals (EDCs) has been rising since the 1990s. EDCs are defined as "an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub)populations" [1][2][3]. EDCs are found in many products comprising plasticizers, personal care products, pesticides… [1]. Humans are constantly exposed to several different EDCs by ingestion, inhalation, and dermal contact. Some classes of EDCs have been studied in detail. Here, we selected three classes of EDCs based on knowledge of their effects on Hypothalamo-Pituitary-Gonadal (HPG) axis: bisphenol A (BPA), phthalates and dichlorodiphenyltrichloroethane (DDT). BPA is one of the most massively produced EDC with over three million tons manufactured annually [4]. It is used in food packaging, toys, resins used in canned, and medical equipment. Because its incomplete polymerization and its release from polycarbonate at high temperature, exposure to BPA is important via food containers [5][6][7]. Phthalates are used as liquid plasticizers found in a wide range of products including plastics, coatings, toys, cosmetics, and medical tubing. They are classified in two groups: high molecular weight phthalates, such as diethylhexyl phthalte (DEHP), and low molecular weight phthalates, such as dibutyl phthalates (DBP) [8]. DDT, an organochlorine pesticide, was largely used after the Second World War for its insecticidal properties. Although it was banned in the 1970s in the Western World, it continues to be used in developing countries. DDT is a synthetic mixture of three isoforms: p,p'DDT, o,p'DDT and p,p'DDD. EDCs are originally thought to act through nuclear hormone receptors, such as estrogen receptor (ER) or androgen receptor (AR) [9]. During the last decade, we, and others, were interested in the effect of EDCs on G-protein-coupled hormone receptors (GPCRs). These studies have shown that there are chemical compounds in the environment capable of binding to GPCRs and disrupting the activity and intracellular signaling pathways of receptor. Moreover, EDCs may alter pathways involved in hormone biosynthesis and/or receptor signaling regulation. This review summarizes the effects of three classes of EDCs on hormones homeostasis and GPCRs signaling involved in the HPG axis. Several molecular mechanisms can be involved in the EDC effects on the HPG axis. All studies cited here were performed in human species.

GPCRs implicated in the HPG axis
The GPCRs are the largest family of cell-surface receptors with over 800 members accounting for 4% of the encoded human genome [10]. About half of them have sensory functions, mediating olfaction, taste, light perception, and pheromone signaling. The other half (~350-400) are called endo-receptors, i.e. receptors that interact with endogenous ligands [11]. These receptors are involved in the detection of many extracellular stimuli (from photons or ions to large hormones proteins). Thus, they have important roles in various physiological systems. Dysfunction of GPCRs contributes to many human diseases and GPCRs represent 34% of all Food and Drug Administration-approved drugs [12].
GPCRs are characterized by a common structure with seven transmembrane helices with an extracellular N terminus and an intracellular C terminus [13]. The N-terminal portion, or transmembrane domain, constitute the ligand binding site while the C-terminal portion and the intracellular loops form a coupling domain with the intracellular effectors [14].
In the classical GPCR signaling pathway, after ligand binding, activated-GPCR binds the intracellular heterotrimeric G proteins, promoting the release of GDP from the Gα subunit, exchanged for GTP and the dissociation of the GTP-bound α subunit from βγ dimers. The activated G proteins can then transduce and amplify GPCR signals via second messengers to produce a variety of cell responses [15]. Briefly, Gαs activates adenylyl cyclases to catalyze the conversion of ATP to cAMP. Members of the Gαi family primarily inhibit cAMP production. The Gα q/11 family converts phosphatidylinositol 4,5-bisphosphate to diacylglycerol and inositol 1,4,5-trisphosphate to activate Protein Kinase C and increases intracellular Ca 2+ levels. Approximately 10% of GPCRs can be coupled with different types of Gs subunit depending on cell type and context [16]. The second messengers then target other enzymes such as cAMP-dependent protein kinase A (PKA), GMP-dependent protein kinase G (PKG), Ca 2+ -dependent protein kinase C (PKC) or calciumsensitive enzymes. The Gβγ subunit can also activate a multitude of effectors (GRKs, ion channels, PI3K, phospholipases, MAP kinases) to induce a variety of cellular effects [17]. G protein-mediated signaling is discontinued when the Gα subunit hydrolyzes GTP to GDP, due to its intrinsic GTPase activity. This then leads to the reassociation of Gα with Gβγ to form the inactive heterotrimer [14]. In addition to canonical signaling through heterotrimeric G proteins, some of GPCRs can use alternative modes of GPCR activation and initiate G protein-independent pathway. The main independent pathway involves a coupling with β-arrestin. Originally, β-arrestin was identified as an essential factor in the endocytosis and arrest of GPCR signaling induced by heterotrimeric G proteins. Today, other functions associated with β-arrestins are being studied and coupling to β-arrestins is increasingly described as "scaffolding" proteins involved in multiple G proteinindependent signaling pathways. Indeed, in addition to clathrin, β-arrestins are able to bind to many proteins involved in different signaling pathways (Src, ERK1/2 and JNK3 kinases protein phosphatases, ubiquitin ligases…) [18]. The activation of β-arrestins signaling pathways can take place at the membrane but also in intracellular after internalization [15]. Indeed, a growing amount of evidence suggests that several molecules have not been known to be regulated by G proteins, suggesting that β-arrestin-mediated signaling pathways may be functioning in parallel with G-protein-mediated pathways enhancing GPCR signaling pathways.
The GnRH, a neuropeptidic hormone, is secreted by hypothalamic GnRHexpressing neurons into the portal blood vessels in rhythmic pulses [19]. It binds to a membrane receptor, the GnRH receptor, also known as the luteinizing hormone releasing hormone receptor (LHRHR), on pituitary gonadotropic cells and stimulates the biosynthesis and secretion of LH and FSH [19]. GnRHR is predominantly coupled to the Gq-protein [20]. GnRH/GnRHR pathway constitutes the initial step in the HPG axis and controls reproduction in both sexes. GnRH loss-of-function mutations are associated to normosmic hypogonadotropic hypogonadism [21]. GnRH neurons appear to be directly regulated by Kisspeptin-1 (KISS1), with Neurokinin B (NKB) and Prokineticin 2 (PROK2). KISS1 is a peptidic hormone mostly expressed in the hypothalamus [22]. It activates GPR54/KISS1R, which results in the activation of phospholipase C via Gq [12]. GPR54 has been described in brain regions, including hypothalamus, but also in peripheral regions [22]. Kisspeptin/GPR54 pathway has a crucial role in the onset of puberty, the regulation of sex hormone mediated secretion of FSH/LH, and in the control of fertility [22,23]. Inactivating and activating mutations in KISS1 or GPR54 genes have been associated with hypogonadotropic hypogonadism and precocious puberty, respectively [23].
Gonadal function is under pituitary control via the gonadotropin hormones: follicle stimulating hormone (FSH) and luteinizing hormone (LH) [24]. FSH and LH are synthesized and secreted by the pituitary gonadotropic cells and work together in the reproductive system. The human chorionic gonadotropin (hCG) is secreted by the placenta and controls ovarian function during gestation. LH and hCG share the same GPCR, the hCG/LHR. The FSH and hCG/LH receptor belong to the glycoprotein-hormone receptor family. Activation of the LH and FSH receptor results in the production of intracellular cyclic AMP (cAMP) via Gα s proteins [25,26]. However, FSHR and LHR can also couple to several other effectors such as Gα q and β-arrestin [26][27][28]. FSHR is expressed in Sertoli and granulosa cells in male and female gonads, respectively, and is required for normal spermatogenesis and growth and maturation of ovarian follicles, as well as for estrogen production [29]. In women, LHR induces luteinization of granulosa cells, progesterone synthesis and corpus luteum maintenance during the luteal phase [30]. In men, LH stimulates testosterone production by Leydig cells [30].
More recently, the INSL3/RXFP2 system pathway was identified for its role in reproduction. Insulin-like peptide-3 (INSL3) belongs to the insulin/relaxin family of peptidic hormones [34,35]. This hormone is mainly produced by testicular Leydig cells and the production is dependent on the state of Leydig cell differentiation [34]. INSL3 is considered as a marker for Leydig cells function. Its best characterized role is in the control of testicular descent since INSL3 gene inactivation males have bilateral cryptorchidism with testis remaining in abdominal position [36,37].

Effects of EDCs on signaling of HPG axis G-protein coupled receptors
Effects of EDCs on the activity of HPG axis GPCR identified in the literature search are summarized in Table 1.

Hypothalamic hormones receptor
Currently, there are no data on the effects of EDCs on the activity of human hypothalamic hormone receptors. However, some studies have been conducted with animal models. Exposure to phthalates leads to a modulation of GnRHR expression (positive or negative depending on the studies) [50,51], as well as an increase in its expression in rat uterus [52].

Gonadotropin hormones receptor
EDCs, like phthalates, increase the FSHR expression in human granulosa cells [38]. DDT has been shown to disturb the FSH induced-cAMP accumulation [39] and aromatase activity in human granulosa cells [40]. Recently, we showed that DDT behaves as an FSHR positive allosteric modulator [41]. DDT interacts with the receptor in the minor binding pocket in the transmembrane domain. DDT acts on the early steps of activation of the FSHR and induces an increase in FSH-stimulated cAMP production. Moreover, the binding of DDT enhances the FSHR response to hCG. The increased response to FSH in the presence of DDT and the gain of sensitivity to hCG may therefore by deleterious. In opposite, BPA is a FSHR negative allosteric modulator [41].
As for FSHR, EDCs, like BPA, disturbes the expression of hCG/LHR in human endometrial stromal cells [42]. In CHO-K1 cells stably transfected with hCG/LHR, DDT reduced the cAMP accumulation induced by hCG [39,41] and hLH (Munier et al., Arch Toxicol, in revision). Moreover, DDT decreases the hCG-and hLHpromoted β-arrestin 2 recruitment (Munier et al., Arch Toxicol, in revision). DDT seems to act as a negative allosteric modulator of the hCG/LHR signaling.

Insl3 receptor, RXFP2
Only one study has very recently focused on the effect if EDCs on receptor signaling to INSL3: RXFP2. In a cellular model of HEK293 transiently expressing human RXFP2, individually, BPA, DEHP and DBP potentiate the cAMP response to INSL3 [43]. Because of their ubiquity, BPA, DEHP and DBP are present in many human biological fluids, as the amniotic liquid. Furthermore, everyone is chronically exposed to mixtures of environmental chemical factors resulting in toxicological interactions that cannot be predicted by reprotoxicological studies of single molecules. The combination of these three molecules, at concentrations found in human amniotic fluid, decreases the basal activity of RXFP2 as well as the response to INSL3. The structural similarity between FSHR and RXFP2 suggests that small hydrophobic molecules, like phthalates and BPA, could use the same binding sites as DDT in FSHR. The binding of one or two compounds to this site could lead to a stabilization of the active state of the receptor driving an increase of agonist activity [53]. In contrast, the binding of three compounds (DEHP+DBP + BPA) likely leads to a steric hindrance that may prevent the conformational changes necessary for the activation of RXFP2 and probably stabilize an inactive state. This study shows that in addition to individual EDC targets, HPG axis GPCRs can also be targeted by EDC cocktails.

Membrane sexual steroid hormones receptor
The G protein-coupled receptor (GPER/GPR30) is a membrane estrogen receptor [31]. Gene inactivation of GPER in mice did not induce major modifications in reproductive function [54]. However, several studies show that this receptor has pro-oncogenic effects in hormone-dependent cancers. Although many EDCs exhibit low binding affinities to the nuclear ERs and often require relatively high concentrations (>1 μM) to affect genomic pathways, several studies have focused on non-genomic signaling mediated by GPER [55].
Various DDT derivatives and BPA bind to GPER with a K d between 1 to 10 μM and are competitors of E2 [46]. The binding affinity of EDCs for GPER is higher than for the nuclear receptors. Nevertheless, low concentrations of o,p'DDE and BPA increased cAMP production by GPER [32,45,46]. BPA and phthalate (MEHP) also affect proliferation and migration in human cervical cancer cells [56], in human seminoma cells [45], human breast cancer cells and cancer-associated fibroblasts that lack nuclear ERs [47,57] as well as the migration and invasion of lung cancer cells [48]. BPA modifies these cellular responses by modulating different intracellular signaling pathways (ERK 1/2 or Akt phosphorylation, gene expression) through GPER activation. In opposite, GPER mediates BPA-induced intracellular stress generation (ROS production and calcium accumulation) and apoptosis (caspase activation and mitochondrial membrane potential decrease) in human granulosa cells [49]. Recently, it has also been shown that BPA increases GPER gene expression in breast cancer cell lines [44]. Finally, bisphenols AF and B, two substitutes of BPA, exert high estrogenic effects via GPER pathway at nanomolar concentrations [58,59].

Effects of EDCs on the synthesis and secretion of HPG axis hormones
Effects of EDCs on the synthesis and secretion of HPG axis hormones identified in the literature search are summarized in Table 2. Interestingly, a study led on 262 mother-child pairs from China found a positive correlation between cord blood levels of BPA and KISS1 mRNA expression in placenta tissue [60].
For phthalates, linear regression analysis showed increasing trend for kisspeptin secretion with the concentration of urinary phthalates [61].

GnRH
No epidemiological or experimental studies are available on the possible link between EDC levels and GnRH concentration in human. This is probably explained by the pulsatile nature of its release and the lack of dosage in clinical practice. However, many effects of EDC on GnRH were observed in rodents [93].

Pituitary level
DDT is rapidly metabolized in the body to DDE. Thus, in epidemiological studies, DDE is dosed in the blood more often than DDT. In a cohort of men of reproductive age, statistically significant positive association was found between the serum level of DDE and LH or FSH [63]. However, others studies did not reveal any association between DDT and FSH or LH levels in adult men [62,64,65]. In peri and postmenopausal women, inverse correlation was found between serum DDT and LH [66]. Moreover, it has been shown that maternal exposure to DDT or DDE, assayed in prenatal serum, induced a reduction of plasma LH in teenage boys, not found for FSH [67]. A study also showed that the serum levels of LH (basal level and after GnRH stimulation) was significantly higher in girls with detectable serum DDE levels than in girls with undetectable DDE [68]. This difference was not found for FSH [68].
For BPA, studies found that higher urinary BPA concentration was associated with significantly higher concentrations of serum LH in healthy young men, with or without association with FSH [69][70][71]. However, these results were not confirmed in others cohorts of fertile men [73]. Conversely, another study found a positive correlation between urinary BPA concentration and FSH level, without change in LH level in a cohort of infertile men [72]. In women, no association was found between urinary bisphenol A and LH or FSH levels in premenopausal women [74][75][76]. Moreover, no  association was found in healthy children for LH and FSH [77,78]. A modest negative correlation was found between urinary BPA concentration and peak of GnRHstimulated FSH levels in girls with idiopathic central precocious puberty, without difference for LH levels [79]. Maternal phthalates exposure (urinary samples collected during second trimester) was not associated with serum LH level or FSH in offspring during mini-puberty in boys and girls [80]. However, positive correlations were observed between different phthalates and serum LH in prepubescent Korean children (for serum DBP or MEHP) [81], in girls with attention-deficit/hyperactivity disorder (for urinary MEP) [78] and in Chinese population (11-88 years, males and females) (for urinary MEHHP levels) [82]. In the same populations, either negative [82,83] or no effects [78,81] were observed on FSH level. In men, urinary phthalate metabolites were positively associated with LH and FSH levels [84] in one study while negative association between urinary phthalates concentrations and levels of FSH was found in American men (for MBzP) [85] and in Danish men (for MEHP or %MiNP) [86] without impact on LH.
Altogether, epidemiological data have linked exposure to EDC and LH and/ or FSH level but evidence were often inconclusive. The inconsistent findings may partly be due to differences in the characteristics and sizes of the cohorts and to the different EDC exposure levels among studies.

Sexual steroid hormones
Many data are already available on the effect of endocrine disruptors on the secretion of sex steroids. Recent reviews list all available studies for DDT [93], BPA [93,94] or phthalates [93,[95][96][97].

INSL3
No data are available on the impact of DDT on INSL3 in humans epidemiological studies.
There is also an inverse correlation between BPA level and concentration of INSL3 [92]. Indeed, in a population of 180 boys born after 34 weeks of gestation (52 cryptorchid and 128 control), cord blood levels of free BPA correlated negatively with INSL3 [92]. In this study, cord blood INSL3 level was also significantly decreased in the cryptorchid group compared with the control group [92].
Ex vivo studies on human testicular explant were performed, to study more precisely the effect of endocrine disruptors on the secretion of INSL3.
No data are available on the impact of DDT on INSL3 in humans experimental studies.

Conclusions
Most epidemiological and experimental studies focus on the effect of EDCs on the expression and secretion of hormones, as well as on the activity of nuclear steroid receptors. However, a few experimental studies have shown that G proteincoupled membrane receptors of the HPG axis are targets of EDCs as well. It can be pointed out that most of the studies analyzing the effects of EDCs on GPCRs of HPG axis have been performed with cell culture systems. In vitro models are valuable tools because they are easily manipulated. But the comparison of the effects of EDCs in wild-type and GPCRs-inactivated animal models could provide additional informations on the mode of action of these compounds.
Mechanisms of GPCR disruption by EDCs include: (1) changes in the expression; (2) interaction with transmembrane domain receptor; (3) modulation of intracellular signaling pathways.
The GPCRs of HPG axis, involved in diverse physiological functions, should be considered as possible contributors of the adverse effects of EDCs on reproduction. How their modulation by EDCs contributes to these deleterious effects should be an important field of investigations in the near future.