Endogenous angiotensin system and AGTR2 in epididymis
The epididymal lumen and efferent ductules contain a complete local renin-angiotensin system (RAS) including renin, angiotensin I (ANGI) and angiotensin II (ANGII) in the seminal fluid, the angiotensin-converting enzyme specific to the testes (tACE), and angiotensin II receptor type 1 (AGTR1) and angiotensin II receptor type 2 (AGTR2) in the basal cells of the epididymis (Leung et al. , 2003; Saez et al. , 2004; Speth et al. , 1999; Wong et al. , 1990; Zhao et al. , 1996). Importantly, ANGII in the epididymal lumen is mainly produced through the cleavage of ANGI by angiotensin I-converting enzyme (ACE) (Langford et al. , 1993; Sibony et al. , 1994). Deficiency in tACE leads to male infertility through impairing the function but not the production of sperm, implying that the RAS plays an important role in sperm maturation (Esther et al. , 1996; Hagaman et al. , 1998; Krege et al. , 1995).
AGTR1 and AGTR2 have been found in a radio-ligand binding assay to be expressed in the epididymal lumen. In particular, AGTR2 was specifically detected in basal cells and found to be required for the proton-secretion function of the epididymal lumen (Figure 1 and 2B, Table 1) (Shum et al. , 2008). Unexpectedly, AGTR2 was absent in clear cells, which regulated proton secretion. Further studies showed that AGTR2 activated the nitric oxide (NO)-cGMP pathway in response to ANGII stimulation in basal cells (Figure 1). NO produced by basal cells quickly diffuses to clear cells, activating soluble guanylate cyclase. Then, the elevation of the cGMP concentration mediated by guanylate cyclase triggers the apical accumulation of V-ATPase in the microvilli, ultimately leading to increased proton secretion (Figure 1) (Shum et al. , 2008). This model is consistent with the essential role of ANGII production and the requirement for tACE in the maintenance of the proper luminal ion/water environment and sperm maturation. Thus, a delicate signaling network between basal cells and adjacent clear cells modulated by the receptor AGTR2 may contribute to the finely tuned microenvironment of the luminal space of the epididymis.
Interestingly, male infertility may result from dysfunction in the proton balance in the efferent ductules without significant impairment of AGTR2 function, suggesting that an AGTR2-targeted treatment may have therapeutic potential. In our recent study, although administration of 1 μM ANGII had no significant effect, applying 100 nM ANGII restored pH homeostasis and fluid reabsorption in efferent ductules derived fromAdgrg2 -/Y mice. This rescue effect was blocked specifically by PD123319, an AGTR2 antagonist, but not by an ANGII antagonist (Zhang et al. , 2018). Therefore, the specific agonists of AGTR2 could be considered as therapeutic drugs to treat male infertility associated with a significant impairment in the pH balance in the efferent ductules or epididymis.
For AGTR2, both peptide-based agonists and small chemical compound agonists have been developed, which have therapeutic potential to treat several human diseases (Table 3) (Bennion et al. , 2018; Hallberg et al. , 2018). Sarile and saralasin are two peptide AGTR2 agonists that have been approved by the FDA to treat hypertension and used in the clinic for a short period (Table 3) (Guimond et al. , 2014; Hallberg et al. , 2018). These peptides inactivate AGTR1 but activate AGTR2. Currently, it remains unknown whether the blockade of AGTR1 activity is dispensable for the normal function of the efferent ductules or epididymis. Therefore, the application of these two peptides for the treatment of sperm obstruction in male infertility requires further evaluation. Recently, β-Pro7AngIII was reported to show high selectivity for the activation of AGTR2 but no significant effect on AGTR1 (Hallberg et al. , 2018), providing an alternative choice for peptide-based AGTR2 activation therapy in male infertility. Small-molecule compounds have also been developed to target AGTR2 activation for clinical treatment. For example, MP-157 was used as an AGTR2 agonist for cardiovascular disease treatment in a phase I clinical trial, whereas C21/M24 was examined in a phase II exploration of idiopathic pulmonary fibrosis (IPF) (Table 3) (Hallberg et al. , 2018). Testing these small-molecule compounds or their derivatives will be of great interest for developing treatment for male infertility related to impaired pH homeostasis in the efferent ductules or epididymis.
LGR4, an essential GPCR for epididymal development
LGR4, also called G protein-coupled receptor 48 (GPR48), is a member of the LGR subgroup of the rhodopsin-like GPCR superfamily, which derives its name from a large extracellular domain consisting of multiple leucine-rich repeats (Figure 2C). LGR4 is widely expressed in multiple human and mouse tissues, with the highest expression levels in the epidermis and hair follicles of the skin, pancreatic islet cells, and epithelial cells in the male and female reproductive organs (Van Schoore et al. , 2005; Yi et al. , 2013).
LRG4 has been shown to play an important role in postnatal epididymal development in mice. In Lgr4 knockout mice, the epididymal tubule, especially the caput region, fails to elongate and convolute, and the resulting duct is surrounded by a thick condensation of mesenchymal cells. This abnormal cellular organization suggests that LGR4 is important for epithelial-mesenchymal interactions (Table 1) (Mendive et al. , 2006). Furthermore, the expression levels of estrogen receptor α (ERα) and androgen receptor (AR) are dramatically reduced in the epididymis of male Lgr4 knockout mice, which in turn leads to decreased expression of Na+-K+-ATPase, Na+/H+hydrogen exchanger 3 (NHE3), and aquaporin 9 (Aqp9) (Li et al. , 2010). LRG4 upregulates ERα expression via the cAMP/PKA signaling pathway (Figure 1). Downstream of the LRG4-cAMP-PKA pathway, CREB binds to a Cre motif in the ERα promotor and activates its expression (Li et al. , 2010).
The pivotal role of LGR4 in the epididymis is further supported by aLgr4 hypomorphic mutant mouse line (Lgr4Gt ) that was developed through gene-trap insertional mutagenesis. Short and dilated epididymal tubules are detected in homozygous Lgr4Gt/Gt mice, which have only one-tenth the normal Lgr4 expression level. Moreover, multilamination and distortion of the basement membranes (BMs) is observed in the caput region, and the initial segment is completely lost (Hoshii et al. , 2007). Lgr4knockout or hypomorphic mice also show deficits in the testes and efferent ductules (Qian et al. , 2013), which together with the epididymal defects eventually lead to male infertility in mice.
Overexpressed LGR4 has been found to activate heterotrimeric Gs proteins to elevate intracellular cAMP levels (Gaoet al. , 2006). Moreover, R-spondins and norrin were identified as LGR4 ligands that could bind LGR4 and stimulate the Wnt signaling pathway (Table 3) (Carmon et al. , 2011; de Lau et al. , 2011; Deng et al. , 2013; Glinka et al. , 2011). Recently, tumor necrosis factor (TNF) superfamily member 11 (TNFSF11, also known as RANKL) was identified as a novel LGR4 ligand (Table 3) (Luo et al. , 2016). TNFRSF11A (also called RANK) was considered to be the sole receptor for TNFSF11 until LGR4 was found to compete with RANK and suppress canonical RANK signaling. TNFSF11 binds to LGR4 and subsequently activates the Gq and glycogen synthase kinase 3 beta (GSK3-β) signaling pathway (Luo et al. , 2016). At present, synthesized agonists or antagonists of LGR4 have not been reported.
Complex functions ofG protein-coupled estrogen receptor 1 ( GPER) in the epididymis
GPER, also known as G protein-coupled receptor 30 (GPR30), was first identified as a receptor that demonstrated MAP kinase (Erk1/2) activation by binding to estrogen (Prossnitz et al. , 2007). Compounds such as the GPER antagonist fulvestrant (ICI 182780) and GPER agonist G-1 can also modulate GPER to induce rapid nongenomic cellular responses (Bologa et al. , 2006; Lucas et al. , 2010; Revankar et al. , 2005). Unlike the other members of the GPCR family that mainly reside on the plasma membrane, GPER is broadly localized on the endoplasmic reticulum and nuclear envelope as well as the plasma membrane (Figure 1) (Funakoshi et al. , 2006; Prossnitz et al. , 2007; Thomas et al. , 2005).
GPER has been detected in many male reproductive structures, such as the testes (Cassault-Meyer et al. , 2014; Gautier et al. , 2016; Lucas et al. , 2010), spermatozoa (Arkoun et al. , 2014; Cassault-Meyer et al. , 2014; Gautier et al. , 2016), and prostate (Rago et al. , 2016). It has also been found in the efferent ductules and epididymis (Cao et al. , 2017; Hess et al. , 2011; Katleba et al. , 2015; Krejcirova et al. , 2018; Lu et al. , 2016; Malivindi et al. , 2018; Martinez-Traverso et al. , 2015; Menad et al. , 2017; Pereira et al. , 2014; Rago et al. , 2018), indicating that GPER may play important roles in sperm maturation, protection and storage (Table 1). For instance, in the corpus epididymis of postnatal pigs, GPER participates in sperm maturation by affecting the formation of the blood-epididymal barrier (Katlebaet al. , 2015). In the caudal epididymal epithelium in immature rats, GPER induces a pathway involved in cAMP-CFTR-chloride secretion to regulate osmotic pressure in response to a perfusion solution and thus affects sperm motility (Figure 1) (Caoet al. , 2017).
In addition, the relative abundance of GPER in the efferent ductules and each part of the epididymis, the cellular localization of GPER, and the molecular weight of the protein differ depending on the species, developmental stage, and physiological cycle studied (Krege et al. , 1995; Krejcirova et al. , 2018; Lu et al. , 2016; Pereira et al. , 2014). Therefore, the role of GPER in the efferent ductules and epididymis appears to be complex. The first GPER-specific agonist, G-1, has been identified through virtual and biomolecular screening (Table 3) (Bologa et al. , 2006). Based on the synthesis of the G-1 analog as well as additional screening, two GPER-specific antagonists, G15 and G36, were also identified, both of which inhibit estrogen- and G-1-stimulated cell proliferation in vivo (Table 3) (Dennis et al. , 2009; Dennis et al. , 2011). Recently, a series of indole-thiazole derivatives were identified as new GPER agonists (O’Dea et al. , 2018). These newly identified agonists and antagonists provide very useful tools for further evaluation of the therapeutic potential of GPER in treating male infertility, given the potential complex function of GPER in male systems. Overall, the evaluation of GPER as a drug target in male infertility requires further investigation, and the new compounds identified for specific regulation of GPER activity will certainly accelerate this assessment.
Two adenosine receptors with opposite functions in the epididymis
Adenosine receptors consist of four members, namely, A1, A2A, A2B, and A3. Adenosine receptors are activated by adenosine and transmit signals through classic G protein-cAMP or β-arrestin pathways (Table 1) (Geldenhuys et al. , 2017). Most adenosine receptors have been suggested to be present in the epididymis (Table 1) (Haynes et al. , 1998b; Minelli et al. , 1995).
The A1 and A2 adenosine receptors have been shown to regulate the contractility of the vas deferens and epididymis (Table 1) (Brownhill et al. , 1996; Haynes et al. , 1998a; Haynes et al. , 1998b). Interestingly, it seems that the A1 and A2 receptors have opposite effects on the contractility of the epididymis: the A1 receptor enhances the contractility, whereas the A2 receptor inhibits the contractility (Haynes et al. , 1998b). This phenomenon might be explained by the difference in their G protein-coupling selectivity (van Galenet al. , 1992). In the epididymis, A2 adenosine receptors increase intracellular cAMP levels (Haynes et al. , 1998b), consistent with the generally accepted view that A2 adenosine receptors are coupled to Gs-protein and activate adenylyl cyclase to increase intracellular cAMP levels (Figure 1) (Fredholm et al. , 1994). Further investigation showed that the A2A receptor mediates potassium channel activation through protein kinases A and G in rat epididymal smooth muscle (Haynes, 2000). This result is consistent with the finding that A2receptor activation stimulated cAMP-dependent protein kinase A, which in turn modulated potassium channel activity in arterial or skeletal muscles (Barrett-Jolley et al. , 1996; Kleppisch et al. , 1995). In contrast, the A1 adenosine receptor is likely coupled to effectors through Gi/o proteins, although confirmative evidence is still lacking (Haynes et al. , 1998b).
Adenosine (and its precursor ATP) has been used for several decades to treat cardiac arrhythmias through activating A1adenosine receptors (Szentmiklosiet al. , 2015). Adenosine is also the gold-standard agent to create maximum coronary hyperemia through activating A2Aadenosine receptors (McGeoch et al. , 2008). However, given that adenosine can activate various adenosine receptors, it inevitably produces some undesirable adverse effects. To avoid nonspecific global adverse reactions, selective agonists of A1, A2A, and A3 adenosine receptors have been developed, some of which are currently undergoing clinical trials (Jacobson et al. , 2019). For example, the A1 adenosine receptor partial agonist trabodenoson (INO-8875) was tested for the treatment of glaucoma and ocular hypertension, but it failed in a phase 3 trial because its primary endpoint was not achieved (Table 3) (Jacobson et al. , 2019). The moderately selective A2A adenosine receptor agonist regadenoson was first approved as a pharmacological stress agent in 2008 and is currently being tested in various clinical trials for cardiovascular treatment and diagnosis (Table 3) (Jacobson et al. , 2019). The moderately selective A3 adenosine receptor agonist IB-MECA (CF101, piclodenoson) is being tested in a phase 3 clinical trial for the treatment of autoimmune anti-inflammatory diseases (Table 3) (Jacobson et al. , 2019).
An important limitation of adenosine receptor agonists is agonist-induced desensitization (Mundellet al. , 2011). The application of either partial agonists or positive allosteric modulators (PAMs) may circumvent desensitization and improve therapies. Currently, only adenosine and regadenoson are approved for human use (Jackson et al. , 2018). However, many adenosine receptor agonists and PAMs (such as the A1 adenosine receptor PAM benzoylthiophenes) are being tested in humans, and it is of great interest to test the effects of these compounds on the regulation of epididymis functions and the treatment of male infertility.
Future questions and perspectives
Numerous GPCRs are expressed in the efferent ductules and epididymis, which consist of various cell types. Thus, the following questions arise. (1) Which GPCRs are expressed in a particular cell type? (2) How do these GPCRs contribute to the development and normal physiological functions of the epididymis and efferent ductules? (3) Can any of these GPCRs functionally compensate for each other? (4) If so, is it possible to activate an alternative GPCR in the epididymis or efferent ductules to rescue the dysfunction of a particular GPCR, such as in cases of infertility caused by ADGRG2 mutations? (5) Is there crosstalk between different GPCRs or between GPCRs and other membrane proteins in specific cell types? (6) Are endogenous ligands of the GPCRs in epididymis and efferent ductules constantly produced in the local environment to actively regulate specific physiological processes of epididymis development and sperm maturation? (7) Do second messengers downstream of GPCRs, such as cAMP and calcium, have distinct functions in different types of cells in the epididymis and efferent ductules, and how are they regulated by different GPCRs? (8) Are location bias (signaling compartments) and effector bias important for the regulation of different GPCRs expressed in the epididymis and efferent ductules? (9) What are the endogenous ligands for ADGRG2, AGTR2, GPER and LGR4 in the local male fertility system? (10) Do FDA-approved drugs targeted to GPCRs with known functions in the epididymis, such as AGTR2 and adenosine receptors, have beneficial effects on male fertility? (11) Are there regional drug delivery systems that can target specific GPCRs in the epididymis to decrease the side effects of GPCR ligands? To answer these questions, a systematically investigation of the GPCR expression in epididymis and efferent ductules by transcriptional analysis and the single cell sequencing; utilization of the conditional knock mice driven by the specific epididymis or efferent ductile marker Cre; combined with the molecular and cellular approaches to delineate the mechanism underlying the specific GPCR functions in male infertility and the usage of the biochemical approach and the proteomics and metabolomics to identify the endogenous ligands for specific GPCR such as the ADGRG2, will lay an important foundation for evaluation of these GPCRs as potential therapeutic targets for male infertility treatment. Moreover, usage of the specific known chemical ligands for these GPCRs, united by the selective drug delivery methods and assessment of the effects of these ligands in male infertility mice models will provide further information for drug development toward these GPCRs.