Function and therapeutic potential of G protein‐coupled receptors in epididymis

Abstract

Infertility rates for both females and males have increased continuously in recent years. Currently, effective treatments for male infertility with defined mechanisms or targets are still lacking. G protein‐coupled receptors (GPCRs) are the largest class of drug targets, but their functions and the implications for the therapeutic development for male infertility largely remain elusive. Nevertheless, recent studies have shown that several members of the GPCR superfamily play crucial roles in the maintenance of ion–water homeostasis of the epididymis, development of the efferent ductules, formation of the blood–epididymal barrier and maturation of sperm. Knowledge of the functions, genetic variations and working mechanisms of such GPCRs, along with the drugs and ligands relevant to their specific functions, provide future directions and a great arsenal for new developments in the treatment of male infertility.

Abbreviations

ADGRG2

adhesion G protein‐coupled receptor G2

Ang I

angiotensin I

Ang II

angiotensin II

AQP 9

aquaporin 9

BMs

basement membranes

CBAVD

congenital bilateral absence of the vas deferens

CFTR

cystic fibrosis transmembrane conductance regulator

GPER

G protein‐coupled estrogen receptor 1

GSK3‐β

glycogen synthase kinase 3 beta

HE6

human epididymal gene product 6

IPF

idiopathic pulmonary fibrosis

LGR4

leucine‐rich repeat containing G protein‐coupled receptor 4

PAMs

positive allosteric modulators

RAS

renin–angiotensin system

tACE

angiotensin‐converting enzyme specific to the testes

1. INTRODUCTION

The infertility rate in humans has continued to increase in recent years and has become a significant social burden (Krausz & Riera‐Escamilla, 2018; Winters & Walsh, 2014). Currently, infertility ranks as the third most common public health concern, after cancer and cardiovascular disease. Problems in males and females contribute equally to the increasing infertility rate, and nearly 7% of the male population has fertility problems (Krausz & Riera‐Escamilla, 2018; Winters & Walsh, 2014). However, few effective treatments are available for male infertility with defined mechanisms. It is now recognized that defects in sperm production, decrease of sperm motility and inability of sperm to interact with the oocyte, all contribute to male infertility (Aitken, 2006; Elzanaty, Richthoff, Malm, & Giwercman, 2002).

After spermatogenesis in the testis, the spermatozoa are morphologically complete but immotile and unable to fertilize an oocyte. They must travel through the efferent ductules and the epididymis to acquire the ability to move, capacitate and migrate through the female tract and finally fertilize an oocyte. The efferent ductules are small, coiled tubules that convey sperm from the testis to the epididymis. In mammals, efferent ductules begin with several discrete wide‐lumen ducts that eventually merge into highly convoluted tubules with a narrow lumen (Hess, 2015; Joseph, Shur, & Hess, 2011). The efferent ductule epithelium contains ciliated cells with long motile cilia and non‐ciliated cells with microvillus brush borders (Hess, 2015; Joseph et al., 2011) (Figure 1). It is now known that the major function of the efferent ductules is reabsorption of luminal fluid, which increases the concentration of sperm before they enter the epididymis (Clulow, Jones, Hansen, & Man, 1998; Hess, 2000; Hess & Nakai, 2000).

FIGURE 1.

Diagram showing GPCR signalling and functions in the epididymis and efferent ductules. Above: The efferent ductules are a series of tubules that connect the rete testis to the epididymis. The epithelia of the efferent ductules are mainly composed of two cell types, ciliated cells and non‐ciliated cells. The epididymis is composed of one highly convoluted tubule. The epididymis is segmented morphologically and functionally into following distinct regions: the initial segment (not existing in human epididymis), the caput, the corpus, and the cauda. Each part consists of several cell types, including principal cells, narrow cells, clear cells, and basal cells. Inset: GPER activates cAMP‐CFTR‐chloride transportation to maintain the osmotic pressure of the perfusion solution. ADGRG2 is located exclusively on the apical membrane in non‐ciliated cells. ADGRG2/β‐arrestin1/G_q/CFTR forms a supercomplex that maintains pH and chloride anion homeostasis. AT₂ receptors are specifically detected in basal cells and are essential for the proton‐secretion function of the epididymal lumen through activation of the NO‐cGMP pathway. Different members of the adenosine receptor family have opposite effects on the contractility of the epididymis. LGR4 activates G_s to increase intracellular cAMP levels, which promote ERα expression

The mammalian epididymis is an exceedingly long, convoluted ductal system connecting the efferent ductules with the vas deferens. Functionally, the epididymis creates an ideal environment to promote the functional transformation of spermatozoa and their later storage before ejaculation. The epididymis is segmented into four functionally distinct segments: the initial segment (not existing in human epididymis), the caput, the corpus, and the cauda (Abou‐Haila & Fain‐Maurel, 1984; W. Zhou, De Iuliis, Dun, & Nixon, 2018) (Figure 1). The initial segment, together with the upstream efferent ductules, is responsible for the resorption of the testicular fluid that enters the duct, resulting in a pronounced concentration of the luminal spermatozoa (Abe, Takano, & Ito, 1984). The caput epididymis is highly active in protein synthesis and hormone secretion and plays important roles in sperm maturation. The sperm passing through this region begin to obtain the ability to swim in a progressive manner and to recognize an oocyte (Aitken et al., 2007; Chevrier & Dacheux, 1992). The functional maturation of the sperm continues in the corpus epididymis and reaches full activity in the distal caudal segment. The caudal segment contains a relatively large lumen, and its surrounding epithelial cells have strong absorptive activity (Hermo, Dworkin, & Oko, 1988). There are four main cell types in the epithelium of the epididymal lumen, namely, narrow cells, clear cells, principal cells, and basal cells. Each cell type has different functions involved in the establishment and regulation of a unique luminal environment (Cornwall, 2009; Shum, Da Silva, Brown, & Breton, 2009).

In general, an appropriate microenvironment established by the efferent ductules and epididymis is required for sperm to undergo maturation and acquire progressive motility and the ability to fertilize oocyte during their transit. To date, the exact molecular mechanism involved in maintaining the effective microenvironment in the efferent ductules and epididymis remains to be defined, which creates significant obstacles to developing effective treatments for male infertility. Therefore, there is an urgent need to understand the regulatory mechanisms in the efferent ductules and epididymis involved in both physiological and pathological processes, and to use this knowledge to provide potential drug targets for developing effective therapies.

G protein‐coupled receptors (GPCRs), also called seven‐transmembrane receptors, are a group of important drug targets, accounting for approximately one third of all clinically marketed drugs (Hauser et al., 2018; Santos et al., 2017). Although the roles of GPCRs in cardiovascular disease, neuronal disease, diabetes, and many other diseases have been extensively investigated (Desimine et al., 2018; Dong et al., 2017; Hauser, Attwood, Rask‐Andersen, Schioth, & Gloriam, 2017; Kim et al., 2020; Lammermann & Kastenmuller, 2019; T. Li et al., 2018; Liu et al., 2017; Srivastava, Gupta, Gupta, & Shukla, 2015), there is very much less knowledge of the functions of GPCRs in the efferent ductules and epididymis.

GPCRs, by definition, carry out their selective functions through coupling to the different G protein subtypes or to arrestins (Bridges & Lindsley, 2008; Cahill et al., 2017; Gilman, 1987; Pierce, Premont, & Lefkowitz, 2002; Ritter & Hall, 2009). In general, the binding of ligands (such as hormones, neurotransmitters, or sensory stimuli) induces conformational changes in the transmembrane and intracellular domains of the receptor, thereby allowing interactions with heterotrimeric G proteins or with arrestins (Weis & Kobilka, 2018; F. Yang et al., 2018). For G protein signalling, activated GPCRs act as guanine nucleotide exchange factors (GEFs) for the α subunits of heterotrimeric G proteins, catalyzing the release of GDP and the binding of GTP for G protein activation. Activation of different G proteins, including G_s/o, G_i, G_12/13 or G_q subtypes, affect various cellular processes through different downstream effectors, such as adenylyl cyclase (AC), RhoGEF, or phospholipase C (PLC) (Flock et al., 2015; Flock et al., 2017; Furness et al., 2016; Gilman, 1987; Isogai et al., 2016; Pierce et al., 2002; Ritter & Hall, 2009; Sounier et al., 2015; Venkatakrishnan et al., 2016). The activated GPCRs are also phosphorylated by the GPCR kinases (GRKs), a family of protein kinases that phosphorylate specific serine/threonine residues of GPCRs. Receptor phosphorylation leads to arrestin recruitment and activation (Homan & Tesmer, 2014; Komolov et al., 2017; Premont & Gainetdinov, 2007; Reiter & Lefkowitz, 2006; F. Yang et al., 2015). Activated arrestins not only desensitize receptors but also mediate a second wave of signalling independent of G proteins (Desimine et al., 2018; Dong et al., 2017; Kumari et al., 2016; Lefkowitz & Shenoy, 2005; Liu et al., 2017; Reiter & Lefkowitz, 2006; Shukla et al., 2014; W. Wang, Qiao, & Li, 2018; F. Yang et al., 2015; F. Yang et al., 2018). Even a single type of GPCR can initiate a broad range of physiological processes through arrestin engagement by scaffolding different downstream effectors (Hara et al., 2011; Liu et al., 2017; Luttrell et al., 1999; Miller et al., 2000; Peterson & Luttrell, 2017; Srivastava et al., 2015; Tobin, Butcher, & Kong, 2008; Xiao et al., 2007; F. Yang et al., 2015; F. Yang et al., 2018). However, the exact roles of the G protein subtype or arrestins downstream of the GPCRs in the epididymis remain undefined.

At present, there are no U.S. Food and Drug Administration (FDA)‐approved drugs targeting the GPCRs in the efferent ductules or epididymis, for the treatment of male infertility. In contrast, there are more than 470 GPCR‐targeted drugs for therapies treating other diseases in clinical markets (Hauser et al., 2018). Nevertheless, recent research has elucidated the expression patterns and functions of several important GPCRs in the efferent ductules and epididymis, such as the adhesion G protein‐coupled receptor G2 (ADGRG2), angiotensin AT₂ receptors and the leucine‐rich repeat containing G protein‐coupled receptor 4 (LGR4), and has successfully developed the corresponding ligands to regulate their functions, generating the possibility of developing treatments for male infertility (Figure 1). Here, we review the progress in our knowledge of GPCRs in epididymis and efferent ductules and suggest potential therapeutic approaches by targeting these GPCRs for male infertility.

2. FUNCTION OF ADGRG2 IN FLUID REABSORPTION AND EPIDIDYMIS DEVELOPMENT

Few GPCRs have tissue‐specific distributions in male reproductive systems. The receptor protein ADGRG2, also called GPR64 or human epididymal gene product 6 (HE6), has attracted substantial attention for its relatively specific expression and essential function in male reproductive systems. It is highly expressed in the efferent ductules and the proximal epididymis (Table 1) (Kirchhoff, Osterhoff, & Samalecos, 2008; Obermann et al., 2003). Expression of ADGRG2 has also been detected in parathyroid, prostate, fibroblasts, neuronal and adipose tissue, as well as various types of cancers (Ahn et al., 2019; Balenga et al., 2017; Hamann et al., 2015; Suchy et al., 2020; Xie et al., 2020). Further studies have confirmed the functional importance of ADGRG2 in male fertility. The human and mouse ADGRG2/Adgrg2 gene is localized on chromosome X (Obermann et al., 2003). Adgrg2 ^−/Y mice exhibit reduced sperm numbers, decreased sperm motility, and increased number of spermatozoa with deficient heads or angulated flagella (Davies et al., 2004). Moreover, dysfunction in the fluid resorption of the efferent ductules is observed, which might eventually lead to the above‐mentioned phenotypes in Adgrg2 ^−/Y mice (Table 1) (Davies et al., 2004; Gottwald, Davies, Fritsch, & Habenicht, 2006; Zhang et al., 2018).

TABLE 1.

GPCRs with known functions in epididymis or efferent ductules

Receptor name	Family name	Expression	Function	Signalling effectors	References
ADGRG2 (GPR64)	Adhesion Class GPCRs	Efferent ductules; proximal epididymis (non‐ciliated cells; principal cells)	Fluid reabsorption; possibly sperm maturation	Gs; Gi; Gq; G12/13; β‐arrestin 1; β‐arrestin 2	Davies et al. (2004), Demberg, Rothemund, Schoneberg, and Liebscher (2015), Peeters et al. (2015), Zhang et al. (2018), and Azimzadeh, Talamantez‐Lyburn, Chang, Inoue, and Balenga (2019)
AT2 receptor	Angiotensin receptors	Basal cells	Proton secretion	Unknown	Krege et al. (1995), Esther et al. (1996), and Shum et al. (2008)
LGR4 (GPR48)	Class A orphans	Epithelial cells in the reproductive organs	Epithelial–mesenchymal interactions	Gs, Gq, Wnt	Mendive et al. (2006), Gao et al. (2006), Li et al. (2010), Carmon, Gong, Lin, Thomas, and Liu (2011), and Luo et al. (2016)
GPER (GPR30)	G protein‐coupled estrogen receptor	Testis; spermatozoa; prostate; efferent ductules; epididymis	Formation of blood–epididymal barrier and regulation of osmotic pressure	Gs; β‐arrestin 2	Filardo, Quinn, Bland, and Frackelton (2000), Katleba, Legacki, Conley, and Berger (2015), and Cao et al. (2017)
Adenosine receptor	Adenosine receptors	Epididymis	Contractility of the epididymis	Gs; Gi; Gq; β‐arrestin	Haynes, Alexander, and Hill (1998b), Geldenhuys, Hanif, Yun, and Nayeem (2017), and Santiago et al. (2020)

ADGRG2/GPR64 knockout (KO) results in down‐regulation of epididymis‐specific expression of cystatin, lipocalins, β‐defensins, Adam28, Crisp1, and Enpp2 among others, which have been shown to be closely related to sperm maturation (Davies et al., 2007). For example, cystatin‐related epididymal spermatogenic (CERS) subgroup members are part of an amyloid matrix and associated with extracellular vesicles in the mouse epididymal lumen and may play a functional role in sperm maturation through coordinating interactions between the luminal fluid and spermatozoa (Whelly et al., 2016). Lipocalin 2 modulates sperm maturation through binding to membrane phosphatidylethanolamine to induce lipid raft movement in a PKA‐dependent manner (Watanabe et al., 2014). Lipocalin 6 is involved in preventing calcium overload and premature acrosome reaction of sperm (Yin et al., 2018). β‐defensin 22 enables spermatozoa to undergo the process of fertilization through its heparin binding activity (Diao, Yu, Sun, Zhang, & Tanphaichitr, 2011), and β‐defensin 15 helps with sperm motility and fertility (Y. Zhao et al., 2011). The transcriptional change of these proteins helps to explain the phenotypes in GPR64 KO mice including reduced and defective sperms (Davies et al., 2004).

ADGRG2 belongs to the adhesion GPCR (aGPCR) subfamily, and all members of this family share a very large N‐terminal domain (Fredriksson, Lagerstrom, Lundin, & Schioth, 2003; Hu et al., 2014; Hamann et al., 2015; Kishore & Hall, 2017; Liebscher, Schoneberg, & Promel, 2013; Paavola, Stephenson, Ritter, Alter, & Hall, 2011; Paavola & Hall, 2012; Sun et al., 2013; X. J. Wang et al., 2014). Another feature of aGPCRs is the presence of the GPCR proteolytic site (GPS) at the GPCR Autoproteolysis‐INducing (GAIN) domain (Arac et al., 2012), which auto‐cleaves aGPCRs into an N‐terminal fragment (NTF) and a C‐terminal fragment (CTF) for most of its family members (Arac et al., 2012; de Graaf, Nijmeijer, Wolf, & Ernst, 2016; Hamann et al., 2015). Many members of this family have been shown to function through G protein coupling, and the NTF has been shown to inhibit the constitutive G protein‐coupling activity of its CTF in some members (Demberg et al., 2015; Folts, Giera, Li, & Piao, 2019; Hamann et al., 2015; Hu et al., 2014; Kishore, Purcell, Nassiri‐Toosi, & Hall, 2016; Purcell & Hall, 2018; Sun et al., 2013; X. J. Wang et al., 2014; Zhang et al., 2018). Activation of aGPCRs could be induced through (1) ligand binding (R. Luo et al., 2014; Paavola, Sidik, Zuchero, Eckart, & Talbot, 2014; Petersen et al., 2015), (2) mechanostimulation (Hilbig et al., 2018; Scholz et al., 2015; Wilde et al., 2016), or (3) removing the NTF through autoproteolysis (Demberg et al., 2015; Liebscher et al., 2014; Okajima, Kudo, & Yokota, 2010; Paavola et al., 2011; Ward et al., 2011). Many aGPCRs have been shown to contain a tethered agonist sequence in the N‐terminal region between the cleavage site and the first transmembrane domain. The exposed N‐terminal‐tethered agonist sequence after auto‐cleavage could act as a certain type of endogenous agonists of aGPCR families. Derived peptides from this tethered agonist sequence of aGPCRs can be used as agonists in vitro and in vivo (Balenga et al., 2017; Brown et al., 2017; Demberg et al., 2015; Demberg et al., 2017; Liebscher et al., 2014; Muller et al., 2015; Stoveken, Hajduczok, Xu, & Tall, 2015; Suchy et al., 2020; Wilde et al., 2016).

The signal transduction of ADGRG2 has been extensively investigated. A suspected G_s or G_q coupling was initially proposed in Xenopus melanophores (Foord, Jupe, & Holbrook, 2002). The coupling of ADGRG2 to the calcium‐sensing CaS receptor, G_12/13, G_s, and G_q was then confirmed (Balenga et al., 2017; Demberg et al., 2015; Peeters et al., 2015). Through generation of ADGRG2 mutants that lack NTF, a constitutive β‐arrestin coupling activity and constitutive internalization of ADGRG2 were observed, and GRKs and dynamin were shown to mediate the constitutive internalization of this specific ADGRG2 mutant (Azimzadeh et al., 2019). Parallel to these observations, our study showed that in cells overexpressing either full‐length ADGRG2 or the ADGRG2‐CTF, significant constitutive G_s or G_q coupling activity was observed (Zhang et al., 2018). These studies suggested that ADGRG2‐mediated G_s or G_q signalling may play important roles in the regulation of fluid resorption in the efferent ductules and epididymis (Figure 1). However, the exact functions of G protein subtypes in maintaining the microenvironment of the efferent ductules or epididymis are still unknown, and the downstream effectors involved in controlling the luminal ion/water homeostasis balance in these tissues also remain elusive. Interestingly, immunostaining assays revealed specific expression of ADGRG2 on the apical membrane only in non‐ciliated cells (in the efferent ductules) and principal cells (in the epididymis), not in ciliated cells (Kirchhoff et al., 2008; Obermann et al., 2003). The non‐ciliated cells in efferent ductules are frequently referred as principal cells in the epididymis (Burkett, Schulte, & Spicer, 1987). Cellular expression specificity of ADGRG2 suggests a cell type‐specific function of ADGRG2 in the regulation of ion/water homeostasis in the efferent ductules and epididymis. The specific expression pattern of ADGRG2 allowed us to develop a non‐ciliated cell‐specific labelling technique by exploiting the promoter of the ADGRG2 gene. Using this newly developed method, we successfully isolated non‐ciliated cells and showed that a diminished constitutive chloride current was the cause of the unbalanced pH state in the efferent ductules and dysfunction in fluid resorption in Adgrg2 ^−/Y mice (Zhang et al., 2018).

Further analysis combining Gq ^−/+ and Adgrg2 ^−/Y mouse models, pharmacological intervention and cell labelling techniques demonstrated that ADGRG2 regulated Cl⁻ and pH homeostasis through G_q‐dependent coupling between the receptor and the anion channel cystic fibrosis transmembrane conductance regulator (CFTR) (Figure 1) (Zhang et al., 2018). CFTR and ADGRG2 co‐localize at the apical membrane of non‐ciliated cells, along with selective high expression of G_q in the same cells. Through coupling to G_q, ADGRG2 maintains the basic CFTR outward‐rectifying current, which is required for fluid resorption and sperm maturation (Figure 1) (Zhang et al., 2018). Importantly, whereas disruption of β‐arrestin2 has no significant effects on the fluid resorption function, β‐arrestin 1 deficiency impaired pH and Cl⁻ homeostasis in the efferent ductules and initial segment of the epididymis (Zhang et al., 2018). Further investigation confirmed the coexistence of ADGRG2, CFTR, β‐arrestin 1, and G_q in the same protein complex (Figure 1), while β‐arrestin 1 deficiency abolished the colocalization of ADGRG2 and CFTR on the apical membrane. These data suggested that the ADGRG2/β‐arrestin 1/ G_q /CFTR supercomplex localizes at the apical membrane of non‐ciliated cells and functions as a regional signalling hub, controlling fluid reabsorption and maintaining pH and Cl⁻ homeostasis in the efferent ductules and initial segment of the epididymis (Figure 1) (Zhang et al., 2018). The ADGRG2/CFTR interaction in the epididymis represents yet another example of the functional divergence between the two β‐arrestin isoforms, already established in several other tissues and organs (Lymperopoulos, 2018; Lymperopoulos et al., 2019; Srivastava et al., 2015). For example, in the heart, β‐arrestins 1 and 2 were initially thought of as functionally interchangeable but actually exerted diametrically opposite effects in the mammalian myocardium. β‐arrestin 1 exerts overall detrimental effects on the heart; in contrast, β‐arrestin 2 is overall beneficial for the myocardium (Lymperopoulos et al., 2019).

Consistent with our findings that inhibition of ADGRG2 or G_q activity caused fluid resorption dysfunction, recent clinical studies have revealed that multiple ADGRG2 mutations are associated with male infertility. For example, p.Glu516Ter, p.Leu668ArgfsTer21, p.Arg814Ter, or p.Lys818Ter results in the absence or truncation of the seven‐transmembrane domain, which might abolish receptor coupling to downstream G_q and G_s proteins and β‐arrestins, and eventually leads to male infertility (Figure 2a, Table 2) (Khan et al., 2018; Patat et al., 2016; Yuan et al., 2019). However, it is worth noting the potential CTF‐independent functions of aGPCRs. For example, Gpr126 ^st49 mutant zebrafish still express a functional fragment of NTF but no CTF (Patra et al., 2013). Therefore, these ADGRG2 mutations might preserve some of the ADGRG2 functions and knock‐in models of these mutations are needed for detailed characterization of these disease‐associated ADGRG2 mutations. Moreover, the p.Cys949AlafsTer81 frameshift mutation, the missense p.Lys990Glu, and p.Arg1008Gln mutations produce a protein with an affected C‐terminal domain of ADGRG2, which may affect the expression level of the receptor or G protein/arrestin‐mediated signalling (Figure 2a, Table 2) (Patat et al., 2016; B. Yang et al., 2017; Yuan et al., 2019). Therefore, different ADGRG2 mutations may cause similar male infertility phenotypes, through quite different cellular signalling mechanisms.

FIGURE 2.

GPCR mutations associated with disease. The approximate positions of different mutations are indicated in the structures of ADGRG2 (A), AT₂ receptor (B), and LGR4 (C).. Abbreviations: GAIN domain: GPCR Autoproteolysis‐INducing domain; GPS, G protein‐coupled receptor proteolytic site; LRR, leucine‐rich repeats

TABLE 2.

Disease‐related SNP analysis in GPCRs

GPCR	dbSNP rs# cluster id	dbSNP allele change	Protein residue change	Amino acid pos	Accession disease names	References
ADGRG2 (GPR64) Xp22.13	rs879255540	‐>T	Glu [E]>*	516	Congenital bilateral absence of vas deferens	Patat et al. (2016)
		G > A	Cys [C] > Tyr [Y]	570	Congenital bilateral absence of vas deferens	Yang et al. (2017)
	rs879255539	CTGTG > AGA	Leu [L] > Arg [R]	668	Congenital bilateral absence of vas deferens	Patat et al. (2016)
		C > T	p.Arg [R]>*	814	Obstructive azoospermia	Khan et al. (2018)
		A > T	p.Lys [K]>*	818	Congenital absence of vas deferens	Yuan et al. (2019)
	rs879255538	T>‐	Cys [C] > Ala [A]	949	Congenital bilateral absence of vas deferens	Patat et al. (2016)
		A > G	p.Lys [K] > Glu [E]	990	Congenital bilateral absence of vas deferens	Yang et al. (2017)
		G > A	p.Arg [R] > Gln [Q]	1008	Congenital absence of vas deferens	Yuan et al. (2019)
AT2 receptorXq23	rs121917810	G > T	Gly [G] > Val [V]	21	X‐linked mental retardation	Vervoort et al. (2002), Bienvenu, (2003), and Ylisaukko‐oja et al. (2004)
		T>‐	Phe [F] > Leu [L]	134	Not specified	Piton, Redin, & Mandel, (2013)
	rs5191	G > A	Arg [R] > Lys [K]	248	Not specified	Bean, Tinker, da Silva, & Hegde, (2013) and Wang et al. (2006)
LGR4 (GPR48) 11p14.1	rs587777005	C > T	Arg [R]>*	126	Low bone mineral density	Styrkarsdottir et al. (2013)

Notably, the human ADGRG2 mutations mentioned above are clinically associated with congenital bilateral absence of the vas deferens (CBAVD). In general, CBAVD involves a complete or partial absence of the Wolffian duct derivatives. In most cases of CBAVD, it is generally presumed that the genital tract abnormality is developed by a progressive atrophy related to abnormal electrolyte ion balance and dysfunction of fluid homeostasis in the male excurrent ducts, rather than agenesis. This model is supported by the link between CBAVD and mutations of the gene encoding the chloride channel CFTR (Patat et al., 2016). In our recent report, we have demonstrated a functional coupling between ADGRG2 and CFTR that serves as the key event in maintenance of Cl⁻ and pH homeostasis in efferent ductules and epididymis, and a persistent dysfunction may finally cause progressive atrophy of the efferent/epididymis ductules (Zhang et al., 2018). Thus, impairment of the ADGRG2/CFTR coupling may directly relate to the CBAVD in male infertility patients. It is worth noting that the infertile patients are usually identified as adults, whereas the animal models normally have a shorter life span. This could explain the fact that ADGRG2 KO mice did not develop CBVAD in their life time. For an ADGRG2‐targeted therapy, as a treatment for male infertility, a systematic screening for male sterility genes, identification of the genetic mutations in ADGRG2 or CFTR, as well as genetic or pharmacological intervention in the early stage of a male patient carrying the mutations could be considered.

ADGRG2 show increased constitutive activity in the absence of the NTF (Demberg et al., 2015; Liebscher et al., 2015). The ADGRG2‐CTF is able to constitutively activate the CFTR current in transfected HEK293 cells (Zhang et al., 2018). Therefore, further investigation is needed to determine whether constitutive ADGRG2 activity is sufficient to maintain the microenvironment of the epididymis and efferent ductules or whether an endogenous ADGRG2 ligand is required in this process. It is worth noting that a 15‐amino acid peptide derived from the N‐terminus of the ADGRG2‐CTF was shown to activate ADGRG2 with low affinity (Table 3) (Balenga et al., 2017; Demberg et al., 2015; Demberg et al., 2017). Further modification of ADGRG2 ligands derived from this peptide might increase the activity of certain ADGRG2 mutants and exhibit therapeutic potential. Of note, the peptide‐based agonists often show non‐selectivity toward aGPCR members; hence, structural analysis of a peptide agonist‐bound aGPCR is necessary to elucidate the detailed activation mechanisms of aGPCRs (Demberg et al., 2017). Alternatively, we have also shown that activation of AT₂ receptors in the efferent ductules can restore the dysfunction in fluid resorption in isolated efferent ductules, derived from Adgrg2 ^−/Y mice (Zhang et al., 2018). Thus, further investigation is warranted to determine whether specific therapeutic methods such as treatment with a selective agonist need to be developed for different ADGRG2 mutants or whether a more general rescue approach, such as activation of AT₂ receptors, is sufficient to treat patients carrying ADGRG2 mutations.

TABLE 3.

Potential therapeutic ligands targeting to GPCRs in epididymis

Receptor	Ligand	Structure (or sequence)	Mode of action	Highest status	References
ADGRG2	Tethered peptide agonist	TSFGILLDLSRTSLP	Agonist		Demberg et al. (2015) and Balenga et al. (2017)
AT2 receptor	Angiotensin II (Ang II)	Asp1‐Arg2‐Val3‐Tyr4‐Ile5‐His6‐Pro7‐Phe8	Agonist	Clinic	Guimond, Hallberg, Gallo‐Payet, and Wallinder (2014) and Hallberg, Sumners, Steckelings, and Hallberg (2018)
	Saralasin	[Sar1,Val5,Ala8]Ang II	Agonist	Clinic	Guimond et al. (2014) and Hallberg et al. (2018)
	Sarile	[Sar1,Ile8]Ang II	Agonist	Clinic	Guimond et al. (2014) and Hallberg et al. (2018)
	MP‐157	No structural formula is disclosed	Agonist	Phase 1	Hallberg et al. (2018)
	C21/M24		Agonist	Phase 2	Hallberg et al. (2018)
	C38/M132		Antagonist		Hallberg et al. (2018)
LGR4	R‐spondins	R‐spondin1‐4(RSPO1‐4)	Agonist		Carmon et al. (2011), de Lau et al. (2011), and Glinka et al. (2011)
	Norrin	MRKHVLAASFSMLSLLVIMGDTDSKTDSSFIMDSDPRRCMRHHYVDSISHPLYKCSSKMVLLARCEGHCSQASRSEPLVSFSTVLKQPFRSSCHCCRPQTSKLKALRLRCSGGMRLTATYRYILSCHCEECNS	Agonist		Deng et al. (2013)
	TNFSF11(RANKL)	Tumor necrosis factor (TNF) superfamily member 11	Agonist		Luo et al. (2016)
GPER	G‐1		Agonist		Bologa et al. (2006)
	G15		Antagonist		Dennis et al. (2009) and Dennis et al. (2011)
	G36		Antagonist		Dennis et al. (2011)
A1AR	Trabodenoson (INO‐8875)		Partial agonist	Phase 3	Jacobson, Tosh, Jain, and Gao (2019)
A2AAR	Regadenoson		Agonist		Jacobson et al. (2019)
A3AR	IB‐MECA		Agonist	Phase 3	Jacobson et al. (2019)

3. ENDOGENOUS ANGIOTENSIN SYSTEM AND AT₂ RECEPTORS IN EPIDIDYMIS

The epididymal lumen and efferent ductules contain a complete local renin–angiotensin system (RAS) including renin, angiotensin I (Ang I), and angiotensin II (Ang II) in the seminal fluid, the angiotensin‐converting enzyme specific to the testes (tACE), and angiotensin AT₁ and AT₂ receptors in the basal cells of the epididymis (Leung, Chan, Fu, Zhou, & Wong, 1997; Leung & Sernia, 2003; Saez, Legare, Laflamme, & Sullivan, 2004; Speth, Daubert, & Grove, 1999; Strittmatter, Thiele, De Souza, & Snyder, 1985; Wong & Uchendu, 1990; W. Zhao, Leung, Chew, Chan, & Wong, 1996). Importantly, Ang II in the epididymal lumen is mainly produced through the cleavage of Ang I by tACE (Langford, Zhou, Russell, Wilcox, & Bernstein, 1993; Sibony, Segretain, & Gasc, 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).

AT₁ and AT₂ receptors have been found in a radio‐ligand binding assay to be expressed in the epididymal lumen. In particular, AT₂ receptors were specifically detected in basal cells and were required for the proton‐secretion function of the epididymal lumen (Figures 1 and 2b, Table 1) (Shum et al., 2008). Unexpectedly, AT₂ receptors were absent in clear cells, which regulated proton secretion. Further studies showed that AT₂ receptors also activated the NO‐cGMP pathway in response to Ang II 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 Ang II production and the requirement for tACE in the maintenance of the proper luminal ion/water environment and sperm maturation. Thus, a delicate signalling network between basal cells and adjacent clear cells modulated by the AT₂ receptor 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 AT₂ receptor function, suggesting that an AT₂ receptor‐targeted treatment may have therapeutic potential. In our recent study, applying 100‐nM Ang II restored pH homeostasis and fluid reabsorption in efferent ductules derived from Adgrg2 ^−/Y mice. This restoration was blocked specifically by PD123319, an antagonist of AT₂ receptors, but not by an AT₁ receptor antagonist (Zhang et al., 2018). Therefore, specific agonists of AT₂ receptors 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. AT₂ receptor agonist treatment might also be applied to male infertility caused by deficits in the ionic exchangers NHE3 and DRA, that modulate pH homeostasis in the efferent ductules and epididymis (Lee, Finnigan‐Bunick, Bahr, & Bunick, 2001; Q. Zhou et al., 2001; Zhang et al., 2018).

For AT₂ receptors, both peptide‐based agonists and low MW non‐peptide agonists have been developed, which have therapeutic potential to treat several human diseases (Table 3) (Bennion, Steckelings, & Sumners, 2018; Hallberg et al., 2018). Sarile and saralasin are two peptide agonists of AT₂ receptors 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 block AT₁ receptors but activate AT₂ receptors. Currently, it remains unknown whether the blockade of AT₁ receptors activity is critical 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 AT₂ receptors, with no significant effect on AT₁ receptors (Hallberg et al., 2018), providing an alternative choice for peptide‐based AT₂ receptor activation therapy in male infertility. Low MW non‐peptide compounds have also been developed as agonists of AT₂ receptors for clinical treatment. For example, MP‐157 was used as an AT₂ receptor agonist for cardiovascular disease treatment in a Phase 1 clinical trial, whereas C21/M24 was tested in a Phase 2 exploration of idiopathic pulmonary fibrosis (IPF) (Table 3) (Hallberg et al., 2018). Testing these low MW 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. Meanwhile, as AT₂ receptors are widely expressed and play important roles in many tissues such as heart, brain, adrenal glands, uterine myometrium, ovarian follicles, kidney, and pancreas, the side effects of AT₂ receptor agonists have to be considered (de Gasparo & Siragy, 1999; Leung, 2001; Verdonk, Danser, & van Esch, 2012). Local application of AT₂ receptor agonists can reduce systemic side effects caused by conventional oral or injection administration. Moreover, appropriate therapeutic dosage should also be considered.

4. 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 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, Mendive, Pochet, & Vassart, 2005; Yi et al., 2013).

LRG4 plays an important role in postnatal epididymal development in mice. In Lgr4 KO 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 the oestrogen receptor α (ERα) and the androgen receptor (NR3C4) are dramatically reduced in the epididymis of male Lgr4 KO mice, which in turn leads to decreased expression of Na⁺‐K⁺‐ATPase, the Na⁺/ H⁺ exchanger NHE3 and aquaporin 9 (AQP 9) (X. Y. Li et al., 2010). LRG4 up‐regulates ERα expression via the cAMP/PKA signalling pathway (Figure 1). Downstream of the LRG4‐cAMP‐PKA pathway, CREB binds to a Cre motif in the ERα promoter and activates its expression (X. Y. Li et al., 2010).

The pivotal role of LGR4 in the epididymis is further supported by a Lgr4 hypomorphic mutant mouse line (Lgr4 ^Gt) that was developed through gene‐trap insertional mutagenesis. Short and dilated epididymal tubules are detected in homozygous Lgr4 ^Gt/Gt mice, which have only one tenth of the normal Lgr4 expression level. Moreover, multi‐lamination and distortion of the basement membranes (BMs) are observed in the caput region, and the initial segment is completely lost (Hoshii et al., 2007). Lgr4 KO 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 G_s proteins to elevate intracellular cAMP levels (Gao et al., 2006). Moreover, R‐spondins and norrin were identified as LGR4 ligands that could bind LGR4 and stimulate the Wnt signalling 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) (J. 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 signalling. TNFSF11 binds to LGR4 and subsequently activates the G_q and glycogen synthase kinase 3 beta (GSK3‐β) signalling pathway (J. Luo et al., 2016). Unlike LRG4/cAMP/PKA pathway, the specific role of R‐spondin/LGR4/Wnt or TNFSF11/LRG4/GSK3‐β pathway in male reproduction has not been characterized yet. Nevertheless, RNAseq revealed that mRNAs encoding R‐spondins, norrin, and TNFSF11 are expressed in human epididymis epithelial cells, albeit at low levels (Browne, Yang, Leir, Eggener, & Harris, 2016).

5. COMPLEX FUNCTIONS OF GPER IN THE EPIDIDYMIS

The G protein‐coupled estrogen receptor 1 (GPER), also known as G protein‐coupled receptor 30 (GPR30), was first identified as a receptor that induced MAP kinase (ERK1/2) activation by binding to estrogen (Carmeci, Thompson, Ring, Francke, & Weigel, 1997; Filardo et al., 2000; O'Dowd et al., 1998; Prossnitz, Arterburn, & Sklar, 2007). Compounds such as the GPER antagonist fulvestrant (ICI 182780) and the GPER agonist G‐1 can also modulate GPER to induce rapid non‐genomic cellular responses (Bologa et al., 2006; Lucas, Royer, Siu, Lazari, & Porto, 2010; Revankar, Cimino, Sklar, Arterburn, & Prossnitz, 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, Yanai, Shinoda, Kawano, & Mizukami, 2006; Prossnitz et al., 2007; Revankar et al., 2005; Thomas, Pang, Filardo, & Dong, 2005).

GPER has been detected in many male reproductive structures, such as the testes (Cassault‐Meyer, Gress, Seralini, & Galeraud‐Denis, 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, Romeo, Giordano, Ferraro, & Carpino, 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, Aquila, & Rago, 2018; Martinez‐Traverso & Pearl, 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 (Katleba et 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) (Cao et 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 analogue as well as additional screening, two GPER‐selective 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, Sondergard, Sweeney, & Arnatt, 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.

6. TWO ADENOSINE RECEPTORS WITH OPPOSITE FUNCTIONS IN THE EPIDIDYMIS

Adenosine receptors consist of four members, namely, A₁, A_2A, A_2B and A₃, and the corresponding genes are ADORA1, ADORA2A, ADORA2B, and ADORA3. Adenosine receptors couple to different G proteins that trigger the activation of distinct intracellular signalling pathways. The A₁ and A₃ adenosine receptors are coupled to G_i/o proteins, and the activation of these receptors inhibits cAMP production. In contrast, A_2A and A_2B adenosine receptors are coupled to G_s/olf proteins, and the activation of these receptors results in increased cAMP production (Chen, Lee, & Chern, 2014). Besides G_s and G_i coupling that modulates cAMP levels, G_q coupling with PLC and subsequent IP₃ and Ca²⁺ modulation downstream of adenosine receptors has also been described (Santiago et al., 2020). Moreover, the A₁ adenosine receptor can also signal via interaction with β‐arrestins, which leads to the activation of ERK1/2 (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, Miscetti, Allegrucci, & Mezzasoma, 1995).

The A₁ and A₂ adenosine receptors regulate the contractility of the vas deferens and epididymis (Table 1) (Brownhill, Hourani, & Kitchen, 1996; Haynes et al., 1998b; Haynes, Alexander, & Hill, 1998a). Interestingly, it seems that the A₁ and A₂ receptors have opposite effects on the contractility of the epididymis, with the A₁ receptors enhancing contractility, whereas A₂ receptors inhibit contractility (Haynes et al., 1998b). This phenomenon might be explained by the difference in their G protein‐coupling selectivity (van Galen, Stiles, Michaels, & Jacobson, 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 G_s protein and activate AC to increase intracellular cAMP levels (Figure 1) (Chen et al., 2014; Fredholm et al., 1994; Santiago et al., 2020). Further investigation showed that the A_2A receptor mediates potassium channel activation through PKA and PKG in rat epididymal smooth muscle (Haynes, 2000). This result is consistent with the finding that A₂ receptor activation stimulated cAMP‐dependent PKA, which in turn modulated potassium channel activity in arterial or skeletal muscles (Barrett‐Jolley, Comtois, Davies, Stanfield, & Standen, 1996; Kleppisch & Nelson, 1995).

Besides important roles in the contractility of epididymis, adenosine receptors also actively participate in spermatogenesis, capacitation, and acrosome reaction (Bellezza & Minelli, 2017). The number of capacitated spermatozoa incubated in human tubal fluid was significantly reduced in Adora1 ^−/− spermatozoa, and the difference between Adora1 ^+/+ and Adora1 ^−/− mouse spermatozoa is mainly in the time necessary to reach the maximum percentage of capacitation (Minelli et al., 2004). Meanwhile, the spermatozoa of Adora1 ^−/− mice are less prone to acrosome reaction (Minelli et al., 2004; Minelli, Bellezza, Collodel, & Fredholm, 2008). Furthermore, a significant reduction of the number of pups produced by Adora1 ^−/− male mice suggests that A₁ adenosine receptors must be fully operative to accomplish the optimal degree of capacitation and thereby fertilization (Minelli et al., 2004). Polydeoxyribonucleotide (PDRN), an agonist of A_2A receptors, significantly restored spermatogenic function in varicocele rats and testicular torsion rats (Minutoli et al., 2012; Minutoli et al., 2015).

Adenosine (and its precursor ATP) has been used for several decades to treat cardiac arrhythmias through activating A₁ adenosine receptors (Szentmiklosi et al., 2015). Adenosine is also the gold‐standard agent to create maximum coronary hyperemia through activating A_2A adenosine receptors (McGeoch & Oldroyd, 2008). However, given that adenosine can activate the range of adenosine receptors, it inevitably produces some undesirable adverse effects. To avoid non‐specific global adverse reactions, selective agonists of A₁, A_2A, and A₃ adenosine receptors have been developed, some of which are currently undergoing clinical trials (Jacobson et al., 2019). For example, the A₁ 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 A_2A 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, piclidenoson) 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 (Mundell & Kelly, 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.

7. FUTURE QUESTIONS AND PERSPECTIVES

Numerous GPCRs are expressed in the efferent ductules and epididymis, which consist of various cell types. In the present review, we focus on the role of ADGRG2, AT₂ receptors, LGR4, GPER and the adenosine receptors in the epididymis. Other GPCRs such as bradykinin receptors and Frizzled receptors have also been shown to be expressed in the epididymis (Belleannée et al., 2009; K. Wang et al., 2015). Moreover, RNAseq suggested that many other GPCRs, including ADGRF1 (GPR110), ADGRG1 (GPR56), GPRC5C, GPR107, GPR108, GPR125, GPR137, and GPR160 are expressed in the epididymis (Browne et al., 2016). The precise role of these GPCRs in the epididymis awaits further investigation and will be reviewed in the future.

At present, the following questions remain. (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 (signalling 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, AT₂ receptor, 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 AT₂ receptor 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 systematic 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 ductule 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, use of the specific known chemical ligands for these GPCRs, together with 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.

8. CONCLUSIONS

Male infertility rates have continuously increased in recent years, and few effective treatments with known targets and defined mechanisms exist. Recently, the identification of mutations in specific GPCR superfamily members related to male infertility and the increased understanding of the detailed molecular mechanisms involving these GPCRs in the regulation of sperm maturation and homeostasis of the microenvironments of the epididymis and efferent ductules have provided new clues on the potential development of therapies to treat male infertility, given that these receptors account for almost 1/3 of current clinical drug targets.

In addition to ADGRG2 and AT₂ receptors, GPCR superfamily members such as LGR4, GPER, and adenosine receptors are known to play important roles in the regulation of postnatal epididymal development, the formation of the blood–epididymal barrier, the maintenance of osmotic pressure in a perfusion solution, and the contractility of the epididymis (Table 1). The repertoire of the physiological roles of these GPCRs and other uncharacterized GPCRs, as well as further detailed studies of these receptor connecting to male infertility development, provide entirely novel therapeutic opportunities for the treatment of male infertility.

Currently, a variety of low MW compounds, peptide ligands and endogenous ligands have been found or developed to target AT₂ receptors, LGR4, GPER, and adenosine receptors (Table 3). It is worth noting that several such compounds or ligands have been approved by the FDA for the treatment of diseases other than male infertility. Therefore, there is great interest in testing these ligands and compounds in male infertility animal models to examine their therapeutic potential. It is also worth noting that endogenous or high‐affinity ligands involved in the regulation of ADGRG2 have not been identified. Such tools are greatly needed to understand the function of ADGRG2 in male fertility and evaluate the potential role of ADGRG2 as a therapeutic target in male infertility.

Only a small number of the signalling pathways downstream of GPCRs have been characterized in detail in the efferent ductules and epididymis, and these pathways have shown unique signalling properties, although they sometimes share signal‐transducing effectors (Figure 1). For example, both ADGRG2 and GPER have been shown to couple to G_s in the epididymis; however, they exhibit distinct subcellular microdomain biases in their signalling. ADGRG2 forms a signal transduction complex with β‐arrestin 1, G_q, and CFTR on the apical membrane, whereas GPER forms a complex with G_s at the endoplasmic reticulum, nuclear envelope, and plasma membrane (Figure 1). Therefore, even when sharing effectors, the location bias of each GPCR may determine its detailed specific functions in the epididymis and efferent ductules. This possibility raises the question of whether activation of an alternative GPCR in the epididymis or efferent ductules will be able to restore the dysfunction of a particular GPCR, such as in cases of infertility caused by the ADGRG2 mutations.

Collectively, the complex signalling of GPCR members in the epididymis and the specific physiological roles of these GPCRs that contribute to male fertility are worthy of further detailed investigation. In addition, the prospect of using their ligands highlights new opportunities for the development of treatments for male infertility.

8.1. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in the IUPHAR/BPS Guide to PHARMACOLOGY (http://www.guidetopharmacology.org) and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 (Alexander, Christopoulos, et al., 2019; Alexander, Cidlowski, et al., 2019; Alexander, Fabbro, et al., 2019; Alexander, Mathie, et al., 2019).

CONFLICT OF INTEREST

The authors declare no conflicts of interests.

Zhang D, Wang Y, Lin H, et al. Function and therapeutic potential of G protein‐coupled receptors in epididymis. Br J Pharmacol. 2020;177:5489–5508. 10.1111/bph.15252