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 on 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 elicit great arsenal for potential therapy development for treating male infertility.
Keywords : G protein-coupled receptor (GPCR); epididymis; male infertility; ADGRG2; AGTR2; LGR4
Abbreviations: GPCR, G protein-coupled receptor; ADGRG2, adhesion G protein-coupled receptor G2; AGTR2, angiotensin II receptor type 2; LGR4, leucine-rich repeat containing G protein-coupled receptor 4; GPR64, G protein-coupled receptor 64; HE6, human epididymal gene product 6; CFTR, cystic fibrosis transmembrane conductance regulator; CBAVD, congenital bilateral absence of the vas deferens; RAS, renin-angiotensin system; ANGI, angiotensin I; ANGII, angiotensin II; tACE, angiotensin-converting enzyme specific to the testes; AGTR1, angiotensin II receptor type 1; NO, nitric oxide; IPF, idiopathic pulmonary fibrosis; GPR48, G protein-coupled receptor 48; ERα, estrogen receptor α; AR, androgen receptor; NHE3, Na+/H+ hydrogen exchanger 3; Aqp9, aquaporin 9; BMs, basement membranes; TNF, tumor necrosis factor; GSK3-β, glycogen synthase kinase 3 beta; GPER, G protein-coupled estrogen receptor 1; GPR30, G protein-coupled receptor 30; PAMs, positive allosteric modulators.
Introduction
The infertility rate of humans has continuously increased in recent years and has become a significant social burden (Krausz et al. , 2018; Winters et al. , 2014). Currently, infertility ranks as the third most common public health concern below cancer and cardiovascular disease. Issues in males and females contribute equally to the increasing infertility rate and nearly 7% of the male population has fertility problems (Krausz et al. , 2018; Winters et al. , 2014). However, few effective treatments are available for male infertility with defined mechanisms. It is now well accepted 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 et al. , 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, 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 et al. , 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 commonly accepted 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 et al. , 1998; Hess, 2000; Hess et al. , 2000).
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 et al. , 1984; Zhou et al. , 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 et al. , 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 et al. , 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 et al. , 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 et al. , 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 elusive, 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 this knowledge will 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 et al. , 2017; Kim et al. , 2020; Lammermann et al. , 2019; Li et al. , 2018; Liu et al. , 2017; Srivastava et al. , 2015), there is a significant knowledge paucity in regard to the functions of GPCRs in the efferent ductules and epididymis. GPCRs were well known for carrying out their selective functions through coupling to different G protein subtypes or arrestins(Mangliket al. , 2020; Staus et al. , 2020; Wingler et al. , 2020). 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 arrestins. For G protein signaling, activated GPCRs act as guanine nucleotide exchange factors (GEFs) for the α subunits of heterotrimeric G proteins, catalysing the release of GDP and the binding of GTP for G protein activation. Different G protein couples to downstream effectors. For example, the Gs couples to adenyl cyclase whereas the Gq connects to the phospholipase C(Flock et al. , 2017; Flock et al. , 2015; Furness et al. , 2016; Isogai et al. , 2016; Ritter et al. , 2009; Sounier et al. , 2015; Venkatakrishnan et al. , 2016). The activated GPCRs are also phosphorylated by a group of GPCR kinases (GRKs)(Homan et al. , 2014; Komolov et al. , 2017; Reiter et al. , 2006), leading to the recruitment of a different type of arrestins. The interaction of GPCRs with arrestins turns on a second wave of signalling(Desimine et al. , 2018; Dong et al. , 2017; Kumari et al. , 2016; Lefkowitz et al. , 2005; Liu et al. , 2017; Reiter et al. , 2006; Shukla et al. , 2014; Wang et al. , 2018; Yang et al. , 2018; Yang et al. , 2015). 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 et al. , 2017; Srivastava et al. , 2015; Tobin et al. , 2008; Xiao et al. , 2007; Yang et al. , 2018; Yang et al. , 2015). However, the exact roles of the G protein subtype or arrestins downstream epididymis GPCRs remain cloudy.
At present, there are no U.S. Food and Drug Administration (FDA)-approved drugs targeting 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 adhesion G protein-coupled receptor G2 (ADGRG2), angiotensin II receptor type 2 (AGTR2), and leucine-rich repeat containing G protein-coupled receptor 4 (LGR4), and has successfully developed the corresponding ligands to regulate their functions, illuminating the possibility of therapeutic developments regarding male infertility (Figure 1). Here, we review the existing progress of GPCRs in epididymis and efferent ductules, and suggest potential therapeutics directions by targeting these GPCRs for male infertility.
Function of ADGRG2 in fluid reabsorption and epididymis development
Few GPCRs have tissue-specific distributions in male reproductive systems. ADGRG2, also called G protein-coupled receptor 64 (GPR64) or human epididymal gene product 6 (HE6), has attracted substantial attention for its specific expression and essential function in male reproductive systems. It is specifically expressed in the efferent ductules and the proximal epididymis, with much lower expression levels in other tissues (Table 1) (Kirchhoff et al. , 2008; Obermann et al. , 2003). Further studies confirmed the functional importance of ADGRG2 in male fertility. The human and mouse ADGRG2/Adgrg2 gene is localized on chromosome X. 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 -/Ymice (Table 1) (Gottwald et al. , 2006; Zhang et al. , 2018).
ADGRG2 belongs to the adhesion GPCR subfamily, and all members of this family share a very large N-terminal domain(Fredriksson et al. , 2003; Hamann et al. , 2015; Hu et al. , 2014; Kishore et al. , 2017; Liebscher et al. , 2013; Paavola et al. , 2012; Paavola et al. , 2011; Sun et al. , 2013; Wang et al. , 2014). Many members of this family have been shown to function through G protein coupling (Folts et al. , 2019; Purcell et al. , 2018). Without known endogenous ligands, these adhesion GPCRs display significant constitutive activity once their N-terminal region is removed by autocleavage (Demberg et al. , 2015; Hamann et al. , 2015; Hu et al. , 2014; Kishore et al. , 2016; Purcell et al. , 2018; Sun et al. , 2013; Wang et al. , 2014; Zhang et al. , 2018). The transmembrane and cytoplasmic regions remained after cleavage are usually referred to as the β subunit. Our data showed that in cells overexpressing either full-length ADGRG2 or the ADGRG2-β subunit, significant constitutive Gs or Gq coupling activity was observed, which was confirmed by several parallel studies assessing artificial ligands or specific cellular contexts (Demberget al. , 2015; Hamann et al. , 2015). These studies suggested that ADGRG2-mediated Gs or Gq signaling 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). The non-ciliated cells in efferent ductules are frequently referred as principal cells in the epididymis (Burkett et al. , 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 labeling technique by exploiting the promoter of theADGRG2 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 imbalanced pH state in the efferent ductules and dysfunction in fluid resorption inAdgrg2 -/Y mice (Zhang et al. , 2018).
Further analysis combining Gq-/+ andAdgrg2 -/Y mouse models, pharmacological intervention and cell labeling techniques demonstrated that ADGRG2 regulated Cl- and pH homeostasis through Gq-dependent coupling between the receptor and the anion channel CFTR (cystic fibrosis transmembrane conductance regulator) (Figure 1)(Zhang et al. , 2018). CFTR and ADGRG2 colocalized at the apical membrane of non-ciliated cells, accompanied by selective high expression of Gq in the same cells. Through coupling to Gq, ADGRG2 maintains the basic CFTR outward-rectifying current, which is required for fluid resorption and sperm maturation (Figure 1) (Zhanget al. , 2018). In addition to G protein signaling downstream of GPCRs, arrestins (members of a family related scaffold proteins) are known not only to mediate endocytosis of these receptors but also to perform many G protein-independent or G protein-cooperative functions (Dong et al. , 2017; Liu et al. , 2017; Smith et al. , 2018; Yang et al. , 2018; Yang et al. , 2017b).
Importantly, whereas disruption of β-arrestin-2 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 Gq 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/Gq/CFTR supercomplex localizes at the apical membrane of non-ciliated cells and functions as a regional signaling 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/organs(Lymperopoulos, 2018; Lymperopoulos et al. , 2019; Srivastava et al. , 2015). For example, in the heart,β-arrestin-1 and -2 initially thought of as functionally interchangeable, actually exert 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 Gq 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 Gq and Gs proteins and eventually lead to male infertility (Figure 2A, Table 2) (Khan et al. , 2018; Patat et al. , 2016; Yuan et al. , 2019). The p.Cys570Tyr missense mutation is located close to the GPS region of ADGRG2, which may affect its autoinhibitory mechanism mediated by the N-terminal subunit (Yang et al. , 2017a). In contrast, the p.Cys949AlafsTer81 frame shift mutation, the missense p.Lys990Glu and p.Arg1008Gln mutations produce a protein with an intact seven-transmembrane domain, but all of these mutations cause changes in the C-terminal region of ADGRG2, which may be involved in arrestin recruitment and the corresponding signaling (Figure 2A, Table 2) (Patat et al. , 2016; Yang et al. , 2017a; Yuan et al. , 2019). Therefore, different ADGRG2 mutations may cause the same male infertility phenotype through distinct cellular signaling mechanisms.
Notably, the mutations of ADGRG2 in human 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 CFTR chloride channel (Patat et al. , 2016). In our recent report, we have demonstrated a functional coupling between the ADGRG2 and the CFTR serves as the key event in maintenance of the Cl- and pH homeostasis in efferent ductules and epididymis,of which a persistent dysfunction may finally cause progressive atrophy of the efferent/epididymis ductules (Zhang et al. , 2018). Thus, the impairment of the ADGRG2/CFTR coupling may directly relate to the CBAVD in the male infertility patients.
It’s worth noting that the infertile patients are usually identified at their adult age, whereas the animal model normally has a shorter life span. This could explain the ADGRG2 knockout mice did not develop the CBVAD in their life time. For an ADGRG2-targeted therapy for treating male infertility, a systematic screening for male sterility gene, and the identification of the genetic mutations in ADGRG2 or CFTR, as well as genetic or pharmacological intervening in the early stage of a male patient carrying the mutations could be considered.
Currently, the endogenous ligands for ADGRG2 are still unknown. However, the ADGRG2 β-subunit itself shows significant constitutive G protein activity and is able to 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 β-subunit was shown to activate ADGRG2 with low affinity (Table 3) (Demberg et al. , 2015). Further modification of ADGRG2 ligands derived from this peptide might increase the activity of certain ADGRG2 mutants and exhibit therapeutic potential. Alternatively, we have also shown that activation of angiotensin II receptor type 2 (AGTR2) in the efferent ductules is able to rescue fluid resorption dysfunction 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 general rescue approach such as AGTR2 activation is sufficient to treat patients carrying ADGRG2 mutations.