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\begin{document}

\title{Function and therapeutic potential of GPCRs in epididymis}



\author[1]{Daolai Zhang}%
\author[2]{Yanfei Wang}%
\author[3]{Hui  Lin}%
\author[3]{Yujing Sun}%
\author[3]{Mingwei  Wang}%
\author[4]{Yingli Jia}%
\author[5]{Xiao Yu}%
\author[6]{Hui Jiang}%
\author[7]{Wenming Xu}%
\author[2]{Zhigang Xu}%
\author[8]{Jinpeng Sun}%
\affil[1]{Binzhou Medical University - Yantai Campus}%
\affil[2]{Shandong University School of Life Science}%
\affil[3]{Shandong University School of Medicine}%
\affil[4]{Peking University School of Basic Medical Sciences}%
\affil[5]{Shandong University}%
\affil[6]{Peking University Third Hospital}%
\affil[7]{Sichuan University West China Second University Hospital}%
\affil[8]{shandong University}%


\vspace{-1em}



  
  \date{May 21, 2024}


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\selectlanguage{english}
\begin{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.%
\end{abstract}%



\sloppy


\textbf{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.

\textbf{Keywords} : G protein-coupled receptor (GPCR); epididymis; male
infertility; ADGRG2; AGTR2; LGR4

\textbf{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; ER\selectlanguage{greek}α, \selectlanguage{english}estrogen
receptor \selectlanguage{greek}α; \selectlanguage{english}AR, androgen receptor; NHE3,
Na\textsuperscript{+}/H\textsuperscript{+} hydrogen exchanger 3; Aqp9,
aquaporin 9; BMs, basement membranes; TNF, tumor necrosis factor;
GSK3-\selectlanguage{greek}β, \selectlanguage{english}glycogen synthase kinase 3 beta; GPER, G protein-coupled
estrogen receptor 1; GPR30, G protein-coupled receptor 30; PAMs,
positive allosteric modulators.

\textbf{Introduction}

The infertility rate of humans has continuously increased in recent
years and has become a significant social burden (Krausz \emph{et al.} ,
2018; Winters \emph{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 \emph{et al.} , 2018; Winters \emph{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 \emph{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 \emph{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
\emph{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 \emph{et al.} , 1998; Hess, 2000; Hess \emph{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 \emph{et al.} , 1984; Zhou
\emph{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 \emph{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 \emph{et al.} ,
2007; Chevrier \emph{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 \emph{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 \emph{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
\emph{et al.} , 2018; Santos \emph{et al.} , 2017). Although the roles
of GPCRs in cardiovascular disease, neuronal disease, diabetes and many
other diseases have been extensively investigated(Desimine \emph{et al.}
, 2018; Dong \emph{et al.} , 2017; Hauser \emph{et al.} , 2017; Kim
\emph{et al.} , 2020; Lammermann \emph{et al.} , 2019; Li \emph{et al.}
, 2018; Liu \emph{et al.} , 2017; Srivastava \emph{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(Manglik\emph{et al.} , 2020; Staus
\emph{et al.} , 2020; Wingler \emph{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 \selectlanguage{greek}α \selectlanguage{english}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 \emph{et
al.} , 2017; Flock \emph{et al.} , 2015; Furness \emph{et al.} , 2016;
Isogai \emph{et al.} , 2016; Ritter \emph{et al.} , 2009; Sounier
\emph{et al.} , 2015; Venkatakrishnan \emph{et al.} , 2016). The
activated GPCRs are also phosphorylated by a group of GPCR kinases
(GRKs)(Homan \emph{et al.} , 2014; Komolov \emph{et al.} , 2017; Reiter
\emph{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 \emph{et al.} , 2018; Dong \emph{et al.} ,
2017; Kumari \emph{et al.} , 2016; Lefkowitz \emph{et al.} , 2005; Liu
\emph{et al.} , 2017; Reiter \emph{et al.} , 2006; Shukla \emph{et al.}
, 2014; Wang \emph{et al.} , 2018; Yang \emph{et al.} , 2018; Yang
\emph{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 \emph{et al.} , 2011;
Liu \emph{et al.} , 2017; Luttrell \emph{et al.} , 1999; Miller \emph{et
al.} , 2000; Peterson \emph{et al.} , 2017; Srivastava \emph{et al.} ,
2015; Tobin \emph{et al.} , 2008; Xiao \emph{et al.} , 2007; Yang
\emph{et al.} , 2018; Yang \emph{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 \emph{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.

\textbf{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 \emph{et al.} , 2008; Obermann
\emph{et al.} , 2003). Further studies confirmed the functional
importance of ADGRG2 in male fertility. The human and mouse
\emph{ADGRG2/Adgrg2} gene is localized on chromosome X. \emph{Adgrg2}
\textsuperscript{-/Y} mice exhibit reduced sperm numbers, decreased
sperm motility and increased number of spermatozoa with deficient heads
or angulated flagella (Davies \emph{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 \emph{Adgrg2} \textsuperscript{-/Y}mice (Table 1) (Gottwald \emph{et
al.} , 2006; Zhang \emph{et al.} , 2018).

ADGRG2 belongs to the adhesion GPCR subfamily, and all members of this
family share a very large N-terminal domain(Fredriksson \emph{et al.} ,
2003; Hamann \emph{et al.} , 2015; Hu \emph{et al.} , 2014; Kishore
\emph{et al.} , 2017; Liebscher \emph{et al.} , 2013; Paavola \emph{et
al.} , 2012; Paavola \emph{et al.} , 2011; Sun \emph{et al.} , 2013;
Wang \emph{et al.} , 2014). Many members of this family have been shown
to function through G protein coupling (Folts \emph{et al.} , 2019;
Purcell \emph{et al.} , 2018). Without known endogenous ligands, these
adhesion GPCRs display significant constitutive activity once their
N-terminal region is removed by autocleavage (Demberg \emph{et al.} ,
2015; Hamann \emph{et al.} , 2015; Hu \emph{et al.} , 2014; Kishore
\emph{et al.} , 2016; Purcell \emph{et al.} , 2018; Sun \emph{et al.} ,
2013; Wang \emph{et al.} , 2014; Zhang \emph{et al.} , 2018). The
transmembrane and cytoplasmic regions remained after cleavage are
usually referred to as the \selectlanguage{greek}β \selectlanguage{english}subunit. Our data showed that in cells
overexpressing either full-length ADGRG2 or the ADGRG2-\selectlanguage{greek}β \selectlanguage{english}subunit,
significant constitutive Gs or Gq coupling activity was observed, which
was confirmed by several parallel studies assessing artificial ligands
or specific cellular contexts (Demberg\emph{et al.} , 2015; Hamann
\emph{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 \emph{et al.} , 2008). The non-ciliated
cells in efferent ductules are frequently referred as principal cells in
the epididymis (Burkett \emph{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 the\emph{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 imbalanced
pH state in the efferent ductules and dysfunction in fluid resorption
in\emph{Adgrg2} \textsuperscript{-/Y} mice (Zhang \emph{et al.} , 2018).

Further analysis combining Gq\textsuperscript{-/+} and\emph{Adgrg2}
\textsuperscript{-/Y} mouse models, pharmacological intervention and
cell labeling techniques demonstrated that ADGRG2 regulated
Cl\textsuperscript{-} and pH homeostasis through Gq-dependent coupling
between the receptor and the anion channel CFTR (cystic fibrosis
transmembrane conductance regulator) (Figure 1)(Zhang \emph{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) (Zhang\emph{et 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 \emph{et al.} , 2017; Liu \emph{et
al.} , 2017; Smith \emph{et al.} , 2018; Yang \emph{et al.} , 2018; Yang
\emph{et al.} , 2017b).

Importantly, whereas disruption of \selectlanguage{greek}β-\selectlanguage{english}arrestin-2 has no significant
effects on the fluid resorption function, \selectlanguage{greek}β-\selectlanguage{english}arrestin-1 deficiency
impaired pH and Cl\textsuperscript{-} homeostasis in the efferent
ductules and initial segment of the epididymis (Zhang \emph{et al.} ,
2018). Further investigation confirmed the coexistence of ADGRG2, CFTR,
\selectlanguage{greek}β-\selectlanguage{english}arrestin-1 and Gq in the same protein complex (Figure 1), while
\selectlanguage{greek}β-\selectlanguage{english}arrestin-1 deficiency abolished the colocalization of ADGRG2 and CFTR
on the apical membrane. These data suggested that the
ADGRG2/\selectlanguage{greek}β-\selectlanguage{english}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\textsuperscript{-} homeostasis in the efferent ductules and initial
segment of the epididymis (Figure 1) (Zhang \emph{et al.} , 2018). The
ADGRG2/CFTR interaction in the epididymis represents yet another example
of the functional divergence between the two \selectlanguage{greek}β-\selectlanguage{english}arrestin isoforms,
already established in several other tissues/organs(Lymperopoulos, 2018;
Lymperopoulos \emph{et al.} , 2019; Srivastava \emph{et al.} , 2015).
For example, in the heart,\selectlanguage{greek}β-\selectlanguage{english}arrestin-1 and -2 initially thought of as
functionally interchangeable, actually exert diametrically opposite
effects in the mammalian myocardium.\selectlanguage{greek}β-\selectlanguage{english}arrestin-1 exerts overall
detrimental effects on the heart, in contrast, \selectlanguage{greek}β-\selectlanguage{english}arrestin-2 is overall
beneficial for the myocardium(Lymperopoulos \emph{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 \emph{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 \emph{et al.} , 2018; Patat \emph{et al.} ,
2016; Yuan \emph{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
\emph{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 \emph{et al.} , 2016; Yang \emph{et al.} , 2017a;
Yuan \emph{et al.} , 2019). Therefore, different \emph{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 \emph{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\textsuperscript{-} and pH homeostasis in efferent
ductules and epididymis,of which a persistent dysfunction may finally
cause progressive atrophy of the efferent/epididymis ductules (Zhang
\emph{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 \selectlanguage{greek}β-\selectlanguage{english}subunit itself shows significant constitutive G protein
activity and is able to activate the CFTR current in transfected HEK293
cells (Zhang \emph{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 \selectlanguage{greek}β-\selectlanguage{english}subunit was shown to activate ADGRG2 with low affinity
(Table 3) (Demberg \emph{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
\emph{Adgrg2\textsuperscript{-/Y}} mice (Zhang \emph{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 \emph{ADGRG2} mutations.

\textbf{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 \emph{et al.} , 2003; Saez \emph{et al.} , 2004;
Speth \emph{et al.} , 1999; Wong \emph{et al.} , 1990; Zhao \emph{et
al.} , 1996). Importantly, ANGII in the epididymal lumen is mainly
produced through the cleavage of ANGI by angiotensin I-converting enzyme
(ACE) (Langford \emph{et al.} , 1993; Sibony \emph{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 \emph{et al.} , 1996; Hagaman
\emph{et al.} , 1998; Krege \emph{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 \emph{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
\emph{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
\selectlanguage{greek}μ\selectlanguage{english}M ANGII had no significant effect, applying 100 nM ANGII restored pH
homeostasis and fluid reabsorption in efferent ductules derived
from\emph{Adgrg2} \textsuperscript{-/Y} mice. This rescue effect was
blocked specifically by PD123319, an AGTR2 antagonist, but not by an
ANGII antagonist (Zhang \emph{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 \emph{et al.} , 2018; Hallberg
\emph{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 \emph{et al.} ,
2014; Hallberg \emph{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, \selectlanguage{greek}β-\selectlanguage{english}Pro\textsuperscript{7}AngIII was
reported to show high selectivity for the activation of AGTR2 but no
significant effect on AGTR1 (Hallberg \emph{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 \emph{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.

\textbf{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
\emph{et al.} , 2005; Yi \emph{et al.} , 2013).

LRG4 has been shown to play an important role in postnatal epididymal
development in mice. In \emph{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 \emph{et al.} , 2006). Furthermore, the expression levels of
estrogen receptor \selectlanguage{greek}α \selectlanguage{english}(ER\selectlanguage{greek}α\selectlanguage{english}) and androgen receptor (AR) are dramatically
reduced in the epididymis of male \emph{Lgr4} knockout mice, which in
turn leads to decreased expression of
Na\textsuperscript{+}-K\textsuperscript{+}-ATPase,
Na\textsuperscript{+}/H\textsuperscript{+}hydrogen exchanger 3 (NHE3),
and aquaporin 9 (Aqp9) (Li \emph{et al.} , 2010). LRG4 upregulates ER\selectlanguage{greek}α
\selectlanguage{english}expression via the cAMP/PKA signaling pathway (Figure 1). Downstream of
the LRG4-cAMP-PKA pathway, CREB binds to a Cre motif in the ER\selectlanguage{greek}α \selectlanguage{english}promotor
and activates its expression (Li \emph{et al.} , 2010).

The pivotal role of LGR4 in the epididymis is further supported by
a\emph{Lgr4} hypomorphic mutant mouse line
(\emph{Lgr4\textsuperscript{Gt}} ) that was developed through gene-trap
insertional mutagenesis. Short and dilated epididymal tubules are
detected in homozygous \emph{Lgr4\textsuperscript{Gt/Gt}} mice, which
have only one-tenth the normal \emph{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 \emph{et al.} , 2007). \emph{Lgr4}knockout or hypomorphic mice
also show deficits in the testes and efferent ductules (Qian \emph{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 (Gao\emph{et 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 \emph{et al.} , 2011; de Lau \emph{et al.} , 2011; Deng \emph{et
al.} , 2013; Glinka \emph{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 \emph{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-\selectlanguage{greek}β\selectlanguage{english}) signaling pathway (Luo
\emph{et al.} , 2016). At present, synthesized agonists or antagonists
of LGR4 have not been reported.

\textbf{Complex functions ofG protein-coupled estrogen receptor 1 (}
\textbf{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 \emph{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 \emph{et al.} , 2006; Lucas \emph{et al.} , 2010;
Revankar \emph{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 \emph{et al.} , 2006;
Prossnitz \emph{et al.} , 2007; Thomas \emph{et al.} , 2005).

GPER has been detected in many male reproductive structures, such as the
testes (Cassault-Meyer \emph{et al.} , 2014; Gautier \emph{et al.} ,
2016; Lucas \emph{et al.} , 2010), spermatozoa (Arkoun \emph{et al.} ,
2014; Cassault-Meyer \emph{et al.} , 2014; Gautier \emph{et al.} ,
2016), and prostate (Rago \emph{et al.} , 2016). It has also been found
in the efferent ductules and epididymis (Cao \emph{et al.} , 2017; Hess
\emph{et al.} , 2011; Katleba \emph{et al.} , 2015; Krejcirova \emph{et
al.} , 2018; Lu \emph{et al.} , 2016; Malivindi \emph{et al.} , 2018;
Martinez-Traverso \emph{et al.} , 2015; Menad \emph{et al.} , 2017;
Pereira \emph{et al.} , 2014; Rago \emph{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\emph{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\emph{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 \emph{et
al.} , 1995; Krejcirova \emph{et al.} , 2018; Lu \emph{et al.} , 2016;
Pereira \emph{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 \emph{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 \emph{in
vivo} (Table 3) (Dennis \emph{et al.} , 2009; Dennis \emph{et al.} ,
2011). Recently, a series of indole-thiazole derivatives were identified
as new GPER agonists (O'Dea \emph{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.

\textbf{Two adenosine receptors with opposite functions in the
epididymis}

Adenosine receptors consist of four members, namely, A\textsubscript{1},
A\textsubscript{2A}, A\textsubscript{2B}, and A\textsubscript{3}.
Adenosine receptors are activated by adenosine and transmit signals
through classic G protein-cAMP or \selectlanguage{greek}β-\selectlanguage{english}arrestin pathways (Table 1)
(Geldenhuys \emph{et al.} , 2017). Most adenosine receptors have been
suggested to be present in the epididymis (Table 1) (Haynes \emph{et
al.} , 1998b; Minelli \emph{et al.} , 1995).

The A\textsubscript{1} and A\textsubscript{2} adenosine receptors have
been shown to regulate the contractility of the vas deferens and
epididymis (Table 1) (Brownhill \emph{et al.} , 1996; Haynes \emph{et
al.} , 1998a; Haynes \emph{et al.} , 1998b). Interestingly, it seems
that the A\textsubscript{1} and A\textsubscript{2} receptors have
opposite effects on the contractility of the epididymis: the
A\textsubscript{1} receptor enhances the contractility, whereas the
A\textsubscript{2} receptor inhibits the contractility (Haynes \emph{et
al.} , 1998b). This phenomenon might be explained by the difference in
their G protein-coupling selectivity (van Galen\emph{et al.} , 1992). In
the epididymis, A\textsubscript{2} adenosine receptors increase
intracellular cAMP levels (Haynes \emph{et al.} , 1998b), consistent
with the generally accepted view that A\textsubscript{2} adenosine
receptors are coupled to Gs-protein and activate adenylyl cyclase to
increase intracellular cAMP levels (Figure 1) (Fredholm \emph{et al.} ,
1994). Further investigation showed that the A\textsubscript{2A}
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 A\textsubscript{2}receptor activation
stimulated cAMP-dependent protein kinase A, which in turn modulated
potassium channel activity in arterial or skeletal muscles
(Barrett-Jolley \emph{et al.} , 1996; Kleppisch \emph{et al.} , 1995).
In contrast, the A\textsubscript{1} adenosine receptor is likely coupled
to effectors through Gi/o proteins, although confirmative evidence is
still lacking (Haynes \emph{et al.} , 1998b).

Adenosine (and its precursor ATP) has been used for several decades to
treat cardiac arrhythmias through activating A\textsubscript{1}adenosine
receptors (Szentmiklosi\emph{et al.} , 2015). Adenosine is also the
gold-standard agent to create maximum coronary hyperemia through
activating A\textsubscript{2A}adenosine receptors (McGeoch \emph{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
A\textsubscript{1}, A\textsubscript{2A}, and A\textsubscript{3}
adenosine receptors have been developed, some of which are currently
undergoing clinical trials (Jacobson \emph{et al.} , 2019). For example,
the A\textsubscript{1} 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 \emph{et al.} , 2019). The
moderately selective A\textsubscript{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 \emph{et al.}
, 2019). The moderately selective A\textsubscript{3} 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 \emph{et al.} , 2019).

An important limitation of adenosine receptor agonists is
agonist-induced desensitization (Mundell\emph{et 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
\emph{et al.} , 2018). However, many adenosine receptor agonists and
PAMs (such as the A\textsubscript{1} 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.

\textbf{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.

\textbf{Conclusions}

(1) 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.

(2) In addition to ADGRG2 and AGTR2, 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.

(3) Currently, various small-molecule compounds, peptide ligands and
endogenous ligands have been found or developed to target AGTR2, 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.

(4) Only a small number of the signaling pathways downstream of GPCRs
have been characterized in detail in the efferent ductules and
epididymis, and these pathways have shown unique signaling properties,
although they sometimes share signal-transducing effectors (Figure 1).
For example, both ADGRG2 and GPER have been shown to couple to Gs in the
epididymis; however, they exhibit distinct subcellular microdomain
biases in their signaling. ADGRG2 forms a signal transduction complex
with \selectlanguage{greek}β-\selectlanguage{english}arrestin-1, Gq and CFTR on the apical membrane, whereas GPER
forms a complex with Gs 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 rescue the dysfunction
of a particular GPCR, such as in cases of infertility caused by the
ADGRG2 mutations.

(5) Collectively, the complex signaling 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 potential therapies development for
male infertility.

\par\null

\textbf{Acknowledgements}

We acknowledge support from the National Key Basic Research Program of
China (2018YFC1003600 to J. S., Z. X., W. X.), the National Natural
Science Foundation of China (81773704 to J. S., 81771001 to Z. X.,
81901548 to D. Z.); the Shandong Provincial Natural Science Foundation
(ZR2019BC078 to D. Z.); the Shandong Provincial Medicine and Health
Science Technology Development Program (2017WS692 to D. Z.); the
National Science Fund for Distinguished Young Scholars (81825022 to J.
S.), the Fundamental Research Funds of Shandong University (2018JC025 to
Z. X.) and the Program for Changjiang Scholars and Innovative Research
Team in University (IRT13028).

\textbf{Conflict of Interest:} The authors declare no competing
interests.

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\textbf{Figure legends}

\textbf{Figure 1. Schematic showing GPCR signaling 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: G protein-coupled estrogen receptor 1
(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/\selectlanguage{greek}β-\selectlanguage{english}arrestin-1/Gq/CFTR forms a supercomplex that maintains pH and
chloride anion homeostasis. AGTR2 is specifically detected in basal
cells and is essential for the proton-secretion function of the
epididymal lumen through activation of the nitric oxide (NO)-cGMP
pathway. Different members of the adenosine receptor family have
opposite effects on the contractility of the epididymis. LGR4 activates
Gs to increase intracellular cAMP levels, which promote ER\selectlanguage{greek}α \selectlanguage{english}expression.

\textbf{Figure 2. GPCR mutations associated with disease.}

Schematic representation of the structures of ADGRG2 (A), AGTR2 (B), and
LGR4 (C). The approximate positions of different mutations are
indicated. Abbreviations: PLL domain,
pentraxin/laminin/neurexin/sex-hormone-binding-globulin-like domain;
GPS, G protein-coupled receptor proteolytic site; LRR, leucine-rich
repeats.

\textbf{Tables}

Table 1. GPCRs with known functions in epididymis or efferent ductules\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
Receptor name & Family (GRAFS) name & Expression & Function & Signaling
effectors (G or arrestin) & References\tabularnewline
\midrule
\endhead
ADGRG2(GPR64) & Adhesion & efferent ductules; proximal epididymis
(non-ciliated cells; principal cells) & fluid reabsorption & Gs; Gq;
\selectlanguage{greek}β-\selectlanguage{english}arrestin-1 & Davies et al., 2004; Gottwald et al., 2006; Zhang et al.,
2018\tabularnewline
AGTR2 & Rhodopsin & basal cells & proton secretion & unknown & Shum et
al., 2008\tabularnewline
LGR4 (GPR48) & Rhodopsin & epithelial cells in the reproductive organs &
epithelial-mesenchymal interactions & Gs & Mendive et al., 2006; Gao et
al., 2006; Li et al., 2010\tabularnewline
GPER (GPR30) & Rhodopsin & testis; spermatozoa; prostate; efferent
ductules; epididymis & formation of blood-epididymal barrier and
regulation of osmotic pressure & Gs; \selectlanguage{greek}β-\selectlanguage{english}arrestin-2 & Katleba et al.,
2015; Cao et al., 2017\tabularnewline
Adenosine receptor & Rhodopsin & epididymis & contractility of the
epididymis & Gs; Gi; \selectlanguage{greek}β-\selectlanguage{english}arrestin & Geldenhuys et al., 2017; Haynes et
al., 1998b\tabularnewline
\bottomrule
\end{longtable}

Table 2. Disease-related SNP analysis in GPCRs\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
GPCR & dbSNP rs\# cluster id & dbSNP allele change & Protein residue
change & Amino acid pos & Accession Disease Names & PMID\tabularnewline
\midrule
\endhead
ADGRG2(GPR64) Xp22.13 & rs879255540 & -\textgreater{}T & Glu
{[}E{]}\textgreater{}* & 516 & Congenital bilateral absence of the vas
deferens & 27476656\tabularnewline
& & G\textgreater{}A & Cys {[}C{]}\textgreater{}Tyr {[}Y{]} & 570 &
Congenital bilateral absence of the vas deferens &
28805948\tabularnewline
& rs879255539 & CTGTG\textgreater{}AGA & Leu {[}L{]}\textgreater{}Arg
{[}R{]} & 668 & Congenital bilateral absence of the vas deferens &
27476656\tabularnewline
& & C\textgreater{}T & p.Arg {[}R{]}\textgreater{}* & 814 & Obstructive
azoospermia & 30389958\tabularnewline
& & A\textgreater{}T & p.Lys {[}K{]}\textgreater{}* & 818 & Congenital
absence of vas deferens & 30811104\tabularnewline
& rs879255538 & T\textgreater{}- & Cys {[}C{]}\textgreater{}Ala {[}A{]}
& 949 & Congenital bilateral absence of the vas deferens &
27476656\tabularnewline
& & A\textgreater{}G & p.Lys {[}K{]}\textgreater{}Glu {[}E{]} & 990 &
Congenital bilateral absence of the vas deferens &
28805948\tabularnewline
& & G\textgreater{}A & p.Arg {[}R{]}\textgreater{}Gln {[}Q{]} & 1008 &
Congenital absence of vas deferens & 30811104\tabularnewline
AGTR2 Xq23 & rs121917810 & G\textgreater{}T & Gly
{[}G{]}\textgreater{}Val {[}V{]} & 21 & X-Linked Mental Retardation &
14598163 12746399 12089445\tabularnewline
& & T\textgreater{}- & Phe {[}F{]}\textgreater{}Leu {[}L{]} & 134 & not
specified & 23871722\tabularnewline
& rs5191 & G\textgreater{}A & Arg {[}R{]} \textgreater{}Lys {[}K{]} &
248 & not specified & 23757202 16642433\tabularnewline
LGR4(GPR48) 11p14.1 & rs587777005 & C\textgreater{}T & Arg
{[}R{]}\textgreater{}* & 126 & Low bone mineral density &
23644456\tabularnewline
\bottomrule
\end{longtable}

Table 3. Potential therapeutic ligands targeting to GPCRs in epididymis\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Receptor\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Ligand\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Structure (or Sequence)\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Mode of action\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Highest status\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
References\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ADGRG2\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Tethered peptide agonist\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
TSFGILLDLSRTSLP\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Agonist\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Demberg et al., 2015\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
AGTR2\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Angiotensin II (ANG II)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Asp\textsuperscript{1}-Arg\textsuperscript{2}-Val\textsuperscript{3}-Tyr\textsuperscript{4}-Ile\textsuperscript{5}-His\textsuperscript{6}-Pro\textsuperscript{7}-Phe\textsuperscript{8}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Agonist\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Clinic\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Guimond et al., 2014; Hallberg et al., 2018\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Saralasin\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
{[}Sar\textsuperscript{1},Val\textsuperscript{5},Ala\textsuperscript{8}{]}Ang
II\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Agonist\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Clinic\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Guimond et al., 2014; Hallberg et al., 2018\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Sarile\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
{[}Sar\textsuperscript{1},Ile\textsuperscript{8}{]}Ang II\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Agonist\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Clinic\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Guimond et al., 2014; Hallberg et al., 2018\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
MP-157\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
No structural formula is disclosed\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Agonist\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Phase I\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Hallberg et al., 2018\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
C21/M24\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Agonist\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Phase II\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Hallberg et al., 2018\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
C38/M132\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Antagonist\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Hallberg et al., 2018\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
LGR4\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
R-spondins\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
R-spondin1-4(RSPO1-4)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Agonist\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Carmon et al., 2011; de Lau et al., 2011; Glinka et al., 2011\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Norrin\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
MRKHVLAASFSMLSLLVIMGDTDSKTDSSFIMDSDPRRCMRHHYVDSISHPLYKCSSKMVLLARCEGHCSQASRSEPLVSFSTVLKQPFRSSCHCCRPQTSKLKALRLRCSGGMRLTATYRYILSCHCEECNS\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Agonist\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Deng et al., 2013\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
TNFSF11(RANKL)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Tumor necrosis factor (TNF) superfamily member 11\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Agonist\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Luo et al., 2016\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
GPER\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
G-1\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Agonist\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Bologa et al., 2006\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
G15\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Antagonist\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Dennis et al., 2009; Dennis et al., 2011\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
G36\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Antagonist\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Dennis et al., 2011\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
A\textsubscript{1}AR\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Trabodenoson (INO-8875)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
partial agonist\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Phase III\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Jacobson et al., 2019\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
A\textsubscript{2A}AR\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Regadenoson\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
agonist\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Jacobson et al., 2019\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
A\textsubscript{3}AR\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
IB-MECA\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
agonist\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Phase III\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Jacobson et al., 2019\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure1-2020-6-20/Figure1-2020-6-20}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure2-2020-6-20/Figure2-2020-6-20}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

