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