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.