![Transverse section of an adult human penis [Yafi et al., 2016]. The corpus cavernosum (paired) and corpus spongiosum constitute the 3 erectile tissues of the penis. The tunica albuginea surrounds the corpora cavernosa. Blood flows into the corpus cavernosum via the cavernous artery, which branches into helicine arteries that supply the sinusoidal spaces. Blood drains from the sinusoidal spaces into the subtunical plexus, which forms the emissary vein that passes through the tunica albuginea. Emissary veins drain directly into the deep dorsal artery or into the circumflex veins which also drain into the deep dorsal artery. The dorsal nerve is a sensory somatic nerve fibre responsible for reflexogenic erections.](https://www.researchgate.net/profile/Samuel-Cripps-3/publication/352473376/figure/fig1/AS:11431281079535100@1660735342825/Transverse-section-of-an-adult-human-penis-Yafi-et-al-2016-The-corpus-cavernosum_Q320.jpg)
![MLCK and MLCP mediate smooth muscle contraction and relaxation, respectively [Mas, 2010]. Ca²⁺ ions bind to calmodulin to form the Ca²⁺-calmodulin complex (Cam-Ca) which then binds to and activates MLCK. Active MLCK phosphorylates MLC, facilitating smooth muscle contraction. Conversely, active MLCP dephosphorylates MLC, causing smooth muscle relaxation and tumescence. Active RhoA activates Rho kinase which deactivates MLCP by phosphorylation. Inactive RhoA allows for the activation of MLCP. Green refers to pathways driving tumescence, red refers to that of detumescence. MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatase; MLC, myosin light chain; Cam-Ca, Ca²⁺-calmodulin complex; P, phosphate group.](https://www.researchgate.net/profile/Samuel-Cripps-3/publication/352473376/figure/fig3/AS:11431281079525414@1660735342928/MLCK-and-MLCP-mediate-smooth-muscle-contraction-and-relaxation-respectively-Mas-2010_Q320.jpg)
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Review Article
Sex Dev 2021;15:187–212
Erectile Dysfunction in Men on the Rise:
Is There a Link with Endocrine Disrupting
Chemicals?
Samuel M. Cripps Deidre M. Mattiske Andrew J. Pask
School of BioSciences, The University of Melbourne, Melbourne, VIC, Australia
Received: November 2, 2020
Accepted: April 18, 2021
Published online: June 16, 2021
Correspondence to:
Andrew J. Pask, ajpask @ unimelb.edu.au
© 2021 S. Karger AG, Basel
karger@karger.com
www.karger.com/sxd
DOI: 10.1159/000516600
Keywords
Differences of sexual development · Early mammalian
development · Endocrine-disrupting chemicals · Erectile
dysfunction · Erection
Abstract
Erectile dysfunction (ED) is one of the most prevalent chron-
ic conditions affecting men. ED can arise from disruptions
during development, affecting the patterning of erectile tis-
sues in the penis and/or disruptions in adulthood that im-
pact sexual stimuli, neural pathways, molecular changes,
and endocrine signalling that are required to drive erection.
Sexual stimulation activates the parasympathetic system
which causes nerve terminals in the penis to release nitric
oxide (NO). As a result, the penile blood vessels dilate, allow-
ing the penis to engorge with blood. This expansion subse-
quently compresses the veins surrounding the erectile tis-
sue, restricting venous outflow. As a result, the blood pres-
sure localised in the penis increases dramatically to produce
a rigid erection, a process known as tumescence. The sym-
pathetic pathway releases noradrenaline (NA) which causes
detumescence: the reversion of the penis to the flaccid state.
Androgen signalling is critical for erectile function through
its role in penis development and in regulating the physio-
logical processes driving erection in the adult. Interestingly,
estrogen signalling is also implicated in penis development
and potentially in processes which regulate erectile function
during adulthood. Given that endocrine signalling has a
prominent role in erectile function, it is likely that exposure
to endocrine disrupting chemicals (EDCs) is a risk factor for
ED, although this is an under-researched field. Thus, our re-
view provides a detailed description of the underlying biol-
ogy of erectile function with a focus on the role of endocrine
signalling, exploring the potential link between EDCs and ED
based on animal and human studies. © 2021 S. Karger AG, Basel
Erectile Dysfunction
Erectile Dysfunction (ED) is defined as the consistent
or repeated inability to acquire or sustain an erection suf-
ficient for satisfactory sexual performance [McCabe et al.,
2016]. The 5-item International Index of Erectile Func-
tion (IIEF-5) self-questionnaire categorises the severity of
ED based on the numerical score (each of the 5 questions
is worth 5 points) as no ED (22–25), mild (17–21), mild
to moderate (12–16), moderate (8–11), or severe (1–7)
[Rhoden et al., 2002]. Erectile function relies on a combi-
nation of organic (structural, neurologic, vascular, and
endocrine) and psychogenic factors. Thus, ED can have a
number of aetiologies which are broadly classified as ei-
ther organic or psychogenic [Johannes et al., 2000]. Psy-
Cripps/Mattiske/Pask
Sex Dev 2021;15:187–212
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DOI: 10.1159/000516600
chogenic risk factors for ED include depression and anx-
iety [Yang et al., 2019], although these are beyond the
scope of this review. Organic risk factors include vascular,
neurologic, and endocrine abnormalities [reviewed in
Ludwig and Phillips, 2014]. Interestingly, since the penile
vascular tissue that is responsible for erection is a compo-
nent of the global vascular system, ED of vascular origin
is often an indicator of systemic endothelial dysfunction
[Virag et al., 1981]. Thus, ED not only disrupts quality of
life but can also be a strong indicator of cardiovascular
disease [Gandaglia et al., 2014].
ED is one of the most prevalent chronic conditions
and negatively impacts the quality of life of men and their
partners [Fisher et al., 2005]. The exact prevalence of ED
is difficult to estimate, because this relies on subjective
data-gathering and self-questionnaires. The Internation-
al Index of Erectile Function (IIEF) and the Massachu-
setts Male Ageing Study (MMAS)-derived questionnaires
are the most commonly used for investigating ED in pop-
ulation studies. However, researchers use a number of
other questionnaires and methods which significantly
limits comparability of study results [Kessler et al., 2019].
In addition, population studies have traditionally focused
on ED prevalence in older men (>40 years) while over-
looking younger men [Feldman et al., 1994; Corona et al.,
2010; Weber et al., 2013]. As a result, it is challenging to
understand the true prevalence of ED in different age
groups.
In addition, different geographical regions and age de-
mographics yield varying results, creating further com-
plications in understanding the epidemiology of ED. For
example, ED was reported at an overall prevalence of 23.2
and 61% in Australian men from the ages of 35 and 45
years, respectively [Weber et al., 2013; Martin et al., 2014],
and as high as 81.5% in Malaysian men over the age of 18
years [Nordin et al., 2019]. The landmark MMAS re-
vealed a prevalence of mild to moderate ED in 52% of
men aged 40–70 years [Feldman et al., 1994], whereas the
European Male Ageing Study (EMAS) found an average
ED prevalence of 30% in men at ages 40–79 years [Co-
rona et al., 2010]. Although there are regional differences,
it was estimated that ED affected 152 million men world-
wide in 1995 and was predicted to increase to 322 million
men globally by 2025 (using the lowest United Nations
population projections) [Ayta et al., 1999; McKinlay,
2000].
Several studies have unequivocally demonstrated that
ED prevalence increases with age [Feldman et al., 1994;
Chew et al., 2008; Corona et al., 2010; Weber et al., 2013;
Martin et al., 2014; Nordin et al., 2019]. Thus, the pre-
dicted increase in ED prevalence between 1995 and 2025
can be linked to an increasing ageing male population; the
global proportion of men aged over 65 years in 1995 was
4.2% and will increase to 9.5% by 2025 [reviewed in Ayta
et al., 1999]. However, the dramatic increase of ED prev-
alence is too rapid to be explained by ageing or genetic
mutation alone. This is further supported by an excep-
tionally high prevalence of ED in younger men. For ex-
ample, a study using the IIEF-5 showed that Swiss men
aged 18–25 years displayed a prevalence of 30% [Mialon
et al., 2012]. Another IIEF-based study demonstrated that
ED prevalence in a population in India ranged from 9.9
to 13% in 18–40-years-old men [Sathyanarayana Rao et
al., 2015]. Similarly, in Western Australia, ED prevalence
among men in their 20s and 30s was reported as 15.7 and
8.7%, respectively [Chew et al., 2008]. It was also found
that 1 in 4 Caucasian European men seeking medical help
for new-onset ED were below 40 years of age [Capogros-
so et al., 2013]. These results are consistent with the in-
crease of younger men (<40 years) consulting for ED in a
Florence clinic from 5 to 15% between 2010 and 2015
[Rastrelli and Maggi, 2017]. The consistently high preva-
lence of ED in young men globally means that it cannot
be due to increased reporting alone; ED prevalence is in-
creasing across all age groups, not just alongside an in-
creasing ageing population.
Thus, it is likely that environmental and lifestyle fac-
tors are responsible for current global trends in ED prev-
alence. Indeed, several of these factors, which include
smoking and diet, are implicated in the development of
ED [McVary et al., 2001; Bacon et al., 2006; Esposito et al.,
2006; Francis et al., 2007; Ramírez et al., 2016]. However,
the role of endocrine-disrupting chemicals (EDCs) in the
aetiology of ED is unclear. The WHO defines an EDC as
“an exogenous substance or mixture that alters function(s)
of the endocrine system and consequently causes adverse
health effects in an intact organism, or its progeny, or
(sub) populations” [Johansson and Svingen, 2020]. The
term EDC in this review refers specifically to chemicals
which are known to alter hormonal pathways and cause
adverse health effects in humans. Although these adverse
health effects are not yet described to include ED, we
present a logical connection between their impact on hor-
monal pathways and the development and regulation of
erectile tissues.
Endocrine signalling, particularly that of androgens,
affects erectile function by driving penis development
and also by regulating pathways in the adult involved in
erection [Murakami, 1987; Foresta et al., 2004; Miyagawa
et al., 2009]. Correct development of the erectile tissues in
Impacts of EDCs on Erectile Dysfunction
189
Sex Dev 2021;15:187–212
DOI: 10.1159/000516600
the penis including the nerves, smooth muscle, vascula-
ture, and other structural features is essential for adult
erectile function. Although the role of androgens in erec-
tile function is established, the role of other hormones in
this process is not well understood. However, endoge-
nous estrogen signalling has a recently discovered role in
penis development [Cripps et al., 2019; Govers et al.,
2019] and may also regulate aspects of adult physiology
driving erection, including penile blood flow (discussed
below). Thus, endogenous estrogen signalling during de-
velopment and adulthood may contribute to erectile
function. This is further supported by the presence of aro-
matase and estrogen receptors (ERs) throughout the rat
and human penis [Jesmin et al., 2002; Dietrich et al.,
2004].
ED is correlated with circulating testosterone and es-
trogen levels in men; a significantly higher level of circu-
lating estrogen and a higher estrogen-testosterone ratio is
found in men with ED [Chen et al., 2020]. Also, ageing is
associated with reduced circulating testosterone, poten-
tially explaining ageing-induced ED [reviewed in Tsame-
tis and Isidori, 2018]. This data suggests that alterations
to hormonal balance, which could potentially occur via
EDCs, is likely to increase the risk of ED. Furthermore,
the rapid increase in ED prevalence has occurred along-
side our increasing exposure to EDCs via multiple sourc-
es, including plastics, plasticizers, and phytoestrogens
[reviewed in Diamanti-Kandarakis et al., 2009]. Due to
the lack of research investigating a link between EDCs
and ED, it is difficult to give an indication of how signifi-
cant a risk factor EDCs are for this condition. Thus, it is
crucial to understand the potential role of EDC exposure
as a risk factor for ED. However, we first need a compre-
hensive understanding of the physiological and develop-
mental mechanisms which govern erectile function, with
a focus on the role of endocrine signalling in this process,
before speculating on how EDCs may impact these pro-
cesses to cause ED.
Physiology of Erectile Function
Neural Stimulation
Penile erection is an involuntary response elicited by a
variety of stimuli and can arise via psychogenic and re-
flexogenic mechanisms. Psychogenic stimulus occurs at
supraspinal centres via the senses, such as visual stimula-
tion and smell, and imaginary factors, such as recall and
sexual fantasies [de Groat, 2017]. These central stimuli
send signals to the sacral parasympathetic or thoroco-
lumbar sympathetic spinal cord nuclei, which in turn
transmit to the pelvic plexus [Reeves et al., 2016; de Groat,
2017]. These signals then travel through the cavernous
nerve, a branch of the pelvic plexus, which innervates the
erectile tissue of the penis [Colombel et al., 1999].
Reflexogenic stimulus involves stimulation of the dor-
sal nerve (Fig.1), a sensory somatic nerve fibre in the pe-
nis, which relays messages to the spinal erection centres
via the pudendal nerve [de Groat, 2017]. In turn, efferent
nerves from the spine innervate the cavernous nerve as
described for the psychogenic response above. Individu-
als with spinal cord injury above the sacral pathways
maintain erectile responses, demonstrating the signifi-
cance of the reflexogenic response in erectile function
[Courtois et al., 1993]. Taken together, psychogenic and
reflexogenic stimulation induce erection (tumescence)
via stimulation of the cavernous nerve, which is com-
posed of both parasympathetic and sympathetic nerve fi-
bres [Yilmaz et al., 2006].
Parasympathetic stimulation of the cavernous nerve
leads to increased blood flow within the penis, in turn
Deep
dorsal vein Dorsal nerve
Tunica
albuginea
Corpus
cavernosum
Sinusoidal
space
Helicine artery
Cavernous
artery
Circumflex
vein
Subtunical
plexus
Emissary vein
Urethra Corpus spongiosum
Fig. 1. Transverse section of an adult human penis [Yafi et al.,
2016]. The corpus cavernosum (paired) and corpus spongiosum
constitute the 3 erectile tissues of the penis. The tunica albuginea
surrounds the corpora cavernosa. Blood flows into the corpus cav-
ernosum via the cavernous artery, which branches into helicine
arteries that supply the sinusoidal spaces. Blood drains from the
sinusoidal spaces into the subtunical plexus, which forms the em-
issary vein that passes through the tunica albuginea. Emissary
veins drain directly into the deep dorsal artery or into the circum-
flex veins which also drain into the deep dorsal artery. The dorsal
nerve is a sensory somatic nerve fibre responsible for reflexogenic
erections.
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Sex Dev 2021;15:187–212
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driving tumescence [Andersson and Wagner, 1995].
Stimulation of the sympathetic nerves reduces blood flow
to the penis, leading to the flaccid state (detumescence)
[Andersson and Wagner, 1995]. Somatic nerves also have
a role in erectile function via contraction of the bulbocav-
ernosus and ishiocavernosus muscles (described below).
Androgen signalling has been implicated in the regula-
tion of nerve structure required for erectile function. For
example, castration in rats leads to a reduction in the
number of NOS-containing nerve fibres of the cavernosal
and dorsal nerves [Baba et al., 2000]; the dorsal nerve is
not purely a sensory somatic nerve but is also composed
of autonomic NOS-containing nerve bundles [Burnett et
al., 1993; Carrier et al., 1995]. This is consistent with the
findings that rat castration leads to an altered structure of
the dorsal nerve [Armagan et al., 2008] and a reduced
density of NANC nerve fibres innervating the erectile tis-
sue [Zvara et al., 1995; Schirar et al., 1997]. These studies
show that androgen signalling maintains the neural cir-
cuitry within the penis which is critical for erectile activ-
ity (Fig.2).
Estrogen is also a known neuroprotective agent, which
is demonstrated by a variety of mechanisms in several
animal and clinical studies [Brann et al., 2007]. For ex-
ample, ERα protects rat neuronal cells in vitro via increas-
ing Bcl-XL mRNA (an anti-apoptotic transcript from
Bcl-X) and downregulating BAD (considered a pro-apop-
totic gene) [Gollapudi and Oblinger, 1999]. In addition,
estrogen inhibits amyloid-beta-induced apoptosis and
modulates apoptotic mechanisms such as maintaining
expression of Bcl2 (an anti-apoptotic gene) in rat hippo-
campal cells in vitro [Nilsen et al., 2006]. Future studies
need to elucidate whether estrogen also exerts neuropro-
tection within the erectile tissue, although the expression
of ERs in the dorsal nerve of the rat glans penis suggests
this may occur [Jesmin et al., 2002].
Anatomy, Vasculature, and Hemodynamics of
Erection
The erectile tissue within the penis comprises 3 cylin-
drical structures: the paired corpus cavernosa which are
dorsal to the urethra and the smaller corpus spongiosum
NANC nerve NOS
Endothelial cell
Androgens
K+ channel
voltage-gated
Ca2+channel
PDE5
Smooth
muscle cell
NOS
Fig. 2. Androgen regulation of erectile tis-
sue and molecular signalling involved in
erectile physiology. Androgen signalling
maintains non-adrenergic, non-choliner-
gic (NANC) nerve fibre and smooth mus-
cle levels in the erectile tissue. Androgens
also activate K+ channels in smooth mus-
cle, and androgen levels correlate with volt-
age-gated Ca2+ channel expression in the
smooth muscle of the erectile tissue. An-
drogens positively regulate phosphodies-
terase 5 (PDE5) in the smooth muscle and
nitric oxide synthase (NOS) enzymes,
which are localised NANC nerves and en-
dothelial cells.
Impacts of EDCs on Erectile Dysfunction
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Sex Dev 2021;15:187–212
DOI: 10.1159/000516600
which encloses the urethra and forms the glans distally
[Kuno et al., 2001]. The penile artery supplies the penis
with blood and branches into the dorsal, bulbourethral,
and the cavernous arteries [Keegan and Penson, 2013]. The
cavernous arteries lie within the corpus cavernosa and
branch into the helicine arteries, which supply the sinusoi-
dal spaces [Smith and Axilrod, 2007; Keegan and Penson,
2013]. Sinusoidal spaces are blood-filled compartments
within the corpus cavernosum which are drained by ve-
nules that coalesce into subtunical plexi which lie beneath
the tunica albuginea [Keegan and Penson, 2013]. The tu-
nica albuginea is a fibroelastic collagen-based structure
which surrounds the corpora cavernosa [Keegan and Pen-
son, 2013]; the collagen and fibroelastic fibres are arranged
in an inner, circular layer and an outer, longitudinal layer
[Goldstein and Padma-Nathan, 1990]. These fibroelastic
properties allow for an increase in girth and elongation of
the penis during tumescence, while also providing enough
resilience for shrinkage to the flaccid state [Bitsch et al.,
1990; Hsu et al., 1994; Iacono et al., 1995].
The subtunical plexi branch into emissary veins which
penetrate the tunica albuginea [Keegan and Penson,
2013]. Superficial to the tunica albuginea, these veins
drain into the deep dorsal vein or circumflex veins from
the corpus spongiosum; the circumflex veins also ulti-
mately drain into the deep dorsal vein (Fig.1) [Quartey,
2006; Hsu et al., 2013].
Upon sexual stimulation, parasympathetic neural sig-
nals cause the smooth muscle surrounding the cavernous
and helicine arteries to relax, leading to dilation of these
blood vessels and thus increased blood flow into the erec-
tile tissue [Kuno et al., 2001]. In addition, trabecular
smooth muscle within the corpus cavernosum relaxes so
that the sinusoidal spaces can expand following their en-
gorgement of blood via the dilated arteries [Kuno et al.,
2001]. The expanding sinusoids then compress the sub-
tunical plexi against the unyielding tunica albuginea, oc-
cluding venous outflow of the penis [Keegan and Penson,
2013]. In addition, the pressure of the expanding sinu-
soids causes the tunica albuginea to stretch and compress
the emissary veins, further restricting venous outflow
[Panchatsharam et al., 2020]. Also, subtunical venules
possess minimal geometric slack in the flaccid state (un-
like the arteries and nerves), so when they elongate during
tumescence, they subsequently narrow which further re-
stricts outflow from the corpus cavernosum [Udelson et
al., 2001]. This overall process is known as veno-occlu-
sion, whereby blood inflow increases and blood outflow
decreases, which in turn drastically increases the intra-
cavernous pressure and results in tumescence.
Complete veno-occlusion occurs when the engorged
corpora cavernosa are compressed at their base by con-
traction of the ishiocavernosal muscles via somatic nerve
stimulation [Edey et al., 2011]. Similarly, the bulbospon-
giosus muscle which surrounds the corpus cavernosum
and spongiosum contracts to force additional blood into
the penis during erection and compress the urethra to ex-
pel semen [Panchatsharam et al., 2020]. The corpus spon-
giosum also contains sinusoidal spaces which expand
during erection and constrict the urethra to cause forceful
ejaculation [Clement and Giuliano, 2015; Panchatsharam
et al., 2020].
Upon sympathetic stimulation, the penile smooth
muscle reverts to the contracted state, constricting the ar-
terioles and sinusoidal spaces which in turn decompress-
es the penile veins [Andersson et al., 2000]. As a result,
venous outflow increases which causes a reduction in in-
tracavernous pressure, inducing detumescence.
Androgen signalling positively regulates smooth mus-
cle content in the penis. Castration of rats, mice, rabbits,
and dogs significantly reduces trabecular smooth muscle
content accompanied by an increase in connective tissue
[Takahashi et al., 1991; Shabsigh, 1997; Traish et al., 1999;
Palese et al., 2003; Shen et al., 2003]. Furthermore, andro-
gens stimulate the differentiation of mouse pluripotent
mesenchymal cells into smooth muscle cells in vitro
[Singh et al., 2003]. The smooth muscle content within
the erectile tissue is correlated with the degree to which
the corpus cavernosum can expand [Nehra et al., 1998].
Thus, the loss of smooth muscle induced by androgen
deprivation is likely to disrupt erectile function. Indeed,
several animal studies have shown that a loss of androgen
signalling leads to attenuated erectile responses in vivo
[Müller et al., 1988; Takahashi et al., 1991; Heaton and
Varrin, 1994; Mills et al., 1994; Bivalacqua et al., 1998;
Traish et al., 1999, 2003; Palese et al., 2003; Suzuki et al.,
2007; Traish, 2009]. This is consistent with the reduction
of penile smooth muscle content in patients with ED
[Mersdorf et al., 1991; Claro et al., 2005] and those under-
going androgen deprivation [Tomada et al., 2013]. Inter-
estingly, mice exposed to excess androgen levels also ex-
hibit smooth muscle loss in the corpus cavernosa in vivo
[Hiremath et al., 2020]. Therefore, a balance of androgen
signalling maintains smooth muscle content (Fig. 2),
which in turn promotes erectile function.
Androgen signalling also maintains the structural in-
tegrity of the tunica albuginea; castrated rats have re-
duced density of elastic fibres in the tunica albuginea
which are replaced by collagen [Shen et al., 2003]. A re-
duction of elastic fibres may reduce the tunica albuginea’s
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Sex Dev 2021;15:187–212
192
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ability to expand, in turn disrupting veno-occlusion and
causing ED [Akkus et al., 1997]. Indeed, rats with surgical
injury to the tunica albuginea exhibit impaired erectile
function following electric stimulation of the cavernous
nerve [Bivalacqua et al., 2000]. Taken together, andro-
gens also promote erectile function by maintaining the
fibroelastic properties of the tunica albuginea.
Estrogen signalling within the vasculature of the erec-
tile tissue may maintain the structural integrity of the en-
dothelium, a key signalling centre for the regulation of
vasodilation/vasorelaxation. Indeed, ERβ expression in
the male rat aorta is increased in the endothelium and
smooth muscle cells following vascular injury [Lindner et
al., 1998]. In addition, estrogen signalling inhibits TNFα-
and oxidized low-density lipoprotein (oxLDL)-induced
apoptosis of human endothelial cells in vitro [Spyrido-
poulos et al., 1997; Florian and Magder, 2008]. Further-
more, estrogen-mediated activation of Notch1 protects
human umbilical vein endothelial cells from TNFα-
induced apoptosis in vitro [Fortini et al., 2017]. Interest-
ingly, siRNA-knockdown of ERβ, although not ERα,
eliminated the anti-apoptotic effect of estrogen [Fortini
et al., 2017].
Estrogen also increases the expression of Bcl2 and
Bcl-XL in human endothelial cells in vitro, potentially
generating a protective effect on this tissue [Florian and
Magder, 2008]. Thus, estrogen signalling has a role in
maintaining the structural integrity of the endothelium,
although this has not yet been demonstrated in the penile
endothelium. However, the expression of ERs within the
vasculature of the rat penis raises this possibility [Jesmin
et al., 2002].
Calcium-Mediated Penile Smooth Muscle
Contraction/Relaxation and RhoA/Rho Kinase-
Mediated Calcium Sensitisation
Smooth muscle contraction and relaxation is mediated
by 2 critical proteins: myosin light chain kinase (MLCK)
and myosin light chain phosphatase (MLCP). MLCK
phosphorylates myosin light chains (MLCs), causing
smooth muscle contraction [Kamm and Stull, 1985; An-
dersson, 2001]. Conversely, MLCP dephosphorylates
MLCs, driving smooth muscle relaxation [Jin and Bur-
nett, 2006]. In smooth muscle cells, cytosolic calcium ion
(Ca2+) concentrations regulate MLCK and MLCP activity
which facilitates contraction and relaxation, respectively.
Detumescence initiates with the increase in cytosolic Ca2+
concentrations of smooth muscle cells; Ca2+ then binds to
calmodulin to form the Cam-Ca2+ complex which subse-
quently binds to and activates MLCK, leading to smooth
muscle contraction [Andersson, 2001]. However, the rise
in Ca2+ concentration is short-lived and quickly drops to
baseline levels. Therefore, this process alone does not sus-
tain chronic smooth muscle contraction required for the
flaccid state [Mills et al., 2003; Hill-Eubanks et al., 2011].
To achieve this, the protein RhoA activates Rho-ki-
nase, which in turn deactivates MLCP by phosphoryla-
tion. Since MLCP is deactivated and cannot dephosphor-
ylate MLC and thus drive smooth muscle relaxation, the
MLCs can stay phosphorylated at basal Ca2+, increasing
Ca2+ sensitivity of smooth muscle cells [Mills et al., 2003].
Ca2+ sensitivity refers to the dependence of MLC phos-
phorylation on Ca2+ concentrations; sensitivity is high
when small increases in Ca2+ drive a greater degree of
MLC phosphorylation (as in the flaccid state). In contrast,
low sensitivity occurs when larger increases in Ca2+ con-
centration are needed for a lesser degree of MLC phos-
phorylation, which is when MLCP actively dephosphory-
lates MLC [Rembold, 1992].
Inhibition of RhoA/Rho kinase-mediated calcium
sensitization induces erectile activity in the rat, demon-
strating the importance of this pathway in maintaining
the flaccid state [Chitaley et al., 2001; Lasker et al., 2013].
Interestingly, RhoA expression is 17-fold higher in the
rabbit corpus cavernosum compared to the ileum smooth
muscle, which is consistent with the chronic state of
smooth muscle contraction in the corpus cavernosum
compared to other parts of the vascular system [Wang et
al., 2002].
Conversely, during tumescence, Ca2+ concentration in
the smooth muscle cell drops so that MLCK cannot bind
Cam-Ca2+ and induce contraction [Andersson, 2001].
However, reducing Ca2+ concentration is not sufficient to
drive erection because the contractile machinery is sensi-
tised to lower calcium concentrations through RhoA/
Rho-kinase inactivation of MLCP. Thus, inhibition of the
RhoA/Rho kinase pathway must also occur so that MLCP
can activate and dephosphorylate MLC, thereby decreas-
ing Ca2+ sensitivity and driving smooth muscle relaxation
[Mills et al., 2003]. In summary, detumescence and tu-
mescence depend on a simple switch mechanism on
whether MLC is phosphorylated (Fig.3). However, the
signalling pathways that regulate this switch by altering
Ca2+ concentration and Ca2+ sensitivity in the smooth
muscle cells of the erectile tissue are extremely complex.
Nitric Oxide (NO)-cGMP Mediated Tumescence
Nitric oxide (NO) is a non-noradrenergic, non-cho-
linergic (NANC) neurotransmitter and is essential for tu-
mescence, as evidenced by several animal and human
Impacts of EDCs on Erectile Dysfunction
193
Sex Dev 2021;15:187–212
DOI: 10.1159/000516600
studies [Saenz de Tejada, 2002]. Upon parasympathetic
stimulation, NO is released within the penis and activates
soluble guanylyl cyclase which enhances production of
cyclic guanosine monophosphate (cGMP). In turn, cGMP
activates protein kinase G (PKG) which reduces Ca2+
concentration through several mechanisms [Ghalayini,
2004; Krassioukov and Elliott, 2017]. This includes phos-
phorylation of K+ channels, which leads to an efflux of K+
and subsequent hyperpolarization of smooth muscle cells
within the penis [Archer, 2002]. Hyperpolarization closes
voltage-dependent Ca2+ channels, thereby decreasing the
influx of Ca2+ into smooth muscle cells [Andersson and
Wagner, 1995]. In addition, PKG activates cation-ATPase
pumps in the plasma membrane of smooth muscle cells
and the sarcoplasmic reticulum, leading to Ca2+ efflux out
of the cell and sequestration of Ca2+ in the sarcoplasmic
reticulum, respectively (Fig.4) [Lucas et al., 2000]. Acti-
vated PKG can also inhibit the inositol triphosphate 3
(IP3) receptor, which blocks the influx of Ca2+ into the
cytoplasm from the sarcoplasmic reticulum [Lucas et al.,
2000].
Hormonal signalling modulates cellular ion flux; tes-
tosterone treatment of human corpus cavernosum
smooth muscle cells in vitro activates K+ channels [Han
et al., 2008]. Also, castration of rats leads to reduced levels
of voltage-gated Ca2+ channels in the corpus cavernosum
smooth muscle, thus androgen signalling positively cor-
relates with voltage-gated Ca2+ channel expression
(Fig.2) [Luo et al., 2009]. In addition, rapid estrogen sig-
nalling via membrane-bound ERs (discussed below) is
known to modulate intracellular Ca2+ [Zhang et al., 2009;
Rainville et al., 2015; Puglisi et al., 2019]. Therefore, an-
drogens and estrogens may have a role in regulating the
ion flux of smooth muscle cells which is a critical compo-
nent of tumescence. Indeed, androgen treatment was re-
ported to induce rapid relaxation of human cavernosal
arteries and corpus cavernosum in vitro, although this
was not associated with increased levels of cGMP [Wald-
kirch et al., 2008], suggesting androgens can regulate ion
channels via alternate mechanisms.
In addition to lowering cytosolic Ca2+ concentrations,
the NO/cGMP/PKG pathway is thought to inhibit the
RhoA/Rho-kinase pathway, allowing for the activation of
Smooth
muscle
relaxation
Smooth
muscle
contraction
Tumescence
Detumescence
inactive
inactive
active
active
active
inactive
RhoA
RhoA
Rho
kinase
MLCP
MLC
MLCK Cam-Ca
MLCK
MLC
MLCP
P
P
Fig. 3. MLCK and MLCP mediate smooth
muscle contraction and relaxation, respec-
tively [Mas, 2010]. Ca2+ ions bind to
calmodulin to form the Ca2+-calmodulin
complex (Cam-Ca) which then binds to
and activates MLCK. Active MLCK phos-
phorylates MLC, facilitating smooth mus-
cle contraction. Conversely, active MLCP
dephosphorylates MLC, causing smooth
muscle relaxation and tumescence. Active
RhoA activates Rho kinase which deacti-
vates MLCP by phosphorylation. Inactive
RhoA allows for the activation of MLCP.
Green refers to pathways driving tumes-
cence, red refers to that of detumescence.
MLCK, myosin light chain kinase; MLCP,
myosin light chain phosphatase; MLC, my-
osin light chain; Cam-Ca, Ca2+-calmodulin
complex; P, phosphate group.
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MLCP which is essential for tumescence (Fig.4) [Mills et
al., 2003]. This is consistent with rodent studies which
disrupt the NO/cGMP/cGMP pathway: in vitro organ
bath experiments show that the corpus cavernosum of
PKG-null mice have diminished smooth muscle relax-
ation [Hedlund et al., 2000]. Also, inhibition of guanylyl
cyclase in the corpus cavernosum of rats in vivo signifi-
cantly reduces the erectile response after electrostimula-
tion of the cavernous nerve [Martinez-Piñeiro et al.,
1993]. The disrupted erectile responses observed in these
studies are likely due to the RhoA/Rho-kinase pathway
remaining active. Furthermore, PKG inhibits Ca2+ sensi-
tization induced by RhoA in rat aortic smooth muscle
cells in vitro by phosphorylation of RhoA [Sauzeau et al.,
2000]. However, a direct effect of PKG on RhoA specifi-
cally in penile smooth muscle has not yet been proven.
NO/cGMP signalling is also considered the primary path-
way for increasing blood flow into the clitoris and vagina
during sexual arousal [Giuliano et al., 2002]. Thus, sexual
function in the female and male genitalia arise from sim-
ilar molecular pathways.
The phosphodiesterase (PDE) protein family inhibits
tumescence by breaking down secondary messenger mol-
ecules such as cGMP and cAMP (discussed below) (Fig.4)
[Turko et al., 1999]. Interestingly, PDE5 (which breaks
down cGMP) mRNA is present in the human corpus cav-
ernosum at levels 10- to 100-fold higher compared to oth-
er non-reproductive tissues in males [Morelli et al., 2004].
Alongside higher levels of RhoA in the corpus caverno-
sum, this likely serves to maintain the penis in a chroni-
cally contracted state to maintain flaccidity.
Androgen signalling is thought to upregulate PDE5 ex-
pression; castrated rabbits and rats display reduced PDE5
expression and activity, which is restored by testosterone
replacement [Morelli et al., 2004; Zhang et al., 2005; Ar-
magan et al., 2006]. Also, transsexual individuals in a hy-
pogonadal state also exhibit decreased PDE5 expression
and activity in the corpus cavernosum [Morelli et al.,
2004]. In addition, treatment with a PDE5 inhibitor alone
has little effect on the erectile function of castrated ani-
mals, demonstrating that PDE5 expression relies on an-
drogen signalling [Traish et al., 2003; Zhang et al., 2005].
NO
Diffusion
sGC
SR
cGMP
PDEs
Ca2+
Ca2+
Ca2+
Ca
2+
Ca2+
Cation ATP-ase
pump Ca2+
HP K+ channel
K+
K+
Ca2+
Ca2+
Ca2+ channel
PKG
RhoA/Rho-kinase
Smooth muscle cell
Fig. 4. NO-cGMP mediated smooth mus-
cle relaxation. Extracellular nitric oxide
(NO) diffuses through the smooth muscle
cell membrane and activates soluble gua-
nylyl cyclase (sGC), producing cGMP as a
result. This activates protein kinase G
(PKG) which then activates K+ channels
causing an efflux of K+ from the cell. This
results in hyperpolarization (HP) which
blocks Ca2+ channels so Ca2+ influx is re-
duced. In addition, PKG also activates cat-
ion ATPase pumps in the cell membrane
and sarcoplasmic reticulum (SR), driving
an efflux of Ca2+ out of the cell and seques-
tration of Ca2+ in the SR, respectively. PKG
also suppresses the RhoA/Rho-kinase
pathway, thereby decreasing Ca2+ sensitiv-
ity. NO-mediated reduction in cytosolic
Ca2+ and increased Ca2+ sensitivity drives
relaxation of the smooth muscle cell. The
phosphodiesterase proteins (PDEs) break
down cGMP.
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However, androgens have no effect on PDE5 expres-
sion in cavernous smooth muscle cells in vitro, suggesting
an indirect effect of androgens on PDE5 expression in
vivo [Yang et al., 2009]. This is supported by the fact that
no androgen response element has been found in the rat
Pde5a gene [Lin et al., 2013] and that genome-wide
searches for genes regulated by androgens in human cells
did not yield PDE5A as a candidate [Bolton et al., 2007;
Massie et al., 2007]. Rather than directly upregulating
PDE5, androgens may provide the cellular context for
PDE5 expression in the smooth muscle as these hor-
mones are critical for the development and maintenance
of vasculature within the erectile tissue (Fig.2). Indeed,
castration of rats leads to the simultaneous reduction of
cavernous smooth muscle and PDE5 expression [Liu et
al., 2005; Yang et al., 2009].
NO Production by Activation of Nitric Oxide Synthase
Isoforms
NO in the penis is derived from 2 main sources: NANC
parasympathetic nerves and the endothelium lining
blood vessels and sinusoids [Cartledge et al., 2001]. This
is evident by the spatial expression of the nitric oxide syn-
thase (NOS) enzymes, of which there are several isoforms
that differ in tissue distribution. The neuronal NOS
(nNOS) isoform is localised within penile nerve cells in
rodents and humans [Gonzalez-Cadavid et al., 2000;
Dashwood et al., 2011], and the endothelial NOS (eNOS)
isoform is expressed in the endothelium of the mouse and
human erectile tissues (Fig.5, 6) [Burnett et al., 1996; Sef-
tel et al., 1997]. Although the NOS enzymes are referred
to as isoforms, they are encoded by separate genes and not
splice variants from a single gene [Huang et al., 1993].
Upon sexual stimulation of the parasympathetic sys-
tem, NANC nerves within the penis depolarize via an in-
flux of Ca2+ which then forms the Cam-Ca2+ complex,
activating nNOS [Bredt and Snyder, 1990]. As a result,
nNOS produces NO which relaxes smooth muscles,
thereby dilating penile blood vessels and initiating the
erectile response. Despite this, nerve cell depolarization
via Ca2+ influx is transitory and nNOS quickly deacti-
vates, thus relaxing smooth muscles only briefly [Hurt et
al., 2012]. However, this initial increase in blood flow and
shear stress on the endothelium activates phosphoinosit-
ide 3-kinase (PI3K) which stimulates protein kinase B
(Akt), in turn activating eNOS by phosphorylation
(Fig.6) [Hurt et al., 2002; Musicki et al., 2005; Wen et al.,
2011]. Phosphorylation activates NOS considerably lon-
ger than by depolarization, and thus phosphorylated
eNOS can continually produce NO to sustain smooth
muscle relaxation (Fig.6) [Hurt et al., 2012].
NO
transcription
phosphorylation
eNOSPI3K/Akt
mER mER
Rapid, non-genomic signalling
P
ERE NOS3
Nucleus
Endothelial cell
ER ER
Fig. 5. Estrogen-mediated positive regula-
tion of eNOS expression/activation. In the
endothelial cell, when the estrogen recep-
tor (ER) binds to the estrogen ligand (en-
dogenous or exogenous estrogen or estro-
gen-mimicking EDCs; green circle), it di-
merises and translocates to the nucleus
where it binds to an estrogen-response ele-
ment (ERE) in the NOS3 promoter. This
induces transcription of NOS3 which leads
to production of endothelial nitric oxide
synthase (eNOS). In addition, the associa-
tion of membrane-bound estrogen recep-
tors (mERs) with estrogen initiates rapid,
non-genomic signalling. This involves ac-
tivation of the phosphoinositide 3-kinase/
protein kinase B (PI3K/Akt) pathway,
which in turn activates eNOS by phosphor-
ylation so that it produces NO. NO is the
same as shown in Figure 4.
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In addition, sexual stimulation increases production
of cyclic adenosine monophosphate (cAMP) (discussed
further below), which activates protein kinase A (PKA).
In turn, PKA phosphorylates nNOS so it also continually
produces NO (Fig.6) [Hurt et al., 2012]. These findings
demonstrate that while nNOS initiates NO-mediated
erection upon parasympathetic stimulation, both nNOS
and eNOS sustain erection via their phosphorylated state
[Hurt et al., 2012]. Administration of mice with the non-
specific NOS inhibitor (i.e., inhibits all NOS isoforms)
L-nitroarginine methyl ester (L-NAME) abolishes or sig-
nificantly attenuates erection, revealing the critical nature
of the NO-cGMP pathway for tumescence [Burnett et al.,
1996; Mizusawa et al., 2001; Cashen et al., 2002].
Androgens positively regulate the NOS enzymes; nu-
merous animal studies have demonstrated that androgen
NANC nerve
cAMP
cAMP cGMP
PKA
PKA
PGI2IP
PGE2EP
PKG
Ca2+
VIP
VIP-R
nNOS
nNOS
sAC sGC
SMC relaxation
Shear stress Estrogen
Endothelial cell
Smooth muscle cell
RhoA/Rho-kinase
NO
mAChR
Cholinergic nerve
Acetylcholine
PI3K/Akt eNOS
eNOS
P
P
Cam-Ca
Cam-Ca
Fig. 6. NO sources and other factors which drive smooth muscle
relaxation. The NO-cGMP pathway reduces cytosolic Ca2+ and in-
hibits the RhoA/Rho-kinase pathway as depicted in Figure 4.
When the NANC nerves are stimulated (lightning bolt), Ca2+
binds to calmodulin to form the calmodulin-Ca2+ (Cam-Ca2+)
complex. This subsequently binds to and activates neuronal NOS
(nNOS), driving NO production. Also, stimulation of NANC
nerves drives production of cAMP in these cells. This activates
protein kinase A (PKA) which in turn activates nNOS by phos-
phorylation (P). The initial production of NO by the NANC nerves
leads to smooth muscle cell (SMC) relaxation, in turn leading to
shear stress on the endothelial cells. This triggers the PI3K/Akt
pathway, which then activates eNOS by phosphorylation. Acetyl-
choline released from cholinergic nerves binds to the muscarinic
acetylcholine receptor (mAChR), which increases Ca2+ in the en-
dothelial cell. This leads to formation of Cam-Ca2+, which binds to
and activates eNOS. Endogenous estrogen signalling also activates
eNOS by stimulating the PI3K/Akt pathway and upregulates ex-
pression of eNOS (see Fig. 5). In addition to the NO-cGMP path-
way, vasoactive intestinal peptide (VIP) in the NANC nerves may
bind to its receptor (VIP-R) on the smooth muscle cell to stimulate
soluble adenylyl cyclase (sAC). This leads to production of cAMP
in the smooth muscle cell, activating PKA to reduce cytosolic Ca2+
concentration. cAMP may also mediate smooth muscle cell relax-
ation via activation of PKG. The prostanoids prostaglandin E2
(PGE2) and prostacyclin (PGI2) can also drive cAMP production
via association with the EP and IP receptors on the smooth muscle
cell, respectively. NANC is the same as shown in Figure 2sGC, PKG
and NO are the same as shown in Figure 4. PI3K/Akt and eNOS are
the same as shown in Figure 5.
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signalling is associated with increased NOS activity/ex-
pression in the erectile tissue and nerves innervating the
penis [Lugg et al., 1995; Penson et al., 1996; Reilly et al.,
1997; Schirar et al., 1997; Marin et al., 1999; Park et al.,
1999; Seo et al., 1999; Armagan et al., 2006]. However,
androgen deprivation was found to increase eNOS mRNA
and have no effect on nNOS mRNA levels in rat erectile
tissue [Shen et al., 2000a]. Furthermore, androgen with-
drawal was reported to increase eNOS levels in the hu-
man corpus cavernosum [Tomada et al., 2013]. The con-
flicting data suggests that a precisely controlled balance
of androgen signalling is required for maintaining nor-
mal levels of NOS expression and activity (Fig.2). Also,
cGMP can stimulate expression of the human PDE5A
gene [Lin et al., 2001, 2006]. Thus, androgens may indi-
rectly upregulate PDE5 via positive regulation of the NOS
enzymes which in turn drives cGMP production.
Estrogen signalling may also promote smooth muscle
relaxation by stimulating NOS expression and activity in
the erectile tissue. Indeed, in humans and animals, ERs
upregulate eNOS via an estrogen-response element in the
eNOS promoter (Fig.5) [MacRitchie et al., 1997; Yang et
al., 2000; McNeill Anne et al., 2002; Min, 2007]. Interest-
ingly, in human endothelial cell cultures, activated mem-
brane-bound ERs rapidly stimulate the PI3K/Akt path-
way via a non-genomic mechanism, which in turn acti-
vates eNOS by phosphorylation (Fig.5, 6) [Haynes et al.,
2000, 2003]. This is consistent with the significantly high-
er basal release of endothelium-derived NO in the male
mouse aorta compared to that of the male estrogen recep-
tor knockout (ERKO) mouse, suggesting that ER levels
are related to basal NO production in endothelium
[Rubanyi et al., 1997].
Furthermore, estrogen-deficient post-menopausal
women have reduced levels of ERα, eNOS, and phosphor-
ylated eNOS in endothelial cells of the antecubital vein
compared to premenopausal women [Gavin et al., 2009].
Postmenopausal women also display reduced endotheli-
al-dependent dilation of the brachial artery, suggesting
that a loss of estrogen leads to a reduction in NO bioavail-
ability [Gavin et al., 2009]. Taken together, estrogen sig-
nalling in the endothelium can upregulate and activate
eNOS via genomic and non-genomic mechanisms, re-
spectively.
Estrogen may also promote tumescence by positively
regulating nNOS activity/expression. The treatment of
human nNOS-expressing neuroblastoma cell lines with
estrogen was reported to cause a rapid increase in NO
production via activation of eNOS and nNOS in vitro
[Wen et al., 2004; Xia and Krukoff, 2004]. Also, the injec-
tion of estrogen into ovariectomized rats increases nNOS
mRNA in the hypothalamus and hippocampus [Ceccatel-
li et al., 1996; Grohe et al., 2004]. The stimulation of neu-
ronal NO production by estrogen may also explain the
neuroprotective properties of estrogen as NO is a known
neuroprotective agent [Chiueh, 1999; Wen et al., 2004].
Thus, estrogen signalling may positively regulate nNOS
in nerves innervating the erectile tissue. However, to the
best of our knowledge this remains to be proven.
Disruptions of NO-cGMP Pathway and Compensatory
Mechanisms
The NO-cGMP pathway has a profound impact on tu-
mescence, and compensatory mechanisms exist if it is
disrupted. For example, mice with a mutation for nNOS
display normal mating behaviour and erectile function;
eNOS is upregulated in these mice which may compen-
sate for disrupted NO production [Burnett et al., 1996].
Although eNOS is defined by its localisation to the endo-
thelium, it may also localize to neural cells within the pe-
nis, potentially substituting the function of nNOS [Cash-
en et al., 2002]. This remains to be proven, although eNOS
is localised in the dendritic spines of primary culture cor-
tical and hippocampal neurons from rats at embryonic
day 18 [Caviedes et al., 2017].
Mice with mutations for eNOS also display normal
erectile function and retain about 60% of the NOS activ-
ity in the penis compared to that of WT mice [Burnett et
al., 2002]. This shows that other NOS isoforms synthesise
NO in mice lacking eNOS, compensating for erectile
function [Burnett et al., 2002]. In addition, although
nNOS is defined by its neuronal localization, its expres-
sion in endothelial cells within the penis may also com-
pensate for a loss of eNOS [Cashen et al., 2002]. This is
reinforced by the co-expression of nNOS with eNOS in
the human umbilical vein endothelial cells in vitro [Ba-
chetti et al., 2004].
Overall, the activity of NOS isoforms can compensate
for each other if one is mutated, thereby allowing for tu-
mescence despite disruption of the NO-cGMP pathway.
Further compensation may arise by potential overlap of
eNOS and nNOS localisation in the erectile tissue.
Additional Pro-Erectile Signalling Pathways
While parasympathetic signalling mediated by the
NO-cGMP pathway is primarily responsible for tumes-
cence, other signalling pathways modulate erectile func-
tion through stimulation of cGMP and cAMP produc-
tion. These factors may also compensate for deficiencies
in NO-signalling, potentially explaining normal erectile
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function in NOS mutant mice from the studies men-
tioned above.
cAMP/PKA Pathway
The second messenger cAMP is produced by adenylyl
cyclase and activates PKA [Sassone-Corsi, 2012]. In addi-
tion to cGMP signalling, cAMP/PKA signalling is thought
to mediate smooth muscle relaxation in the penis. Indeed,
several studies have identified cAMP signalling in the
corpus cavernosum smooth muscle [Lin et al., 2005]. In
addition, forskolin (adenylyl cyclase activator) adminis-
tration relaxes the human corpus cavernosum in vitro;
the magnitude of relaxation correlates with the level of
cAMP accumulation induced by forskolin in human cor-
poral smooth muscle cells in vitro [Palmer et al., 1994].
The mechanism by which cAMP/PKA signalling relaxes
penile smooth muscle cells likely involves the activation
of K+ channels on the smooth muscle cell membrane, hy-
perpolarizing the smooth muscle cell and thereby de-
creasing cytosolic Ca2+ levels. This is illustrated by the
ablation of PGE1 (a relaxing factor discussed below) in-
duced activation of K+ channels in human corporal
smooth muscle cells in vitro by a PKA inhibitor [Lee et
al., 1999].
In contrast, the treatment of rats with an adenylyl cy-
clase inhibitor does not affect the erectile response in vivo
following electrostimulation of the cavernous nerve
[Martinez-Piñeiro et al., 1993]. There is also little evi-
dence to suggest that the cAMP/PKA pathway reduces
Ca2+ sensitivity to the contractile machinery in penile
smooth muscle through inhibition of the RhoA/Rho-ki-
nase pathway, a critical component for tumescence.
Therefore, it is likely that the NO/cGMP/PKG pathway is
the key driver for tumescence while cAMP/PKA signal-
ling has a relatively minor role by reducing cytosolic Ca2+
concentration (Fig.6).
Importantly, these pathways are not mutually exclu-
sive; crosstalk exists between cAMP and cGMP signal-
ling. This is partially discussed above with cAMP/PKA-
mediated phosphorylation of nNOS. In addition, both
cAMP and cGMP can activate PKG in cavernosal smooth
muscle cell cultures from young (16 weeks) and old (28
months) rats [Lin et al., 2002]. Therefore, while activation
of the cAMP pathway may have minor direct effects on
tumescence, it may also indirectly contribute to it by re-
inforcing the cGMP/PKG-signalling pathway (Fig.6).
Vasoactive Intestinal Peptide
Before NO-cGMP signalling was established as the key
pathway for tumescence, considerable attention was giv-
en to the relaxant effects of vasoactive intestinal peptide
(VIP) in this process. Nerves innervating the erectile tis-
sue in humans and rabbits contain VIP, and thus it may
function as a neurotransmitter in the penis to promote
tumescence [Polak et al., 1981; Willis et al., 1983]. This is
supported by the presence of VIP receptors in smooth
muscle cells of the rat corpus cavernosum in vitro [Gui-
done et al., 2002]. Furthermore, administration of a VIP-
antagonist to the rabbit corpus cavernosum reduces re-
laxation in vitro following electric stimuli [Kim et al.,
1995] and also to the rat penis in vivo following cavernous
nerve stimulation [Suh et al., 1995]. In addition, tumes-
cence is associated with increased VIP concentration in
the cavernous blood in humans, and administration of
VIP drives erection [Ottesen et al., 1984]. The mechanism
of VIP-mediated tumescence most likely involves elevat-
ing cAMP levels in smooth muscle cells of the erectile tis-
sue; VIP dose-dependently induces production of cAMP
in rat corpus cavernosum smooth muscle cells [Guidone
et al., 2002].
However, VIP is not the primary relaxant agent in tu-
mescence: injection of VIP into the rat penis enhances erec-
tion in vivo but does not induce a full erection [Suh et al.,
1995]. Also, electrical field stimulation-induced relaxation
of the human corpus cavernosum in vitro is not diminished
by VIP inactivation [Pickard et al., 1993]. For a rigid erec-
tion sufficient for penetration, it is likely that VIP functions
alongside other pro-erectile pathways. Indeed, VIP and
NOS are colocalised in nerves innervating the erectile tissue
in animals and humans, suggesting that NO and VIP com-
plement each other to facilitate erectile activity (Fig. 6)
[Ehmke et al., 1995; Andersson, 2001]. This is supported by
injection of a combination of VIP and SIN-1 (NO-releasing
compound) into the rabbit corpus cavernosum which aug-
ments the erectile response in vivo compared to those in-
jected with SIN-1 or VIP alone, revealing an additive effect
of the NO and VIP pathways [Sazova et al., 2002]. Overall,
VIP is indisputably a smooth muscle relaxant and most
likely complements NO-cGMP signalling via the cAMP
pathway to promote tumescence (Fig.6).
VIP signalling appears to be independent of androgen
signalling; men with chemical castration display no sig-
nificant change in VIP levels in the corpus cavernosum
compared to non-castrated individuals [Cormio et al.,
2005]. Also, castrated rats display no significant change
of VIP mRNA levels in the corpus cavernosum [Shen et
al., 2000b]. However, the erectile function of castrated
rats display greater responsiveness to VIP, suggesting that
androgens negatively regulate the VIP/cAMP pathway
[Zhang et al., 2011].
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Prostanoids (Involved in Tumescence and
Detumescence)
Prostanoid metabolites are produced via several syn-
thases from arachidonic acid in the endothelium [Minhas
et al., 2000]. The prostanoid metabolites identified in the
human and animal corpus cavernosum are the prosta-
glandins (PGI2, PGF2α, PGE2, PGD2) and thromboxane
A2 (TXA2) [Khan et al., 1999; Andersson, 2011]; these
mediate their functions via the IP, FP, EP, DP, and TP
receptors, respectively (Fig.6, 7) [Andersson, 2011]. The
prostanoids exert dual functions on erectile function as
some contract or relax penile smooth muscle: administra-
tion of a TP receptor agonist and PGF2α to isolated hu-
man corpus cavernosum preparations results in contrac-
tion [Hedlund and Andersson, 1985b]. On the other
hand, treatment with PGE2 and PGE1 leads to relaxation
[Hedlund and Andersson, 1985b].
Prostanoid-induced relaxation is supported by studies
which show that injection of PGE1 leads to relaxation of
the monkey [Bosch et al., 1989] and rat corpus caverno-
RhoA/Rho-kinase
MLCP
DAG
IP3
PKC
CPI-17
Ca2+
Ca2+ channel
Smooth muscle cell
Ca2+
Ca2+
DP
Ca2+
Ca2+
IP3R
TRPC3
Ca2+ P
Endothelial cell
Adrenergic nerve
NA
ET-1
Ang-II
TXA2
PLC
SR
Receptor (α1, ETA, AT1, TP)
Fig. 7. Smooth muscle contraction pathways. Upon stimulation of
adrenergic nerves (lightning bolt), noradrenaline (NA) is released
and binds to the α1-adrenoreceptor (α1). Endothelin-1 (ET-1), an-
giotensin-II (Ang-II), and the prostanoid thromboxane A2 (TXA2)
released from the endothelial cell bind to their receptors ETA, AT1,
and TP, respectively, on the smooth muscle cell. Association of
these ligands with their receptors leads to activation of phospholi-
pase C (PLC), which then produces inositol triphosphate 3 (IP3)
and diacylglycerol (DAG). IP3 associates with the IP3 receptor
(IP3R) on the sarcoplasmic reticulum (SR), which acts as a channel
to release Ca2+ from the SR. The activated IP3Rs couple with mem-
brane-bound transient receptor potential canonical 3 (TRPC3)
channels, leading to an influx of extracellular Ca2+. Increased cy-
tosolic Ca2+ in the smooth muscle cell causes depolarization (DP),
activating Ca2+ channels in the smooth muscle cell membrane
which leads to a further influx of Ca2+. DAG leads to activation of
protein kinase C (PKC), which activates CPI-17 by phosphoryla-
tion. This then inhibits MLCP. Association of NA, ETA, Ang-II,
and TXA2 with their receptors may also drive the RhoA/Rho-ki-
nase pathway to inhibit MLCP. Thus, these signalling factors drive
smooth muscle cell contraction by increasing cytosolic Ca2+ and
increasing Ca2+ sensitivity. MLCP is the same as shown in Figure
3.
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sum in vivo [Chen et al., 1992]. In addition, the EP recep-
tors are known to mediate PGE1- and PGE2-induced re-
laxation of the human corpus cavernosum in vitro [An-
gulo et al., 2002]. In fact, the documented relaxant effects
of PGE1 has led to its use as a treatment for ED and results
in greater satisfaction in sexual performance [Linet and
Neff, 1994; Urciuoli et al., 2004]. Prostanoids may con-
tribute to tumescence by stimulating cAMP production;
Gs-protein coupled EP and IP receptors (for PGE2 and
PGI2) are known to stimulate adenylyl cyclase (Fig. 6)
[Ricciotti and FitzGerald, 2011]. This is supported by
PGE1 administration in combination with an inhibitor of
a cAMP-specific PDE which leads to relaxation and in-
creased cAMP levels in primary culture human cavernosal
smooth muscle cells [Bivalacqua et al., 1999]. Further-
more, in equine penile arteries, treatment of a PKA in-
hibitor decreases the relaxant effects of PGE1, demonstrat-
ing that this prostaglandin relaxes penile blood vessels via
the cAMP/PKA pathway [Ruiz Rubio et al., 2004].
Interestingly, treatment of rats with PGE1 dose-de-
pendently increases NO production and increases n/
eNOS expression in the rat corpus cavernosum in vivo,
revealing that PGE1 may also relax erectile tissue through
the NO-cGMP pathway [Escrig et al., 1999]. This contra-
dicts the finding that inhibition of NOS did not affect
PGE1-mediated relaxation of equine penile arteries in vi-
tro [Ruiz Rubio et al., 2004]. However, the same authors
demonstrated that the combined inhibition of PKA and
PKG reduced PGE1-mediated relaxation, suggesting
PGE1 primarily influences cAMP signalling and poten-
tially the cGMP pathway. It should be noted that to the
best of our knowledge, PGE1 has not been identified as a
naturally occurring prostaglandin in the penis. Thus, the
relaxant effects of PGE1 described above do not necessar-
ily reflect that of the native prostaglandins.
In contrast to relaxant prostanoids, the TP receptor
(for TXA2) can activate phospholipase C (PLC), an en-
zyme which hydrolyses phosphatidylinositol 4,5-biphos-
phate (PIP2) into diacylglycerol (DAG) and IP3, intracel-
lular messengers which increase cytosolic Ca2+ concen-
tration (discussed further below) (Fig.7) [Feletou, 2010].
In addition, the TP receptor can activate RhoGEF, which
in turn activates RhoA [Feletou, 2010]. Thus, TXA2
through its receptor may drive smooth muscle contrac-
tion in the penis by elevating cytosolic Ca2+ and promot-
ing the RhoA/Rho-kinase pathway (Fig.7). Indeed, TP
receptors are identified as contractile factors of human
penile arteries and trabecular smooth muscle in vitro
[Angulo et al., 2002]. Also, treatment of rat cavernous ar-
teries with the TXA2 analogue U46619 led to increased
Ca2+ concentration and contraction in vitro [Grann et al.,
2016]. The authors also found that treatment of the Rho-
kinase inhibitors Y27632 and glycyl-H1152P dose-de-
pendently attenuated U46619-induced contraction, pro-
viding further evidence that TXA2 mediates contraction
via activating the RhoA/Rho-kinase pathway. DP recep-
tors (for PGF2α) can also increase Ca2+ concentration and
inhibit production of cAMP, potentially explaining its
contractile properties in the penis [Ricciotti and FitzGer-
ald, 2011].
Contradictory findings have been reported on the ef-
fects of PGI2 on penile smooth muscle: PGI2 treatment to
isolated human corpus cavernosum was reported to cause
contraction [Hedlund and Andersson, 1985b], while a
separate study found that treatment of iloprost (PGI2 an-
alogue) to isolated rat corpus cavernosum leads to relax-
ation [Bassiouni et al., 2019]. Another study found that
PGI2 relaxed 4 of 6 isolated human corpus cavernosum
samples but had no effect on the remaining 2 [Kirkeby et
al., 1993]. Thus, it is unclear from these results as to
whether PGI2 has a contractile, relaxant, or neutral effect
on smooth muscle cells in the corpus cavernosum.
This is unexpected as PGI2 is an established vasodila-
tor in blood vessels. Lue [2011] suggests that this discrep-
ancy arises from varying distribution of IP receptors (for
PGI2) within the penis. Indeed, it is unlikely the IP recep-
tor is present in trabecular smooth muscle because PGI2
fails to relax trabecular smooth muscle in human corpus
cavernosum in vitro [Angulo et al., 2002]. However, PGI2
is a potent vasodilator in human penile arteries in vitro,
which is confirmed by the presence of IP receptors in this
tissue [Angulo et al., 2002]. Thus, the specific distribution
of prostanoid receptors in the vascular bed of the penis
can coordinate the effects of prostanoids on smooth mus-
cle relaxation.
Taken together, prostanoid signalling relaxes and con-
tracts penile smooth muscle, thus contributing to tumes-
cence and detumescence, respectively (Fig.6, 7).
Interestingly, in addition to the role that prostanoids
have in erectile physiology, the mechanism by which an-
drogens masculinize mouse embryos involves the arachi-
donic acid cascade which leads to prostaglandins [Gupta
and Goldman, 1986]. Thus, androgen-mediated pros-
tanoid signalling may also drive development of the erec-
tile tissue, although more research is required to elucidate
this.
Acetylcholine
In addition to NANC nerves which release NO, the
mammal penis is also innervated by cholinergic nerves
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which release acetylcholine to drive erection (Fig.6) [An-
dersson, 2001]. This is further supported by the high den-
sity of acetylcholinesterase (which breaks down acetyl-
choline) in the monkey corpus cavernosum [Stief et al.,
1989]. Acetylcholine mediates its effects through 2 types
of acetylcholine receptors (AChRs): nicotinic (nAChR)
and muscarinic (mAChR) [Pohanka, 2012].
Four mAChR subtypes (M1–M4) are identified in the hu-
man corpus cavernosum [Traish et al., 1995]. Acetylcho-
line administration to the equine deep dorsal penile vein
induces relaxation in vitro and is mediated by the mA-
ChRs (particularly M3) [Martínez et al., 2003]. Thus, ace-
tylcholine signalling through mAChRs can promote tu-
mescence (Fig.6). This is consistent with the administra-
tion of atropine (mAChR antagonist) to the human
corpus cavernosum which partially blocks relaxation in
response to electrical stimulation in vitro [Saenz de Te-
jada et al., 1988]. However, as atropine only partially im-
paired erectile function, acetylcholine-mAChR signalling
cannot be the predominant pathway which mediates tu-
mescence [Saenz de Tejada et al., 1988].
Acetylcholine may also drive tumescence via the
nAChRs, which are expressed in nerves innervating the
rat corpus cavernosum [Faghir-Ghanesefat et al., 2017].
Furthermore, administration of nicotine (α7-nAChR ag-
onist) increases relaxation of the rat corpus cavernosum
induced by electric-field stimulation in vitro [Faghir-
Ghanesefat et al., 2017]. Thus, acetylcholine signalling
through nAChRs, in addition to mAChRs, may promote
tumescence. This is further supported by the blockade of
mAChRs which only attenuates acetylcholine-induced
erection in monkeys in vivo, while blockade of both
mAChRs and nAChRs abolished it [Stief et al., 1989]. In
contrast, nicotine administration can contract (and relax)
the rabbit corpus cavernosum in vitro via nAChRs [Nguy-
en et al., 2015]. Therefore, acetylcholine signalling may
also have a role in detumescence.
Nevertheless, cholinergic signalling in the penis is pri-
marily recognised as a driver for tumescence by modulat-
ing pro-erectile signalling pathways [Saenz de Tejada et
al., 1988]. For example, the administration of NOS in-
hibitors to the rabbit and rat corpus cavernosum in vitro
abolishes acetylcholine-induced relaxation, suggesting
acetylcholine drives tumescence by modulating the NO-
cGMP pathway [Knispel et al., 1991; Faghir-Ghanesefat
et al., 2017]. This is consistent with administration of ace-
tylcholine to the rabbit corpus cavernosum in vitro which
leads to elevated cGMP levels [Azadzoi et al., 1992].
Acetylcholine-mediated tumescence is endothelium
dependent, illustrated by the failure of acetylcholine to
relax human corpus cavernosum lacking endothelium in
vitro (successful with endothelium) [Saenz de Tejada et
al., 1988]. This is consistent with the reduced relaxation
response to acetylcholine of the rabbit corpus caverno-
sum denuded of endothelium [Azadzoi et al., 1992]. En-
dothelial-dependent acetylcholine signalling is further
supported by the presence of mAchRs in endothelial cells
of the human corpus cavernosum (Fig.6) [Traish et al.,
1990].
The effect of acetylcholine on penile endothelium like-
ly involves stimulation of NO. The administration of car-
bachol (mAchR agonist) augments the erectile response
from electrostimulation in wild-type mice, but in contrast
it has no effect to that of eNOS-deficient mice [Burnett et
al., 2002]. Thus, eNOS mediates the pro-erectile effects of
the cholinergic agent carbichol, which likely reflects en-
dothelium-dependent acetylcholine signalling in the pe-
nis. Acetylcholine is known to increase intracellular Ca2+
concentration in the endothelium via mAchRs so that
Ca2+ binds to calmodulin to form the Cam-Ca2+ complex,
in turn activating eNOS, which is a distinct activation
pathway compared to eNOS phosphorylation following
shear stress described above (Fig.6) [Zhao et al., 2015;
Behringer, 2017]. To the best of our knowledge, this has
yet to be proven in the endothelium within the erectile
tissue, although it is likely.
These studies show that the interaction of acetylcho-
line with its receptors mediates tumescence indirectly by
promoting the NO-cGMP pathway within the endothe-
lium of the erectile tissue (Fig.6).
Noradrenaline-Mediated Detumescence
The sympathetic pathway is responsible for detumes-
cence, and several studies have demonstrated that adren-
ergic nerves of the sympathetic nervous system innervate
the human and rodent erectile tissue [Andersson et al.,
2000]. These nerves release the neurotransmitter nor-
adrenaline (NA) which is recognised as the primary agent
for detumescence (Fig.7). Several studies have demon-
strated that NA contracts strips of corpus cavernosum,
cultured corpus cavernosum cells, and penile artery seg-
ments [Andersson and Wagner, 1995]. This is further
supported by the presence of α1-adrenoreceptors on
smooth muscle cells of the human and rat corpus caver-
nosum [Costa et al., 1993; Véronneau-Longueville et al.,
1998].
Upon release from adrenergic nerve terminals within
the erectile tissue, NA binds to α-adrenoreceptors 1 and
2 [Traish et al., 2000]. These receptors facilitate smooth
muscle contraction and thus detumescence; administra-
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tion of phenylephrine (α1-adrenoreceptor agonist) and
clonidine (α2-adrenoreceptor agonist) contract the hu-
man corpus cavernosum, corpus spongiosum, and cav-
ernous artery in vitro [Hedlund and Andersson, 1985a;
Christ et al., 1990]. In addition, administration of ago-
nists for α-adrenoreceptors 1 and 2 induce contraction of
the rabbit corpus cavernosum in vitro [Gupta et al., 1998].
It is thought that the α1-adrenoreceptor is primarily
responsible for contraction of the penile smooth muscle;
contraction of the human and rabbit corpus cavernosum
in organ chambers by electrical field stimulation is atten-
uated by prazosin, an α1-adrenoreceptor antagonist, but
not by rauwolscine, an α2-adrenoreceptor antagonist
[Saenz de Tejada et al., 1989]. Furthermore, the contrac-
tile response of the α2-adrenoreceptor agonist UK 14,304
was approximately half that of phenylephrine in the rab-
bit corpus cavernosum in vitro [Gupta et al., 1998]. Thus,
the α1-adrenoreceptor is the NA receptor primarily re-
sponsible for smooth muscle contraction in the corpus
cavernosum.
The functional differences between the
α-adrenoreceptors 1 and 2 may arise from their differen-
tial localisation within the erectile tissue. Indeed, both
phenylephrine and clonidine contract the human corpus
spongiosum and cavernous artery in vitro, although
phenylephrine is more potent in corpus spongiosum
whereas clonidine is more potent in the cavernous artery
[Hedlund and Andersson, 1985a]. Thus, it is likely that
α1-adrenoreceptors are the predominant α-adrenergic
receptors in the corpus spongiosum smooth muscle,
whereas α2-adrenoreceptors are predominant in the
smooth muscle surrounding the cavernous artery. It is
also thought that postsynaptic α2-adrenoreceptors in the
penile smooth muscle are positioned more distally from
adrenergic nerve terminals in comparison to α1-
adrenoreceptors, potentially explaining the dominant
contractile effect of α1-adrenoreceptors [Saenz de Tejada
et al., 2000]. However, further studies are needed to con-
firm this hypothesis. Taken together, these studies show
that the α-adrenoreceptors are responsible for NA-medi-
ated detumescence.
The mechanism of noradrenergic-induced smooth
muscle contraction involves the increase of intracellular
Ca2+ concentration (Fig.7). This is demonstrated by the
reduction of NA-induced contraction of the human cor-
pus cavernosum/spongiosum in vitro by either removal
of extracellular Ca2+ or administration of Ca2+ channel
blockers [Fovaeus et al., 1987]. In addition, smooth mus-
cle cells of the rabbit corpus cavernosum exhibit increased
Ca2+ concentration following exposure to NA in vitro
while an α1-adrenoreceptor antagonist inhibits the in-
crease of Ca2+ concentration in these cells [Sato and
Kawatani, 2002].
Adrenergic signalling via α1-adrenoreceptors raises
intracellular Ca2+ levels by activating PLC, which in turn
produces IP3 and DAG from PIP2. IP3 subsequently binds
to the IP3-receptors (IP3Rs) which are membrane-bound
receptors located in the sarcoplasmic reticulum. They act
as Ca2+ channels and upon activation by IP3 release Ca2+
sequestered in the sarcoplasmic reticulum into the cyto-
plasm [Boittin et al., 1999; Bastin and Heximer, 2011; Na-
rayanan et al., 2012]. In addition, IP3R-mediated release
of Ca2+ activates membrane-bound transient receptor
potential canonical 3 (TRPC3) channels which causes an
influx of extracellular Ca2+. This results in depolarization
which subsequently activates voltage-dependent Ca2+
channels, leading to further Ca2+ influx (Fig.7) [Naray-
anan et al., 2012]. It is important to note that these signal-
ling pathways have been identified primarily in vascular
tissue outside the erectile tissue of the penis.
Nevertheless, it is likely that activation of these signal-
ling pathways by NA also drives smooth muscle contrac-
tion in the penis to cause detumescence. Indeed, admin-
istration of NA to the rabbit corpus cavernosum leads to
accumulation of inositol phosphates in vitro, including
IP3, suggesting the α-adrenoreceptors activate PLC [Hol-
mquist et al., 1992]. However, the onset of this reaction is
slow as there is a significant increase in inositol phosphate
levels only after 15 min of NA exposure, contradicting the
rapid contraction of smooth muscle induced by NA [Hol-
mquist et al., 1990, 1992]. This may be due to method-
ological complexities; further work is required to eluci-
date the role of IP3 signalling in detumescence [Hol-
mquist et al., 1992].
DAG, the other product of PLC, activates protein ki-
nase C (PKC) which can also drive smooth muscle con-
traction [Hilgers and Webb, 2005]. PKC phosphorylates
the CPI-17 protein, which in turn inhibits MLCP (drives
smooth muscle relaxation) and thus increases Ca2+ sensi-
tization (Fig.7) [Li et al., 1998; Nunes et al., 2010]. CPI-17
is expressed in the human and rabbit corpus cavernosum,
although to the best of our knowledge, smooth muscle
contraction in the penis mediated by PKC/CPI-17 signal-
ling has yet to be proven [Jiang and Chitaley, 2012]. How-
ever, exposure of the rat corpus cavernosum to phorbol
12-myristate13-acetate (PMA) (PKC activator) potenti-
ates phenylephrine-induced contractions in vitro, while
exposure to chelerythine chloride (PKC inhibitor) inhib-
its it [Husain et al., 2004]. Thus, adrenergic signalling
may drive detumescence via activation of PKC. This is
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also supported by the exposure of the rat corpus caverno-
sum to phenylephrine in vitro which leads to increased
levels of PKC isozymes [Husain et al., 2004].
In contrast, studies on the mouse corpus cavernosum
show that exposure to chelerythrine (PKC inhibitor) had
no significant effects on phenylephrine-induced contrac-
tions in vitro, although it significantly reduced phenyl-
ephrine-induced contractions in the mouse aorta in vitro
[Jin et al., 2008]. This suggests PKC activity does not have
a significant role in smooth muscle contraction within the
penile vascular bed, in contrast to other areas of the vas-
cular system. Taken together, these data show that PKC
may have a role in adrenergic-induced detumescence, al-
though this is not fully resolved, and future studies should
address this knowledge gap.
Moreover, Y-27632 (Rho-kinase inhibitor) reduces
noradrenergic contractions of human and rabbit corpus
cavernosum in vitro in a dose-dependent manner [Rees
et al., 2001]. Also, Y-27632 inhibits the contractile effect
of methoxamine (α1-adrenoreceptor agonist) in the rat
penis in vivo following autonomic stimulation [Mills et
al., 2001a]. Therefore, NA signalling may also activate
Rho-kinase (sensitises smooth muscle cells to Ca2+) to
contract smooth muscle. In summary, NA signalling is a
prominent factor driving detumescence via several path-
ways which increase intracellular Ca2+ concentration and
Ca2+ sensitivity within smooth muscle cells (Fig.7).
Other Signalling Pathways Involved in Detumescence
Endothelin-1
In addition to NA, several other factors promote detu-
mescence (including some prostanoids described above).
Endothelin-1 (ET-1) is considered the most potent con-
tractile agent of smooth muscle within the corpus caver-
nosum and is produced in human penile smooth muscle
cells and endothelial cells in vitro [Saenz de Tejada et al.,
1991; Andersson, 2001; Davenport, 2002; Granchi et al.,
2002]. In addition, the ET-1 receptors (ETA and ETB)
have been identified in the corpus cavernosum of humans
and other mammals [Carneiro et al., 2008]. Research on
isolated cavernosal strips from rats and mice has shown
that the association of ET-1 with the ETA receptor causes
smooth muscle contraction and thus mediates detumes-
cence [Carneiro et al., 2008].
ET-1/ETA signalling mediates smooth muscle con-
traction by increasing cytosolic Ca2+ concentration, con-
firmed in smooth muscle cells of the human corpus cav-
ernosum in vitro [Zhao and Christ, 1995]. This is sup-
ported by ET-1 treatment of the rabbit corpus cavernosum
which leads to accumulation of inositol phosphates in vi-
tro, suggesting that ET-1 also activates PLC in this tissue
[Holmquist et al., 1992]. Also, endothelin-induced con-
tractions of the rabbit and human corpus cavernosum are
reduced in Ca2+-free solution, or after treatment with ni-
modipoine (Ca2+ channel blocker) [Holmquist et al.,
1990]. This demonstrates that ET-1 signalling partly re-
lies on Ca2+ influx to drive smooth muscle contraction.
Furthermore, treatment of the rabbit corpus cavernosum
with H7 (PKC inhibitor) reduces ET-1-mediated contrac-
tion in vitro and abolishes it in Ca2+-free solution [Hol-
mquist et al., 1990]. These results provide evidence that
ET-1 drives smooth muscle contraction in the penis by
increasing intracellular Ca2+ levels and increasing Ca2+
sensitivity via PKC activation (Fig.7).
Also, pre-treatment of the Rho-kinase inhibitor
Y-27632 prior to intracavernous injection of ET-1 in the
rat penis inhibits ET-1-mediated contraction in vivo
[Mills et al., 2001a]. Thus, ET-1 may also drive smooth
muscle contraction via activation of Rho-kinase (Fig.7).
This is supported by ET-1 administration to the rat cor-
pus cavernosum which leads to dose-dependent contrac-
tions in vitro that are relaxed by Y-27632 [Wingard et al.,
2003].
Interestingly, combined treatment of ET-1 and phe-
nylephrine (α1-adrenoreceptor agonist) augmented the
contractile response in the rat corpus cavernosum in vi-
tro, compared to ET-1 or phenylephrine treatment alone.
Also, the combined ET-1 and phenylephrine treatment
correlated with an increase in membrane-RhoA in rat
cavernosal tissue homogenates [Wingard et al., 2003].
These studies suggest that ET-1 and NA mediate detu-
mescence in an additive fashion, potentially via increas-
ing activity and levels of RhoA in smooth muscle cells.
Also, ET receptor antagonists do not affect smooth mus-
cle contraction of the rabbit corpus cavernosum in vitro
induced by the α1-adrenoreceptor, further suggesting
that NA and ET-1 mediate detumescence separately to
produce an additive effect [Mumtaz et al., 2006]. During
tumescence, it is likely that NO signalling inhibits
ET-1-mediated vasoconstriction; injection of ET-1 into
the rat corpus cavernosum during neural stimulation- or
NO donor-induced erection diminishes its ability to con-
tract smooth muscle in vivo [Mills et al., 2001b].
Interestingly, ET-1 signalling via the ETB receptor me-
diates smooth muscle relaxation. This is evident by injec-
tion of ET-1 into the rat corpus cavernosum which in-
duces both vasodilation and vasoconstriction [Ari et al.,
1996]. Furthermore, administration of an ETB agonist
leads to relaxation of the rat and mouse corpus caverno-
sum in vitro [Carneiro et al., 2008]. Also, ETB signalling
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increases formation of NO in human fetal endothelial and
smooth muscle cells of the penis, potentially explaining
the relaxant effect of ET-1/ETB signalling [Filippi et al.,
2003]. These results demonstrate that a single signalling
factor can exert opposite effects on erectile function.
However, smooth muscle contraction is considered the
predominant role of ET-1 in erectile physiology (Fig.7).
This is consistent with administration of ET-1 on the bo-
vine retractor penis muscle and penile artery which in-
duces contraction and not relaxation [Parkkisenniemi
and Klinge, 1996].
Angiotensin II
The peptide hormone angiotensin II is present in the
human penile endothelium and smooth muscle cells at
physiologically relevant levels (Fig.7) [Kifor et al., 1997;
Ertemi et al., 2011]. Angiotensin II promotes detumes-
cence which is supported by its contraction of the human
corpus cavernosum in vitro, an effect blocked by admin-
istration of losartan, an antagonist of angiotensin type 1
receptor (AT1) [Becker et al., 2001; Ertemi et al., 2011].
Furthermore, intra-cavernosal injection of angiotensin II
in canines abolishes spontaneous erections whereas ad-
ministration of losartan increases intracavernous pres-
sure [Kifor et al., 1997]. Also, transfection of short hairpin
RNA to silence the angiotensin II gene using an adenovi-
rus (Ad-Ang-2) in rats with diabetes mellitus-induced
erectile dysfunction (DMED) prolonged erectile function
in vivo compared to DMED rats without angiotensin II
silencing [Zhang et al., 2018]. Also, angiotensin II silenc-
ing with this method led to reduced contraction of the
corpus cavernosum in vitro of rats with DMED [Zhang et
al., 2018].
Angiotensin II promotes detumescence via activation
of the RhoA/Rho-kinase pathway (Fig.7); the expression
of RhoA and ROCK2 is lower in the penises of DMED rats
exposed to Ad-Ang-2 shRNA compared to DMED con-
trols [Zhang et al., 2018]. Also, similar to NA and ET-1,
it is thought that angiotensin II signalling via the AT1 re-
ceptor mediates vasoconstriction via activation of PLC,
thus generating IP3 and DAG which causes increased
Ca2+ and activation of PKC, respectively (Fig.7) [Wynne
et al., 2009]. However, to the best of our knowledge this
remains to be proven specifically in the erectile tissue of
the penis.
Angiotensin II-induced smooth muscle contraction
may also occur via modulation of NA signalling; it was
reported that losartan inhibits phenylephrine-induced
contraction of the canine corpus cavernosum in vitro
[Comiter et al., 1997]. Thus, angiotensin II may interact
with NA signalling to promote detumescence. Indeed,
angiotensin II can act at sympathetic nerve endings to
promote neurotransmission [Reid, 1992]. Therefore, an-
giotensin II signalling may drive the release of NA from
penile sympathetic nerves. Again, to the best of our
knowledge this remains to be proven in the penis. Also,
administration of the NO donor sodium nitroprusside
abolishes angiotensin II-induced contraction of the ca-
nine corpus cavernosum in vitro [Comiter et al., 1997].
Thus, NO signalling may also promote tumescence by
negatively regulating angiotensin II signalling.
Overall, the balance of relaxant and contractile factors
mediated by parasympathetic and sympathetic systems,
respectively, determines the contractile state of penile
smooth muscle. In addition, multiple other signalling
pathways add further complexity to this process.
Link between Endocrine Disrupting Chemicals and
Erectile Dysfunction
Effects of Estrogenic-EDCs and Endogenous Estrogen
Signalling on Erectile Function
Exposure to EDCs with estrogenic properties (estro-
genic-EDCs) during development may impact pattern-
ing of erectile tissues in the penis resulting in ED in the
adult. Neonatal administration of the estrogenic EDCs
diethylstilbestrol (DES) and estradiol valerate (EV) to rat
pups during postnatal days 1-12 leads to the accumula-
tion of fat cells in the corpus cavernosum, reduced cav-
ernous spaces, loss of smooth muscle, reduction in weight
of the skeletal muscles required for tumescence, and dis-
rupted fertility in adulthood [Goyal et al., 2004a, b, 2005a,
b]. A reduction in cavernosal spaces and smooth muscle
of the penis is also seen following administration of DES
to rats during prepuberty and puberty [Goyal et al.,
2004a]. Similarly, treatment of the estrogen 17α-ethinyl
estradiol (EE) to neonatal rats results in penile malfor-
mations, including the accumulation of fat cells in the
penis, reduction of the bulbospongiosus muscle, and im-
paired fertility in adulthood [Mathews et al., 2009]. This
is consistent with BPA administration at toxic levels over
12 days to 8–12 weeks old rabbits (juvenile), which leads
to increased thickness of the penile tunica albuginea,
subtunical fat deposition, and reduced sinusoidal spaces
[Moon et al., 2001]. It is likely that these structural chang-
es to the penis tissues of BPA-exposed rabbits cause the
attenuated contraction and relaxation of the corpus cav-
ernosum in vitro that they exhibit in adulthood [Moon
et al., 2001].
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In addition to their developmental impacts, estrogen-
ic-EDC exposure during adulthood may also disrupt
erectile function by altering the penile architecture or
erectile physiology. Daily treatment of estradiol for 2
weeks in adult rats leads to increased contraction and de-
creased relaxation of the corpus cavernosum smooth
muscle in vitro, as well as disrupted erectile function in
vivo [Kataoka et al., 2013]. This is consistent with daily
EV administration for 1 and 12 weeks to sexually mature
rats which impairs erectile responses in vivo and leads to
replacement of corpus cavernosum smooth muscle fibres
with loose connective tissue after 12 weeks of EV expo-
sure [Lewis et al., 2002; Adaikan and Srilatha, 2003].
Thus, chronic exposure to EDCs in adults may also lead
to smooth muscle atrophy in the penis. In addition, daily
administration of EV or the phytoestrogen daidzein over
12 weeks to adult rabbits potentiates contraction of the
corpus cavernosum in vitro, as well as decreases the mag-
nitude of relaxation [Srilatha and Adaikan, 2004].
Although estrogenic-EDC exposure during develop-
ment and adulthood has deleterious effects on erectile
function, endogenous estrogen also has a role in penis
development which may promote erectile function in the
adult. Mice with disrupted endogenous estrogen signal-
ling exhibit accelerated delamination of the penis tissues,
impacting the timing of the separation of the prepuce
from the glans [Cripps et al., 2019; Govers et al., 2019].
This may in turn disrupt the development of tissues re-
quired for erectile function; delamination events are
known to cause physical keratin partitions between tis-
sues which block diffusion of patterning and growth fac-
tors [Salas et al., 2016; Liu et al., 2017]. Thus, endogenous
estrogen signalling during development may be required
for the diffusion of growth factors across the developing
penis, in turn driving development of the erectile tissue.
This is consistent with the potential pro-erectile role of
endogenous estrogen during adulthood, which may in-
volve maintaining the structural integrity of erectile tis-
sues, regulating smooth muscle cell ion flux and regulat-
ing NO production (described above).
Taken together, it is likely that a balance of endoge-
nous estrogen signalling during development and also in
adulthood is required for optimal erectile function. Thus,
any EDC which results in an increase or decrease to es-
trogen signalling may alter this balance to cause ED.
Indirect and Direct Mechanisms for ED Induced by
Estrogenic-EDCs
Estrogenic-EDCs may disrupt penis development and
erectile function indirectly via the suppression of testos-
terone production; there is evidence that estrogen inhib-
its the luteinizing hormone (LH) responsiveness of Ley-
dig cells in immature rats, which may in turn decrease
circulating testosterone produced in these cells [van
Beurden et al., 1978; Schulster et al., 2016]. This is sup-
ported by estrogen administration to rabbits and rats
which leads to decreased testosterone levels [Adaikan and
Srilatha, 2003; Goyal et al., 2004a, 2005a, b; Srilatha and
Adaikan, 2004] and by estrogen therapy in male-to-fe-
male transsexuals which is associated with a decline in
circulating testosterone [Dittrich et al., 2005]. Thus, ex-
ogenous estrogen may disrupt erectile function via inhi-
bition of testosterone production, thereby functioning as
an anti-androgen.
However, estrogenic-EDCs may also impact erectile
function directly as the ERs are expressed throughout the
embryonic human and rodent penis [Jesmin et al., 2002;
Dietrich et al., 2004; Baskin et al., 2020]. Indeed, testos-
terone treatment fails to restore erectile function in rats
exposed to high levels of estrogen during adulthood,
which demonstrates that estrogenic-EDCs may disrupt
this process via pathways independent of androgen sig-
nalling [Kataoka et al., 2013]. Furthermore, estrogen ex-
posure to the developing tammar wallaby inhibits phallus
growth but does not impair normal androgen synthesis,
also suggesting a direct action of estrogen in the penis
[Chen et al., 2018].
Overall, estrogenic-EDCs may cause ED through inhi-
bition of testosterone production and also by activation
of ERs localised in the penis. This may occur during penis
development and in adult physiology.
Potential Role of EDCs in Human ED and Other
Aspects of Male Reproductive Health
Although animal studies have made a link between
EDCs and ED, it is difficult to investigate this in human
populations as the wide range of ED risk factors may con-
found results [Rambhatla and Mills, 2016]. Nonetheless,
there is some evidence that human exposure to EDCs is
linked to ED. Factory workers in China exposed to high
levels of BPA in the workplace displayed higher risk of
male sexual dysfunction, including risk of ED, compared
to non-exposed factory workers [Li et al., 2009]. In addi-
tion, high BPA levels in urine and blood are an indicator
for deteriorating sexual function in men, including erec-
tile function [Li et al., 2010]. Furthermore, EDCs are as-
sociated with disruption of several other aspects of male
reproductive health. For example, treatment of develop-
ing rats with EDCs leads to reduced sperm count [Axels-
tad et al., 2018], and workplace exposure to BPA in hu-
Cripps/Mattiske/Pask
Sex Dev 2021;15:187–212
206
DOI: 10.1159/000516600
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Conclusion
ED is extremely prevalent globally and presents major
lifestyle and health problems for affected individuals and
their partners. The rapid increase in prevalence cannot be
accounted for by genetics and age alone; environmental
factors must also play a role. Thus, it is critical to under-
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mechanisms which govern erectile function; disruptions
to any of these factors are considered risk factors for ED.
However, the role of EDCs as risk factors for ED is stark-
ly under-researched. This is despite established knowl-
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pervasive in our environment, and multiple animal stud-
ies strongly suggest EDCs are among the risk factors for
human ED. Thus, this area needs far greater attention in
order to reduce ED prevalence and avert the plethora of
health hazards presented by EDCs.
Conflict of Interest
The authors have no conflict of interest to declare.
Funding Sources
Aspects of this paper were supported by a project grant from
the National Health and Medical Research Council to A.J.P.
Author Contributions
All authors contributed to the writing and editing of the docu-
ment.
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