Themed Section: Cannabinoids in Biology and Medicine, Part II
(D9-THC) attenuates mouse
sperm motility and male
Daniel J Morgan1,2, Charles H Muller3, Natalia A Murataeva1,2,
Brian J Davis1,2and Ken Mackie1,2
1Gill Center for Biomolecular Science, Indiana University, Bloomington, IN, USA,2Department of
Psychological and Brain Sciences, Indiana University, Bloomington, IN, USA, and3Department of
Urology, University of Washington School of Medicine, Seattle, WA, USA
Daniel J Morgan, Gill Center for
Biomolecular Science, Indiana
University, Bloomington, IN
47405, USA. E-mail:
sperm motility; cannabinoid; CB1
receptor; D9-THC; sperm
energetics; male reproduction
2 December 2010
13 April 2011
10 May 2011
BACKGROUND AND PURPOSE
Numerous studies have shown that N-arachidonoylethanolamine (AEA) can inhibit sperm motility and function but the
ability of cannabinoids to inhibit sperm motility is not well understood. We investigated the effects of WIN 55,212-2, a CB1
cannabinoid receptor agonist, and D9-tetrahydracannabinol (D9-THC) on the ATP levels and motility of murine sperm in vitro.
In addition, the effects of acute administration of D9-THC on male fecundity were determined.
Effects of D9-THC on basal sperm kinematics were determined using computer-assisted sperm analysis (CASA). Stop-motion
imaging was performed to measure sperm beat frequency. The effect of D9-THC on sperm ATP was determined using a
luciferase assay. Male fertility was determined by evaluating the size of litters sired by D9-THC-treated males.
Pretreatment of sperm for 15 min with 1 mM D9-THC reduced their basal motility and attenuated the ability of bicarbonate
to stimulate flagellar beat frequency. Treatment with 5 mM WIN 55,212-2 or 10 mM D9-THC for 30 min reduced sperm ATP
levels. In sperm lacking CB1 receptors this inhibitory effect of WIN 55,212-2 on ATP was attenuated whereas that of D9-THC
persisted. Administration of 50 mg·kg-1D9-THC to male mice just before mating caused a 20% decrease in embryonic litter
CONCLUSIONS AND IMPLICATIONS
D9-THC inhibits both basal and bicarbonate-stimulated sperm motility in vitro and reduces male fertility in vivo. High
concentrations of WIN 55,212-2 or D9-THC inhibit ATP production in sperm; this effect of WIN 55,212-2 is CB1 receptor-
dependent whereas that of D9-THC is not.
This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section
visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit
D9-THC, D9-tetrahydrocannabinol; AEA, N-arachidonoylethanolamine; CASA, computer-assisted sperm analysis;
CB1, cannabinoid receptor 1; DAGL, sn-1-diaglycerol; KO, knockout; MAGL, monoacylglycerol lipase; Me-AEA,
methanadamide; SACY, soluble adenylyl cyclase; tmAC, transmembrane adenylyl cyclase; VAP, average path velocity;
VCL, curvilinear velocity; VSL, straight-line velocity; WIN 55,212-2 or WIN-2, (R)-(+)-[2,3-dihydro-5-methyl-3-(4-
morpholinylmethyl)pyrrolo[1,2,3-de]-1, 4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate; WIN 55,212-3 or
WIN-3, (S)-(–)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1, 4-benzoxazin-6-yl]-1-
British Journal of
British Journal of Pharmacology (2012) 165 2575–25832575
© 2011 The Authors
British Journal of Pharmacology © 2011 The British Pharmacological Society
Sperm capacitation refers to ‘the change undergone by sperm
in the female reproductive tract that enables them to pen-
etrate and fertilize an egg’ (Chang et al., 1976). Capacitation
occurring either in vitro or in the female reproductive tract
involves a series of changes in sperm physiology including
phospholipid remodelling of the plasma membrane, redistri-
bution of membrane cholesterol, tyrosine phosphorylation of
sperm proteins, increased motility, hyperactivation and the
acrosome reaction. Early-stage capacitation events include
increased synthesis of cAMP, which activates PKA, causing
the onset of bicarbonate-stimulated sperm motility (Wenne-
muth et al., 2003; Nolan et al., 2004; Morgan et al., 2008).
Sperm exhibit limited basal motility in the absence of bicar-
bonate. The presence of bicarbonate leads to the acquisition
of rapid and progressive motility caused by elevated flagellar
beat frequency (Wennemuth et al., 2003). Late-stage capaci-
tation events such as hyperactivated motility, tyrosine phos-
phorylation and the acrosome reaction require prolonged
exposure to bicarbonate and BSA in vitro (Byrd, 1981; Lee and
Storey, 1986; Boatman and Robbins, 1991; Visconti et al.,
1995a,b; Harrison, 1996).
Previous work has found a complete endocannabinoid
signalling system in sperm. Sperm from mice, humans, pigs
and frog express the CB1 receptor, while CB2 has been
detected in boar and human sperm (Maccarrone et al., 2005;
Rossato et al., 2005; Cobellis et al., 2006; Francavilla et al.,
2009; Aquila et al., 2010b). However, CB2 protein appears
absent from mouse sperm (Grimaldi et al., 2009). The TRPV1
channel, an ion-channel receptor for capsaicin that is also
activated by N-arachidonoylethanolamine (AEA) is detected
in boar and human sperm (Schuel et al., 2002a; Maccarrone
et al., 2005; Francavilla et al., 2009; Grimaldi et al., 2009). The
endocannabinoid AEA as well as NAPE-PLD, one of the
enzymes responsible for its synthesis have been detected in
human sperm (Francavilla et al., 2009). AEA has been
detected in the female reproductive tract and seminal fluid at
concentrations as high as 10–12 nM (Schuel et al., 2002a;
Schuel, 2006). The enzymes, sn-1-diaglycerol (DAGL) and
monoacylglycerol lipase (MAGL), that are responsible for the
synthesis and degradation of 2-AG, respectively, are detected
in epididymal sperm (Cobellis et al., 2010). The enzyme
FAAH hydrolyzes AEA and is detected in sperm from frog,
boar and human (Maccarrone et al., 2005; Cobellis et al.,
2006; Francavilla et al., 2009). Consistent with an important
role for FAAH in reproduction, male mice lacking FAAH
exhibit decreased litter size (Sun et al., 2009). Sperm from
these mice have reduced motility, decreased ability to
undergo the acrosome reaction and lower capacity for in vitro
fertilization (Sun et al., 2009). Previous work has shown that
D9-THC and AEA inhibit the fertilizing capacity (capacitation)
of sea urchin sperm (Chang et al., 1991; 1993; Schuel et al.,
1991; 1994). More recent work has shown decreased progres-
sive motility and a reduced ability to undergo the acrosome
reaction in human sperm treated with sub-micromolar con-
centrations of D9-THC (Whan et al., 2006). Additional studies
have found that AEA inhibits sperm motility, hyperactiva-
tion, mitochondrial function, plasma membrane voltage
potential, as well as the zona pellucida-stimulated acrosome
reaction (Schuel et al., 2002b; Maccarrone et al., 2005;
Rossato et al., 2005). Exposure to 1 mM methananamide (Me-
AEA), a non-hydrolyzable analogue of AEA, also inhibits
sperm motility and mitochondrial membrane potential in a
CB1-dependent manner (Barbonetti et al., 2010). Treatment of
human sperm with either D9-THC or D8-THC reduces mito-
chondrial O2 production indicating that both endocannab-
inoids as well as phytocannabinoids such as D9-THC can
impair mitochondrial respiration (Badawy et al., 2009).
Antagonism of CB1 with rimonabant has been shown to
enhance sperm motility, sperm energy metabolism, survival,
protein tyrosine phosphorylation and the capacity to
undergo the acrosome reaction (Aquila et al., 2010a). Inhibi-
tion of the TRPV1 receptor increases the incidence of spon-
taneous acrosome reaction in human and boar sperm
suggesting that TRPV1-mediated AEA signalling is important
for correct timing of the acrosome reaction (Maccarrone
et al., 2005; Francavilla et al., 2009). However, despite the
large number of recent studies, the effects of cannabinoids on
basal and bicarbonate-stimulated flagellar beat frequency,
male fertility and sperm energetics are still not well under-
stood. In particular, very little is known about the possible
effects of D9-THC on the bicarbonate-stimulated motility that
occurs within the female reproductive tract. Therefore, in this
study, we have investigated whether D9-THC inhibits sperm
ATP levels as well as basal and bicarbonate-stimulated motil-
ity in vitro. We have also given male mice a single injection of
50 mg·kg-1D9-THC, just prior to mating, to determine
whether acute exposure to D9-THC inhibits litter size in vivo.
All animal care and experimental procedures were approved
by the institutional animal care and use committees at the
University of Washington or Indiana University and were
conducted in accordance with the National Institutes of
Health Guide for the Care and Use of Laboratory Animals.
CD1 male mice were obtained from Charles River (Wilming-
ton, MA). CB1 knockout mice in a CD1 background were
generously provided by Catherine Ledent and bred in our
facility (Ledent et al., 1999). Nomenclature for receptors
follows BJP’s Guide to Receptors and Channels (Alexander et al.,
2011). All mice used in these experiments were housed under
a 12:12 h light–dark cycle (lights on 06h 00min, lights off 18h
00min) and provided with standard mouse chow ad libitum.
In order to harvest sperm, mice were killed by CO2asphyxi-
ation followed by cervical dislocation.
entia were excised and cleaned in HS medium containing:
135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 1 mM
pyruvic acid, 20 mM lactic acid, 5 mM glucose and 20 mM
HEPES (pH 7.4). Sperm were harvested by a 15 min ‘swim out’
in medium HS supplemented with 5 mg BSA mL-1. Released
sperm were sedimented, then resuspended in BSA-free HS
medium. Bicarbonate stimulation of motility was measured
in HS medium supplemented with 15 mM NaHCO.
The caudal epididymides and vasa defer-
Analysis of sperm motility.
ined in individual sperm as previously described (Wennemuth
Flagellar beat frequency was exam-
DJ Morgan et al.
2576British Journal of Pharmacology (2012) 165 2575–2583
et al., 2003; Nolan et al., 2004; Morgan et al., 2008). Motility
was analysed for sperm bathed with HS medium alone or HS
medium containing D9-THC for 15 min. No sperm possessing
a well-defined sinusoidal waveform necessary for estimation
of beat frequency were observed for sperm treated with 10 mM
D9-THC for 15 min. Therefore, data for sperm treated with
10 mM D9-THC for 13 min, the longest period of treatment for
which beat frequency could be determined, were shown in
Supporting Information Figure S1. Bicarbonate-stimulated
motility was analysed in sperm perfused with HS medium
containing 15 mM bicarbonate for 60 s. Briefly, stop-motion
images were collected at 20–40 ms intervals for sperm loosely
tethered to a glass surface at the head. A solenoid-controlled
gravity-driven local perfusion device produced rapid changes
in medium composition. Images were processed, and motility
was determined using MetaMorph (Universal Imaging, Down-
ington, PA). Computer-assisted sperm analysis (CASA) was
performed using a Hamilton-Thorne Research IVOS sperm
described (Hamilton-Thorne, Danvers, MA) (Burton et al.,
1999). Sperm were treated with a range of D9-THC concentra-
tions (0.001, 0.01, 0.1, 1 and 10 mM) in HS medium. For each
D9-THC concentration examined, sperm were pretreated with
HS medium containing the appropriate concentration of
D9-THC for 30 min prior to CASA. In separate experiments,
sperm were incubated with 10 mM D9-THC for 15, 30, 60 or
90 min prior to CASA. CASA was performed on sperm placed
Products B.V., Nieuw-Vennep, the Netherlands). Analysis was
restricted to 15–100 track points at a 60 Hz frame rate using
Olympus ‘negative phase’ optics. Standard kinematics were
calculated by the CASA programme. Cells exhibiting less than
10 mm·s-1average path velocity were considered to be non-
motile. Sperm velocity was measured as straight-line velocity
(VSL; the straight-line distance from beginning to end of track
divided by the elapsed time), average path velocity (VAP; the
five-point smoothed average path distance divided by time
elapsed) and curvilinear velocity (VCL or track speed; the total
distance between all detected head centroids divided by the
Sperm ATP levels were determined using a luciferase-based
ATP Determination Kit from Molecular Probes (Eugene, OR)
and a Lmax II microplate reader (Molecular Devices, Sunny-
vale, CA). Prior to assaying for ATP, sperm were treated with
HS medium containing D9-THC, WIN 55,212-2 or the inactive
enantiomer, WIN 55,212-3. The concentration curve exam-
ining the effects of D9-THC on ATP was determined in sperm
treated with 0, 0.001, 0.01, 0.1, 1 and 10 mM D9-THC for
30 min. Experiments investigating the amount of time
required for 10 mM D9-THC or 5 mM WIN 55,212-2 to reduce
ATP levels in sperm were performed using sperm treated with
HS medium containing drug for 15, 30, 60 or 90 min. Quan-
tification of ATP was determined by measuring luminescence.
Analysis of D9-THC effects on litter size
Sexually mature wild-type and CB1knockout (KO) male CD1
mice (12–18 weeks) were given i.p. injections of either
50 mg·kg-1D9-THC (n = 9 wild-type males and 17 litters) or
18:1:1 vehicle containing 0.9% saline, 5% cremaphor and 5%
ethanol (n = 7 wild-type males and 20 litters). Litter size was
also examined for CB1 KO males treated with 50 mg·kg-1
D9-THC (n = 5 males and 6 litters) or vehicle (n = 5 males and
6 litters). The volume of vehicle or 5 mg·mL-1D9-THC injected
was 10 mL·g-1of body weight. Injections were administered
just prior to the onset of the dark cycle, and injected males
were bred overnight with wild-type CD1 females. Plug-
positive CD1 females were removed from breeding cages the
following morning. New cages of wild-type male and female
breeders were set up daily due to possible desensitization of
sperm CB1receptors. Males treated with D9-THC were reused
after being allowed to recover for 2 weeks. Plug-positive
females were killed by CO2asphyxiation on the 12th day of
gestation, and litter size was determined by counting the
number of e12.5 embryos present.
Data are presented as means ? SEM. Statistical analyses were
performed using Microsoft Excel (unpaired t-tests) (Redmond,
WA, USA) or GraphPad Prism 4 (La Jolla, CA, USA) (two-way
ANOVA with Bonferroni’s post hoc test). Unpaired t-tests were
used to analyse data shown in Figures 1–3. A two-way
repeated-measures ANOVA with Bonferroni’s post hoc test was
used to analyse the data in Figure 4.
Activation of sperm motility
We examined the effects of 1 and 10 mM D9-THC on basal and
bicarbonate-stimulated motility in sperm from wild-type
CD1 mice. The percentage of motile wild-type sperm
decreased from 82% (untreated sperm) to 35% when sperm
were bathed in HS medium containing 1 mM D9-THC for
15 min (Figure 1A). However, the slow resting beat frequency
of motile sperm (2.63 ? 0.09 Hz) was only slightly decreased
to 2.33 ? 0.08 Hz (P < 0.01) during 15 min of exposure to
1 mM D9-THC (Figure 1B). While perfusion of HS medium
containing 15 mM NaHCO3 for 1 min caused a threefold
increase in beat frequency (7.8 ? 0.6 Hz) in wild-type sperm,
perfusion of sperm exposed to 1 mM D9-THC in the same
medium increased their beat frequency significantly less (6.24
? 0.29 Hz; P < 0.01) (Figure 1C). Treatment of sperm with
10 mM D9-THC for 15 min reduced the percentage of motile
sperm to 5% (Figure 1A). The basal beat frequency of sperm
treated with 10 mM D9-THC for 13 min was reduced to 1.02 ?
0.06 Hz (Supporting Information Figure S1). Bicarbonate-
stimulated motility was completely abolished in the few
10 mM D9-THC-treated sperm that did possess a sinusoidal
waveform (Figure 1C). Thus, 15 min of treatment with 1 mM
D9-THC reduces the activating effects of bicarbonate on beat
frequency by 20%, while 10 mM D9-THC completely abolishes
this form of motility (Figure 1C).
Sperm motility was also evaluated by CASA in wild-type
mouse sperm treated with increasing concentrations of
D9-THC for 30 min. We found that D9-THC at 1 mM and above
inhibited curvilinear velocity (Figure 2E), while only 10 mM
Cannabinoid inhibition of sperm motility
British Journal of Pharmacology (2012) 165 2575–25832577
D9-THC inhibited VSL (Figure 2A) and VAP (Figure 2C). Addi-
tional CASA analyses were performed to determine the
amount of time required for 10 mM D9-THC to inhibit sperm
motility. In these experiments, treatment of wild-type sperm
with 10 mM D9-THC for 15 min or more generally decreased
the VSL (Figure 2B), VAP (Figure 2D) and VCL (Figure 2F).
In multiple studies, AEA or Me-AEA has been shown to
inhibit sperm motility (Schuel et al., 2002b; Maccarrone et al.,
2005; Rossato et al., 2005; Barbonetti et al., 2010). Therefore,
we hypothesized that decreased ATP production due to mito-
chondrial dysfunction might account for the reduced basal
motility of D9-THC-treated sperm. We found that treatment
with either 5 mM WIN 55,212-2 (high-efficacy CB1agonist) or
10 mM D9-THC (low-efficacy CB1agonist) reduced sperm ATP
levels (Figure 3). A concentration-effect curve for D9-THC
inhibition of sperm ATP levels was determined, and 10, 30
and 100 mM
(Figure 3A). Treatment of wild-type sperm with 10 mM
D9-THC reduced ATP levels by 91% relative to untreated con-
trols (Figure 3B) within 60 min. The inhibitory effect of
D9-THC on ATP persisted in sperm lacking CB1 receptors,
suggesting this effect was not mediated by CB1 receptors
(Figure 3C). Exposure to 5 mM WIN 55,212-2 for 30 min
caused a 35% decrease in sperm ATP (Figure 3D). The inhibi-
tory effect of 5 mM WIN 55,212-2 on sperm ATP was attenu-
ated in sperm lacking the CB1 receptor (Figure 3E). The
non-CB1interacting enantiomer, WIN 55,212-3, had no effect
on sperm ATP levels in either wild-type or CB1deficient sperm
(Figure 3D, E), suggesting that the effects of 5 mM WIN
55,212-2 on sperm ATP levels are mediated by CB1receptors,
while those of D9-THC are not.
To determine if acute exposure of sperm to D9-THC inhibited
male fertility, 50 mg·kg-1D9-THC was administered to male
CD1 mice just prior to breeding at the onset of the dark cycle.
Litter size from vehicle- or drug-treated males was determined
by counting the number of embryos from plug-positive CD1
females on the 12th day of gestation. Acute administration of
50 mg·kg-1D9-THC reduced litter size from 14.7 ? 0.6
(vehicle-treated wild-type males, n = 7) to 11.8 ? 0.8 (THC
treated wild-type males, n = 9) embryos per litter (P < 0.01).
Litter size was also examined in male CB1knockout mice to
determine whether the reduction in litter size observed in
wild-type mice treated with 50 mg·kg-1D9-THC was CB1-
mediated. Acute administration of 50 mg·kg-1D9-THC had no
effect on the sizes of litters sired by CB1knockout males (13.5
? 0.2, n = 6 males) when compared with vehicle-treated CB1
knockout males (13.7 ? 0.7, n = 6 males).
Discussion and conclusions
The primary objective of this study was to employ multiple
methodological approaches to better understand the effects
of D9-THC on sperm ATP levels and motility. Numerous pre-
vious studies have demonstrated that AEA inhibits basal
Basal and bicarbonate-stimulated motility is inhibited by D9-THC. (A)
Treatment with 1 mM D9-THC or 10 mM D9-THC for 15 min progres-
sively reduced the percentage of motile sperm. (n = 111–138 cells).
At least eight cells were examined from each animal in two to three
independent experiments. #P < 0.05 (untreated vs. 1 mM THC), *P <
0.05 (untreated vs. 10 mM THC). (B) Averaged flagellar beat fre-
quency was determined for wild-type sperm that were bathed in HS
medium containing 1 mM D9-THC (THC) for 15 min. (n = 22–47
cells). At least eight cells were examined from each animal in two to
three independent experiments. #P < 0.05 (untreated vs. 1 mM THC).
(C) Sperm were bathed in HS medium alone or HS medium contain-
ing 1 mM or 10 mM D9-THC (THC) for 15 min and subsequently
perfused with HS medium containing 15 mM HCO3-(BC) for 1 min.
Bicarbonate-stimulated beat frequency was reduced in sperm treated
with 1 mM D9-THC relative to sperm treated with HS medium con-
taining bicarbonate. #P < 0.05 (BC vs. BC+1 mM THC). Treatment of
sperm with 10 mM D9-THC completely blocked the stimulating effect
of bicarbonate on beat frequency. *P < 0.001 (BC vs. BC +10 mM
THC). The number of sperm used for each condition is designated in
parentheses. Unpaired t-tests were used to calculate P values. Error
bars represent SEM.
DJ Morgan et al.
2578 British Journal of Pharmacology (2012) 165 2575–2583
sperm motility as well as other sperm functions such as the
acrosome reaction that are required for fertilization of the
oocyte. However, the ability of exogenous cannabinoids such
D9-THC to inhibit sperm motility is not well understood.
Earlier studies investigating the role of cannabinoid signal-
ling in sperm motility have focused mostly on the ability of
AEA, Me-AEA or D9-THC to reduce the percentage of motile
sperm. However, a recent study using CASA demonstrated
that the motility of human sperm is inhibited by 5 and 10 mM
Me-AEA (Barbonetti et al., 2010). Our study confirms previous
work showing that activation of cannabinoid signalling
increases the percentage of immotile sperm. However,
extending previous studies, we have used CASA and stop-
motion videos to determine whether D9-THC reduces the beat
frequency and swimming speed (kinematics) of the remain-
ing fraction of sperm that are motile. Interestingly, we find
that while 1 mM D9-THC dramatically reduces the percentage
of sperm that are motile, the beat frequency of sperm that
retain their motility is only slightly affected (12% reduction).
Measurement of sperm motility using CASA indicates 1 mM
D9-THC reduces sperm ‘swimming speed’ by 46% (VSL), 42%
(VAP) and 30% (VCL).
Sperm kinematics are rapidly inhibited by 10 mM D9-THC. Dose–response curves over a range of D9-THC concentrations were constructed for CASA
kinematic analysis (A, C, E). Treatment of sperm with 10 mM D9-THC for 30 min significantly inhibited VSL (A) and average path velocity (C), while
VCL (E) was inhibited by both 1 and 10 mM D9-THC. (n = 2113–4498 cells for A, C, and E.) *, P < 0.05 (untreated vs. 10 mM THC and 1 mM THC).
Onset of the D9-THC effect was rapid, with treatment of wild-type sperm with 10 mM D9-THC decreasing the VSL (B), VAP (D) and VCL (F) within
15 min. (n = 791–1603 cells for HS in B, D, and F). (n = 1240, 886, 252, 113 and 620 cells at 0, 15, 30, 60 and 90 min for HS +THC in B, D and
F) *P < 0.05 (HS vs. THC). Unpaired t-tests were used to calculate P-values. Error bars represent SEM.
Cannabinoid inhibition of sperm motility
British Journal of Pharmacology (2012) 165 2575–25832579
Inhibition of sperm ATP production is one way that D9-THC
might reduce basal motility. Recent work has demonstrated
the ability of Me-AEA to disrupt mitochondrial function in
sperm (Rossato et al., 2005; Barbonetti et al., 2010). However,
blockade of electron transport with the respiratory chain
complex I inhibitor, rotenone, does not significantly impair
sperm motility when glucose is present, and glycolysis is able
to occur (Barbonetti et al., 2010). Sperm motility and ATP
levels were also normal when oxidative phosphorylation was
inhibited using carbonyl cyanide m-chlorophenylhydrazone
(Mukai and Okuno, 2004). In contrast, sperm from mice
lacking glyceraldehyde-3-phosphate dehydrogenase-S, an
enzyme required for glycolysis in sperm, fail to exhibit pro-
gressive motility (Miki et al., 2004). Cumulatively, these
earlier studies suggest that glycolysis rather than oxidative
phosphorylation produces most of the ATP needed to sustain
motility in sperm. In order to determine whether D9-THC
impairment of mitochondrial function might disrupt energy
production, we investigated ATP levels in sperm treated with
D9-THC. In this study we find that 10 mM D9-THC severely
D9-THC and WIN 55,212-2 reduce sperm ATP levels. Treatment of sperm for 60 min with 10, 30 and 100 mM D9-THC reduced ATP levels (A). 10 mM
D9-THC reduced ATP levels in sperm from wild-type mice relative to untreated controls (HS) in a time-dependent fashion (B). Significantly, the
effect of D9-THC on ATP levels was present in sperm lacking CB1receptors, suggesting that the inhibitory effect of D9-THC on ATP levels was not
CB1mediated (C). Treatment with 5 mM WIN 55,212-2 (WIN2) causes a 35% reduction in ATP levels in wild-type sperm. WIN 55,212-3 (WIN3),
which does not bind with high affinity to CB1receptors, had no effect on sperm ATP levels (D). The effect of WIN 55,212-2 on sperm ATP was
absent in sperm lacking the CB1 cannabinoid receptor (E). Student’s unpaired t-test was used to calculate significance (*P < 0.05). Error bars
DJ Morgan et al.
2580British Journal of Pharmacology (2012) 165 2575–2583
decreases sperm ATP levels in a CB1 receptor-independent
manner. Since this effect is present in CB1-/-sperm it is likely
that 10 mM D9-THC reduces ATP via a non-CB1-mediated
mechanism. Previous work has shown that 10 mM AEA
reduces sperm viability, raising the possibility that 10 mM
D9-THC might be reducing ATP levels in our study via CB1-
independent cytotoxicity (Barbonetti et al., 2010). In con-
trast, treatment with 1 mM D9-THC does not decrease sperm
ATP levels despite the ability of this concentration to inhibit
basal motility. These results suggest that the inhibition of
basal motility by 1 mM D9-THC is not caused by THC-induced
decreases in ATP availability. However, treatment with 5 mM
WIN 55,212-2 does cause moderate reductions in sperm ATP
levels that are absent in CB1knockout sperm or sperm treated
with the inactive enantiomer WIN 55,212-3, suggesting an
efficacious CB1agonist can inhibit ATP production.
To date, the ability of endo- or exo- cannabinoids to inhibit
bicarbonate-stimulated motility has not been studied. This
type of motility is best characterized by an increase in pro-
gressive forward motility due to increased flagellar beat
frequency. Studies of mutant sperm lacking either the sperm-
specific PKA catalytic subunit (Ca2) (Nolan et al., 2004) or the
soluble form of adenylyl cyclase (SACY) (Esposito et al., 2004;
Hess et al., 2005; Xie et al., 2006) have provided definitive
evidence that both proteins are required for the acceleration
of the flagellar beat frequency that characterizes the rapid
activation of motility by the HCO3-anion. However, neither
cAMP production (SACY) nor PKA activation in sperm (Ca2)
are required for the maintenance of a slow basal flagellar beat.
Mice possessing sperm that are unable to synthesize cAMP in
response to bicarbonate are infertile demonstrating the neces-
sity of this signalling pathway for fertility (Esposito et al.,
2004; Hess et al., 2005; Xie et al., 2006). The ability of sperm
treated with D9-THC to undergo bicarbonate-stimulated
motility was investigated in this study. We find that 15 min
of treatment with 1 mM D9-THC attenuates bicarbonate
enhancement of beat frequency by 20%. However, despite
the slightly reduced response to bicarbonate these sperm do
respond to bicarbonate by substantially increasing their beat
frequency from 2.64 Hz (resting basal motility) to 6.24 Hz.
Previous work has shown that activation of the Gi/o-coupled
CB1inhibits the production of cAMP by transmembrane ade-
nylyl cyclases (tmACs) (Howlett et al., 1986; 1990; 2004).
However, the increase in cAMP synthesis that drives
bicarbonate-stimulated motility in sperm is catalysed by
SACY rather than tmAC. The finding that 10 mM D9-THC does
not appear to block bicarbonate-stimulated motility supports
the conclusion that bicarbonate-stimulated cAMP signalling
via SACY is not substantially modulated by CB1receptors.
In vivo male reproduction
Previous work demonstrated that chronic treatment with
cannabinoids causes a reduction in spermatogenesis, circulat-
ing testosterone and male fertility (Dalterio et al., 1982). This
early study raised the possibility that chronic exposure to
cannabinoids might inhibit male fertility via endocrine medi-
ated down-regulation of spermatogenesis. In order to deter-
mine whether D9-THC might inhibit male fertility via an
acute, non-endocrine mechanism on sperm function, male
mice were treated with 50 mg·kg-1D9-THC just before mating
(onset of the dark phase of the light–dark cycle). Treatment of
wild-type CD1 males with 50 mg·kg-1D9-THC reduced their
litter size by 20% (11.8 ? 0.8) relative to vehicle-treated males
(14.7 ? 0.6). The effect of 50 mg·kg-1D9-THC on decreased
litter size was absent in CB1knockout males, suggesting that
effects of D9-THC on litter size is CB1mediated. Interestingly,
the 20% reduction in litter size from males treated acutely
with 50 mg·kg-1D9-THC is similar in magnitude to the
reduced litter size for FAAH-/-males that has been previously
reported (Sun et al., 2009). Our result raises the possibility
that acute administration of D9-THC inhibits male fertility by
a mechanism involving reduced sperm function.
Taken together, the results of the current study provide
significant new insight into the ability of cannabinoid signal-
ling to partially inhibit bicarbonate-stimulated motility while
providing additional evidence that cannabinoids can inhibit
basal motility. We found that 10 mM D9-THC inhibits ATP
levels in sperm through a non-CB1 mechanism since the
reduction of ATP by 10 mM D9-THC is retained in CB1knock-
out sperm. In contrast, treatment with 5 mM WIN 55,212-2,
an efficacious CB1 agonist, caused a more modest 42%
decrease in sperm ATP levels that was absent in CB1knockout
sperm or sperm treated with the inactive analogue WIN
55,212-3. This finding suggests that CB1 activation can
disrupt sperm energetics and ATP levels under certain condi-
tions. Finally, we also determined that a single acute injection
of 50 mg·kg-1D9-THC to male mice just prior to mating can
the reduce size of litters sired by those males.
This research was supported by NIH grants DA11322 and
DA021696, theIndiana UniversityMetaCyt Initiative
Acute administration of D9-THC reduces male fertility. The acute
effect of D9-THC on male fertility was determined by measuring
embryonic (e12.5) litter sizes sired by vehicle treated CD1 wild-type
(WT) males (n = 7 males with 20 litters) or CD1 wild-type males
treated with 50 mg·kg-1D9-THC (n = 9 males with 17 litters). The
effect of vehicle (n = 5 males with 6 litters) and 50 mg·kg-1D9-THC
(n = 5 males with 6 litters) on litter size was also examined in mice
lacking CB1 receptors (KO). Error bars indicate SEM and P-values
were calculated using two-way repeated measures ANOVA with Bon-
ferroni’s post test (*P < 0.01).
Cannabinoid inhibition of sperm motility
British Journal of Pharmacology (2012) 165 2575–25832581
(funded in part by a grant from the Lilly Foundation), and the
Linda and Jack Gill Center for Biomolecular Science. We
would like to thank Donner Babcock for assistance capturing
stop-motion images for determining sperm flagellum beat
Conflicts of interest
Alexander SPH, Mathie A, Peters JA (2011). Guide to Receptors and
Channels (GRAC), 5th Edition. Br J Pharmacol 164 (Suppl. 1):
Aquila S, Guido C, Santoro A, Gazzerro P, Laezza C, Baffa MF et al.
(2010a). Rimonabant (SR141716) induces metabolism and
acquisition of fertilizing ability in human sperm. Br J Pharmacol
Aquila S, Guido C, Santoro A, Perrotta I, Laezza C, Bifulco M et al.
(2010b). Human sperm anatomy: ultrastructural localization of the
cannabinoid1 receptor and a potential role of anandamide in sperm
survival and acrosome reaction. Anat Rec (Hoboken) 293: 298–309.
Badawy ZS, Chohan KR, Whyte DA, Penefsky HS, Brown OM,
Souid AK (2009). Cannabinoids inhibit the respiration of human
sperm. Fertil Steril 91: 2471–2476.
Barbonetti A, Vassallo MR, Fortunato D, Francavilla S,
Maccarrone M, Francavilla F (2010). Energetic metabolism and
human sperm motility: impact of CB1 receptor activation.
Endocrinology 151: 5882–5892.
Boatman DE, Robbins RS (1991). Bicarbonate: carbon-dioxide
regulation of sperm capacitation, hyperactivated motility, and
acrosome reactions. Biol Reprod 44: 806–813.
Burton KA, Treash-Osio B, Muller CH, Dunphy EL, McKnight GS
(1999). Deletion of type IIalpha regulatory subunit delocalizes
protein kinase A in mouse sperm without affecting motility or
fertilization. J Biol Chem 274: 24131–24136.
Byrd W (1981). In vitro capacitation and the chemically induced
acrosome reaction in bovine spermatozoa. J Exp Zool 215: 35–46.
Chang MC, Austin CR, Brown J (1976). Mammalian fertilization.
Res Reprod 8: chart.
Chang MC, Berkery D, Laychock SG, Schuel H (1991). Reduction of
the fertilizing capacity of sea urchin sperm by cannabinoids derived
from marihuana. III. Activation of phospholipase A2 in sperm
homogenate by delta 9-tetrahydrocannabinol. Biochem Pharmacol
Chang MC, Berkery D, Schuel R, Laychock SG, Zimmerman AM,
Zimmerman S et al. (1993). Evidence for a cannabinoid receptor in
sea urchin sperm and its role in blockade of the acrosome reaction.
Mol Reprod Dev 36: 507–516.
Cobellis G, Cacciola G, Scarpa D, Meccariello R, Chianese R,
Franzoni MF et al. (2006). Endocannabinoid system in frog and
rodent testis: type-1 cannabinoid receptor and fatty acid amide
hydrolase activity in male germ cells. Biol Reprod 75: 82–89.
Cobellis G, Ricci G, Cacciola G, Orlando P, Petrosino S, Cascio MG
et al. (2010). A gradient of 2-arachidonoylglycerol regulates mouse
epididymal sperm cell start-up. Biol Reprod 82: 451–458.
Dalterio S, Badr F, Bartke A, Mayfield D (1982). Cannabinoids in
male mice: effects on fertility and spermatogenesis. Science 216:
Esposito G, Jaiswal BS, Xie F, Krajnc-Franken MA, Robben TJ,
Strik AM et al. (2004). Mice deficient for soluble adenylyl cyclase
are infertile because of a severe sperm-motility defect. Proc Natl
Acad Sci USA 101: 2993–2998.
Francavilla F, Battista N, Barbonetti A, Vassallo MR, Rapino C,
Antonangelo C et al. (2009). Characterization of the
endocannabinoid system in human spermatozoa and involvement
of transient receptor potential vanilloid 1 receptor in their
fertilizing ability. Endocrinology 150: 4692–4700.
Grimaldi P, Orlando P, Di Siena S, Lolicato F, Petrosino S,
Bisogno T et al. (2009). The endocannabinoid system and pivotal
role of the CB2 receptor in mouse spermatogenesis. Proc Natl Acad
Sci USA 106: 11131–11136.
Harrison RA (1996). Capacitation mechanisms, and the role of
capacitation as seen in eutherian mammals. Reprod Fertil Dev 8:
Hess KC, Jones BH, Marquez B, Chen Y, Ord TS, Kamenetsky M
et al. (2005). The ‘soluble’ adenylyl cyclase in sperm mediates
multiple signaling events required for fertilization. Dev Cell 9:
Howlett AC, Qualy JM, Khachatrian LL (1986). Involvement of Gi
in the inhibition of adenylate cyclase by cannabimimetic drugs.
Mol Pharmacol 29: 307–313.
Howlett AC, Champion TM, Wilken GH, Mechoulam R (1990).
Stereochemical effects of 11-OH-delta 8-tetrahydrocannabinol-
dimethylheptyl to inhibit adenylate cyclase and bind to
the cannabinoid receptor. Neuropharmacology 29:
Howlett AC, Breivogel CS, Childers SR, Deadwyler SA, Hampson RE,
Porrino LJ (2004). Cannabinoid physiology and pharmacology: 30
years of progress. Neuropharmacology 47 (Suppl. 1): 345–358.
Ledent C, Valverde O, Cossu G, Petitet F, Aubert JF, Beslot F et al.
(1999). Unresponsiveness to cannabinoids and reduced addictive
effects of opiates in CB1 receptor knockout mice. Science 283:
Lee MA, Storey BT (1986). Bicarbonate is essential for fertilization
of mouse eggs: mouse sperm require it to undergo the acrosome
reaction. Biol Reprod 34: 349–356.
Maccarrone M, Barboni B, Paradisi A, Bernabo N, Gasperi V,
Pistilli MG et al. (2005). Characterization of the endocannabinoid
system in boar spermatozoa and implications for sperm
capacitation and acrosome reaction. J Cell Sci 118 (Pt 19):
Miki K, Qu W, Goulding EH, Willis WD, Bunch DO, Strader LF
et al. (2004). Glyceraldehyde 3-phosphate dehydrogenase-S, a
sperm-specific glycolytic enzyme, is required for sperm motility and
male fertility. Proc Natl Acad Sci USA 101: 16501–16506.
Morgan DJ, Weisenhaus M, Shum S, Su T, Zheng R, Zhang C et al.
(2008). Tissue-specific PKA inhibition using a chemical genetic
approach and its application to studies on sperm capacitation. Proc
Natl Acad Sci USA 105: 20740–20745.
Mukai C, Okuno M (2004). Glycolysis plays a major role for
adenosine triphosphate supplementation in mouse sperm flagellar
movement. Biol Reprod 71: 540–547.
DJ Morgan et al.
2582 British Journal of Pharmacology (2012) 165 2575–2583
Nolan MA, Babcock DF, Wennemuth G, Brown W, Burton KA, Download full-text
McKnight GS (2004). Sperm-specific protein kinase A catalytic
subunit Calpha2 orchestrates cAMP signaling for male fertility. Proc
Natl Acad Sci USA 101: 13483–13488.
Rossato M, Ion Popa F, Ferigo M, Clari G, Foresta C (2005). Human
sperm express cannabinoid receptor Cb1, the activation of which
inhibits motility, acrosome reaction, and mitochondrial function. J
Clin Endocrinol Metab 90: 984–991.
Schuel H (2006). Tuning the oviduct to the anandamide tone. J
Clin Invest 116: 2087–2090.
Schuel H, Chang MC, Berkery D, Schuel R, Zimmerman AM,
Zimmerman S (1991). Cannabinoids inhibit fertilization in sea
urchins by reducing the fertilizing capacity of sperm. Pharmacol
Biochem Behav 40: 609–615.
Schuel H, Goldstein E, Mechoulam R, Zimmerman AM,
Zimmerman S (1994). Anandamide (arachidonylethanolamide), a
brain cannabinoid receptor agonist, reduces sperm fertilizing
capacity in sea urchins by inhibiting the acrosome reaction. Proc
Natl Acad Sci USA 91: 7678–7682.
Schuel H, Burkman LJ, Lippes J, Crickard K, Forester E, Piomelli D
et al. (2002a). N-Acylethanolamines in human reproductive fluids.
Chem Phys Lipids 121: 211–227.
Schuel H, Burkman LJ, Lippes J, Crickard K, Mahony MC,
Giuffrida A et al. (2002b). Evidence that anandamide-signaling
regulates human sperm functions required for fertilization. Mol
Reprod Dev 63: 376–387.
Sun X, Wang H, Okabe M, Mackie K, Kingsley PJ, Marnett LJ et al.
(2009). Genetic loss of Faah compromises male fertility in mice.
Biol Reprod 80: 235–242.
Visconti PE, Bailey JL, Moore GD, Pan D, Olds-Clarke P, Kopf GS
(1995a). Capacitation of mouse spermatozoa. I. Correlation
between the capacitation state and protein tyrosine
phosphorylation. Development 121: 1129–1137.
Visconti PE, Moore GD, Bailey JL, Leclerc P, Connors SA, Pan D
et al. (1995b). Capacitation of mouse spermatozoa. II. Protein
tyrosine phosphorylation and capacitation are regulated by a
cAMP-dependent pathway. Development 121: 1139–1150.
Wennemuth G, Carlson AE, Harper AJ, Babcock DF (2003).
Bicarbonate actions on flagellar and Ca2+ -channel responses:
initial events in sperm activation. Development 130:
Whan LB, West MC, McClure N, Lewis SE (2006). Effects of
delta-9-tetrahydrocannabinol, the primary psychoactive
cannabinoid in marijuana, on human sperm function in vitro.
Fertil Steril 85: 653–660.
Xie F, Garcia MA, Carlson AE, Schuh SM, Babcock DF, Jaiswal BS
et al. (2006). Soluble adenylyl cyclase (sAC) is indispensable for
sperm function and fertilization. Dev Biol 296: 353–362.
Additional Supporting Information may be found in the
online version of this article:
Figure S1 Treatment with 10 mM D9-THC reduces basal beat
frequency. Averaged flagellar beat frequency was determined
for wild type sperm that were bathed in HS medium contain-
ing 1 mM D9-THC (red circles and solid line) or 10 mM D9-THC
(red squares and dashed line) (THC) for up to 15 min. (n =
3–47 cells from 2–3 independent experiments). *P < 0.05
(Untreated vs. + 10 mM THC). #P < 0.05 (untreated vs. 1 mM
THC). Error bars represent the SEM and P-values were calcu-
lated by unpaired Student’s t-tests.
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting materials supplied
by the authors. Any queries (other than missing material)
should be directed to the corresponding author for the article.
Cannabinoid inhibition of sperm motility
British Journal of Pharmacology (2012) 165 2575–25832583