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Effects of delta-9-tetrahydrocannabinol, the primarypsychoactive cannabinoid in marijuana, on humansperm function in vitroLynne B. Whan, Ph.D.,a Mhairi C. L. West, Ph.D.,a Neil McClure, M.R.C.O.G.,a,b

and Sheena E. M. Lewis, Ph.D.a

a Obstetrics & Gynaecology, School of Medicine, Queen’s University, Belfast; and b Regional Fertility Centre, Royal-JubileeMaternity Service, Belfast, United Kingdom

Objective: To investigate effects of delta-9-tetrahydrocannabinol (THC) on human sperm function in vitro.Design: Laboratory analysis of sperm motility after exposure to THC using computer-assisted semen analysis andacrosome reaction by fluoroscein isothiocyanate–labeled peanut agglutinin staining.Setting: An assisted reproductive technology unit.Patient(s): Seventy-eight male patients.Intervention(s): Sperm were divided into 90% (the best fertilizing potential used in assisted conception) and 45%(the poorer subpopulation) fractions by density centrifugation and incubated with THC at concentrationsequivalent to therapeutic (0.032 �M) and recreational (0.32 and 4.8 �M) plasma levels at 37°C for 3 h.Main Outcome Measure(s): Sperm motility and spontaneous and induced acrosome reactions.Result(s): Percentage progressive motility was decreased dose dependently in the 90% fraction (by 2%–21%;P�.05; P�.001). The 45% fraction showed a greater decrease in percentage progressive motility (by 28% at0.032 �M; 56% at 4.8 �M; P�.004 and P�.01 res). Straight line velocity and the average path velocity also werereduced (by 10%, in the 90% LAYER) in both fractions. Spontaneous acrosome reactions were reduced in the90% (17% at 0.032 �M, 35% at 4.8 �M P�.004 and P�.001 resp) and more markedly in the 45% fractions(17%–35%; P�.001). When the acrosome reaction was artificially induced (90% fraction) by A23187, THC (4.8�M) resulted in a 57% inhibition (P�.001).Conclusion(s): The use of THC as a recreational drug may adversely affect male fertility. (Fertil Steril� 2006;85:653–60. ©2006 by American Society for Reproductive Medicine.)

Key Words: Cannabis, sperm, tetrahydrocannabinol, motility, acrosome reaction

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t least 14% of the reproductive population worldwide isffected by infertility (1). Male reproductive health is al-eady under threat from a range of environmental factorsncluding endocrine disrupters, toxic pollutants, ionizing ra-iation, and lifestyle factors such as sexually transmittednfections, alcoholism, smoking, and anabolic steroid use.urther hazards such as fast food, recreational drugs, andtress levels also may impair male fertility.

Marijuana is the most commonly used recreational drugorldwide (2– 4). The term “marijuana” refers to a mix-

ure of the leaves and flowering tops of the cannabis plantannabis sativa. Key findings from the British Crime Sur-ey (2001) revealed that around 3 million (11%) of 16–59-ear-olds used the drug, with a prevalence of 26.9% among6–24-year-olds (5). Marijuana contains at least 20 activeannabinoids with the primary psychoactive cannabinoideing delta-9-tetrahydocannabinol (THC) (3, 6, 7).

eceived November 10, 2004; revised and accepted August 10, 2005.resented in part at the British Fertility Society meeting, Cheltenham,England, April 2004.

eprint requests: Sheena E. M. Lewis, Obstetrics & Gynaecology, Insti-tute of Clinical Science, Grosvenor Road, Belfast BT12 6BJ, UK (FAX:

s44 2890328247; E-mail: [email protected]).

015-0282/06/$32.00oi:10.1016/j.fertnstert.2005.08.027 Copyright ©2006 American Soc

The Government reclassification of cannabis from a classto a class C drug in 2004 has led many people to believe

hat the drug is now legal and therefore not harmful to health.t is imperative that its effects on male fertility are estab-ished so that men can make an informed choice about usinghe drug if it is found to be a risk to reproductive health. Aecond reason to look at its effects on male fertility is thatHC is currently in phase 3 clinical trials as a therapy forultiple sclerosis (7–9). Already in the United States, THC

s prescribed under the name Dronabinol (synthetic THC inesame oil) and used clinically in the treatment of cachexiassociated with AIDS and to alleviate the nausea associatedith cancer chemotherapy (7). Its future therapeutic use may

nvolve many men of reproductive age.

The endocannabinoid system regulates many functions inhe human body, including reproduction. Two cannabinoideceptors have thus far been identified—CB1 and CB2 (3,0–13)—as well as a family of lipid signaling ligands; ofhich anandamide, or arachidonyl ethanoamide (AEA), is

he most abundant in reproductive fluids (14). The activesychotrophic ingredient of marijuana, tetrahydrocannabinolTHC), acting as an antagonist and binding to these canna-inoid receptors, may upset the endogenous cannabinoid
ignaling pathways and thus disrupt the normal functions of
653Fertility and Sterility� Vol. 85, No. 3, March 2006iety for Reproductive Medicine, Published by Elsevier Inc.

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eproduction (15). CB1 receptors have been found in ratestes and mouse vas deferens (16). Therefore, they may play

possible role in the control of spermatogenesis and maleertility (17–19).

Indeed, Schuel et al. have reported evidence for the pres-nce of cannabinoid receptors on sea urchin (20) and human21) sperm, and in the sea urchin these receptors have beenhown to mediate THC inhibition of the acrosome reaction22, 23). Gerard et al. (24) have identified and cloned auman cannabinoid receptor which is expressed in the testis.

Both CB1 and CB2 receptors have been found also inouse uterus (25). The endogenous cannabinoid system has

een suggested to mediate chemical communication betweenterus and embryo as evidenced by presence of CB1 andB2 receptors in embryos from one-cell through to blasto-yst stage. Owing to its inhibitory effect on embryo cellivision, anandamide may act as a negative signal for em-ryo development and implantation (26). This is evidencedy the higher and lower levels of anandamide synthetase andmide hydrolase, respectively, detected when the uterus iseast receptive to implantation. The uterus may, therefore,se anandamide to direct both the location and the timing ofmbryo implantation.

The small number of human studies investigating the effectsf marijuana on male reproductive function have producedaried results. Most of the original studies on the effects of THCn male reproduction were performed in the mid-1970s andarly 1980s. Some did not control for potential confounders,thers were uncontrolled (27), and all had small numbers andsed only basic technologies (27, 28).

Reported reductions in testosterone, sperm production,nd sperm motility and increased abnormalities in sperm29) were contradicted shortly thereafter by a larger wellontrolled study of chronic heavy users which failed to findny difference in plasma testosterone at study entry or afterhree weeks of heavy daily cannabis use (30). Decreased serumH levels also have been observed in males after smokingannabis (27, 31). Reduced sperm counts have been associatedith chronic high-dose cannabis smoking (27, 28).

Hembree et al. (28) reported that high-dose marijuana use8–20 cigarettes per day) was associated with a significantecrease in sperm concentration, which was preceded by aecrease in sperm motility and a reduction in normal spermorphology. Other studies on the effects of THC on motility

nd metabolism of human monkey, rabbit (28, 32–35), andull (36) sperm have produced varying results.

However, in many of these studies only subjective mea-ures of sperm motility using light microscopy were avail-ble (28, 37). Computer-assisted semen analysis (CASA)llows the automated analysis of the quality of sperm move-ent and, in particular, the following motility parameters to

e assessed: percentage progressive motility, straight-line
elocity (VSL), average path velocity (VAP), curvilinear s
654 Whan et al. Cannabis and sperm function

elocity (VCL), amplitude of lateral head displacementALH), beat cross frequency (BCF), and linearity (38).

In this study we aimed to determine the THC-inducedffects on sperm motility quantitatively by CASA and theffects of THC on sperm acrosome reaction—an exocytoticvent imperative for sperm penetration into the egg duringertilization (39). The acrosome reaction can be induced initro in capacitated sperm by incubation with a divalentation ionophore (40). These parameters are both known toave strong prognostic value as biomarkers of male fertility41, 42). To date, neither the effect of cannabis on theomputer-determined parameters of sperm function nor theffect on the acrosome reaction status has been reported foruman sperm.

ATERIALS AND METHODSubjectsemen samples (n � 78) were obtained after a recommended

wo to five days of sexual abstinence from caucasian men;ged 26–42 years, attending the Andrology Laboratory,oyal Maternity Hospital, Belfast, for infertility investiga-

ions. According to World Health Organization referencealues (38), their semen profiles were normozoospermicn � 13) or showed a combination of oligozoospermia (con-entration �20 � 106/mL; n � 18), asthenozoospermiaprogressive motility �25, n � 19; 25–49, n�32) and/orligoteratoasthenozoospermia (n � 12). Informed writtenonsent for participation was obtained. The project waspproved by the Queen’s University Belfast Research andthics Committee and was in accordance with the Declara-

ion of Helsinki as revised in 1983.

reparation of Semen Using Density Centrifugationemen was prepared using a discontinuous (90.0%-45.0%)ercoll gradient (42). One milliliter of 90% Percoll was

ayered under the 45% layer with a syringe. The semenample was layered on top of the 45% layer (max 1 mL). Theube was spun at 450g for 12 min. Two hundred millilitersas carefully removed from the bottom of the tube andlaced in a clean tube. Two hundred microliters of Biggershitten Whittingham buffer (BWW) (43) supplementedith 600 mg human albumen (albutein; Alpha TherapeuticK, Thetford, Norfolk, UK) was added to wash the sperm

nd subsequently spun for 6 min at 200g. Biggers Whittenhittingham buffer is a bicarbonate-buffered medium con-

aining albumen and is used for culturing the sperm to induceapacitation (38). The supernatant was removed and theesulting pellet resuspended in 200 �L BWW (90% frac-ion). Two hundred microliters was also carefully removedrom the middle layer of the tube and placed in a clean tube.wo hundred microliters of BWW was added to wash theperm and subsequently spun for 6 min at 200g. The super-atant was removed and the resulting pellet resuspended in00 �l BWW (45% fraction). Thus two subpopulations of
perm were prepared: that with the best fertilizing potential
Vol. 85, No. 3, March 2006

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s used in assisted conception treatments (90% fraction), andhe poorer subpopulation (45% fraction) similar to the spermrofile of men with male infertility (44).

ffect of THC on Sperm Motilityrepared sperm were divided into four aliquots. Three ali-uots were incubated with a soluble form of THC (suppliedn 95% ethanol; GW Pharmaceuticals, Salisbury, Wilts, UK)t concentrations of 0.032 (n � 23), 0.32 (n � 27), or 4.8n � 40) �M. The final concentration of ethanol to whichperm were exposed was 0.0095% and was shown not toave any toxic effects on sperm function. One aliquot wasncubated in the absence of THC (control) by adding theame volume of BWW. The test and control samples werencubated at 37°C for 3 h to induce capacitation (38), andperm motility was assessed using a Hamilton Thorne Inte-rated Visual Optical System sperm analyzer version 10.7Beverly, Massachusetts).

ffect of THC on the Acrosome Reaction, Observedy FITC-PNA Stainingrepared sperm were divided into four aliquots: three ali-uots were incubated with THC at concentrations of 0.032n � 24), 0.32 (n � 24), or 4.8 �M (n � 33). One aliquotas incubated in the absence of THC (control) by adding the

ame volume of BWW. After incubation of the test andontrol samples for 3 h at 37°C, sperm acrosome status wasssessed by fluoroscein isothiocyanate–labeled peanut ag-lutinin (FITC-PNA; Sigma Aldridge Co, Poole, Dorset,K) (41) staining and epifluorescence microscopy. Peanut

gglutinin, from Arachis hypogea, is specific for �-D-galac-ose residues and therefore binds to and labels the outercrosomal membrane (45).

Briefly, 20 �L of sperm suspension was spread over alean microscope slide and allowed to air dry. The smearas fixed in 95% ethanol for 5 min and allowed to air dry.he fixed slides were stained in FITC-PNA (600 �L FITC-NA in 15.4 mL reagent water in a foil-covered Coplin jar)or 15 min at ambient temperature. Slides were rinsed byipping in PBS twice before fixing for 15 min in parafor-aldehyde at ambient temperature. Slides were air dried,ounted, and stored in the dark until scoring. Between 100

nd 250 sperm were counted per slide and scored into threelasses for PNA labeling (41):

I: acrosome intact—whole acrosome labeling denotes anintact outer acrosomal membrane;

II: partially acrosome-reacted—patchy acrosome labelingsuggests a transition stage where the outer acrosomalmembrane is fenestrating;

II: acrosome-reacted—equatorial segment–only labelingdenotes a normally acrosome-reacted spermatozoa thathas lost the outer acrosomal membrane over the ante-rior cap of the acrosome but has retained the equatorial
segment of the acrosome intact.
ertility and Sterility�

ffect of THC on the Artificially Inducedcrosome Reactionhe effect of THC on the artificial induction of the acrosome

eaction by the ionophore A23187 was also investigated.gain, sperm were prepared as above and the 90% fractionas incubated with THC at a final concentration of 4.8 �M

1.5 �g/mL) for 3 h at 37°C (n � 22). The ionophore23187 (Sigma) was then added to a final concentration of.5 �M and incubated at 37°C for 15 min (38, 41). Spermcrosome status was assessed by FITC-PNA staining andpifluorescence microscopy (41).

tatistical Analysisotility and acrosome reaction results were analysed using

PSS version 11.0 (www.SPSS.com). Student t test foraired samples was employed to compare the results ofontrol and THC-treated samples.

FIGURE 1

(A) Effect of THC, at recreational and therapeuticconcentrations, on the numbers of progressivelymotile sperm in the 90% fraction. (B) Effect ofTHC, at recreational and therapeuticconcentrations, on the numbers of progressivelymotile sperm in the 45% fraction. Blackbar � control; yellow bar � 0.032 �M; red bar �0.32 �M; blue bar � 4.8 �M THC.

Whan. Cannabis and sperm function. Fertil Steril 2006.

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ESULTSffects of THC on Sperm Motilityhree concentrations of THC were chosen to mimic plasmaoncentrations obtained after recreational (46–48) (4.8 and.32 �M) or therapeutic (9, 46, 49) (0.032 �M) use of the drug.xposure of human sperm to THC produced a dose-dependent

eduction in the numbers of progressively motile sperm in the0% fraction (Fig. 1A). At 0.032 �M there was no significantifference in progressive motility (P�.80). At 0.32 �M aignificant decrease of 14% (P�.03) was noted in progressiveotility, and at 4.8 �M there was a 21% decrease (P�.001). In

he 45% fraction (Fig. 1B) there was a significant decrease inrogressive motility at 0.032 �M of 28% (P�.004), at 0.32 �Mf 23% (P�.04), and at 4.8 �M of 56% (P�.01).

The effects of THC on the quality of sperm motility werelso assessed using CASA (Fig. 2). The VAP was signifi-antly (decreased �10%; P�.03) in the 90% fraction at 4.8M. Similarly, VSL was significantly decreased (�10%;�.05). No significant difference in these parameters wasbserved in the 45% fraction. The ALH was found to beignificantly decreased (�9%; P�.05) by treatment of theperm in the 90% fraction with THC at 4.8 �M (data nothown). In the 45% fraction there was no significant differ-nce observed in the ALH (P�.06) at this higher concentra-ion. There was no significant effect of THC at a concentra-ion of 4.8 �M on the linearity of sperm in the 90% fractionP�.07). There was, however, a significant decrease�12%; P�.02) in linearity in sperm in the 45% fraction athis higher concentration. There was no significant effect ofHC at the lower concentrations (0.032 and 0.32�M) on anyf these motility parameters for both the 90% and the 45%ractions (data not shown).

FIGURE 2

Effect of THC, at the higher therapeuticconcentration, on the quality of sperm motility(VAP and VSL) in the 90% fraction. Blackbar � control; blue bar � 4.8 �M THC.

nWhan. Cannabis and sperm function. Fertil Steril 2006.


ffects of THC on the Spontaneous Acrosome Reaction

n the 90% fraction (Fig. 3A), sperm showed a significantnhibition of the spontaneous acrosome reaction (�15%;�.04) with THC at a concentration of 4.8 �M. No signif-

cant difference was observed in spontaneous acrosome re-ction in the 90% fraction at 0.32 �M (P�.38) or 0.032 �MP�.31). However, sperm in the 45% fraction (Fig. 3B)howed a significant dose-dependent inhibition in spontane-us acrosome reaction at all three concentrations (�35%P�.001), �25%, (P�.001) and �17% (P�.004) at 4.8,.32, and 0.032 �M, respectively). A similar trend wasbserved in the percentage partial acrosome reaction (data

FIGURE 3

(A) Effect of THC, at recreational and therapeuticconcentrations, on the acrosome reaction ofsperm in the 90% fraction. (B) Effect of THC, atrecreational and therapeutic concentrations, onthe acrosome reaction of sperm in the 45%fraction. Black bar � control; yellow bar � 0.032�M; red bar � 0.32 �M; blue bar � 4.8 �MTHC.


ot shown).

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ffects of THC on the Acrosome Reaction Artificiallynduced by A23187he addition of the ionophore A23187 to sperm in the 90%

raction resulted in a significant induction of the acrosomeeaction (�55%; P�.001). The concomitant addition ofHC (4.8 �M) substantially inhibited this A23187-in-uced acrosome reaction (�57%; P�.001; Fig. 4).

ISCUSSIONuman cannabinoid receptors and their endogenous ligands,

ollectively known as the endocannabinoid system, haveumerous physiologic functions at both organ and cellularevels (49). Two subtypes of cannabinoid receptors haveeen identified, CB1 and CB2 (50, 51). The CB1 receptor isresent in the nervous system and in peripheral tissues. Inontrast, the CB2 receptor is expressed primarily on T-cellsf the immune system (52, 53). Both endogenous and anver-increasing range of exogenous agonists exist for botheceptors (16). Humans have a functional endogenous can-abinoid system throughout their reproductive tract as well,ncluding the ovary, endometrium, testis, epididymis, pros-ate, and sperm. The endogenous agonists AEA, palmi-oylethanolamide, and oleoylethanolamide have now beeneported to occur naturally in reproductive fluids (14).

In females, cannabinoid receptors have already been re-orted to influence the menstrual cycle, embryo develop-ent, implantation (26, 54), and maintenance of pregnancy

nd lactation (2, 55). In contrast, there is a paucity ofnformation on the role of cannabinoid receptors in maleertility. However, recent research has given indirect evi-ence for the presence of cannabinoid receptors on humanperm similar to those found in the brain by showing bindingf labeled agonists (20, 21). Information about the receptor

FIGURE 4

Effect of THC, at the higher recreationalconcentration, on the artificially induced acrosomereaction of sperm in the 90% fraction.


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The effects of THC on sperm function are important forwo reasons: firstly, because of its increasing use as a rec-eational drug in society, and, secondly, because it has theotential to be a significant disrupter of this ubiquitoushysiologic system, given that it is a strong exogenousgonist for the CB1 receptor (49). In the present study, weetermined the effects of THC on two subpopulations ofperm—the 90% and the 45% fractions—and found thatHC reduced the number of progressively motile sperm inoth fractions, although the 45% fraction exhibited moreamage than sperm in the 90% fraction. This subpopulations inferior in morphology, motility, and DNA integrity andppears to be more susceptible to damage from exogenousssault. In support of this suggestion, reductions in theumbers of progressively motile sperm in the 45% fractionere caused by the lower therapeutic as well as higher

ecreational concentrations of the drug, whereas the higherecreational dose was necessary to impair the function of thetronger 90% subpopulation (Figs. 1 and 3).

Prior to the present study, our knowledge of the effects ofHC on sperm function has been largely due to the work ofchuel’s group in Buffalo, New York (20, 23, 47). Schuelorked initially with sea urchins to determine the direct

ffects of THC on sperm fertilizing potential (47). Sea urchinggs are particularly useful because they are readily avail-ble for research, unlike human eggs which are tightly reg-lated by the Human Fertilization and Embryology Authority

n the United Kingdom. In addition, sea urchin fertilization takeslace in water so eggs can be cultured and observed in theiratural environment within the laboratory. Schuel’s grouphowed that THC altered motility in sea urchin sperm (23,7, 56). They also studied in vitro effects of exogenousannabinoid agonists (AM-356 and THC) on human spermapacitation and hemi-zona binding during 1–6 h incubationnd evaluated the effects of AM-356 on hyperactivatedperm motility (21). Although swimming behavior was af-ected by AM-356, no change in the percentage of motileperm was observed. However, they did not determine theffects of THC on human sperm motility.

Sperm motility is energized by adenosine triphosphateATP) production in the mitochondria by glycolysis andxidative phosphorylation. Inhibition of glycolysis and therebs cycle may result in the depletion of ATP owing to aecrease in the supply of reduced nicotinamide-adenineinucleotide (60). Our group has previously observed that aeduction in mitochondrial function, reflected in alteredembrane potential, is associated with a reduction in motil-

ty (44). Delta-9-tetrahydrocannabinol has been shown toisrupt mitochondrial function in other cell types by alteringitochondrial membrane potential (57). This, in turn, un-

ouples electron transport, resulting in an inhibition of theynthesis of ATP and explains the decrease in sperm motilitybserved in the present study. Sarafian et al. (57) havereviously shown that a decrease in mitochondrial mem-rane potential occurred after only 1 h exposure to THC. The
ffects were persistent, with function remaining diminished
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or 30 h after THC exposure. This disruption of mitochon-rial membrane potential and decrease in ATP productionay affect further cell functions, including cell signaling

athways and membrane ion transport, which may also beertinent in regulating sperm acrosome reaction, which alsoas impaired by exposure to THC in the present study

Fig. 3).

Impaired sperm motility may also be due to deficientxidative phosphorylation as a result of damaged or absentroteins in the electron transport chain subunits caused byitochondrial deletions or base substitutions (58). In a pre-

ious study we reported that in low-density fractions, i.e., the5% fraction of poorly motile sperm, only a small proportionf sperm contained wild-type mitochondrial DNA (mtDNA)44). This low-density fraction also has a greater incidencef large-scale deletions, reflected in the significantly greaterize of mtDNA deletions, suggesting that sperm with goodotility also have better-quality mtDNA. Also of note, the

evels of mtDNA damage in sperm are much higher thanhose observed in somatic cells (59–61), suggesting thatperm may be more vulnerable to damage. Owing to the lackf histone proteins to protect the DNA against attack byeactive oxygen species (ROS), mitochondria have littlerotection against oxidative assault, so their DNA accumu-ates large numbers of mutations (62). The most immediateffect of ROS damage is in the mtDNA as it is continuallyxposed to a high steady-state level of ROS and free radicalsithin the mitochondrial matrix (63). It is also vulnerableecause of the mitochondria’s lack of repair mechanisms.e have shown previously that sperm with reduced motility

evels generate more ROS (42) and that they have lessntioxidant protection available to them (64). Therefore,hese sperm are doubly susceptible to damage (44). Sarafiant al. (65) reported that marijuana smoke containing THC ispotent source of cellular oxidative stress that could con-

ribute to cellular damage. Because the respiratory chain hashe capacity for ROS production, an up-regulation by THCay cause significant oxidative damage to mtDNA. Con-

ersely, it has been reported that cannabinoids have anntioxidant effect on cortical neurones (66). If this is true forther cell types, this antioxidant potential may actually pro-ect sperm from oxidative stress. Studies are needed toetermine whether cannabinoids act as pro-oxidants or anti-xidants under physiologic and recreational conditions.

The present study shows that THC alters another essentialperm function, that of the acrosome reaction, at higherecreational concentrations in the 90% fraction. The inhibi-ion of the acrosome reaction was greater in the 45% fractiont both recreational and therapeutic concentrations, suggest-ng again that THC has more pronounced effects on spermhat are of poorer quality. In the present study, the low levelsf acrosome reaction observed in the controls are consistentith previous reports (2%–7%) (21, 39, 67, 68). Schuel et al.

20) reported that THC reduced sperm hemi-zona binding inea urchins by inhibiting the acrosome reaction. They
howed that THC inhibited the acrosome reaction although r

t a much higher concentration of THC (25 �M) than wasmployed in the present study. The group (21) also assessedhe effects of AM-356 (1.0 and 2.5 nmol/L) and THC (0.15nd 1.5 �M) on acrosome status in human sperm and foundhat both blocked the acrosome reaction in a concentration-ependant manner.

A report from Chang et al. (22) stated that THC reduceshe fertilizing capacity of sea urchin sperm by blocking thecrosome reaction that normally is stimulated by a specificigand in the egg’s jelly coat. Our study supports this work,uggesting that THC may impair fertilization by modulatingcrosome reactions at recreational concentrations. One func-ion of endocannabinoid receptors appears to be in regulatingecond messenger systems such as Ca2� signalling (50, 14,9), including the mechanism by which the acrosome reac-ion occurs. THC binds to cannabinoid receptors, which arepecific guanine nucleotide–binding (G) protein–coupledransmembrane receptors and are an integral part of the lipidilayer in sperm. THC is a lipophilic molecule that perme-tes the lipid bilayer and disorders its fluidity (70), thusausing a persistent volume change of the receptor molecule.

minimal change in the receptor molecule structure isssociated with major alteration in signal transduction (70),lthough, because the primary signal transduction targets forannabinoids in sperm have not yet been identified, this ispeculative. Binding of THC to cannabinoid receptors onperm may affect signal transduction by inhibition of ade-ylate cyclase, which in turn may result in inhibition ofyclic adenosine monophosphate (cAMP) production. Sub-equently, this may reduce cAMP-dependent protein kinase

levels, decreasing phosphorylation of Ca2� channels (70)nd preventing calcium-gated ion channels from opening.his action of calcium-gated ion channels, the influx ofa2�, and the efflux of H� ions increase intracellular pHhich is essential to triggering the acrosome reaction

71, 72).

The acrosome reaction can be induced in vitro in capac-tated sperm by incubation with A23187, a Ca2�/H� iono-hore (40). The present study shows that THC causes aarked inhibition (�57%) in acrosome reactions that are

rtificially induced by the ionophore A23187. This is ingreement with Schuel et al. (56) although to a lesser extent20%–30%). It has been reported that A23187 bypasses theeceptor activation–signal transduction components of thecrosome reaction (73). Indeed, Schuel et al. (56) reportedhat THC does not inhibit the acrosome reaction artificiallynduced by other ionophores such as ionomycin that inducehe acrosome reaction by directly opening Ca2� channels inhe cell membrane. This would suggest that in Schuel et al.’sork (23) and in the present study THC is inhibiting some

vent prior to the opening of Ca2� channels. It should beoted that A23187 is also known to act as an uncoupler ofxidative phosphorylation and inhibitor of mitochondrialTPase activity. Therefore, it may be the case that THC isreventing the elevation of intracellular calcium, either by
educing ATP production (as discussed earlier) necessary for
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a2� transport across the membrane or by reducing theompetence of receptors on sperm to initiate the secondessenger pathways necessary for initiation of the acrosome

eaction.

In conclusion, the use of THC as a recreational drug maympair crucial sperm functions and adversely affect maleertility, especially in those who are already on the border-ine of infertility. Further studies are ongoing to determine ifhe impairment of sperm motility is due to damage of spermitochondria at a genetic or functional level.

cknowledgements: The authors would like to acknowledge the donation ofoluble THC by GW Pharmaceuticals, UK, for this study. Thanks are alsoue to Mrs. Margaret Kennedy for preparation of semen samples in thetudy.

EFERENCES1. Fishel S, Dowell K, Thornton S. Reproductive possibilities for infertile

couples: present and future. In: Bentley GR, Mascie-Taylor CGN,Infertility in the modern world. Cambridge (UK): Cambridge Univer-sity Press; 2000. p. 17–45.

2. Park B, McPartland JM, Glass M. Cannabis, cannabinoids and repro-duction. Prostaglandins Leukot Essent Fatty Acids 2004;70:189–97.

3. Sharpe P, Smith G. Cannabis: Time for scientific evaluation of thisancient remedy. Anesth Analg 2000;90:237–40.

4. Hall W Solowij N. Adverse effects of cannabis. Lancet 1998;352:1611–16.

5. Bolling K, Clemens S, Phelps A, Smith P. 2001 British Crime Surveyin England and Wales Technical Report. London: BMRB; 2002.

6. Ashton H. Pharmacology and effects of cannabis: a brief review. Br JPsychiatry 2001;178:101–6.

7. Royal Society and the Academy of Medical Sciences. Use of cannabisand its derivatives for medical and recreational purposes. Interdisc SciRev 1998;23:325–28.

8. Consroe P, Musty R, Rein J, Tillery W, Pertwee RG. The perceivedeffects of smoked cannabis on patients with multiple sclerosis. EurNeurol 1997;38:44–8.

9. Zajicek J, Fox P, Sanders H, Wright D, Vickery J, Nunn A, et al.Cannabinoids for treatment of spasticity and other symptoms related tomultiple sclerosis (CAMS study): multicentre randomised placebo-controlled trial. Lancet 2003;362:1517–26.

0. Hirst RA, Lambert DG, Notcutt WG. Pharmacology and potentialtherapeutic uses of cannabis. Br J Anaesth 1998;81:77–84.

1. Felder CC, Glass M. Cannabinoid receptors and their endogenousagonists. Annu Rev Pharmacol Toxicol 1998;38:179–200.

2. Pertwee RG. Cannabis and cannabinoids: pharmacology and rationalefor clinical use. Pharm Sci 1997;3:539–45.

3. Pertwee RG. Pharmacology of cannabinoid CB1 and CB2 receptors.Pharmacol Ther 1997;74:129–80.

4. Schuel H, Burkman LJ, Lippes J, Crickard K, Forester E, Piomelli D,et al. N-Acylethanolamines in human reproductive fluids. Chem PhysLipids 2002;121:211–27.

5. Salzet M, Breton C, Bisogno T, Di Marzo V. Comparative biology ofthe endocannabinoid system. Possible role in the immune response. EurJ Biochem 2000;267:4917–27.

6. Pertwee RG, Ross RA, Craib SJ, Thomas A. Cannabidiol antagonizescannabinoid receptor agonists and noradrenaline in the mouse vasdeferens. Eur J Pharmacol 2002;456:99–106.

7. Gerard C, Mollereau C, Vassart G, Parmentier M. Nucleotide sequenceof a human cannabinoid receptor cDNA. Nucleic Acids Res 1990;18:7142.

8. Sugiura T, Kondo S, Sukagawa A, Tonegawa T, Nakane S, YamashitaA, et al. Enzymatic synthesis of anandamide, an endogenous cannabi-
noid receptor ligand, through N-acylphosphatidylethanolamine path-

way in testis: involvement of Ca(2�)-dependent transacylase andphosphodiesterase activities. Biochem Biophys Res Commun1996;218:113–7.

9. Pertwee RG, Joe-Adigwe G, Hawksworth GM. Further evidence for thepresence of cannabinoid CB1 receptors in mouse vas deferens. EurJ Pharmacol 1996;296:169–72.

0. Schuel H, Goldstein E, Mechoulam R, Zimmerman AM, ZimmermanS. Anandamide (arachidonylethanolamide), a brain cannabinoid re-ceptor agonist, reduces sperm fertilizing capacity in sea urchins byinhibiting the acrosome reaction. Proc Natl Acad Sci U S A 1994;91:7678 – 82.

1. Schuel H, Burkman LJ, Lippes J, Crickard K, Mahony MC, GiuffridaA, et al. Evidence that anandamide-signaling regulates human spermfunctions required for fertilization. Mol Reprod Dev 2002;63:376–87.

2. Chang MC, Berkery D, Schuel R, Laychock SG, Zimmerman AM,Zimmerman S, et al. Evidence for a cannabinoid receptor in sea urchinsperm and its role in blockade of the acrosome reaction. Mol ReprodDev 1993;36:507–16.

3. Schuel H, Chang MC, Berkery D, Schuel R, Zimmerman AM, Zim-merman S. Cannabinoids inhibit fertilization in sea urchins by reducingthe fertilizing capacity of sperm. Pharmacol Biochem Behav 1991;40:609–15.

4. Gerard C, Mollereau C, Vassart G, Parmentier M. Molecular cloning ofa human cannabinoid receptor which is also expressed in testis. Bio-chem J 1991;279:129–34.

5. Das SK, Paria BC, Chakraborty I, Dey SK. Cannabinoid ligand-receptor signaling in the mouse uterus. Proc Natl Acad Sci U S A1995;92:4332–36.

6. Paria BC, Das SK, Dey SK. The preimplantation mouse embryo is atarget for cannabinoid ligand-receptor signaling. Proc Natl Acad SciU S A 1995;92:9460–64.

7. Kolodny RC, Masters WH, Kolodner RM, Toro G. Depression ofplasma testosterone levels after chronic intensive marihuana use.N Engl J Med 1974;290:872–4.

8. Hembree WC, Nahas GG, Zeidenberg P, Huang HFS. Changes inhuman spermatozoa associated with high dose marijuana smoking. In:Nahas GG, Paron M, Marihuana: biological effects. Oxford (UK):Pergamon Press;1980. p. 429–39.

9. Kolodny RC, Lessin P, Toro G, Masters WH, Cohen J. Depressionof plasma testosterone with acute administration. In: Braude MC,Szara S, The pharmacology of marijuana. New York: Raven Press;1974. p.217–25.

0. Mendelson JH, Kuehnle J, Ellingboe J, Babor TF. Plasma testosteronelevels before, during and after chronic marihuana smoking. N EnglJ Med 1974;291:1051–5.

1. Cone EJ, Johnston RE, Moore JD, Roache JD. Acute effects of smokingmarijuana on hormones, subjective effects and performance in malehuman subjects. Pharmacol Biochem Behav 1976;24:1749–54.

2. Hong CY, Chaput de Saintonge DM, Turner P. Delta 9-tetrahydrocannabinolinhibits human sperm motility. J Pharm Pharmacol 1981;33:746–7.

3. Hong CY, Chaput de Saintonge DM, Turner P, Fairbairn JW. Compar-ison of the inhibitory action of delta-9-tetrahydrocannabinol and petro-leum spirit extract of herbal cannabis on human sperm motility. HumToxicol 1982;1:151–4.

4. Perez LE, Smith CG, Asch RH. Delta 9-tetrahydrocannabinol inhibitsfructose utilization and motility in human, rhesus monkey, and rabbitsperm in vitro. Fertil Steril 1981;35:703–5.

5. Husain S. THC effects on fructose utilization and sperm motility. FertilSteril 1982;37:448.

6. Shahar A, Bino T. In vitro effects of delta9 tetrahydrocannabinol (THC)on bull sperm. Biochem Pharmacol 1974;23:1341–2.

7. Hembree WC, Zeidenberg P, Nahas GG. Marihuana’s effects on humangonadal function. In: Nahas GG, Marijuana: chemistry, biochemistryand cellular effects. New York: Springer Verlag; 1976. p. 521–32.

8. World Health Organization. WHO laboratory manual for the examina-tion of human semen and sperm-cervical mucus interaction. Cambridge
(UK): Cambridge University Press; 1999.
659

3

4

4

4

4

4

4

4

4

4

4

5

5

5

5

5

5

5

5

5

5

6

6

6

6

6

6

6

6

6

6

7

7

7

7

9. Plachot M, Mandelbaum J, Junca AM. Acrosome reaction of humansperm used for in vitro fertilization. Fertil Steril 1984;42:418–23.

0. Breitbart H, Naor Z. Protein kinases in mammalian sperm capacitationand the acrosome reaction. Rev Reprod 1999;4:151–9.

1. Mortimer D. Sperm fertilizing ability testing. In: Mortimer D, Practical labo-ratory andrology. Oxford: Oxford University Press; 1994. p. 199–240.

2. Donnelly ET, Lewis SE, McNally JA, Thompson W. In vitro fertiliza-tion and pregnancy rates: the influence of sperm motility and morphol-ogy on IVF outcome. Fertil Steril 1998;70:305–14.

3. Biggers JD, Whitten WK, Whittingham DG. The culture of mouseembryos in vitro. In: Daniel JD, Methods in mammalian embryology.San Francisco: WH Freeman; 1971. p. 86–116.

4. O’Connell M, McClure N, Powell LA, Steele EK, Lewis SE. Differ-ences in mitochondrial and nuclear DNA status of high-density andlow-density sperm fractions after density centrifugation preparation.Fertil Steril 2003;79(Suppl 1):754–62.

5. Mortimer D, Curtis EF, Miller RG. Specific labelling by peanut agglu-tinin of the outer acrosomal membrane of the human spermatozoon.J Reprod Fertil 1987;81:127.

6. Huestis MA, Sampson AH, Holicky BJ, Henningfield JE, Cone EJ.Characterisation of the absorption phase of marjuana smoking. ClinPharmacol Ther 1992;52:31–41.

7. Schuel H, Schuel R, Zimmerman AM, Zimmerman S. Cannabinoidsreduce fertility of sea urchin sperm. Biochem Cell Biol 1987;65:130–6.

8. Chang MC, Berkery D, Laychock SG, Schuel H. Reduction of the fertilizingcapacity of sea urchin sperm by cannabinoids derived from marihuana. III.Activation of phospholipase A2 in sperm homogenate by delta 9-tetrahydro-cannabinol. Biochem Pharmacol 1991;42:899–904.

9. Pertwee RG. Cannabinoid receptors and pain. Prog Neurobiol 2001;63:569–611.

0. Matsuda LA. Molecular aspects of cannabinoid receptors. Crit RevNeurobiol 1997;11:143–66.

1. Pertwee RG. Evidence for the presence of CB1 cannabinoid receptorson peripheral neurones and for the existence of neuronal nonCB1cannabinoid receptors. Life Sci 1999;65:597–605.

2. Munro S, Thomas KL, Abu-Shaar M. Molecular characterization of aperipheral receptor for cannabinoids. Nature 1993;365:61–5.

3. Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI. Struc-ture of a cannabinoid receptor and functional expression of the clonedcDNA. Nature 1990;346:561–4.

4. Schmid PC, Paria BC, Krebsbach RJ, Schmid HH, Dey SK. Changes inanandamide levels in mouse uterus are associated with uterine recep-tivity for embryo implantation. Proc Natl Acad Sci U S A 1997;94:4188–92.

5. Asch RH, Smith CG. Effects of delta 9-THC, the principal psychoactivecomponent of marijuana, during pregnancy in the rhesus monkey.J Reprod Med 1986;31:1071–81.

6. Schuel H, Berkery D, Schuel R, Chang MC, Zimmerman AM, Zim-merman S. Reduction of the fertilizing capacity of sea urchin sperm bycannabinoids derived from marihuana. I. Inhibition of the acrosomereaction induced by egg jelly. Mol Reprod Dev 1991;29:51–9.

7. Sarafian TA, Kouyoumjian S, Khoshaghideh F, Tashkin DP, Roth MD.


Delta 9-tetrahydrocannabinol disrupts mitochondrial function and cellenergetics. Am J Physiol Lung Cell Mol Physiol 2003;284:L298–306.

8. Cummins JM, Jequier AM, Martin R, Mehmet D, Goldblatt J. Semenlevels of mitochondrial DNA deletions in men attending an infertilityclinic do not correlate with phenotype. Int J Androl 1998;21:47–52.

9. Hayakawa M, Hattori K, Sugiyama S, Ozawa T. Age-associated oxy-gen damage and mutations in mitochondrial DNA in human hearts.Biochem Biophys Res Commun 1992;189:979–85.

0. Corral-Debrinski M, Horton T, Lott MT, Shoffner JM, McKee AC,Beal MF, et al. Marked changes in mitochondrial DNA deletion levelsin Alzheimer brains. Genomics 1994;23:471–76.

1. Fromenty B, Carrozzo R, Shanske S, Schon EA. High proportions ofmtDNA duplications in patients with Kearns-Sayre syndrome occur inthe heart. Am J Med Genet 1997;71:443–52.

2. Wiseman H, Halliwell B. Damage to DNA by reactive oxygen andnitrogen species: role in inflammatory disease and progression to can-cer. Biochem J 1996;313:17–29.

3. Wei YH, Lu CY, Lee HC, Pang CY, Ma YS. Oxidative damage andmutation to mitochondrial DNA and age-dependent decline of mito-chondrial respiratory function. Ann N Y Acad Sci 1998;854:155–70.

4. Lewis SE, Boyle PM, McKinney KA, Young IS, Thompson W. Totalantioxidant capacity of seminal plasma is different in fertile and infer-tile men. Fertil Steril 1995;64:868–70.

5. Sarafian TA, Magallanes JAM, Shau H, Tashkin DP, Roth MD. Oxi-dative stress produced by marijuana smoke. An adverse effect enhancedby cannabinoids. Am J Respir Cell Mol Biol 1999;20:1286–93.

6. Hampson AJ, Grimaldi M, Axelrod J, Wink D. Cannabidiol and(�)delta9-tetrahydrocannabinol are neuroprotective antioxidants. ProcNatl Acad Sci U S A 1998;95:8268–73.

7. Boyers SP, Tarlatzis BC, Stronk JN, DeCherney AH. Fertilizationand cleavage rates of heparin-exposed human oocytes in vitro, andthe effect of heparin on the acrosome reaction. Fertil Steril 1987;48:628 –32.

8. Yudin AI, Gottlieb W, Meizel S. Ultrastructural studies of the earlyevents of the human sperm acrosome reaction as initiated by humanfollicular fluid. Gamete Res 1988;20:11–24.

9. Zoratti C, Kipmen-Korgun D, Osibow K, Malli R, Graier WF. Anan-damide initiates Ca(2�) signaling via CB2 receptor linked to phospho-lipase C in calf pulmonary endothelial cells. Br J Pharmacol 2003;140:1351–62.

0. Nahas GG, Harvey DJ, Sutin KM. Psychoactive cannabinoids andmembrane signaling. Hum Psychopharmacol 2000;15:535–49.

1. Aitken RJ, Ross A, Hargreave T, Richardson D, Best F. Analysis ofhuman sperm function following exposure to the ionophore A23187.Comparison of normospermic and oligozoospermic men. J Androl1984;5:321–29.

2. Aitken RJ, Buckingham DW, Fang HG. Analysis of the responses ofhuman spermatozoa to A23187 employing a novel technique for as-sessing the acrosome reaction. J Androl 1993;14:132–41.

3. Aitken RJ. The extragenomic action of progesterone on human sper-
matozoa. Hum Reprod 1997;12(Suppl):38–42.
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