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Jekyll and Hyde: Two Faces of Cannabinoid Signaling in Male and Female Fertility

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Mammalian reproduction is a complicated process designed to diversify and strengthen the genetic complement of the offspring and to safeguard regulatory systems at various steps for propagating procreation. An emerging concept in mammalian reproduction is the role of endocannabinoids, a group of endogenously produced lipid mediators, that bind to and activate cannabinoid receptors. Although adverse effects of cannabinoids on fertility have been implicated for years, the mechanisms by which they exert these effects were not clearly understood. With the identification of cannabinoid receptors, endocannabinoid ligands, their key synthetic and hydrolytic pathways, and the generation of mouse models missing cannabinoid receptors, a wealth of information on the significance of cannabinoid/endocannabinoid signaling in spermatogenesis, fertilization, preimplantation embryo development, implantation, and postimplantation embryonic growth has been generated. This review focuses on various aspects of the endocannabinoid system in male and female fertility. It is hoped that a deeper insight would lead to potential clinical applications of the endocannabinoid signaling as a target for correcting infertility and improving reproductive health in humans.
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Jekyll and Hyde: Two Faces of Cannabinoid Signaling in
Male and Female Fertility
Haibin Wang, Sudhansu K. Dey, and Mauro Maccarrone
Departments of Pediatrics, Cell and Developmental Biology, and Pharmacology (H.W., S.K.D.), Division of Reproductive and
Developmental Biology, Vanderbilt University Medical Center, Nashville, Tennessee 37232; Department of Biomedical
Sciences (M.M.), University of Teramo, Teramo 64100, Italy; and Mondino-Tor Vergata Neuropharmacology Center (M.M.),
University of Rome Tor Vergata, 00133 Rome, Italy
Mammalian reproduction is a complicated process designed
to diversify and strengthen the genetic complement of the
offspring and to safeguard regulatory systems at various steps
for propagating procreation. An emerging concept in mam-
malian reproduction is the role of endocannabinoids, a group
of endogenously produced lipid mediators, that bind to and
activate cannabinoid receptors. Although adverse effects of
cannabinoids on fertility have been implicated for years, the
mechanisms by which they exert these effects were not clearly
understood. With the identification of cannabinoid receptors,
endocannabinoid ligands, their key synthetic and hydrolytic
pathways, and the generation of mouse models missing can-
nabinoid receptors, a wealth of information on the signifi-
cance of cannabinoid/endocannabinoid signaling in spermat-
ogenesis, fertilization, preimplantation embryo development,
implantation, and postimplantation embryonic growth has
been generated. This review focuses on various aspects of the
endocannabinoid system in male and female fertility. It is
hoped that a deeper insight would lead to potential clinical
applications of the endocannabinoid signaling as a target for
correcting infertility and improving reproductive health in
humans. (Endocrine Reviews 27: 427– 448, 2006)
I.
Lipid Signaling in Reproduction
II. The Endocannabinoid System
A. Introduction
B. Metabolic routes
C. Molecular targets and signaling pathways
III. Endocannabinoids and Male Fertility
A. Introduction
B. The endocannabinoid system in Sertoli cells
C. The endocannabinoid system in sperm
IV. Endocannabinoids and Female Fertility: Embryo
Implantation
A. Introduction
B. Endocannabinoids and preimplantation embryo
development
C. Endocannabinoids and oviductal embryo transport
D. Biphasic endocannabinoid sensor in blastocyst
implantation
V. Endocannabinoids and Female Fertility: Immunoregulation
A. Th1/Th2 cytokines and fertility
B. The endocannabinoid system in lymphocytes of preg-
nant women
C. FAAH as a molecular integrator of fertility signals
VI. Endocannabinoids and Clinical Implications
VII. Conclusions and Future Direction
I. Lipid Signaling in Reproduction
S
EXUAL PROCREATION IS initiated by interactions be-
tween a sperm and an egg leading to fertilization (1–4).
The fertilized egg (embryo) undergoes several mitotic cell
divisions, ultimately producing the blastocyst with two dis-
tinct cell types: the inner cell mass (ICM) and the trophec-
toderm (5–9). The nurturing of an offspring within the body
and production of a live birth is an enduring task, requiring
safeguard regulatory systems at various critical steps. De-
spite success in producing embryos and initiating embryonic
development outside the womb by in vitro fertilization and
embryo transfer, there is still a significant knowledge gap in
understanding the mechanisms by which a successful preg-
nancy is achieved. A deeper insight into these processes will
help to generate new ideas and concepts for improving fer-
tility and pregnancy-associated health issues in humans. It is
difficult to define the hierarchical landscape of the molecular
pathways during human pregnancy, because of experimen-
tal difficulties and ethical restrictions on research with hu-
man embryos. It is hoped that experiments on mice and other
animal models that bear certain reproductive similarities
with humans combined with those feasible experiments in
humans would generate meaningful information to address
this critical issue. Over the past several years, molecular and
genetic studies have provided evidence that lipid mediators
serve as important signaling molecules in coordinating a
series of events during early pregnancy.
First Published Online May 8, 2006
Abbreviations: AA, Arachidonic acid; AdR, adrenergic receptor;
AEA, N-arachidonoylethanolamine (also known as anandamide); 2-AG,
2-arachidonoylglycerol; AMT, AEA membrane transporter; CB1, brain-
type cannabinoid receptor; CB2, spleen-type cannabinoid receptor;
COX, cyclooxygenase; cPLA
2
, cytosolic PLA
2
; DAG, diacylglycerol;
DAGL, DAG lipase; E
2
,17
-estradiol; FAAH, fatty acid amide hydro-
lase; ICM, inner cell mass; INF-
, interferon-
; LIF, leukemia inhibitory
factor; LPA, lysophosphatidic acid; MAGL, monoacylglycerol lipase;
NAPE, N-acylphosphatidylethanolamine; NAT, N-acyltransferase; NK,
natural killer; NO, nitric oxide; P
4
, progesterone; PG, prostaglandin; PL,
phospholipase; Th1, type 1 T-helper; Th2, type 2 T-helper; THC, 9-
tetrahydrocannabinol; TRPV1, transient receptor potential vanilloid 1
(vanilloid receptor); ZP, zona pellucida.
Endocrine Reviews is published by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
0163-769X/06/$20.00/0 Endocrine Reviews 27(5):427–448
Printed in U.S.A. Copyright © 2006 by The Endocrine Society
doi: 10.1210/er.2006-0006
427
at Semmelweis Univ Medicine Central Library on November 19, 2008 edrv.endojournals.orgDownloaded from
Under pathophysiological conditions when a cell is acti-
vated in response to a stimulus, membrane phospholipids
generate numerous lipid-signaling molecules, such as eico-
sanoids and lysophospholipids. Prostaglandins (PGs), one of
the major group of eicosanoid lipid mediators, are generated
from arachidonic acid (AA), which is released from mem-
brane phospholipids by phospholipase (PL)A
2
. AA thus re-
leased is transformed by cyclooxygenases (COXs) to PGH,
which is then converted to various PGs by specific PG syn-
thases (10). Distinct expression profiles of cytosolic PLA
2
(cPLA
2
), COX-1, and COX-2 in the ovary and uterus at
different stages of pregnancy implicate their differential
functions (11–14). In mice, COX-1-derived PGF
2
,asalu-
teolytic hormone acting on the corpus luteum, is critical for
the onset of parturition (15–18), whereas PGI
2
and PGE
2
generated by COX-2 are essential for ovulation, fertilization,
implantation, and decidualization (11, 13, 19 –22). The role of
PG during pregnancy is further illustrated by poor fertility,
resulting from deferred implantation, in mice lacking
cPLA
2
(12). Collectively, these studies in mice establish the
importance of lipid signaling through the cPLA
2
-COX axis
during early pregnancy (23, 24). Observations of COX-2 ex-
pression in the periovulatory ovary and uterus during im-
plantation as well as delayed follicular rupture and increased
incidence of miscarriages upon pharmacological inhibition
of COX further suggest that cPLA
2
-COX-derived PG sig-
naling is also operative in human ovulation and pregnancy
maintenance (25–29). Moreover, PGE
2
and PGF
2
are also
thought to play important roles in human parturition by
directly acting on the myometrium (30, 31).
Another example of the critical role of lipid signaling in
reproduction is the influence of lysophosphatidic acid (LPA),
a small lipid molecule belonging to the lysophospholipid
group. LPA influences a range of processes through its cell-
surface G protein-coupled receptors, LPA
1–4
(32). A recent
study in mice shows that LPA
3
is expressed in the uterine
luminal epithelium, with peak expression occurring during
the periimplantation period. Its expression overlaps with
cPLA
2
and COX-2 at the site of blastocyst implantation (33).
More importantly, mice missing LPA
3
exhibit remarkably
similar defects as cPLA
2
-deficient mice, such as deferred
on-time implantation, retarded fetal development, embryo
crowding, and sharing of one placenta by several embryos
(12, 33). This study adds another genetic link between lipid
signaling and female fertility. Restoration of on-time implan-
tation in LPA
3
-deficient females by PG supplementation fur-
ther suggests a fundamental interaction between LPA-LPA
3
and cPLA
2
-COX-2-PG signaling pathways (33). These find-
ings establish a new concept that a short delay in the attach-
ment of blastocysts to the uterine surface during early preg-
nancy adversely affects later developmental processes (24,
34, 35).
Increasing evidence points toward the pathophysiological
significance of endocannabinoids, another group of bioactive
lipid-signaling molecules, in both female and male fertility.
These endogenous cannabinoid ligands mimic the action of
natural cannabis compound
9
-tetrahydrocannabinol (THC)
in many aspects of central and peripheral functions, includ-
ing reproductive events (23, 36 –51). THC is thought to ac-
count for the majority of the reproductive hazards in mar-
ijuana users (Table 1). For example, chronic marijuana use is
associated with decreased plasma testosterone levels (52),
reduced sperm counts, and impotency in men (52–56). In
women, the chronic use of marijuana is often associated with
fetal abnormalities and early pregnancy termination (57–66).
In addition, early studies indicate that embryotoxicity and
specific teratological malformation in animal models are cor-
related with exposure to natural cannabis extracts during
pregnancy (67–73). With the characterization of cannabinoid
receptors and their ligands endocannabinoids, the key syn-
thetic and hydrolytic pathways of endocannabinoids, and
the generation of gene mutation mouse models, a large body
of molecular, genetic, and physiological evidence has been
generated that supports key roles of cannabinoid ligand-
receptor signaling in spermatogenesis, fertilization, preim-
plantation embryo development, implantation, and postim-
plantation embryonic growth. This review focuses on the
roles of the endocannabinoid system in male and female
fertility. A better understanding of this field will help in
developing strategies for potential clinical applications of the
endocannabinoid-targeted drugs as the next generation of
therapeutics to treat human infertility.
II. The Endocannabinoid System
A. Introduction
Two main molecular targets of THC (Fig. 1), the psycho-
active component of Cannabis sativa, are brain-type (CB1) and
spleen-type (CB2) cannabinoid receptors (74 –78). Over the
last few years, a number of endogenous ligands for CB re-
ceptors have been identified; they are collectively called en-
docannabinoids. They are amides, esters, and ethers of long-
chain polyunsaturated fatty acids, isolated from brain and
peripheral tissues (79 82). Two arachidonate derivatives,
N-arachidonoylethanolamine (AEA, know as anandamide)
and 2-arachidonoylglycerol (2-AG) (Fig. 1), are the endocan-
nabinoids whose biological activity has been best character-
ized to date (75, 81, 83–85). Also 2-AG-ether (noladin ether),
an ether-type endocannabinoid (Fig. 1), is now included in
the cohort of these lipid mediators (86), but its actual phys-
iological relevance is still debatable (87). More recently, O-
arachidonoylethanolamine (virodhamine), an “inverted
AEA” (Fig. 1), has been identified and shown to behave as
a partial agonist and a full agonist for CB1 and CB2, respec-
tively (88). In addition, N-oleoylethanolamine, N-palmi-
toylethanolamine, and N-stearoylethanolamine are consid-
ered endocannabinoid-like molecules and may have an
“entourage effect”, i.e., they may potentiate the activity of
AEA or 2-AG by inhibiting their degradation (82, 89, 90).
B. Metabolic routes
1. AEA synthesis and degradation. It is now widely accepted
that AEA is produced by a transacylase-phosphodiesterase-
mediated synthesis, starting from the precursor N-arachido-
noylphosphatidylethanolamine (NArPE). The latter com-
pound originates from the transfer of AA from the sn-1
position of 1,2-sn-di-arachidonoyl- phosphatidylcholine to
phosphatidylethanolamine, catalyzed by a calcium-depen-
428 Endocrine Reviews, August 2006, 27(5):427– 448 Wang et al. Endocannabinoid Signaling in Fertility
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dent N-acyltransferase [trans-acylase (NAT)] (81, 91). NArPE
is then cleaved by a recently characterized N-acylphosphati-
dylethanolamine (NAPE)-specific phospholipase D (NAPE-
PLD), which belongs to the zinc metallo-hydrolase family of
the
-lactamase fold (92) and releases AEA and phosphatidic
acid. The biological activity of AEA at CB receptors is ter-
minated by its removal from the extracellular space, which
occurs through a two-step process: cellular uptake by a high-
affinity transporter, followed by intracellular degradation by
a fatty acid amide hydrolase (FAAH), N-arachidonoyleth-
anolamine amidohydrolase (EC 3.5.1.4) (93, 94). Several
properties of a selective AEA membrane transporter (AMT)
have been characterized, although its molecular structure
remains unknown (95–98). In fact, there is controversy re-
garding the existence of endocannabinoid transporters, and
the mechanism by which AEA is taken up by cells is currently
being debated (99, 100). The uptake of AEA has the features
of a facilitated transport; it is dependent on the concentration,
time, and temperature, and independent of external Na
ions
or ATP hydrolysis. However, the molecular and genetic
identity of AMT still remains unknown (100, 101). In par-
ticular, the relationship between AMT and FAAH is still
under debate, because FAAH may not need a transporter to
get in contact with AEA (102), and AMT perhaps exports,
rather than imports, AEA across the plasma membrane (103).
Increasing pharmacological, biochemical, and morphologi-
cal evidence seems to favor for the existence of an AMT
different from FAAH (96, 97). The development of new drugs
able to inhibit AMT selectively without affecting FAAH cor-
roborates this speculation (104). However, it has recently
been suggested that AEA uptake is driven by nonprotein-
mediated diffusion and is regulated by its degree of hydro-
lysis by FAAH in specific cell types (105). The target of some
of the novel transport inhibitors recently developed seems
not to be the membrane transporter, but rather FAAH or an
uncharacterized intracellular component that delivers AEA
to FAAH (105). Once taken up by cells, AEA is a substrate for
FAAH that breaks the amide bond and releases AA and
ethanolamine (106). Mammalian FAAH is a membrane-
bound enzyme with a globular shape. It has 28
-helices and
11
-sheets, which account for approximately 53 and 13% of
the whole protein structure, respectively (102). This enzyme
uses an unusual serine-serine-lysine (S241-S217-K142) cata-
lytic triad (106).
Together with AEA and congeners, CB and non-CB re-
ceptors, NAT, NAPE-PLD, AMT, and FAAH, along with the
enzymes that metabolize 2-AG (see Section II.B.2), constitute
the “endocannabinoid system” (80, 82, 107–111). This system
is schematically depicted in Fig. 2.
2. 2-AG synthesis and degradation. 2-AG acts as a potent and
full agonist for both CB1 and CB2 and, like AEA, is not stored
in intracellular compartments but is produced on demand.
The biosynthetic pathway of 2-AG provides for rapid hy-
TABLE 1. Comparative adverse impacts of marijuana usage on reproductive events
Animal models Human
Male fertility Blocks hypothalamic GnRH release, decreasing serum levels
of FSH (266) and LH (144, 266 –274)
Decreases serum LH (52, 296, 297) and
testosterone levels (52)
Inhibits prolactin secretion (144, 275–277) Induces gynecomastia (298)
Decreases testicular testosterone production (266, 270, 272,
278–283)
Decreases spermatogenesis and mobility
(oligospermia), induces sperm anomalies,
and blocks acrosome reaction (52–56)
Causes testis lesions (284–287), reduces weights of testes
and accessory reproductive organs (279, 281, 288, 289)
and even induces demasculinization (290)
Disrupts normal spermatogenesis (153, 154, 286–289, 291–
293) and reduces fertilization (146, 147, 149, 150, 152,
171)
Reduces copulatory behavior (144, 294, 295)
Female fertility Blocks hypothalamic GnRH release, thus decreasing serum
levels of FSH (299–301) and LH (299–308)
Suppresses or increases serum LH levels in a
menstrual stage-specific manner (328, 329)
Inhibits prolactin secretion (277, 300, 309, 310) Inhibits prolactin secretion (330, 331)
Causes impaired ovarian function with reduced
progesterone secretion (280, 311, 312)
Increases serum testosterone level (331)
Disrupts normal reproductive cycle and ovulation (300, 306,
311–315)
Disrupts menstrual cycle (331)
Delays sexual maturation (316, 317) Poor oocyte retrieval rate when undergoing
IVF treatment (66)
Facilitates sexual behavior (318–320) Causes intrauterine fetal growth restriction
(57, 58)
Exerts embyrotoxicity and inhibits early embryo
development (68 –73)
Increases the incidence of preterm birth (59,
61, 63, 64) and prematurity with low fetal
birth weight (59 66)
Induces implantation failure (191) Induces greater difficulty at delivery (332–
334)
Increases incidence of miscarriage, stillbirths and term
pregnancy failure (312, 321–323)
Reduces fetal birth weight (323–325)
Delays the onset of parturition (326)
Inhibits milk ejections during lactation (327)
IVF, In vitro fertilization.
Wang et al. Endocannabinoid Signaling in Fertility Endocrine Reviews, August 2006, 27(5):427–448 429
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drolysis of inositol phospholipids by a specific PL, PLC; this
enzyme generates diacylglycerol (DAG), which is converted
to 2-AG by a sn-1-DAG lipase (85). Recently, two sn-1-specific
DAG lipases (DAGL) responsible for 2-AG synthesis have
been cloned by confronting human genome with Penicillium
DAGL sequence (112). These two isoforms (
and
) have
molecular masses of 120 and 70 kDa, respectively, with four
transmembrane domains and are members of the serine-
lipase family with serine and aspartic acid (S443-D495) par-
ticipating in the catalytic triad. The
isoform is predominant
in the adult brain, whereas the
isoform is expressed in
developing brain (112).
The pharmacological effects of 2-AG depend on its life
span in the extracellular space, which in turn is limited by a
rapid transport through the membrane. It is proposed that
the 2-AG membrane transporter is the same as AMT (113).
In fact, 2-AG accumulation is reduced by an AMT inhibitor,
AM404, and indirectly by high concentrations of AA (113).
The effect of AM404 is due to the inhibition of AMT, but not
due to FAAH activity, because the level of 2-AG remains
unaltered in the presence of two strong FAAH inhibitors,
URB597 and AM374 (114).
Once accumulated in the cell, 2-AG can be degraded by
FAAH (115), but FAAH is not the only enzyme responsible
for its metabolism. In fact, mice lacking FAAH are unable to
metabolize AEA but can still hydrolyze 2-AG (116). An en-
zyme responsible for 2-AG degradation, monoacylglycerol
lipase (MAGL), has been isolated from the porcine brain (115)
and cloned and characterized in rat (117) and human brain
(118). Rat brain MAGL is a 33-kDa protein and, unlike FAAH,
is localized in the cytosol (117). MAGL and DAGL are mem-
bers of the endocannabinoid system as shown in Fig. 2.
C. Molecular targets and signaling pathways
1. Cannabinoid receptors. AEA and 2-AG differentially activate
cannabinoid receptors. CB1 are most abundant in the central
nervous system but are present in peripheral tissues includ-
ing the heart, uterus, embryo, testis, liver, small intestine, and
peripheral cells like lymphocytes. CB2 are predominantly
expressed in astrocytes, spleen, and immune cells (23, 74 –76,
111, 119). Both CB1 and CB2 belong to the rhodopsin family
of G protein-coupled seven trans-membrane spanning re-
ceptors. They show 44% overall identity, with 68% identity
within the transmembrane regions, and are coupled mainly
to the G
i/o
family of G proteins (74). Signal transduction
FIG. 1. Chemical structures of natural and endogenous cannabinoids.
430 Endocrine Reviews, August 2006, 27(5):427– 448 Wang et al. Endocannabinoid Signaling in Fertility
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pathways regulated by CB-coupled G
i/o
proteins include the
inhibition of adenylyl cyclase (69, 77), regulation of ionic
currents (inhibition of voltage-gated L, N, and P/Q-type
Ca
2
channels as well as activation of K
channels) (120
125), the activation of focal adhesion kinase (126) and MAPK
(125, 127, 128). In addition, activation of CB1 or inhibition of
CB2 alters nitric oxide (NO) synthase (74, 119). However, a
recent report shows that WIN55,212–2, an aminoalkylindole
cannabinoid agonist, increases intracellular Ca
⫹⫹
via CB1
coupling to Gq/11 G proteins (129), suggesting diversity of
CB1 signaling pathways. Furthermore, there is some evi-
dence that endocannabinoids induce a biological activity via
other CB receptors, like a purported CB3 (GPR55) receptor
(130–132). GPR55 is an orphan G protein-coupled receptor
that has low sequence homology (10 –15%), compared to that
of CB1 or CB2, and is expressed in the testis at approximately
a 15-fold higher level than in the brain (130, 131). GPR55 does
not appear to couple with G
i
or G
s
proteins, suggesting that
it activates different signal transduction pathways from
those executed by CB1 and CB2 (130, 131). Among the
non-CB receptors, the type-1 vanilloid receptor [now called
transient receptor potential vanilloid 1 (TRPV1)] has at-
tracted great interest as a new molecular target of AEA.
2. Vanilloid receptors. TRPV1 is a six-trans-membrane span-
ning protein with intracellular N and C terminals and a
pore-loop between the fifth and sixth transmembrane helices
(133). TRPV1 is a ligand-gated and nonselective cationic
channel that is activated by molecules derived from plants,
such as capsaicin (the pungent component of “hot” red pep-
pers) and resinferatoxin, and also by stimuli like heat and
protons (134). In the last few years, a number of studies have
suggested a physiological role for AEA as a TRPV1 agonist,
leading to the concept that AEA, besides being an endocan-
nabinoid, is also a true “endovanilloid” (135, 136). In con-
trast, 2-AG is unable to bind to and activate TRPV1 (134, 136).
The interaction of AEA with TRPV1 occurs at a cytosolic
binding site (135, 137), triggering activation of nonselective
ion channels, activation of protein kinases, increased intra-
cellular Ca
⫹⫹
concentration, mitochondrial uncoupling, and
release of cytochrome c (138, 139). TRPV1 is expressed in
peripheral sensory fibers (134) and in several nuclei of the
central nervous system (140), suggesting the existence of
central endogenous agonists for its activation. In this context,
N-arachidonoyldopamine, an endogenous capsaicin-like
substance (Fig. 1), has been identified; it activates TRPV1
with high potency and is also a potent cannabimimetic com-
pound (141). Collectively, there seems to be an overlap be-
tween the endogenous cannabinoid system and the vanilloid
system.
Activation of the different molecular targets by AEA or
2-AG leads to several biological activities, some of which are
summarized in Table 2. It appears that virtually all central
and peripheral systems in mammals are affected by endo-
cannabinoids. Among the peripheral activities of AEA, the
regulation of reproduction is an emerging interest (23, 36
49). The effects of AEA are under metabolic control, so that
within a very narrow concentration range AEA regulates
blastocyst function and implantation (125, 142, 143). A more
detailed account of this regulation is described in the fol-
lowing sections.
III. Endocannabinoids and Male Fertility
A. Introduction
Although there is evidence that chronic administration of
THC to animals induces male impotency (144) and reduces
testosterone secretion, sperm production, motility, and via-
bility as well as acrosome reaction and fertilization (51, 145–
158), a role for the endocannabinoid system in male fertility
is still largely unexplored. Recent reports show that
N-acylethanolamines are present in human reproductive flu-
ids at low nanomolar ranges (49), and that AEA influences
human sperm functions (48, 146). For example, in vitro stud-
ies demonstrate that the AEA congener N-palmitoylethano-
lamine affects the time-course of capacitation of human sper-
matozoa by modulating their membrane properties (159,
160). Furthermore, the rat testis is able to synthesize AEA
(161), and human seminal plasma contains AEA (49). The
presence of CB1 in Leydig cells (162) and its association with
testosterone secretion have also been observed in mice (163).
There is evidence that THC alters Sertoli cell function (164),
although the underlying molecular mechanism is not known.
FIG. 2. The endocannabinoid system. The synthesis of AEA from
membrane N-arachidonoylphosphatidylethanolamines is catalyzed
by the sequential activity of NAT and NAPE-PLD, which releases
AEA and phosphatidic acid. AEA is transported in both directions
through the cell membrane by a selective AMT and, once taken up,
is hydrolyzed by FAAH to ethanolamine (EtNH
2
) and AA. The main
targets of AEA are CB1 and CB2 receptors (CBR), showing an ex-
tracellular binding site, and type-1 vanilloid receptors (TRPV1),
showing an intracellular binding site. 2-AG is also released from
membrane lipids through the activity of DAGL. 2-AG can also be
hydrolyzed by FAAH or, more importantly, by MAGL, releasing glyc-
erol and AA. The transport of 2-AG across the cell membrane may be
mediated by AMT or a related transporter, and CBR (but not TRPV1)
is the target of this endocannabinoid.
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Because Sertoli cells are involved in the regulation of germ
cell development by providing nutrients and hormonal sig-
nals needed for spermatogenesis, their ability to bind and
degrade AEA seems important in controlling the spermat-
ogenic output. Because AEA can serve as a proapoptotic
factor (138), it may also be involved in the survival and death
of Sertoli cells. In the same context, the possible interplay of
AEA with FSH could be of interest, because FSH dramati-
cally impacts fetal and early neonatal Sertoli cell proliferation
and is critical for regulating spermatogenesis in adult males
(165). These aspects of Sertoli cell biology with relation to
endocannabinoid signaling are further elaborated below.
B. The endocannabinoid system in Sertoli cells
In mice, Sertoli cells have the biochemical machinery to
bind and degrade AEA at various developmental stages (4 to
24 d) (166). For example, immature Sertoli cells express func-
tional CB2 on their surface, but the receptor level does not
fluctuate much during aging (166). Instead, FAAH activity
declines age-dependently due to reduced transcription, and
so does the uptake of AEA through AMT. A typical AMT was
found to uptake AEA in Sertoli cells and, like AMT of other
human peripheral cells, this uptake was significantly in-
creased by NO donors (166). This effect might be relevant in
vivo, because NO plays roles in regulating male fertility (167,
168). In particular, NO regulates the contribution of Sertoli
cells to fertility and to inflammation-mediated infertility
(169). A faster removal of AEA from the extracellular space
might be the rationale for some effects of NO.
An interesting observation is that AEA can force Sertoli
cells to undergo apoptosis and that this process is more
evident upon aging (166). The proapoptotic effect of AEA is
not mediated by CB1, CB2, or TRPV1. Instead, CB2 expressed
by Sertoli cells have a protective role against the harmful
effects of AEA, and so does FSH. In fact, FSH dose-depen-
dently inhibits apoptosis, and this event is correlated with
increased FAAH activity (166). The finding that the endo-
cannabinoid system is operative in Sertoli cells opens up a
new perspective to the understanding and treatment of male
infertility. In particular, the observation that FAAH modu-
lates the biological effects of AEA on Sertoli cells and that this
FAAH-mediated control is under hormonal regulation con-
stitutes a concept that AEA hydrolysis is an important check-
point in human fertility.
C. The endocannabinoid system in sperm
AEA signaling is implicated in regulating sperm functions
required for fertilization in invertebrates and mammals, in-
cluding humans (48, 145–152). It has been shown in sea
urchin (Strongylocentrotus purpuratus) that sperm synthesizes
AEA (170) and AEA binds to CB receptors and reduces
fertilizing capacity of sperm (48, 146–152, 171). Unlike lower
animals, ejaculated sperm from mammals must undergo
functional maturation for fertilizing an egg. Sperm acquire
fertilization competence because they reside in the female
genital tract where, after a series of physiological changes,
they become “capacitated”, i.e., able to fertilize an egg (1–3).
Capacitation consists of changes occurring at two sites: 1) on
the sperm head that enables it to bind to the zona pellucida
(ZP) and induces acrosome reaction; and 2) in the flagellum,
where hyperactivated sperm motility is facilitated. The con-
trol of this crucial process involves modifications of intra-
cellular ions (172, 173), plasma membrane fluidity (174), me-
tabolism, and motility (175). However, the sequence of these
changes and local regulatory mechanisms that allow capac-
itation to progress as sperm become closer to an oocyte still
remains poorly understood. Recently, AEA has been shown
to reduce human sperm motility by reducing mitochondrial
activity (176). In addition, AEA inhibits capacitation-induced
acrosome reaction, and its effects are prevented by the CB1
antagonist SR141716 (176). These data led to the suggestion
that the activity of AEA on sperm function requires CB1
activation. We investigated whether sperm cells of boar (Sus
scropha) are able to bind and metabolize AEA and whether
TABLE 2. Effects of endocannabinoids and congeners in the central nervous and peripheral systems
Central nervous system Peripheral systems
Thalamus, hypothalamus, hippocampus Cardiovascular system
Control of pain initiation Profound decrease in blood pressure (hypotension) and heart
rate (bradycardia)
Control of wake/sleep cycles Induction of hypotension during hemorrhagic shock or
endotoxic shock
Control of thermogenesis Vasodilation
Control of food intake Platelet aggregation
Impairment of working memory Immune system
Impairment of memory consolidation Alteration of synthesis and secretion of ILs
Inhibition of long-term potentiation Down-regulation of rat mast cell activation
Inhibition of glutamatergic transmission Stimulation of hematopoietic cell growth
Basal ganglia, striatum, globus pallidus Inhibition of leukemia inhibitory factor (LIF) release
Control of psychomotor disorders Inhibition of neutrophil recruitment
Interference with dopaminergic transmission Digestive tract
Inhibition of
-aminobutyric acid (GABA)ergic transmission Inhibition of peristalsis
Potentiation of
-aminobutyric acid (GABA)-mediated
catalepsy
Inhibition of intestinal motility
Cortex, cerebellum, spinal cord Liver
Blockade of N-methyl-D-aspartate (NMDA) receptors Control of lipogenesis and peripheral energy balance
Control of tremor and spasticity
Retina
Control of scotopic vision
432 Endocrine Reviews, August 2006, 27(5):427– 448 Wang et al. Endocannabinoid Signaling in Fertility
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this endocannabinoid modulates their function. Boar sperm
biology in some aspects resembles that of humans, and boar
spermatozoa are available in a greater amount than human
spermatozoa. Thus, boar sperm were used as a model to fully
characterize the endocannabinoid system (177–179).
Boar spermatozoa have the biochemical machinery to bind
(CB1 and TRPV1), synthesize (NAPE-PLD), and degrade
(AMT and FAAH) AEA (180). It was also shown that acti-
vation of CB1 by an AEA-stable analog, methanandamide,
inhibits capacitation and hence the ability of sperm cells to
react to ZP proteins with acrosome exocytosis through a
cAMP-dependent pathway, although CB1 was ineffective on
spontaneous acrosome reaction (180). It was also noted that
once the capacitation is completed, AEA stabilizes the acro-
some membranes by activating TRPV1, thus reducing spon-
taneous acrosome reaction. These results are schematically
depicted in Fig. 3.
Taken together, sperm function seems to be regulated by
endocannabinoids that exert a dual stage-dependent effect.
On one hand, AEA, present in both seminal plasma and
uterine fluids, may prevent premature capacitation in freshly
ejaculated sperm via a CB1-mediated mechanism for trav-
eling along the uterine tract without any fertilizing potential.
Conversely, a few hours later when sperm have reached the
oviduct (a condition that corresponds to in vitro capacitation),
this inhibitory brake becomes less stringent. It is speculated
that spermatozoa are exposed to a progressively reduced
concentration of AEA in the proximal female genital tract
(49), and sperm capacitation occurs as a consequence of re-
lease from CB1 inhibition. The observation that the endo-
cannabinoid system is operative in sperm adds a new di-
mension to the intricate endocannabinoid network
regulating mammalian fertility. Overall, these findings
present new perspectives to the understanding and treat-
ment of male fertility problems.
IV. Endocannabinoids and Female Fertility: Embryo
Implantation
A. Introduction
The onset of a new life begins with fertilization of a mature
oocyte with capacitated sperm (1– 4). The one-cell fertilized
zygote, now termed embryo, undergoes several mitotic cell
divisions, eventually forming the blastocyst with two distinct
cell populations, the ICM and a layer of trophectoderm cells
surrounding the ICM (7–9). The embryo proper is derived
exclusively from the ICM, whereas the placenta and extraem-
bryonic membranes are generated from cells contributed by
the trophectoderm (181, 182). A two-way interaction between
the blastocyst and the maternal uterine luminal epithelium
initiates the process of implantation. A considerable amount
of early pregnancy loss occurs due to either preimplantation
embryonic death or implantation failure resulting from asyn-
chronous embryonic development and/or failure of the
uterus to differentiate to the receptive stage (183, 184). Un-
derstanding the mechanism of preimplantation embryonic
development and implantation in the uterus is a fundamen-
FIG. 3. The endocannabinoid system in sperm function. Binding of AEA to the extracellular site of CB1 (CB1R) leads to inhibition of sperm
motility, capacitation, and ZP-induced acrosomal reaction (AR), without affecting spontaneous AR. In contrast, binding of a lipoxygenase-
catalyzed oxygenation of arachidonylethanolamide to the intracellular site of vanilloid receptors (TRPV1) inhibits spontaneous AR. Sperm also
possess the AMT, the AEA-synthesizing phospholipase D (NAPE-PLD), and the AEA-hydrolyzing FAAH. FAAH cleaves AEA into ethanolamine
(EtNH
2
) and AA.
Wang et al. Endocannabinoid Signaling in Fertility Endocrine Reviews, August 2006, 27(5):427–448 433
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tal challenge to reproductive biologists with the goal of al-
leviating the problems of human infertility and ensuring the
birth of quality offspring. Such knowledge will also help in
developing novel contraceptive approaches to restrict rap-
idly growing world population.
Development of the preimplantation embryo to the blas-
tocyst stage and differentiation of the uterus to the receptive
stage are basic requirements for the initiation of implantation
in all species (5, 6, 8). Although the precise sequence and
details of the molecular interactions involved in these pro-
cesses have not yet been defined, increasing evidence from
gene expression and transgenic mouse studies during the last
two decades shows that coordinated integration of a range
of signaling pathways in paracrine, autocrine, and/or jux-
tacrine manners participates in embryo-uterine dialogue
during implantation (5, 6, 8, 185, 186). Among these signaling
molecules, endocannabinoid signaling has recently been
highlighted as an important player in directing preimplan-
tation embryo development and the timely homing of em-
bryos into a receptive uterus for implantation.
B. Endocannabinoids and preimplantation embryo
development
THC, the major psychoactive component in marijuana, has
been shown to exert a wide array of adverse effects on human
health, including reproduction (23, 36 49). With the identi-
fication of CB1 and CB2 (77, 78) and two major endocan-
nabinoids, AEA and 2-AG (83– 85), as ligands for these re-
ceptors in the early 1990s, we and others using mice as an
animal model have been pursuing experiments to explore the
pathophysiological significance of cannabinoid/endocan-
nabinoid ligand-receptor signaling during early pregnancy.
In mice, only CB1 is expressed in the oviduct and uterus,
whereas both CB1 and CB2 are expressed in preimplantation
embryos (69, 187–189). CB1 mRNA is primarily detected
from the four-cell stage through the blastocyst stage, whereas
CB2 is present from the one-cell through the blastocyst stage
(69). Autoradiographic binding sites of [
3
H]AEA are also
evident from the one-cell through blastocyst stages. Inter-
estingly, the majority of AEA binding sites are noted in outer
cells of embryos at the eight-cell, morula, and blastocyst
stages. Scatchard analysis of binding kinetics in d 4 blasto-
cysts (d 1 vaginal plug) showed that AEA binds to a single
class of high-affinity receptors. The presence of CB1 mRNA
and protein as detected by immunocytochemistry in preim-
plantation embryos correlates well with AEA binding sites
(188, 190). Furthermore, blastocyst CB1 is biologically active,
because both THC and AEA inhibit forskolin-stimulated
cAMP formation, and this inhibition is prevented by per-
tussis toxin pretreatment (69, 187). The presence of biolog-
ically active CB1 in the blastocyst suggested that the mouse
embryo is a potential target for both endocannabinoids and
natural cannabinoids. In fact, synthetic (CP 55940, WIN
55212–2), natural (THC), or endogenous (AEA and 2-AG)
cannabinoids arrest the development of two-cell embryos
into blastocysts in culture (69, 191). A reduction in trophec-
todermal cell numbers is noted in those blastocysts that es-
cape the developmental arrest in the presence of cannabinoid
agonists (190). Furthermore, these adverse effects are re-
versed by simultaneous addition of selective antagonists to
CB1 [SR 141716 (192)] with cannabinoid agonists (191), but
not by a selective CB2 antagonist [SR 144528 (193)]. Recent
observations of CB2 expression in early embryos and em-
bryonic stem cells by microarray analysis (194), and the ab-
sence of its expression in trophoblast stem cells derived from
preimplantation blastocysts (H. Wang and S. K. Dey, un-
published data) suggest that CB2 expression is restricted to
blastocyst ICM cells. Collectively, these results suggest that
cannabinoids mediate their actions on preimplantation em-
bryos via CB1. The role of CB2 in the early embryo is yet to
be defined.
With the availability of cannabinoid receptor knockout
mice in the late 1990s (195, 196), the physiological relevance
of cannabinoid receptor signaling during early embryo de-
velopment was further examined. It was observed that
CB1/, CB2/,orCB1/⫺⫻CB2/ double mutant
embryos recovered from the oviduct on d 3 and from the
uterus ond4ofpregnancy show asynchronous development
compared with wild-type embryos (188, 189). This impaired
in vivo embryo development is rescued by mating CB mutant
females with wild-type males, producing all heterozygous
embryos in a mutant maternal environment. This indicates
that embryonic CB receptors, but not maternal factors, direct
early synchronous embryonic development (189). These
studies also provide genetic evidence that CB1 and CB2 are
critical for preimplantation embryo development, although
asynchronous development of CB2/ embryos still re-
mains puzzling. Because CB2 is expressed in the embryonic
stem cells, but not in trophectoderm-derived trophoblast
stem cells, it is conceivable that CB2 plays a role in specifying
pluripotent ICM cell lineage during blastocyst formation. To
ascertain whether embryos deficient in cannabinoid recep-
tors respond to endocannabinoids in vitro, two-cell wild-type
or mutant embryos were cultured in the presence or absence
of AEA. Although a comparable development of wild-type
and mutant embryos was observed in the absence of AEA in
culture, CB1/ and CB1/⫺⫻CB2/ mutant embryos,
but not CB2/ or wild-type embryos, were resistant to the
inhibitory action of AEA (188). This observation reinforces
the tenet that CB1 is the functional receptor for ensuring
normal embryo growth and differentiation to blastocysts.
Collectively, these studies provide pharmacological, molec-
ular, and genetic evidence that the preimplantation embryo
is indeed a target for cannabinoid ligand-receptor signaling.
C. Endocannabinoids and oviductal embryo transport
During early pregnancy, another critical event occurring
in parallel with preimplantation embryonic development is
the timely transport of embryos from the oviduct into the
uterus. In mice, embryos at the late morula or early blastocyst
stage enter the uterus, where they develop and differentiate
to achieve implantation competency, escape from the ZP,
and implant into the receptive uterus. Thus, normal oviduc-
tal embryo transport is one of the prerequisites for on-time
implantation in the uterus, whereas a dysfunctional regula-
tion of this process resulting from oviductal embryo reten-
tion may increase the incidence of pregnancy failure or cause
tubal pregnancy in humans.
434 Endocrine Reviews, August 2006, 27(5):427– 448 Wang et al. Endocannabinoid Signaling in Fertility
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During the course of our study over the past several years
exploring the potential physiological roles of endocannabi-
noid signaling during early pregnancy, we have consistently
observed that approximately 40% of CB1/ mice show
pregnancy loss (188, 189). Because these mutant mice have
normal ovulation and fertilization when compared with
wild-type mice (189), we initially thought that asynchronous
embryo development could be the major cause of this preg-
nancy loss. We speculated that normal pregnancy in
CB1/ mice would be restored by mating mutant females
with wild-type males to generate all heterozygous embryos
with normal preimplantation growth. However, we still ob-
served that about 40% of CB1/ mothers did not yield any
embryos in the uterus when examined in the midmorning on
d 4 of pregnancy, suggesting that a maternal, but not em-
bryonic, loss of CB1 is the determining cause for pregnancy
failure.
To determine the underlying cause for this pregnancy
failure, we examined oviductal embryo transport in
CB1/, CB2/, and CB1/⫺⫻CB2/ double mutant
females. No embryos were found to be trapped in the ovi-
ducts in wild-type or CB2/ mice, which was expected
because only CB1 is expressed in the mouse oviduct and
uterus. However, a substantial number of CB1/ and
CB1/⫺⫻CB2/ mice showed impaired oviductal trans-
port with retention of embryos at the morula and blastocyst
stages within the oviduct on d 4. These trapped embryos
appeared morphologically normal and implanted upon
transfer into d 4 pseudopregnant receptive uteri, suggesting
that they remained implantation-competent. These results
indicate that maternal expression of CB1 in the reproductive
tracts plays a fundamental role in ensuring normal oviduct
to uterine transport of embryos, and its deficiency results in
embryo retention in the oviduct for an extended period,
causing reduced fertility in CB1/ mice. This observation
was confirmed in our reciprocal embryo transfer experi-
ments between CB mutant and wild-type mice. Indeed, only
CB1/ recipients show oviductal embryo retention and
implantation failure, irrespective of the genotypes of donor
embryos (189). We further observed that wild-type pregnant
mice treated with a CB1-selective antagonist (SR141716) ex-
hibit impaired embryo transit through the oviduct, but this
defect did not occur in mice treated with vehicle or a CB2-
selective antagonist (SR144528). Interestingly, wild-type fe-
males exposed to a stable AEA analog (methanandamide) or
natural THC showed pregnancy loss with embryos retained
in the oviduct (189). These observations demonstrate that
aberrant cannabinoid signaling, either silenced or enhanced,
impairs embryo transport. This suggests that there is an
endocannabinoid tone mediated via CB1 in the oviduct that
regulates normal embryo transport into the uterus for im-
plantation. Collectively, these observations provide evidence
that whereas embryonic CB1 primarily contributes to normal
embryo development, oviductal CB1 directs timely oviductal
transport of embryos. Although there is no evidence of ec-
topic pregnancy in animals other than humans, these find-
ings may have clinical importance because embryo retention
in the fallopian tube as a result of dysfunctional muscular
contraction is one cause of ectopic pregnancy in women (197,
198). The ability of trapped blastocysts within CB1/ ovi-
ducts to implant after transfer into wild-type pseudopreg-
nant mouse uteri suggests that similar embryo retention in
the fallopian tube would result in ectopic pregnancy in
women.
Previous studies have established that the journey of the
embryo from the oviductal isthmus into the uterus in rodents
is aided by a wave of regulated contraction and relaxation of
the oviduct muscularis. It is thought that the sympathetic
neuronal circuitry, under the direction of ovarian hormones,
coordinates the “closing and opening” of the sphincter at the
isthmus-uterine junction, thereby regulating the timely pas-
sage of embryos from the oviduct into the uterus (199, 200).
During pregnancy, rising progesterone (P
4
) levels from the
newly formed corpus luteum decrease the turnover rates and
thus the levels of noradrenaline, a ligand with higher affinity
for the
-adrenergic receptor (AdR) than the
-AdR at the
adrenergic nerve endings (201). In contrast, the sensitivity of
the
-AdR is increased in the circular muscle of the oviduct
isthmus under P
4
dominance, causing muscle relaxation and
facilitating embryo transport through the oviduct (199). Ob-
servations of embryo retention within the oviduct in wild-
type females after exposure to an
1-AdR agonist phenyl-
ephrine and/or a
2-AdR antagonist butoxamine (189) led us
to speculate that CB1-mediated endocannabinoid signaling
is functionally coupled to adrenergic signaling to regulate
oviductal motility conducive to embryo transport. Indeed,
CB1 expression is colocalized with that of
1- and
2-AdRs
in mouse oviductal muscularis at the isthmus region, and the
-AdR agonist isoproterenol restores normal embryo trans-
port in CB1/ mice (189). Experiments with in vitro [
3
H]no-
radrenaline release provided further evidence that genetic or
pharmacological loss of oviductal CB1 increases noradren-
aline release from the adrenergic nerve terminals, maintain-
ing a smooth muscle contractile tone through
-AdR, and
thereby impeding oviductal embryo transport. In contrast,
exposure to excessive natural or synthetic cannabinoid li-
gands leads to predominant relaxation phase of the oviductal
muscularis due to attenuated noradrenaline release, impair-
ing embryo transport (189). Collectively, these findings re-
inforce the concept that aberrant cannabinoid signaling, aris-
ing from either silencing or amplification, impedes the highly
coordinated oviductal smooth muscle contraction and relax-
ation critical to embryo transport during early pregnancy.
The potential mechanism creating this endocannabinoid tone
in the oviduct during early pregnancy remains to be ex-
plored. A differential regional expression of NAPE-PLD and
FAAH with higher expression of NAPE-PLD in the isthmus
and FAAH in the ampulla may contribute to generate an
appropriate level of AEA conducive to preimplantation em-
bryo growth and transportation (H. Wang and S. K. Dey,
unpublished data).
D. Biphasic endocannabinoid sensor in blastocyst
implantation
Upon a successful journey through the oviduct, the em-
bryo at the late morula or early blastocyst stage encounters
a new microenvironment in the uterus for its attainment of
implantation competency. It is thought that blastocyst acti-
vation and uterine receptivity are two distinct events in the
Wang et al. Endocannabinoid Signaling in Fertility Endocrine Reviews, August 2006, 27(5):427–448 435
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process of implantation (202). In mice, ovarian P
4
and 17
-
estradiol (E
2
) are the primary factors that coordinate these
two events (5). Under P
4
priming, the closure of the uterine
lumen occurs and coincides with the escape of the blastocyst
from the ZP, bringing the blastocyst trophectoderm in close
contact with the uterine luminal epithelium (blastocyst ap-
position). Superimposition of the P
4
-primed uterus with pre-
implantation ovarian E
2
secretion and its catechol metabolite,
4-hydroxy-17
-estradiol (4-OH-E
2
), produced from primary
E
2
in the uterus, differentially regulate uterine preparation
and blastocyst activation, respectively. For example, the pri-
mary ovarian E
2
, via its interaction with nuclear E
2
receptors,
participates in the preparation of the P
4
-primed uterus to the
receptive state in an endocrine manner, whereas its metab-
olite 4-OH-E
2
, mediates blastocyst activation for implanta-
tion in a paracrine manner (203). These coordinated actions
of P
4
and E
2
set up the window of implantation. The first
event in the process of implantation is the attachment of the
blastocyst trophectoderm with the uterine luminal epithe-
lium that occurs within a narrow time frame when an inti-
mate two-way dialogue occurs between the implantation-
competent blastocyst and the receptive uterus. In mice, this
attachment reaction is initiated around 2400 h ofd4of
pregnancy (204). However, elimination of preimplantation
E
2
secretion by ovariectomy on the morning of d 4 results in
implantation failure with blastocyst dormancy within the
quiescent uterine lumen (205, 206). This condition is referred
to as delayed implantation and can be maintained for many
days by continued P
4
treatment. However, implantation with
blastocyst activation is rapidly initiated by a single injection
of E
2
in the P
4
-primed uterus (205, 206). This physiologically
relevant delayed implantation model has been widely used
to identify signaling pathways mediating embryo-uterine
cross-talk during blastocyst implantation. Using normal
pregnancy and delayed implantation model, we have elu-
cidated a unique association of endocannabinoid-CB1 sig-
naling in embryo-uterine interactions during implantation.
As stated earlier, natural, synthetic, or endogenous can-
nabinoids can dramatically inhibit preimplantation embryo
development and blastocyst zona-hatching in culture (69,
191, 207). This observation correlates well with higher levels
of AEA in the nonreceptive uterus (142, 188, 207). On the
other hand, lower levels of AEA in the receptive uterus and
at the implantation site suggest that regulated AEA levels are
conducive to normal embryo development and implanta-
tion. There is evidence that cannabinoid effects are differ-
entially executed, depending on the embryonic stage and
cannabinoid levels in the uterine environment. Blastocysts
exposed in culture to low levels of AEA exhibit accelerated
trophoblast differentiation and outgrowth, whereas inhibi-
tion of trophoblast differentiation is observed at higher doses
of AEA (143, 208), suggesting dual functions of AEA de-
pending on its local concentration (143, 207). Thus, uterine
AEA levels are critical in regulating the “window” of im-
plantation by synchronizing trophoblast differentiation and
uterine preparation to the receptive state.
To gain further insight into the underlying causes of these
biphasic effects of AEA, the status of anandamine binding in
preattachment and attachment-competent blastocysts imme-
diately before implantation ond4ofpregnancy was studied.
Similar expression studies were also performed using dor-
mant and estrogen-activated blastocysts. The results showed
that normal blastocysts collected in the early morning of d 4
have higher levels of AEA binding, but this binding remark-
ably declines in blastocysts recovered ond4inlate afternoon
before the attachment reaction (188). These observations sug-
gest that down-regulation of AEA binding to the blastocyst
is important for achieving implantation competence. Simi-
larly, dormant blastocysts also show increased levels of AEA
binding sites, but this binding significantly decreases by 12 h
after termination of dormancy by an E
2
injection (188). The
immunoreactive CB1 protein parallels AEA binding in dor-
mant and activated blastocysts (125, 188). These results col-
lectively suggest that coordinated down-regulation of blas-
tocyst CB1 and uterine AEA levels in the receptive uterus are
important for implantation. It is interesting to note that the
peripheral AEA levels remain relatively low during implan-
tation, whereas the levels increase before and during partu-
rition in humans (209).
Increasing evidence suggests that the bioeffectiveness of
AEA depends on its concentration in the extracellular space,
which is regulated by its synthesis by NAPE-PLD, its trans-
port across the plasma membrane, and its degradation by
FAAH (92–98, 210). To further address the underlying mech-
anism by which differential uterine AEA levels are spatio-
temporally established under different pregnancy status, we
examined the expression profiles of NAPE-PLD and FAAH
in the mouse uterus during early pregnancy. Correlating
with higher levels of AEA in the nonreceptive uterus and
interimplantation sites, higher levels of Nape-pld mRNA and
NAPE-PLD activity were detected in these tissues compared
with the implantation site and receptive uterus (142). It is
interesting to note that the implanting blastocyst exerts an
inhibitory effect on uterine Nape-pld expression (142). There
is also evidence that blastocysts can up-regulate uterine
FAAH activity by releasing a lipid “FAAH activator” (211).
These observations suggest a potential role of the implanting
embryo in regulating uterine AEA levels, perhaps to serve as
a protective mechanism against exposure to detrimental lev-
els of AEA. This is further confirmed by the observation of
higher FAAH expression and activity in the implanting em-
bryo (212, 213). Therefore, the differential and dynamic ex-
pression and activity of NAPE-PLD and FAAH in the em-
bryo and uterus create optimal levels of AEA beneficial to
blastocyst activation and uterine receptivity for implanta-
tion. This tight regulation of AEA synthesis and hydrolysis
in the pregnant uterus further indicates that endocannabi-
noid ligand-receptor signaling plays an important role in
implantation.
These studies clearly establish the concept that whereas
lower levels of AEA and CB1 are beneficial for implantation,
higher levels are detrimental. Using the delayed implanta-
tion mouse model, we have provided further evidence that
AEA at low concentrations confers blastocyst competency to
implantation via CB1 (125), whereas experimentally elevated
natural or synthetic cannabinoid levels interfere with im-
plantation. These findings are consistent with our previous
observations of stimulation and inhibition of trophoblast
growth at low and high AEA levels, respectively (143). To
reveal the underlying mechanism of this biphasic AEA action
436 Endocrine Reviews, August 2006, 27(5):427– 448 Wang et al. Endocannabinoid Signaling in Fertility
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in blastocyst implantation, we further explored the potential
signaling pathways that are coupled with CB1 under differ-
ent AEA concentrations. We found that AEA-induced stim-
ulatory and inhibitory influences on blastocyst function and
implantation are executed by different signal transduction
pathways: the ERK and Ca
⫹⫹
signaling pathways. AEA at a
low concentration activates ERK signaling in dormant blas-
tocysts via CB1. In contrast, at higher AEA levels, it fails to
achieve ERK activation, but instead inhibits Ca
⫹⫹
mobiliza-
tion (125). This finding provided for the first time a potential
“cannabinoid sensor” mechanism to influence crucial steps
during early pregnancy. An association of spontaneous preg-
nancy loss with elevated peripheral AEA levels in women
(214, 215) is consistent with the observations in mice (see
Section V). These findings in mice and humans reinforce the
concept that endocannabinoid signaling is at least one of the
pathways determining the fate of embryo implantation. In
this regard, there is evidence that activation of CB1 inhibits
human decidualization and promotes apoptosis of decidual
cells in vitro (216), thus adding a new role of endocannabi-
noids in human pregnancy. The possible physiological con-
sequence of the different signaling pathway triggered by
AEA through GPR55 (130, 131, 217) deserves further inves-
tigation. In addition, the pathophysiological impact of other
endocannabinoids or “endocannabinoid-like” compounds
(218) on various reproductive events warrants further
investigation.
Taken together, these studies demonstrate that under nor-
mal physiological conditions, endocannabinoid signaling
through CB1 is crucial to various female reproductive events
that include development of embryos, their oviductal trans-
port, and ultimately their homing and implantation in the
receptive uterus; conversely, an aberration in endocannabi-
noid signaling, either silenced or enhanced, derails these
processes (Fig. 4). These observations add a new dimension
to the concern that the adverse effects of maternal use of
cannabinoids on offspring may be seeded very early in preg-
nancy. There is now evidence that defective implantation
creates an adverse ripple effect during the subsequent course
of pregnancy both in humans and mice (12, 33, 35). Therefore,
our findings in mice raise a cautionary note for women of
reproductive ages regarding chronic abuse or medicinal con-
sumption of marijuana or other endocannabinoid system-
oriented drugs. More importantly, they raise caution against
the use of CB1 antagonists to treat obesity in humans.
V. Endocannabinoids and Female Fertility:
Immunoregulation
A. Th1/Th2 cytokines and fertility
There is evidence for the role of peripheral lymphocytes in
embryo implantation and successful pregnancy in humans
(219). In fact, normal gestation is based on an early immu-
nological adaptation that involves peripheral T lymphocytes
in pregnant women (40, 219, 220). These cells produce type
1 T-helper (Th1) and type 2 T-helper (Th2) cytokines, which
have opposite effects on trophoblast growth, as schemati-
cally depicted in Fig. 5. Th2 cytokines (IL-3, IL-4, and IL-10)
favor blastocyst implantation and successful pregnancy by
promoting trophoblast growth either directly or indirectly
through the inhibition of natural killer (NK) cell activity and
the stimulation of natural suppressor cells. Conversely, Th1
cytokines [IL-2, IL-12, and interferon-
(INF-
)] impair ges-
tation by causing a direct damage to the trophoblast through
stimulation of NK cells and secretion of TNF-
by macro-
phages. The latter cells play a role in this network, but several
aspects of their contribution to the balance of Th1 and Th2
cytokines remain to be elucidated. The trophoblast stimu-
lates the release of profertility Th2 cytokines from T lym-
phocytes (so-called “Th2 bias”) through IL-4, whereas the
antifertility Th1 bias is signaled by IL-2. In addition, P
4
plays
a role in this network, and in fact it induces a Th2 bias by
binding to the intracellular P
4
receptor in T cells (219, 221).
FIG. 4. Endocannabinoid signaling in blastocyst activation and implantation. Evidence suggests that regulated levels of endocannabinoids,
primarily AEA, in the receptive uterus and CB1 in activated blastocysts, are beneficial for implantation, whereas higher levels are detrimental
to this process. This biphasic role of AEA is further supported by findings that AEA within a very narrow range regulates blastocyst activation
and implantation by differentially modulating ERK signaling and Ca
2
channel activity via CB1. Uterine AEA levels conducive to implantation
are primarily regulated by the coordinated expression and activity of N-acylphosphatidylethanolamine-hydrolyzing PLD (NAPE-PLD) that
generates AEA and by FAAH that degrades AEA in the uterus during early pregnancy. In addition, the implanting blastocyst down-regulates
uterine NAPE-PLD expression, but enhances uterine FAAH activity via releasing a putative FAAH activator, thus contributing to rapid turnover
of AEA at the implantation site. Ge, Glandular epithelium; IS, implantation site; INTER-IS, interimplantation site; Le, luminal epithelium;
Myo, myometrium; S, stroma; Tr, trophectoderm.
Wang et al. Endocannabinoid Signaling in Fertility Endocrine Reviews, August 2006, 27(5):427–448 437
at Semmelweis Univ Medicine Central Library on November 19, 2008 edrv.endojournals.orgDownloaded from
The resulting hormone-cytokine network is a key element at
the fetal-maternal interface, and a defect in its integrity may
result in fetal loss (219, 221–224). Additionally, T lympho-
cytes produce leukemia inhibitory factor (LIF), which favors
embryo implantation and survival (221–224). IL-4 stimulates,
whereas IL-2 inhibits LIF release (Fig. 5). Clinical observa-
tions that women with unexplained recurrent abortions have
reduced expression of LIF production suggest that this cy-
tokine is indeed critical for implantation and pregnancy
maintenance in humans (219, 220, 225). In this context, P
4
-
induced Th2 bias has been found to stimulate LIF release
from T lymphocytes (Fig. 5), and IL-4 has been shown to
mediate this P
4
effect (219).
B. The endocannabinoid system in lymphocytes of pregnant
women
The FAAH activity is known to regulate the endogenous
tone and the biological activity of AEA in vivo (226). In this
respect, lymphocyte FAAH has been shown to influence
pregnancy outcome by regulating AEA level at the fetal-
maternal interface (40), which appears to interfere with the
lymphocyte-dependent cytokine network. Thus, decreased
activity and expression of FAAH in peripheral lymphocytes is
associated with pregnancy loss and may serve as an early (8
wk gestation) marker of human spontaneous abortion; AMT
activity and cannabinoid binding are not altered (214, 227).
Interestingly, defective FAAH in maternal lymphocytes is also
associated with failure to achieve an ongoing pregnancy after
in vitro fertilization and embryo transfer (215). Therefore, it
seems that FAAH, but not AMT or cannabinoid receptors, is
important in lymphocyte-mediated regulation of the hormone-
cytokine network at the fetal-maternal interface in natural and
medically-assisted gestation. In addition, analysis of the lym-
phocyte-endocannabinoid system during human ovulatory cy-
cles has shown the highest FAAH activity and the lowest AEA
concentrations on d 21 of the cycle (Table 3), a period that
temporally coincides with the putative window of uterine re-
ceptivity for implantation (228); binding to CB1 and activities of
AMT and NAPE-PLD were similar in T cells at all stages of the
ovulatory cycle (Table 3).
C. FAAH as a molecular integrator of fertility signals
FAAH expression in T lymphocytes has been shown to be
regulated by Th1/Th2 cytokines: IL-4 and IL-10 enhance
FIG. 5. Th1/Th2 cytokines in human fertility. Th2 cytokines (IL-3, -4, and -10) are released by T cells and favor blastocyst implantation by
promoting trophoblast growth through inhibition of NK cell activity. Conversely, Th1 cytokines (IL-2, IL-12, and INF-
), also released by T cells,
impair gestation by damaging the trophoblast through stimulation of NK cells. Macrophages contribute to NK activity by secreting TNF
. The
trophoblast induces a Th2 bias through IL-4, whereas IL-2 stimulates the release of Th1 cytokines. Also, P
4
induces the Th2 bias by binding
to T lymphocytes. Finally, these cells release LIF under positive or negative control of IL-4 or IL-2, respectively.
T
ABLE 3. The endocannabinoid system in human lymphocytes during the ovulatory cycle
Parameter Day 7 Day 14 Day 21
CB1 binding (cpm per mg protein) 20,000 2,030 (100%) 20,000 2,050 (100%) 17,400 1,795 (87%)
FAAH activity (pmol/minmg protein) 115 12 (100%) 46 5 (40%)
a
253 22 (220%)
a
AMT activity (pmol/minmg protein) 50 5 (100%) 43 4 (86%) 45 5 (90%)
NAPE-PLD activity (pmol/minmg protein) 130 15 (100%) 117 12 (90%) 130 15 (100%)
AEA content (pmol per mg protein) 2.15 0.20 (100%) 3.76 0.35 (175%)
b
1.29 0.14 (60%)
b
a
P 0.01 vs. d7.
b
P 0.05 vs. d7(P 0.05 in all other cases).
438 Endocrine Reviews, August 2006, 27(5):427– 448 Wang et al. Endocannabinoid Signaling in Fertility
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FAAH activity, whereas IL-2 and INF-
attenuate its activity
(227). In particular, IL-4, known to mediate favorable effects
of P
4
on pregnancy (221, 229), also partially mediates the
effect of P
4
on FAAH expression. Unlike FAAH, little alter-
ation is noted for AMT, NAT, and NAPE-PLD activities and
CB1 expression in intact lymphocytes by this steroid (227,
230). P
4
also modulates the effects of THC on sexual recep-
tivity (227). It appears that this effect of P
4
occurs through
increased level of the transcription factor Ikaros, which in
turn increases FAAH gene expression by binding to a specific
sequence in the promoter region (230). Profertility Th2 cy-
tokines potentiate the activation of FAAH by P
4
, whereas
antifertility Th1 cytokines have the opposite effect (227). Lep-
tin is also an important regulator of fertility (231) and im-
mune response (232), and leptin knockout (ob/ob/) mice
are infertile (231). Leptin, too, enhances FAAH gene tran-
scription through a signal transducer and activator of tran-
scription 3-mediated up-regulation of the FAAH promoter
(233). Leptin alone or synergistically with P
4
reduces AEA
levels in T cells, without affecting the other players in the
endocannabinoid system (233). Overall, the up-regulation of
lymphocyte FAAH by profertility signals strengthen the
speculation that this enzyme affects human fertility by mod-
ulating the AEA levels. Also, uterine FAAH is regulated by
sex hormones (234), but the implications and molecular de-
tails of this regulation are still elusive. Nonetheless, FAAH
activity seems to be important for embryo-uterine cross-talk,
because mouse blastocysts release a soluble “FAAH activa-
tor”, which contributes to AEA disposal at the implantation
site (211). The initial biochemical characterization of this
activator shows that it is neutralized by lipase activity,
whereas PLA
2
, PLC or PLD, DNAse I, or RNAse A are
ineffective. Additionally, the FAAH activator does not re-
semble platelet-activating factor, leukotriene B
4
,orPGs
known to be present in blastocysts (211); its molecular iden-
tity is under investigation. In addition, there is molecular
evidence that uterine NAPE-PLD is a major player in regu-
lating the levels of AEA in the uterus during early pregnancy
(142). In fact, NAPE-PLD activity was higher at the interim-
plantation site and lower at the implantation site, and E
2
and
P
4
down-regulated its expression (142). Thus, P
4
seems to
up-regulate lymphocyte FAAH activity, whereas it down-
regulates uterine NAPE-PLD expression. This would suggest
that regulated AEA levels are critical to successful implan-
tation and pregnancy establishment. On a final note, it is also
possible that interplay between endocannabinoids and eico-
sanoids (prostanoids, leukotrienes, or lipoxins) contributes
to immunoregulation of fertility, but this speculation awaits
experimental support.
VI. Endocannabinoids and Clinical Implications
Implications of the endocannabinoid system working via
cannabinoid and vanilloid receptors in many central and
peripheral aspects of human pathophysiology have been
proposed. We describe here the endocannabinoid signaling
pathways that impact both male and female fertility with the
hope of designing and developing endocannabinoid-ori-
ented drugs for the treatment of infertility. In this respect, the
biphasic roles of endocannabinoid signaling in fertilization,
preimplantation embryo development, oviductal embryo
transport, and implantation have major clinical implications
(69, 125, 142, 143, 146, 148, 180, 187–189, 191, 207, 211–213,
215, 227, 230, 234, 235). The incidence of spontaneous abor-
tion, the most common adverse outcome of pregnancy, as-
sociated with considerable sufferings and medical costs (236,
237), is increased by cigarette smoking and the use of illicit
drugs (238, 239). It is interestingly to note that the early stages
( 8 wk) of spontaneous abortion in humans are associated
with decreased activity and expression of FAAH in maternal
T lymphocytes and increased blood levels of AEA. This cor-
relation of down-regulation of FAAH in women who are
prone to miscarriage appears specific, because other mem-
bers of the endocannabinoid system are not affected (214,
215, 227). This suggests that high FAAH activity with low
AEA levels are among the factors that are important for
successful pregnancy and raises concerns about marijuana
use by pregnant women.
We suggest that FAAH is an important player in coordi-
nating hormone-cytokine-endocannabinoid networks criti-
cal to reproduction. Thus, the FAAH level may serve as a
marker to monitor the early stages (8 wk) of human ges-
tation. Because low FAAH levels correlate with pregnancy
failure, it is possible that drugs that enhance FAAH activity
may improve human fertility. Regulating endocannabinoid
levels via metabolic pathways could be an advantage over
the use of CB receptor agonists and antagonists to eliminate
their psychotropic effects (109, 240 –242). However, the de-
velopment and use of selective FAAH activators as thera-
peutics for fertility regulation may significantly reduce or
eliminate AEA signaling via CB receptors and increase the
incidence of reproductive disturbances (188, 189). Further-
more, FAAH regulates the levels of several other endoge-
nous endocannabinoid-like compounds whose functions are
not yet known, leaving open the question of the biological
consequences of their enhanced degradation upon treatment
with FAAH activators. Other enzymes that catalyze the hy-
drolysis of N-palmitoylethanolamine (243) and hydrolysis
(117) and synthesis (112) of 2-AG have recently been iden-
tified. It remains to be seen how these pathways contribute
to the overall endocannabinoid tone relevant to reproductive
functions. In addition, the development and use of FAAH
activators calls for attention to “the other side of the coin,”
because there is effort to develop FAAH inhibitors (rather
than activators) as novel therapeutic agents for the treatment
of pain (226), neurodegenerative disorders (111), cancer (244,
245), and anxiety (246).
VII. Conclusions and Future Direction
Mammalian reproduction is a complex process, involving
spermatogenesis, oogenesis, fertilization, preimplantation
embryo development, timely passage of embryos through
the oviduct, and their implantation in the uterus, eventually
establishing a functional placenta for successful pregnancy.
Each step in this process requires spatiotemporally regulated
various networks of endocrine, paracrine, juxtacrine, and
autocrine modulators. Although previous and prevailing
Wang et al. Endocannabinoid Signaling in Fertility Endocrine Reviews, August 2006, 27(5):427–448 439
at Semmelweis Univ Medicine Central Library on November 19, 2008 edrv.endojournals.orgDownloaded from
studies have placed much emphasis on the roles of canna-
binoids/endocannabinoids on neuronal functions (109, 111,
247–249), there were sporadic reports of adverse effects of
cannabinoids on pregnancy outcome, including retarded
embryo development and pregnancy failure. Emerging ev-
idence now points toward important roles of the endocan-
nabinoid system in mammalian reproduction. In this review,
we present molecular, genetic, physiological, and pharma-
cological evidence that cannabinoid/endocannabinoid sig-
naling is functionally operative in both male and female
reproductive events.
With respect to male reproductive functions, endocan-
nabinoids show biphasic roles in regulating Sertoli cell ap-
optosis via differential receptor signaling mechanisms. Fur-
thermore, we describe here a stage-dependent effect of AEA
on sperm function in that it prevents premature capacitation
via a CB1 to ensure normal transit of sperm through the
uterus to oviduct. It is tempting to suggest that such an
inhibitory brake may become less stringent when sperm
reaches the oviduct, the site of fertilization. Activation of CB1
by AEA (extracellular signaling) leads to inhibition of sperm
motility, capacitation, and ZP-induced but not spontaneous
acrosome reaction. In contrast, binding of AEA to TRPV1
receptors (intracellular signaling) inhibits spontaneous ac-
rosome reaction.
Upon fertilization, one-cell embryos initiate cell division
and undergo preimplantation development. As described
above, endocannabinoid signaling through CB1 is crucial to
various female reproductive events that include develop-
ment of embryos, their oviductal transport, and ultimately
their homing and implantation in the receptive uterus; con-
versely, an aberration in endocannabinoid signaling, either
silenced or enhanced, derails these processes. Furthermore,
decreased activity and expression of FAAH in maternal T
lymphocytes and resulting increased AEA levels are asso-
ciated with spontaneous abortion in women. These findings
add a new dimension to the concern that the adverse effects
of maternal use of cannabinoids on offspring may be seeded
very early in pregnancy, thus raising a cautionary note for
women of reproductive ages regarding chronic abuse or
medicinal consumption of marijuana or other endocannabi-
noid system-oriented drugs. More importantly, they raise
caution against the use of CB1 antagonists to treat obesity in
humans. Further in-depth investigation is warranted to bet-
ter understand pathophysiological significance of the can-
nabinoid/endocannabinoid signaling pathway in mamma-
lian reproduction.
Synchronous development of the preimplantation embryo
to the blastocyst stage before implantation is one of the pre-
requisites for normal “on-time” implantation. We have ob-
served that either silencing or amplifying CB signaling leads
to asynchronous preimplantation embryo development (188,
189). However, the underlying molecular mechanisms re-
main unknown. Recently, global gene expression profiles
during preimplantation mouse development analyzed by
microarray techniques have generated a comprehensive data
set covering nearly all mouse genes during early embryo-
genesis (194, 250–256). Therefore, strategies comparing
global gene expression, proteomic, and/or lipidomic profiles
between normal and defective embryos may help to identify
novel genes and gene products regulated by endocannabi-
noid signaling in the periimplantation embryo development.
Furthermore, it is crucial to gather in-depth information on
how the endocannabinoid signaling is differentially regu-
lated in peripheral and central systems. It is especially an
important concern for developing endocannabinoid system-
oriented drugs for selectively targeting central or peripheral
tissues to avoid adverse effects in unrelated tissue types.
Therefore, more efforts should be directed to explore in detail
the tissue- and cell-specific effects of endocannabinoid sig-
naling using conditional transgenic mouse models. In fact,
there is a recent study showing functional dissociation of
FAAH between central and peripheral tissues using brain
tissue-selective mutant mice (257). On the other hand, efforts
should also be directed to elucidate the common link that
encompasses the central and peripheral endocannabinoid
signaling. For example, increasing evidence suggests that
AEA-CB1 signaling exerts a regulatory role on hypothalamic
neuronal activity for the pulsatile release of GnRH (GnRH)
(258–260), a central hormone that controls reproductive per-
formances in both males and females.
Although a wealth of knowledge on the roles of lipid
mediators including endocannabinoids, LPA and PGs, and
protein signaling molecules, such as growth factors, cyto-
kines, homeotic genes, and transcription factors in embryo-
uterine interactions during implantation, has been generated
(5, 6, 8, 23, 36, 183), their hierarchical blueprint in directing
uterine and embryonic functions during pregnancy remains
to be deciphered. We need to understand whether these
pathways function independently or in parallel, or converge
to a common signaling pathway to establish a network of
lipid-protein signaling cross-talk between the embryo and
uterus that is necessary for implantation. In this respect,
another area of attention in endocannabinoid research has
evolved from the finding that the COX and lipoxygenase
enzymes can use AEA and 2-AG as substrates to generate
novel PGs and hydroxy-endocannabinoids (261–264), sug-
gesting a close link between endocannabinoids and eico-
sanoids (265). The potential function and signaling pathways
of these metabolic products in reproductive events remain to
be determined.
Acknowledgments
The authors thank Prof. A. Finazzi-Agro` (Department of Experimen-
tal Medicine and Biochemical Sciences, University of Rome “Tor Ver-
gata”) for continuing support and all colleagues who over the years gave
their valuable contribution to the fertility studies. We also thank G.
Bonelli for excellent production of the artwork and Susanne Tranguch
for her critical reading of the manuscript. We apologize for unintended
omission of any relevant references.
Address all correspondence and requests for reprints to: S. K. Dey,
Division of Reproductive and Developmental Biology, Departments of
Pediatrics and Cell & Developmental Biology and Pharmacology,
Vanderbilt University Medical Center, Nashville, Tennessee 37232.
E-mail: sk.dey@vanderbilt.edu; or M. Maccarrone, Department of Bio-
medical Sciences, University of Teramo, 64100 Teramo, Italy. E-mail:
mmaccarrone@unite.it
Studies by the authors incorporated in this review were supported by
National Institutes of Health Grants (DA06668, HD12304, and HD33994,
to S.K.D.); Ministero dell’Istruzione, dell’Universita` e della Ricerca (CO-
440 Endocrine Reviews, August 2006, 27(5):427– 448 Wang et al. Endocannabinoid Signaling in Fertility
at Semmelweis Univ Medicine Central Library on November 19, 2008 edrv.endojournals.orgDownloaded from
FIN 2002 and 2003, to M.M.); and Fondazione TERCAS (Research Pro-
gram 2004, to M.M.). S.K.D. is a recipient of the Method to Extend
Research in Time Awards from the National Institute of Child Health
and Human Development and the National Institute on Drug Abuse.
H.W. is a recipient of the Solvay/Mortola Research Award from the
Society for Gynecologic Investigation.
The authors have nothing to declare.
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