From the Department of Pediatrics and the
§ Department of Molecular and Integrative Physiology, Ralph
L. Smith Research Center, University of Kansas Medical Center, Kansas
City, Kansas 66160-7338, the ¶ Hormel Institute, University of
Minnesota, Austin, Minnesota 55912, the
Laboratory of Genetics,
National Institute of Mental Health, Bethesda, Maryland 20892, and the
** Laboratory of Molecular Neurobiology, Clinic of Psychiatry,
University of Bonn, 53105 Bonn, Germany
Received for publication, January 24, 2001
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ABSTRACT |
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The mechanisms by which synchronized embryonic
development to the blastocyst stage, preparation of the uterus for the
receptive state, and reciprocal embryo-uterine interactions for
implantation are coordinated are still unclear. We show in this study
that preimplantation embryo development became asynchronous in mice that are deficient in brain-type (CB1) and/or spleen-type
(CB2) cannabinoid receptor genes. Furthermore,
whereas the levels of uterine anandamide (endocannabinoid) and
blastocyst CB1 are coordinately down-regulated with the onset of
uterine receptivity and blastocyst activation prior to implantation,
these levels remained high in the nonreceptive uterus and in dormant
blastocysts during delayed implantation and in pregnant, leukemia
inhibitory factor (LIF)-deficient mice with implantation
failure. These results suggest that a tight regulation of
endocannabinoid signaling is important for synchronizing embryo
development with uterine receptivity for implantation. Indeed this is
consistent with our finding that while an experimentally induced,
sustained level of an exogenously administered, natural cannabinoid
inhibited implantation in wild-type mice, it failed to do so in
CB1 Previous investigation suggested that cannabinoid exposure has
adverse effects on pregnancy outcome (reviewed in Refs. 1-5). Cannabinomimetic drugs interact with two types of cannabinoid receptors, brain-type (CB1) and spleen-type (CB2) (6-9). These receptor subtypes are negatively coupled to adenylate cyclase and to
N-type and P/Q-type calcium channels and positively coupled to
mitogen-activated protein kinase and to A-type potassium channels through Gi/o proteins (7, 10). CB1 is expressed in brain and other peripheral tissues (7-11). CB2 is expressed primarily in
immune tissues including the spleen, leukocytes, and tonsils (12). We
have previously shown that CB1 is expressed in the preimplantation
mouse embryo at much higher levels than in the brain (1, 2). The
discovery of cannabinoid receptors led to the identification of
endogenous cannabinoid ligands, N-arachidonoylethanolamine (anandamide), and 2-arachidonoylglycerol (13-17). We observed that anandamide is synthesized in the pregnant mouse oviduct and uterus (18)
and that its levels are far higher in the uterus than in any other
mammalian tissue examined (3). However, anandamide levels are
significantly lower at implantation sites than at interimplantation sites, suggesting endocannabinoid ligand-receptor signaling during implantation (3). Indeed anandamide at higher levels adversely affects
embryo development and implantation, whereas at lower levels it
stimulates embryo growth and differentiation via CB1 (1-5).
Interestingly, 2-arachidonoylglycerol is present in the mouse
uterus at amounts similar to or lower than the lowest anandamide levels
(1-5 nmol/g of tissue), and its level does not vary significantly between implantation and interimplantation
sites.1 Using cannabinoid
receptor mutant mice (19) and physiological approaches, we further
defined the role of ligand-receptor signaling with cannabinoids in
preimplantation embryo development and in implantation.
Animals--
Adult CD-1 (Charles River Breeding Laboratories),
CB1, and CB2 mutant mice on a C57BL/6J background
or (LIF)2 mutant
mice on a mixed background were housed in the animal care facility at
the University of Kansas Medical Center. Story Landis (NINDS, National
Institutes of Health) kindly provided LIF mutant mice that
were originally generated by Philippe Brulet (Pasteur Institute). We
have characterized these mice previously (20). Heterozygous breeding
was performed to generate littermate (+/+), (+/ Embryo Culture--
Two-cell embryos recovered on day 2 were
cultured in 25 µl of Whitten's medium in groups of 5-10 under
silicon oil in an atmosphere of 5% CO2 in air for 72 h with or without anandamide (7.5 nM) (1, 2). The control
cultures contained the same concentration of the vehicle. The embryos
were observed every 24 h to monitor their development. At the
termination of culture the number of embryos forming blastocysts was recorded.
Anandamide Binding--
Autoradiographic ligand binding in
blastocysts was performed as previously described (1, 2). Blastocysts
were incubated with 4.5 nM [3H]anandamide
(specific activity 221 Ci/mmol, PerkinElmer Life Sciences) in the
presence or absence of a 500-fold molar excess of unlabeled anandamide.
After incubation, blastocysts were washed, fixed in 2%
paraformaldehyde, cytospun onto slides, air-dried, and subjected to
autoradiography using a liquid emulsion. Blastocysts were poststained
in hematoxylin. The autoradiographic signals were quantitated under
dark field conditions using an OPTIMA II program with an image analysis
system. Statistical analysis was performed using one-way analysis of
variance followed by a Newman-Keuls test.
CB1 Immunostaining--
Blastocysts were cytospun onto slides,
fixed in 2% formalin for 15 min, washed in phosphate-buffered saline,
and incubated in blocking solution for 10 min. They were then incubated
with an affinity-purified rabbit antipeptide antibody specific to CB1 (1 µg/ml) overnight at 4 °C (2). Immunostaining was performed using a Zymed Laboratories Inc. Histostain-SP kit (2).
Blastocysts were counterstained with hematoxylin. Red deposits
indicated the sites of immunoreactive CB1 protein. Control blastocysts
were incubated parallel with the antibody that was preneutralized with excess antigenic peptide.
Analysis of Anandamide and Other
N-Acylethanolamines--
Extraction, isolation, and analysis of
N-acylethanolamine (NAE) were done essentially as previously
described (3). Uteri were homogenized in chloroform/methanol (2:1), and
the homogenate was partitioned against 2.5% aqueous NaCl. Internal
standards (N-acyl-1,1,2,2-2H4-ethanolamines
with chain lengths of 16:0, 17:0, 18:0, 18:1n-9, 18:2n-6,
and 20:4n-6; d4 NAEs, 0.1 µg each)
were added to the extract. The NAEs were isolated using solid phase
silica extraction columns (Alltech Associates). Neutral lipids were
eluted with 4 ml of chloroform followed by elution of the NAEs with 4 ml of chloroform:methanol (98:2). The NAE fractions were taken to
dryness and treated with 50 µl of
tert-butyldimethylchlorosilyl/imidazole reagent (Alltech) at
80 °C for 1 h. The
tert-butyldimethylchlorosilyl/imidazole derivatives were
extracted into 50 µl of hexane for analysis by gas
chromatography-mass spectrophotometry in the selected ion monitoring
mode. Endogenous NAEs were quantitated by comparing the peak size of
their (M Administration of
Drugs--
( Embryo Development Is Asynchronous in Cannabinoid
Receptor-deficient Mice--
Because synchronized embryo development
and uterine preparation are important for successful implantation, we
first compared the in vivo developmental potential of
cannabinoid receptor mutant embryos with wild-type embryos. We observed
that the development of CB1 Mutant Embryos Are Resistant to Anandamide--
Two-cell wild-type
or mutant embryos were cultured in the presence or absence of
anandamide as previously described (1, 2). As observed previously (1,
2), most (>79%) of the 2-cell wild-type embryos failed to develop to
the blastocyst stage in the presence of anandamide. In contrast, more
than 80% of the CB1 Anandamide Levels Are Higher in the Nonreceptive Uterus--
In
the mouse, progesterone (P4) and estrogen sequentially
program the uterus into prereceptive, receptive, and nonreceptive phases during pregnancy or pseudopregnancy (23, 25). Blastocysts implant only in the receptive uterus. The P4-primed uterus
becomes receptive on day 4 when it is superimposed with preimplantation ovarian estrogen. Subsequently the receptive uterus proceeds to the
nonreceptive phase, and blastocysts can no longer implant (21, 25).
Similar uterine changes are experimentally produced during delayed
implantation induced by ovariectomy before preimplantation ovarian
estrogen secretion on day 4. Under this condition, blastocysts undergo
dormancy and fail to implant. This condition is terminated by an
injection of estrogen with activation of dormant blastocysts, attainment of the receptive uterus, and implantation (21, 23, 25). The
mechanism by which estrogen initiates these events in the
P4-primed uterus is not clearly understood.
The mouse uterus is receptive on day 4, whereas it was considered to be
nonreceptive on day 5 (21). We previously observed that not only are
uterine anandamide levels higher on day 5 of pseudopregnancy, the
levels are lower at implantation sites than at interimplantation sites
in day 5 pregnant mice (3). Thus, the regulated levels of anandamide
correlate with uterine receptivity and implantation. However, we have
recently discovered that the mouse uterus on day 5 of pseudopregnancy
is still receptive to implantation. For example, most (86%) of the day
5 pseudopregnant recipients receiving transfer of day 4 blastocysts
showed implantation. In contrast, transferred blastocysts completely
failed to implant in day 6 pseudopregnant recipient
uteri.3 To examine
whether uterine anandamide levels correlate with gradual progression of
a receptive uterus to a nonreceptive state, we measured uterine
anandamide levels on days 4-6 of pseudopregnancy. The results show
that anandamide levels are lower on day 4 but increase gradually
reaching maximum levels on day 6 (Fig.
2A). These results show that
uterine nonreceptivity results in higher anandamide levels, which could
be a cause of implantation failure. In fact, experimentally induced,
sustained, and higher levels of exogenous cannabinoids postpone
implantation via activation of CB1 (4). Higher levels of anandamide
during the nonreceptive phase could be due to lower levels of
anandamide hydrolase activity in the uterus. Indeed the lower
anandamide level at the implantation site is accompanied by higher
anandamide hydrolase activity (18). A correlation between decreased
anandamide hydrolase activity and increased pregnancy loss in women has
recently been reported, suggesting an adverse effect of higher
anandamide levels during pregnancy (26).
During delayed implantation blastocysts undergo dormancy, and
implantation does not occur (21, 23, 25). We surmised that if the
down-regulation of uterine anandamide levels is important for the
receptive state, then its levels should be higher in the P4-treated delayed implant uterus and lower in the
estrogen-induced receptive uterus. Indeed, uterine anandamide levels
were considerably higher in the delayed implant uterus than in the
estrogen-induced receptive uterus (Fig. 2B). These results
again suggest that uterine anandamide levels are critical to implantation.
Uterine Anandamide Levels Are Higher in Leukemia Inhibitory
Factor-deficient Mice--
In the mouse, LIF is expressed
in a biphasic manner, first in endometrial glands on the morning of day
4 and then in stromal cells adjacent to blastocysts during the
attachment reaction on the night of day 4 (20), suggesting its role in
implantation. Indeed, mice deficient in LIF show implantation failure,
and blastocysts undergo dormancy (27). This is similar to delayed
implantation produced by ovariectomy during normal pregnancy. However,
LIF Cannabinoid Receptors Are Higher in Dormant
Blastocysts--
Because an intimate interaction between the
blastocyst and the uterus is essential for implantation, we examined
whether uterine anandamide levels at different phases of receptivity
correlate with cannabinoid receptor expression in the blastocyst. As
previously reported (1, 2), significant levels of
[3H]anandamide binding were observed in normal
blastocysts collected at 0900 h on day 4 of pregnancy (Fig.
3, A, panel a, and
B). This binding remarkably decreased in blastocysts
recovered at 2000 h on day 4 just prior to the attachment reaction
between the blastocyst and the uterine luminal epithelium (Fig. 3,
A, panel c, and B). These results suggest that
down-regulation of anandamide binding to the blastocyst is important
for the initiation of implantation. If this speculation is correct,
then anandamide binding should remain high in dormant blastocysts
during delayed implantation but diminish with blastocyst activation.
Indeed, dormant blastocysts exhibited an increased population of
anandamide binding sites (Fig. 3, A, panel e, and
B), which significantly diminished by 12 h after
termination of the delay by an estrogen injection (Figs. 3, A,
panel g, and B). The CB1 immunoreactive protein
paralleled anandamide binding in dormant (delayed) and activated
blastocysts (Fig. 3C). As stated before, blastocysts fail to
implant and undergo dormancy in LIF Cannabinoid Receptor Mutant Mice Are Resistant to
Cannabinoid-induced Implantation Failure--
We speculated that if
tight regulation of ligand-receptor signaling is important for
implantation, maintaining a sustained level of exogenously administered
cannabinoids should disrupt uterine receptivity for implantation in
wild-type mice but not in mutant mice. An active natural cannabinoid,
( The mechanism(s) by which the development of preimplantation
embryos into active blastocysts is synchronized with uterine receptivity for implantation is not clearly understood. Our present observations of the asynchronous development of embryos deficient in
cannabinoid receptors during the preimplantation period and of the
coordinated down-regulation of both uterine anandamide levels and
blastocyst cannabinoid receptors prior to implantation in wild-type
mice suggest that ligand-receptor signaling with endocannabinoids
locally helps in regulating the "window" of implantation. Although
the Mendelian frequency of offspring resulting from heterozygous crossings of CB1-null mice is skewed resulting in a somewhat
reduced number of homozygous offspring (19), and although the pregnancy rate in mutant mice resulting from homozygous mating is somewhat lower,
the birth of viable CB1 On day 5 of pseudopregnancy when the uterus is still receptive,
LIF expression persists in uterine glands. In contrast,
LIF expression is undetectable or extremely low in the
nonreceptive day 6-uterus.3 Moreover, estrogen is
essential for the induction of uterine LIF in mice (20, 28). This is
consistent with the absence of LIF expression in the
P4-primed delayed implant mouse uterus and its rapid
induction after an estrogen injection (20). Thus, the virtual
absence of uterine LIF on day 6 and during delayed implantation
correlates with higher uterine anandamide levels and implantation
failure. However, we do not know whether the absence of LIF is the
cause of higher uterine anandamide levels or whether the higher levels
are the consequence of implantation failure in the absence of LIF.
The physiological significance of anandamide in the uterus and
cannabinoid receptors in the blastocyst is still not fully understood.
Although it is clear that higher uterine anandamide and blastocyst
cannabinoid receptor levels are detrimental to the implantation
process, uterine anandamide and blastocyst cannabinoid receptors still
persist, albeit at lower levels, at the time of implantation. This
suggests that lower levels of anandamide and cannabinoid receptors are
beneficial to implantation. This suggestion is consistent with our
recent observation that whereas higher anandamide levels are
detrimental to blastocyst outgrowth in culture, lower levels stimulate
this event (5). Similar biphasic (inhibitory and stimulatory) effects
of anandamide at high and low concentrations are evident for other
neural and behavioral functions (29), although the definitive cause of
these biphasic effects of anandamide is not yet clearly understood
(29). More recently, a bidirectional regulation of airway
responsiveness by endogenous cannabinoids has been documented (30).
Nonetheless, it is envisioned that a biphasic paracrine signaling via
anandamide and cannabinoid receptors influences the fate of the
embryo-uterine interactions during implantation and that aberrant
levels of uterine anandamide and/or embryonic cannabinoid receptors are
likely to adversely affect embryonic development and implantation. This
could be a mechanism to prevent implantation of abnormal embryos
resulting from exposure to aberrant levels of cannabinoids. In
conclusion, the present study highlights the importance of the
ligand-receptor signaling with cannabinoids in female fertility and
places the embryo and/or the uterus as targets for this signaling.
/
/CB2
/
double mutant mice. The present study is clinically important because
of the widely debated medicinal use of cannabinoids and their reported
adverse effects on pregnancy.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
), and (
/
) mice of
specific mutation. Homozygous males and females with specific null
mutations were crossed to generate homozygous embryos with respective
mutations or double mutation. Females were mated with fertile or
vasectomized males of the same strain to induce pregnancy or
pseudopregnancy (day 1 = vaginal plug), respectively. To induce
conditions of delayed implantation, mice were ovariectomized on the
morning (0800-0900) of day 4 of pregnancy and maintained with a daily
injection (subcutaneously) of progesterone (P4, 2 mg/mouse)
from days 5-7. To activate dormant blastocysts and initiate
implantation, P4-primed delayed implanting pregnant mice
were injected with estradiol-17
(E2, 25 ng/mouse) (21).
57) ions to those of the corresponding stable
isotope-labeled internal standards (22). Anandamide accounted for over
90% of total NAEs.
)-
9-Tetrahydrocannabinol ((
)-THC) or
its inactive stereoisomer (+)-THC was delivered at a constant rate via
miniosmotic pumps (Alzet Corp., Palo Alto, CA) to study their effects
on implantation in wild-type and
CB1
/
/CB2
/
pregnant mice. Miniosmotic pumps (mean pumping rate of 0.52 µl/h and
fill volume of 96 µl) containing either (
)THC or (+)THC were placed
under the back skin of mice on day 2 (1000 h) of pregnancy and
continued through day 5. These pumps released THC at a rate of 20 µg/h. Cytochrome P450 inhibitors, metyrapone and
clotrimazole (50 mg/kg body weight), were first injected
intraperitoneally 2 h before the installation of pumps and
injected twice daily until day 4 of pregnancy (4). On day 5 (1000 h),
implantation sites were determined by the blue dye method (21). If
implantation sites were absent, uterine horns were flushed with saline
to recover unimplanted blastocysts. Mice without implantation sites or
blastocysts were excluded from the experiments.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
,
CB2
/
, or
CB1
/
/CB2
/
double
mutant embryos recovered from the oviduct on day 3 (Table I) and from the uterus on day 4 (Table
II) of pregnancy was asynchronous. On the
morning of day 3, about 84% of the wild-type embryos recovered were at
the 8-cell stage. In contrast, only about 24% of
CB1
/
, 42% of
CB2
/
, and 50% of the
CB1
/
/CB2
/
double
mutant embryos were at the 8-cell stage, resulting in an increased
population of 2-, 4-, and 6-cell embryos (Table I). On the morning of
day 4, about 98% of the wild-type embryos were blastocysts. However,
only about 62% of CB1
/
, 71% of
CB2
/
, or 61% of
CB1
/
/CB2
/
embryos
were at the blastocyst stage; a considerable number of embryos were at
the morula stage. However, these morulae were apparently viable and
normal because they rapidly developed into blastocysts in culture. We
speculated that developmentally retarded embryos eventually form
blastocysts and implant. Thus, we next compared the status of
implantation in the wild-type and mutant mice on day 5 of pregnancy.
Increased localized endometrial vascular permeability at the site of
the blastocyst is one of the early markers of implantation (23). This
can be monitored by an intravenous injection of a blue dye (Chicago
blue B) solution which specifies implantation sites as distinct blue
bands along the uterus (4, 21, 23). We observed that whereas all of the
vaginal plug-positive wild-type or CB2
/
mice
(n = 7) showed an average of 9 or 7 implantation sites, respectively, 6 of 10 (60%) CB1
/
and 14 of
18 (78%)
CB1
/
/CB2
/
plug-positive mice showed an average of 7 and 9 implantation sites per
mouse, respectively. These results suggest that the loss of CB1 had
modest, if any, adverse effects on implantation. This is perhaps
because of the implantation of blastocysts that eventually formed from
the slowly growing embryos. Our next objective was to examine whether
the mutant embryos are responsive to an endocannabinoid exposure
in vitro.
Developmental stages of embryos on day 3 of pregnancy
Developmental stages of embryos on day 4 of pregnancy
/
or
CB1
/
/CB2
/
double
mutant embryos developed into blastocysts in the presence of anandamide
(Fig. 1). However, in vitro
development of CB2
/
embryos, like wild-type
embryos, was severely compromised in the presence of anandamide. These
results provided genetic confirmation to our previous findings that
CB1, but not CB2, in wild-type blastocysts responds to cannabinoid
agonists in a stage- and dose-dependent manner in
vitro (1-5) although these embryos express CB2
mRNA (1). Whereas the developmental response of
CB2
/
embryos to anandamide is similar to
that of wild-type embryos in vitro (1, 2), early
asynchronous development of CB2
/
embryos
in vivo, like CB1
/
or
CB1
/
/CB2
/
embryos, is surprising. It is possible that the reproductive tract
environment differentially modulates the development of mutant embryos
in response to endocannabinoids in vivo but not during their
development in vitro. In this respect, the mouse uterus
expresses the CB1 but not the CB2 gene (24), and
thus uterine CB1 may influence embryonic development in vivo
in the absence of embryonic CB2. Another possibility could be that
uterine anandamide levels in mutant mice are different from those of
the wild-type mice. However, uterine anandamide levels in
CB1
/
/CB2
/
double
mutant mice are not significantly different from the wild-type uterine
levels.1 Nonetheless, the present genetic evidence
suggests that cannabinoid receptors have some role in synchronizing
embryo development in the reproductive tract in vivo.
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Fig. 1.
Effects of anandamide on in vitro
development of preimplantation embryos. Two cell embryos
were recovered from oviducts of wild-type,
CB1 /
, CB2
/
,
or CB1
/
/CB2
/
double mutant mice mated with males of the same genotypes and were
cultured in the presence or absence of anandamide for a period of
72 h. Embryonic development was assessed every 24 h under a
dissecting microscope. The numbers at the top of
each bar indicate the number of blastocysts developed per
total number of 2-cell embryos cultured.
View larger version (18K):
[in a new window]
Fig. 2.
Levels of endogenous anandamide in the mouse
uterus. A, uterine levels on days 4-6 of
pseudopregnancy. Levels in day 4 uteri were significantly
(p < 0.05; n = 4 or 5) lower than
those on days 5 and 6 of pseudopregnancy. B,
anandamide levels in delayed implanting and receptive uterus. Levels in
P4-primed delayed uterus were significantly
(p < 0.05) higher than in E2-treated
P4-primed receptive uterus (n = 6 or 7).
C, levels in day 4 pregnant uteri of
LIF /
and LIF+/+ mice.
Levels in LIF+/+ uteri were significantly
(p < 0.05) lower than in
LIF
/
uteri (n = 5 or 6).
Statistical analysis was performed by analysis of variance followed by
Student's t test. A uterine anandamide assay for each
experiment within the same group was run simultaneously. Variations in
uterine anandamide levels among the three groups are likely due to
different experimental conditions and the use of mice of different
genetic backgrounds. CD-1 mice were used in experiments A and B,
whereas mice on a mixed background were used in experiment C.
/
blastocysts implant successfully after
transfer into wild-type uteri (27), establishing LIF as an essential
maternal factor for uterine preparation prior to implantation. We
speculated that uterine anandamide levels should remain high in
LIF
/
uteri as compared with wild-type uteri
on day 4 of pregnancy. Indeed, uterine levels in
LIF
/
mice were significantly higher than
those in wild-type mice (Fig. 2C). Collectively, the above
results suggest that regulated levels of anandamide are associated with
various phases of uterine receptivity for implantation.
/
mice
(20, 27). We observed that the levels of anandamide binding to dormant
LIF
/
blastocysts were also higher as
compared with active wild-type blastocysts (data not shown).
Collectively, these results demonstrate that the decreased levels of
cannabinoid receptors in active blastocysts just prior to implantation
correlate with decreased levels of anandamide in the receptive
uterus.
View larger version (115K):
[in a new window]
Fig. 3.
Cannabinoid receptors in the
blastocyst. A, autoradiographic localization of
[3H]anandamide binding sites in the normal, dormant
(delayed), and activated blastocysts. Autoradiographic signals appear
as black grains in these bright field photomicrographs of blastocysts.
Panel a, day 4 at 0900 h; panel c, day 4 at
2000 h; panel e, P4-treated day 7 dormant
(delayed); and panel g, P4 + E2-treated day 7 activated. Corresponding nonspecific
binding is shown in panels b, d, f,
and h. ICM, inner cell mass; Tr,
trophectoderm. B, quantitation of anandamide binding to
blastocysts. Nonspecific bindings were subtracted from total bindings
to determine specific bindings. Each experiment used 10-15 blastocysts
and was repeated 3-4 times with similar results. Results are mean ± S.E.; asterisk signifies p < 0.05. C, immunolocalization of CB1 protein in blastocysts. Reddish
brown deposits indicate the sites of immunoreactive CB1.
Photomicrographs of three representative blastocysts for each
experiment are shown. Whereas intense immunostaining is observed in
P4-primed dormant blastocysts (upper
panels), staining is remarkably lower in P4 + E2-treated activated blastocysts (lower
panels). Blastocysts incubated with preneutralized
antibodies showed no positive signals (data not shown). These
experiments were repeated three times.
)-THC, or its inactive stereoisomer, (+)-THC, was delivered
subcutaneously at a constant rate via miniosmotic pumps in wild-type or
CB1
/
/CB2
/
double
mutant mice from days 2-5 of pregnancy. To inhibit rapid systemic
degradation of THC by cytochrome P450 enzymes, mice were injected twice daily with P450 inhibitors as previously
described (4). The mouse uterus accumulates (
)-THC when its infusion accompanies P450 inhibitors; the levels are below the limit
of detection when (
)-THC alone is infused (4). Mice were examined for
implantation on day 5 by the blue dye method. As observed previously
(4), (
)-THC in the presence of P450 inhibitors inhibited
implantation in wild-type mice, and morphologically dormant-looking
blastocysts were recovered from these mice. In contrast, similar
treatments failed to inhibit implantation in CB1
/
/CB2
/
double
mutant mice (Table III). Administration
of (+)-THC plus the P450 inhibitors or the inhibitors alone
had no adverse effects on implantation in wild-type or double mutant
mice. These results, indeed, demonstrate that sustained and higher
levels of cannabinoids are inhibitory to implantation and that this
effect is mediated by cannabinoid receptors because the double mutant
mice, but not the wild-type mice, are resistant to this effect.
Effects of infusion of ()-THC or (+)-THC on implantation in wild-type
and CB1
/
× CB2
/
double mutant mice
)-THC or (+)-THC was administrated via miniosmotic pumps in the
presence of cytochrome P450 inhibitors, metyrapone (Met) and
clotrimazole (Clot) from days 2-5 of pregnancy. On day 5 (1000 h),
implantation sites (IS) were determined by the blue dye method. Uteri
without IS were flushed with saline to recover unimplanted blastocysts.
Mice without IS or blastocysts were excluded from the experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
,
CB2
/
, or
CB1
/
/CB2
/
double
mutant offspring suggests that the absence of embryonic and/or uterine
cannabinoid receptors is not indispensable for embryonic development or
implantation. However, the observed increased mortality of
CB1
/
offspring perhaps could be due to
inferior fetal development resulting from the implantation of slowly
developing embryos (19). The major phenotypes of
CB1
/
or
CB1
/
/CB2
/
double
mutant mice are their resistance to exogenous cannabinoid exposure with
respect to embryo development and implantation in vitro and
in vivo. This suggests that embryonic development and implantation are likely to be affected by aberrant levels of exogenous or endogenous cannabinoids in the uterus and/or aberrant embryonic expression of cannabinoid receptors during early pregnancy.
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ACKNOWLEDGEMENT |
---|
We thank Dr. H. Lim for the critical and constructive discussions we had with her during the course of this work.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants DA 06668, HD 12304, and HD 29968 (to S. K. D.), HD 37394 (to B. C. P.), and GM 45741 (to H. H. O. S.) and by the Hormel Foundation (to H. H. O. S.). Center grants in Reproductive Biology (HD 33994) and Mental Retardation (HD 02528) provided access to various core facilities.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
NICHD/National Institutes of Health Method to Extend Research
in Time (MERIT) awardee.
§§ To whom correspondence should be addressed: Dept. of Molecular and Integrative Physiology, 3901 Rainbow Blvd., Kansas City, KS 66160-7338. Tel.: 913-588-6213; Fax: 913-588-5677; E-mail: sdey@kumc.edu.
Published, JBC Papers in Press, March 8, 2001, DOI 10.1074/jbc.M100679200
1 B. C. Paria, P. C. Schmid, R. J. Krebsbach, H. H. O. Schmid, and S. K. Dey, unpublished results.
3 H. Song, B. C. Paria, and S. K. Dey, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
LIF, leukemia
inhibitory factor;
NAE, N-acylethanolamine;
THC, 9-tetrahydrocannabinol.
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REFERENCES |
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