Effects of anandamide on embryo implantation in the mouse
Introduction
Anandamide (ANA) is one of the endogenous ligands for cannabinoid receptors, isolated from brain and peripheral tissues [1], [2]. This compound binds with high affinity to brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors and mimics most of the effects of (−)Δ9-tetrahydrocannabinol [(−)THC], a psychoactive derivative of marijuana [3], [4], [5]. Marijuana has been reported to have adverse effects on reproduction, including retarded embryo development, fetal loss and pregnancy failure [6], [7]. However, mouse uterus contains the highest levels of anandamide, and the levels are lower at the implantation sites, but higher at the interimplantation sites [8]. Furthermore, in mouse uterus, down-regulation of anandamide levels has been associated with uterine receptivity, while up-regulation correlates with uterine refractoriness to embryo implantation. Altogether, the above findings suggest that anandamide might play an important role during embryo implantation in the mouse.
Preimplantation mouse embryos expressed both CB1-R and CB2-R mRNA, the levels of the former in the embryo is much higher than those found in the brain [6], [9]. Also, activation of CB1-R by cannabinoid ligands interferes with preimplantation embryo development, and the specific CB1 receptor antagonist might block this effect [10]. These studies suggested that embryonic arrest in response to cannabinoid might be mediated by CB1 receptor. However, both the CB1-R and CB2-R genes are expressed in the preimplantation mouse embryos, there is little information of which receptor is functional in the embryo attachment and outgrowth in the co-culture system.
Under culture conditions that plastic Petri dishes are precoated with fibronectin, 7 nM anandamide may accelerate the mouse embryonic trophoblast differentiation, but can inhibit differentiation at higher doses [11]. To further investigate the effects of anandamide on embryo implantation, in this work, an in vitro implantation model was established to compare the effects of different doses of anandamide on attachment and outgrowth of blastocysts on the monolayer of uterine epithelial cell, and to detect which receptor is involved in the embryo attachment and outgrowth in the co-culture system.
Section snippets
Material
Anandamide was purchased from Sigma. SR141716 and SR144528 were gifts from Prof. Madeleine Mosse and Dr. Francis Barth (Sanofi Recherche, France), respectively. All test agents were dissolved in ethanol and diluted with Ham's F-12 medium (Gibco, Rockville, MD, USA). The final ethanol concentration was less than 0.01%.
Animal
Kunming white strain mice [12], [13], [14] (Experimental Animal Center, the Institute of Genetic Science, CAS) were housed in the animal facility of the State Key Laboratory of
14 nM anandamide promotes blastocyst attachment
To determine the pharmacological effect of anandamide on blastocyst attachment, an in vitro co-culture system was used to test the effect of 14 nM anandamide on attachment of mouse blastocysts to the monolayers of uterine epithelial cell. Data were collected at different times (12, 24, 36 h) after hatched embryos were transferred onto the monolayer of the uterine epithelial cells. Blastocysts began to adhere to a monolayer of uterine epithelial cells after 6 h of co-culture in both the control
Discussion
More and more evidence indicates that anandamide, working as local mediator, is closely involved in regulation of embryo implantation [8]. The production of anandamide by the endometrium may be associated with various phase of uterine receptivity.
In this study, we show that 14 nM anandamide can significantly promote blastocyst attachment (Fig. 2) and outgrowth (Fig. 3). However, 56nM anandamide delays attachment and inhibits outgrowth of blastocysts. Wang J et al. [11] reported previously that
Acknowledgements
We thank Prof. Madeleine Mosse and Dr. Francis Barth for generously providing us with SR141716 and SR144528, respectively. This research was supported by the Special Funds for Major State Basic Research Project (Grant No. G1999055903), the “Climbing Project of China” program (Grant No. 970211019-3), and the 100-Scientist-program of the Chinese Academy of Sciences.
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