Differential regulation of endocannabinoid synthesis and degradation in the uterus during embryo implantation

Abstract

Preimplantation embryo development to the blastocyst stage and uterine differentiation to the receptive state are prerequisites for embryo implantation. Burgeoning evidence suggests that endocannabinoid signaling is critical to early pregnancy events. Anandamide (N-arachidonoylethanolamine) and 2-AG (2-arachidonoylglycerol) are two major endocannabinoids that bind to and activate G-protein coupled cannabinoid receptors CB1 and CB2. We have previously shown that a physiological tone of anandamide is critical to preimplantation events in mice, since either silencing or amplification of anandamide signaling causes retarded development and oviductal retention of embryos via CB1, leading to deferred implantation and compromised pregnancy outcome. Whether 2-AG, which also influences many biological functions, has any effects on early pregnancy remains unknown. Furthermore, mechanisms by which differential uterine endocannabinoid gradients are established under changing pregnancy state is not clearly understood. We show here that 2-AG is present at levels one order of magnitude higher than those of anandamide in the mouse uterus, but with similar patterns as anandamide, i.e. lower levels at implantation sites and higher at interimplantation sites. We also provide evidence that region- and stage-specific uterine expression of N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH), and sn-1-diacylglycerol (DAG) lipase alpha (DAGLalpha) and monoacylglycerol lipase (MAGL) for synthesis and hydrolysis of anandamide and 2-AG, respectively, creates endocannabinoid gradients conducive to implantation. Our genetic evidence suggests that FAAH is the major degrading enzyme for anandamide, whereas COX-2, MAGL and to some extent COX-1 participate in metabolizing 2-AG in the pregnant uterus. The results suggest that aberrant functioning of these pathways impacting uterine anandamide and/or 2-AG levels would compromise pregnancy outcome.

Fig. 1

Levels of anandamide (A) and 2-AG (B) in the periimplantation mouse uterus. Anandamide and 2-AG concentrations at interimplantation sites (Inter-IS) were significantly higher (*P<0.05) than those from implantation sites (IS) (unpaired t test). Uterine tissues from

Fig. 2

Spatiotemporal expression of DAGLα in the periimplantation mouse uterus. Uterine expression of DAGLα was analyzed by immunohistochemistry. Photomicrographs of representative uterine cross sections are shown (100X). le, luminal epithelium; ge, glandular epithelium; s, stroma; myo, myometrium; bl, blastocyst; em, embryo; IS, implantation site; Inter-IS, interimplantation site.

Fig. 3

Uterine cell-specific expression of MAGL during early pregnancy in mice. MAGL protein expression was analyzed using immunohistochemistry. Photomicrographs of representative uterine cross sections are shown (100X). le, luminal epithelium; ge, glandular epithelium; s, stroma; myo, myometrium; bl, blastocyst; em, embryo; IS, implantation site; Inter-IS, interimplantation site.

Fig. 4

Cell and stage-specific expression of NAPE-PLD in pregnant mouse uteri. The spatiotemporal accumulation of NAPE-PLD in the periimplantation uterus was examined by immunohistochemistry. Photomicrographs of representative uterine cross sections are shown (100X). le, luminal epithelium; ge, glandular epithelium; s, stroma; myo, myometrium; bl, blastocyst; em, embryo; IS, implantation site; Inter-IS, interimplantation site.

Fig. 5

Cell and stage-specific expression of FAAH in mouse uteri during early pregnancy. FAAH localization in uterine cells of various pregnancy stages was examined by immunohistochemistry. Photomicrographs of representative cross uterine sections are shown (100X). le, luminal epithelium; ge, glandular epithelium; s, stroma; myo, myometrium; bl, blastocyst; em, embryo; IS, implantation site; Inter-IS, interimplantation site.

Fig. 6

Region-specific uterine induction of COX-2 in mice during the periimplantation period. Immunohistochemistry was employed to determine COX-2 protein localization in the pregnant uterus. Photomicrographs of representative cross uterine sections are shown (100X). le, luminal epithelium; ge, glandular epithelium; s, stroma; myo, myometrium; bl, blastocyst; em, embryo; IS, implantation site; Inter-IS, interimplantation site; M, mesometrial site; AM, anti-mesometrial site.

Fig. 7

Uterine levels of anandamide (A, C) and 2-AG (B, D) in day 7 pregnant wild-type, FAAH^−/−, COX-2^−/−, cPLA2α^−/− and COX-1^−/− mice. We noted that FAAH deficiency increases anandamide levels at the implantation sites, while the absence of COX-2 derails the normal uterine 2-AG accumulation pattern. The loss of cPLA2α or COX-1 shows no obvious changes in uterine patterns anandamide and 2-AG levels. *P<0.05 (unpaired t test).