Expression and regulation of the fatty acid amide hydrolase gene in the rat uterus during the estrous cycle and peri-implantation period

Molecular Human Reproduction Vol.8, No.7 pp. 651–658, 2002 Expression and regulation of the fatty acid amide hydrolase gene in the rat uterus during the estrous cycle and peri-implantation period A.Z.Xiao1,2, Y.G.Zhao1 and E.K.Duan1,3 1The State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100080 and 2Department of Physiology, Jinzhou Medical College, Jinzhou, 121001, China 3To whom correspondence should be addressed. E-mail: Fatty acid amide hydrolase (FAAH) is involved in embryo development and implantation. Sex hormones down-modulate FAAH activity in the mouse uterus. However, the regulation of the FAAH gene in the uterus is unknown. Our results showed that FAAH mRNA is localized to uterine epithelial cells and circular myometrium during the estrous cycle. In ovariectomized rats, estradiol (E2) plus progesterone (P4) increased FAAH levels in both epithelial cells and circular myometrium. Interestingly, during the implantation period, FAAH mRNA was detected not only in epithelial cells and circular myometrium, but also in the primary decidual zone surrounding the implanting embryo on day 6 and in whole decidualized stromal cells on day 7. Its levels in the stromal cells were markedly higher at the implantation sites than at the inter-implantation sites on days 6 and 7. When implantation was delayed and then induced by E2 or E2 plus P4, FAAH mRNA levels were significantly increased in subepithelial stromal cells and circular myometrium, indicating that blastocyst activation and initiation of implantation in rats requires higher expression of the FAAH gene in subepithelial stromal cells and circular myometrium. In conclusion, the expression of FAAH mRNA is different in the non-pregnant and pregnant rat uterus and sex hormones up-regulate FAAH gene expression. Key words: estrous cycle/FAAH gene/implantation/steroid hormones/uterus Introduction Anandamide (N-arachidonoylethanolamine, ANA) is an endogenous ligand for both the brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors. It is isolated from brain and peripheral tissues (Devane et al., 1992; Mechoulam et al., 1998), and exerts a wide spectrum of central and peripheral effects (Calignano et al., 1998; Meng et al., 1998; Murillo-Rodriguez et al., 1998). The periimplantation mouse uterus contains high levels of ANA. The levels are highest during the non-receptive phase in pseudopregnant uterus and at the inter-implantation sites, and lowest at the sites of embryo implantation (Schmid et al., 1997). Low levels (7 nmol/l) of ANA accelerate blastocyst trophoblast differentiation and outgrowth, while inhibition of trophoblast differentiation is observed with a higher dose (28 nmol/l) of ANA in vitro (Wang et al., 1999). ANA also inhibits 2-cell embryo development to the blastocyst stage and reduces the rate of zona hatching of blastocysts in vitro. A synthetic cannabinoid (CP 55 940) prevents implantation. All these effects are mediated via CB1-R in embryos and uterus (Paria et al., 1995, 1998; Wang et al., 1999; Maccarrone et al., 2000a). The effectiveness of cannabinoids depends on their metabolism and turnover by the target organ. Fatty acid amide hydrolyase (FAAH) can catalyse ANA hydrolysis to arachidonate and ethanolamine (Di Marzo et al., 1994; Cravatt et al., 1996; Giang and Cravatt, 1997). During human gestation, FAAH is the only critical event controlling ANA level or action (Maccarrone et al., 2002). FAAH expression © European Society of Human Reproduction and Embryology and activity is detected in mouse and human uterine epithelium during the peri-implantation and non-pregnant period, and in preimplantation and implanting embryos (Paria et al., 1996, 1999; Maccarrone et al., 2000a). FAAH activity is higher at the implantation sites and lower at the inter-implantation sites in the mouse uterus (Paria et al., 1996). Sex steroids, progesterone (P4) and estrogen down-modulate mouse uterine FAAH activity (Maccarrone et al., 2000a), but the regulation of the FAAH gene and its expression in rat uterus are not known. In this investigation, we used in-situ hybridization and densitometric analysis to examine the expression of FAAH mRNA in rat uterus during the estrous cycle and peri-implantation period, and study its regulation by ovarian steroid hormones. Materials and methods Chemicals and reagents HindIII, XbaI, Yeast tRNA, P4 and 17β-estradiol (E2) were purchased from Sigma. DNase free RNase and EcoRI were the product of Promega. λDNA/ HindIII markers and pGEM-3Zf (⫹)/HaeIII markers were purchased from Pharmacia. X-Gal, digoxigenin RNA labelling kit (SP6/T7), anti-digoxigeninalkaline phosphatase Fab fragments, 5-bromo-4-chloro-3-indolyl phosophate (BCIP), nitro blue tetrazolium (NBT) chloride and blocking reagent were from Boehringer Mannheim. Salmon sperm DNA was from Gibco. Animal and tissue preparation Sprague-Dawley female rats (220–250 g) were supplied by the Experimental Animal Center of the Institute of Zoology, Chinese Academy of Sciences. 651 A.Z.Xiao, Y.G.Zhao and E.K.Duan They were housed with free chow and water and 12:12 h light:dark cycle for 1 week. The stages of the estrous cycle were identified by vaginal smear. The uterine horns were removed at different stages of the estrous cycle for in-situ hybridization (n ⫽ 3 animals per stage). To examine the effects of ovarian steroid hormones on FAAH gene expression in the non-pregnant rat uterus, rats were ovariectomized without regard to the stage of the estrous cycle. These rats rested for 2 weeks before receiving any treatment. The ovariectomized rats were divided into four groups and respectively injected with sesame oil (0.1 ml/rat), E2 (0.1 mg/rat), P4 (0.4 mg/rat), or E2 (0.1 mg/rat) plus P4 (0.4 mg/rat) (Feng et al., 1998). Steroids were dissolved in sesame oil and injected s.c. with the same volume. These rats were killed at 24 h after injection. Uterine horns were collected and tested (n ⫽ 3 animals per group). Virgin female rats (220–250 g) were mated with fertile males of the same strain. The morning of finding sperm was designated as day 1 of pregnancy. The rats on days 1–5 of pregnancy were killed at 17:00–18:00 and embryos were recovered on days 2–5 from the reproductive tract to confirm pregnancy. The rats on days 6 and 7 of pregnancy were also killed at 17:00–18:00. Implantation sites were visualized with i.v. injections of Trypan Blue dye solution (0.1% in saline, 0.1 ml/rat) on day 6. Implantation sites were distinct on day 7 and blue dye injection was not required. Uterine horns of the rats on days 1–7 of pregnancy (n ⫽ 3 animals per day) were excised, and uterine horns on days 6 and 7 were separated into the implantation and interimplantation sites. To examine the effects of ovarian steroid hormones on FAAH gene expression in the pregnant rat uterus, a delayed implantation rat model was established. Rats were ovariectomized at 8:00–9:00 on day 4 of pregnancy. (...truncated)