Lipid signaling in embryo implantation

Prostaglandins & other Lipid Mediators 77 (2005) 84–102

Review

Lipid signaling in embryo implantation Haibin Wang, Sudhansu K. Dey∗ Departments of Pediatrics, Cell & Developmental Biology and Pharmacology, Division of Reproductive & Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA Received 14 July 2004; accepted 14 September 2004

Abstract A reciprocal interaction between the implantation-competent blastocyst and the receptive uterus is required for successful implantation. Although various molecular pathways are known to participate in this cross-talk, a comprehensive understanding of the implantation process is still missing. Gene expression studies and genetically engineered mouse models have provided evidence that lipid mediators serve as important signaling molecules in coordinating the series of events during early pregnancy including preimplantation embryo formation and development, implantation and postimplantation growth. This review focuses on the roles of two groups of lipid mediators, prostaglandins (PGs) and endocannabinoids, during early pregnancy. Our laboratory has shown that while PGs generated by the cPLA2 –cyclooxygenase (COX) system are essential to ovulation, fertilization, and implantation, endocannabinoids are important for synchronizing preimplantation embryo development with uterine receptivity for implantation. A better understanding of these molecular signaling pathways is hoped to generate new strategies to correct implantation failure and improve pregnancy rates in women. © 2004 Elsevier Inc. All rights reserved. Keywords: Phospholipase A2 ; Cyclooxygenase; Prostaglandin; Endocannabinoid; Embryo implantation

Contents 1. 2. ∗

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The phospholipase A2 and cyclooxygenase systems in prostaglandin biosynthesis . . . . . .

Corresponding author. Tel.: +1 615 322 8642; fax: +1 615 322 4704. E-mail address: [email protected] (S.K. Dey).

1098-8823/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.prostaglandins.2004.09.013

85 86

H. Wang, S.K. Dey / Prostaglandins & other Lipid Mediators 77 (2005) 84–102

3. 4.

5.

6. 7.

Temporal and cell-specific expression of cPLA2 ␣, COX-1 and COX-2 in the periimplantation uterus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . cPLA2 ␣ is crucial for ‘on-time’ embryo implantation that directs subsequent development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Normal ‘window’ of implantation is altered in cPLA2 ␣−/− mice . . . . . . . . . . . . . . . . 4.2. Deferred implantation in cPLA2 ␣−/− mice leads to defective postimplantation development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Embryo spacing is disturbed in cPLA2 ␣−/− mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . COX-2 deficiency leads multiple female reproductive failures . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Ovulation and fertilization are defective in Cox-2−/− mice . . . . . . . . . . . . . . . . . . . . . 5.2. Embryo implantation is impaired in the absence COX-2 . . . . . . . . . . . . . . . . . . . . . . . 5.3. Decidualization is defective in Cox-2−/− mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Compensatory up-regulation of COX-1 rescues female infertility from the loss of COX-2: a function of genetic makeup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endocannabinoid signaling: a biphasic sensor in embryo implantation . . . . . . . . . . . . . . . . . Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

85

87 89 89 90 90 92 92 93 94 94 95 98 99 99

1. Introduction Successful implantation depends upon synchronized development of the blastocyst to the stage when it is competent to implant, and the uterus to the stage when it is receptive to implantation [1,2]. The first step in the process of implantation is the apposition of the blastocyst to the luminal epithelium of the uterus and is initiated by the creation of an implantation chamber surrounding the blastocyst along the uterus [1,3]. This is followed by the attachment of the blastocyst trophectoderm with the luminal epithelium at the antimesometrial pole of the uterus in rodents. This attachment reaction is coincident with an increased endometrial vascular permeability at the sites of the blastocyst. In mice, the attachment reaction occurs in the evening (22:00–23:00 h) of day 4 of pregnancy [4]. This attachment leads to extensive proliferation and differentiation of uterine stromal cells to decidual cells (decidualization) with luminal epithelial cell apoptosis at the attachment site [5], allowing subsequent adherence and penetration by trophoblast cells through the stroma in a regulated manner. The initiation and progression of implantation are the results of coordinated integration of various signaling pathways between the embryo and the uterus. It involves spatiotemporally regulated endocrine, paracrine, autocrine, and juxtacrine modulators that span cell–cell and cell–matrix interactions. Despite identifying numerous molecules involved in embryo–uterine dialogue, there is a significant knowledge gap in understanding the in vivo events of implantation. A better understanding of the molecular signaling pathways that regulate uterine receptivity and implantation competency of the blastocyst may lead to strategies to correct implantation failure and improve pregnancy rates in humans. Gene expression studies and genetically engineered mouse models have provided valuable clues to

86

H. Wang, S.K. Dey / Prostaglandins & other Lipid Mediators 77 (2005) 84–102

the implantation process with respect to specific lipid mediators, growth factors, cytokines, adhesion molecules and transcription factors. Since the process of implantation, a process considered a proinflammatory reaction, is accompanied with an increased endometrial vascular permeability at the sites of blastocysts, it is envisioned that prostaglandins (PGs), by virtue of their vasoactive properties, are involved in implantation and decidualization [6]. In addition, an emerging concept in implantation is the role of endocannabinoids, a group of lipid mediators that are ligands for the cannabinoid receptors [7]. This review primarily focuses on the lipid signaling pathways by PGs and endocannabinoids involving the embryo–uterine “cross-talk” during implantation in mice.

2. The phospholipase A2 and cyclooxygenase systems in prostaglandin biosynthesis PGs are generated from arachidonic acid by phospholipase A2 s (PLA2 s) followed by cyclooxygenases (COXs or prostaglandin endoperoxide H synthases). PLA2 plays crucial roles in diverse cellular functions, including phospholipid metabolism, immune functions and signal transduction by generating bioactive lipid mediators [8,9]. Once activated by a variety of stimuli, PLA2 hydrolyzes the ester bonds of fatty acids at the sn-2 position of phospholipids, producing free fatty acids and lysophospholipids. The mammalian PLA2 superfamily consists of four major subfamilies that include cytosolic (cPLA2 ), secretory (sPLA2 ), Ca2+ -independent (iPLA2 ) and platelet-activating factor (PAF) acetylhydrolase. While PLA2 and sPLA2 participate in various cellular functions by generating free fatty acids, including arachidonic acid, iPLA2 and PAF acetylhydrolase primarily contribute to membrane remodeling and attenuation of PAF bioactivity, respectively [10]. PLA2 -derived arachidonic acid gives rise to various lipid mediators, including PGs, thromboxanes, endocannabinoids and leukotrienes. These mediators via various signaling pathways exert a wide range of cellular functions [11–13]. Among the PLA2 superfamily members, cPLA2 is a key regulator of eicosanoid biosynthesis, because it selectively releases arachidonic acid [11]. Recently, two other cPLA2 isoforms, cPLA2␳ and cPLA2␥ , have been identified in human [14,15], assigning a new name for cPLA2 as cPLA2␣ , although they in fact, unlike cPLA2␣ , do not show substrate preference for arachidonic acid [14]. COX, the rate-limiting enzyme in PG biosynthesis, exists in two isoforms, COX-1 and COX-2, which are encoded by two separate genes and exhibit distinct cell-specific expression, regulation and subcellular localization, yet share similar structural and kinetic properties [16]. COX-1 is considered to be a constitutive enzyme that mediates “housekeeping” functions. In contrast, COX-2 is an inducible enzyme and is induced in a variety of cell types by growth factors, cytokines and inflammatory stimuli [17]. The key regulatory step in PG biosynthesis is the enzymatic conversion of PLA2 -derived arachidonic acid by COXs into PGG2 , which is then reduced to an unstable endoperoxide intermediate, PGH2 . PGH2 is sequentially metabolized to five primary active structurally related prostaglandins, including PGE2 , PGD2 , PGF2␣ , PGI2 and thromboxane A2 (TxA2 ) via cell-specific isomerase and specific PG synthases [17]. PGE1 and PGE2 can be slowly metabolized to the cyclopentenone PGs, PGA1 and PGA2 , in biological fluids [18]. PGD2 , in turn, sequen-

H. Wang, S.K. Dey / Prostaglandins & other Lipid Mediators 77 (2005) 84–102

87

Fig. 1. Biosynthesis of prostaglandins and anandamide. The key step in PG biosynthesis is the enzymatic conversion of phospholipase A2 -derived arachidonic acid by COX enzymes into PGG2 , which is then reduced to an unstable endoperoxide intermediate, PGH2 . PGH2 is sequentially metabolized to five primary active structurally related prostaglandins, including PGE2 , PGD2 , PGF2 ␣, PGI2 , and thromboxane A2 , via cell-specific isomerase and specific PG synthases. Anandamide is generated from its precursor, NAPE, which is released from membrane lipids by a transacylase enzyme. The key enzyme for this pathway has recently been characterized as NAPE-phospholipase D. It remains unknown whether there is a specific anandamide synthase, which catalyzes condensation reaction between arachidonic acid and ethanolamine to generate anandamide in living cells. FAAH is considered a major enzyme for degrading anandamide. There is now evidence that COX-2 can metabolize anandamide and produce prostaglandin-ethanolamines. In addition, anandamide can also be converted into 12(S)hydroxy-arachidonylethanolamide by lipooxygenase enzyme. Abbreviations: PLA2 (phospholipase A2 ); COX (cyclooxygenase); PG (prostaglandin); TX (thromboxane); PPAR (peroxisome proliferators-activated receptor); TAE (transacylase enzyme); PLD (phospholipase D); NAPE (N-arachidonyl-phosphatidyl ethanolamine); FAAH (fatty acid amide hydrolase); PG-Et (prostaglandin-ethanolamine); 12(S) anandamide (12(S)-hydroxy-arachidonyl ethanol amide).

tially forms the J series of cyclopentenone PGs, 9-deoxy-9 PGD2 (PGJ2 ), 12 PGJ2 , and 15-deoxy-12 ,14 PGJ2 (15dPGJ2 ) [19]. PGI2 and TxA2 spontaneously degrade into inactive compounds under physiological conditions (Fig. 1). These prostanoids are involved in a variety of pathophysiologic processes, including modulation of the inflammatory reaction, gastrointestinal cytoprotection and ulceration, angiogenesis, cancer, hemostasis and thrombosis, renal hemodynamic and diseases. Normally PGs exert their function by interacting with cell surface G-protein coupled receptors. These receptors have been named as EP1 –EP4 , FP, DP, IP and TP for PGE2 , PGF2␣ , PGD2 , PGI2 and thromboxanes, respectively. In addition, evidence suggests that a PGD2 metabolite 15-deoxy-12 ,14 PGJ2 (15dPGJ2 ) and PGI2 can serve as a ligand for nuclear peroxisome proliferator activated receptor ␥ (PPAR␥) [20] and PPAR [21], respectively.

3. Temporal and cell-specific expression of cPLA␣, COX-1 and COX-2 in the periimplantation uterus In rodents, increased uterine vascular permeability at sites of blastocyst apposition is one of the earliest events in the implantation process and is preceded by generalized uterine

88

H. Wang, S.K. Dey / Prostaglandins & other Lipid Mediators 77 (2005) 84–102

Fig. 2. Expression of Cox-1, Cox-2 and cPLA2 α in wild-type uteri during implantation. In situ hybridization of Cox-1, Cox-2 and cPLA2 α on days 4–6 (09:00 h) of pregnancy is shown. Arrows indicate the location of blastocysts. ge, glandular epithelium; le, luminal epithelium; s, stroma; myo, myometrium. Reprinted with permission from Ref. [23].

edema and luminal closure. Vasoactive PGs are implicated in these processes. Since PGs are produced by the action of COXs on arachidonic acid derived from cPLA2␣ , we employed gene expression studies and demonstrated unique expression patterns of Cox-1 and Cox-2 genes in the periimplantation mouse uterus [22]. As shown in Fig. 2, Cox-1 is expressed in uterine luminal and glandular epithelial cells on the morning of day 4 of pregnancy, but its expression becomes undetectable in the luminal epithelial cells by the time of the attachment reaction. This Cox-1 expression coincides with the generalized uterine edema required for luminal closure. In contrast, the Cox-2 gene is expressed in the luminal epithelium and subepithelial stromal cells at the anti-mesometrial pole exclusively surrounding the blastocyst at the time of attachment reaction on day 4 midnight and persists through the morning of day 5. The Cox-1 gene that is downregulated from the time of attachment reaction on day 4 is again expressed in the mesometrial and anti-mesometrial secondary decidual beds on days 7 and 8. These results suggest that PGs generated by COX-1 are involved in decidualization and/or continued localized endometrial vascular permeability observed during the postimplantation period. In contrast, the Cox-2 gene, expressed at the anti-mesometrial pole on days 4 and 5, switches its expression to the mesometrial pole from day 6 onward. These results suggest that PGs produced at this site by COX-2 are involved in angiogenesis for the establishment of placenta. Recently, we also demonstrated a cellspecific expression pattern of cPLA2 α relevant to implantation in mice [23]. For example,

H. Wang, S.K. Dey / Prostaglandins & other Lipid Mediators 77 (2005) 84–102

89

cPLA2 α is expressed in the uterine epithelium on day 4 of pregnancy resembling the pattern of Cox-1 (Fig. 2). By the time of attachment reaction on day 4 midnight through day 5 morning, cPLA2 α is expressed in stromal cells surrounding the implanting blastocyst. With the progression of implantation (days 6–8), the expression of cPLA2 α is primarily restricted to the mesometrial pole of the implantation site as that of Cox-2 (Fig. 2). However, the expression of cPLA2 α is more widespread and apparently at lower levels than that of Cox2. These results suggest that cPLA2 α is available as an arachidonic acid provider for uterine PG biosynthesis during implantation.

4. cPLA2 ␣ is crucial for ‘on-time’ embryo implantation that directs subsequent development Of the many PLA2S , cPLA2␣ is known to couple functionally to COX-2 in specific cell types [24,25]. Our previous observations of spatiotemporal expression of CPLA2 ␣ and COX-2 in the periimplantation uterus indicate that cPLA2 ␣–COX-2 coupling is critical to embryo implantation and decidualization. Indeed, mice with null mutation for cPLA2 α exhibit small litters with presumed parturition defects and often show pregnancy failures [26,27].

4.1. Normal ‘window’ of implantation is altered in cPLA2 α−/− mice To reveal the underlying cause of this reduced fertility, we examined in detail the reproductive phenotypes of these females during pregnancy. A modest reduction in ovulation and fertilization rates was noted in these mutant females. However, these reduced rates could not fully account for the reduced litter size and frequent pregnancy failure observed in these mice [26,27], suggesting uterine defects between fertilization and parturition. We thus investigate whether cPLA2 ␣ deficiency impedes implantation and decidualization in mice. As shown in Fig. 3A, a large number of blastocysts fail to show on-time implantation in a significant number of cPLA2 α−/− female mice when examined on day 5 midmorning by blue dye methods [2]. Few implantation sites detected on this day showed a very weak blue reaction, indicating defective vascular permeability changes during the attachment reaction. However, those unimplanted embryos observed in cPLA2 α−/− uteri on day 5 did show blue reaction when examined on day 6, indicating that normal ‘window’ of implantation is deferred in cPLA2 α−/− mice (Fig. 3A and B). It is surmised that lack of cPLA2 ␣ reduces the production of arachidonic acid as a substrate for PG synthesis. Indeed, reduced uterine levels of PGs were noted in the absence of cPLA2 ␣ prior to and during implantation. As expected, exogenous administration of PGE2 and carbaprostacyclin (cPGI, a more stable analogue of PGI2 ) restored the normal implantation timing in cPLA2 α−/− mice (Fig. 3C). These observations reinforce the major role of cPLA2␣ in PG biosynthesis and its critical function in the initiation of attachment reaction. Moreover, by performing reciprocal embryo transfer studies, we provided evidence that maternal cPLA2␣ , but not embryonic, is the primary contributor to on-time implantation.

90

H. Wang, S.K. Dey / Prostaglandins & other Lipid Mediators 77 (2005) 84–102

4.2. Deferred implantation in cPLA2 α−/− mice leads to defective postimplantation development Findings of deferred implantation and small litter size (4.8 ± 0.8 versus 12.2 ± 0.8) in cPLA2 α−/− mice as compared to wild-type mice led us to speculate that there would be midgestational defects in embryonic growth in these mutant mice. Indeed, as shown in Fig. 3D, while most of the implantation sites in wild-type or cPLA2 α+/− mice were well spaced and developed normally, many implantation sites in cPLA2 α−/− mice were smaller and showed signs of resorption. Many of the isolated embryos from cPLA2 α−/− mice exhibited retarded growth at varying degrees (Fig. 3E). Furthermore, defective development of feto-placental unit with hemorrhagic placentas and preponderance of trophoblast giant cells was frequently noted, although decidual defect was not apparent in these null mutant mice (Fig. 3F). These results show that a transient delay in the attachment reaction produces heterogeneous fetoplacental developmental phenotypes ranging from less severe to markedly retarded growth. 4.3. Embryo spacing is disturbed in cPLA2 α−/− mice As mentioned above, although the implantation rate in cPLA2 α−/− mice increased on day 6 after a short delay, embryo spacing became aberrant (Fig. 3B–D). Implantation sites were closely apposed or even fused together (Fig. 3D). Upon dissection, we often observed that two or more embryos were residing in the same decidual envelope or conjoined by a single placenta (Fig. 3G). This could be one reason for retarded embryonic development and resorption in cPLA2 α−/− mice, resulting from crowding of embryos. However, retarded

Fig. 3. cPLA2 ␣ deficiency shifts the normal ‘window’ of implantation, leading to retarded feto-placental development in mice. (A) The number of implantation sites was examined on days 5 and 6 of pregnancy in wild-type and cPLA2 α−/− mice by the blue dye method. The numbers above the bars indicate the number of mice with implantation sites/total number of mice (unpaired t-test, *P < 0.001). The uteri of mice with a few or without implantation sites were flushed to recover unimplanted blastocysts. (B) Representative photographs of uteri with implantation sites (blue bands) on days 5 and 6. Note very few or no implantation sites on day 5, but unevenly spaced implantation sites on day 6 in cPLA2 α−/− mice. Arrowheads and arrows indicate ovaries and implantation sites, respectively. Brackets indicate crowding of implantation sites. (C) Restoration of normal implantation in cPLA2 α−/− mice. cPLA2 α−/− mice were injected with saline or PGE2 plus cPGI (carbarprostacyclin) twice (10:00 and 18:00 h) on day 4 and implantation sites were examined on day 5 (10:00 h). Note increased number of implantation sites with prominent blue reaction after PG treatment. Parentheses indicate crowding of implantation sites. (D) A composite photograph of uteri in wild-type and cPLA2 α−/− mice on day 12 of pregnancy. Resorption sites were often noted (arrows) and many implantation sites were closely apposed and even conjoined (parentheses) to each other in cPLA2 α−/− mice. (E) Photographs of embryos isolated from implantation sites of one representative wild-type 26 and two cPLA2 α−/− mice on day 12. Note retarded and asynchronous development of embryos in cPLA2 α−/− mice. (F) Histological examination of day 12 implantation sites in cPLA2 α−/− mice. Feto-placental units from cPLA2 α−/− mice were examined on day 12. Embryos and placentas show defective development with a preponderance of trophoblast giant cells. Arrowheads and an arrow indicate trophoblast giant cells and degenerating embryo, respectively. (c and d) Higher magnifications of (a and b), respectively, la, labyrinthine trophoblast; sp, spongiotrophoblast; dec, decidua. (G) Representative photographs of conjoined embryos in a placenta (a and c) and three embryos in the same decidual envelope (b and d) from cPLA2α −/− mice on day 12. (c) A histological section of (a) with two embryos; embryos shown in (d) are from (b). Yellow arrows indicate the source of the embryos from the decidual envelope. Reprinted with permission from Ref. [23].

H. Wang, S.K. Dey / Prostaglandins & other Lipid Mediators 77 (2005) 84–102

91

Fig. 3. (Continued).

growth of well-spaced embryos was also noted in cPLA2 α−/− mice. Limited information is currently available regarding cellular and molecular basis of embryo spacing in the uterus [28]. Previous reports using pharmacological inhibitors suggested that PGs are involved in embryo spacing in rats [29]. However, although PG treatment on day 4 of pregnancy restored normal implantation timing in cPLA2 α−/− mice, this treatment did not rescue altered embryo spacing (Fig. 3C), suggesting that other signaling molecules are involved in normal embryo spacing prior to the attachment reaction. Our results provide genetic evidence for a role of cPLA2␣ in this important event.

92

H. Wang, S.K. Dey / Prostaglandins & other Lipid Mediators 77 (2005) 84–102

Our studies demonstrate that timing of implantation is a crucial determinant for normal feto-placental development and pregnancy outcome. It is surmised that an altered uterine environment resulting from the shifting of the normal ‘window’ of implantation cannot efficiently support normal pregnancy. This finding provides a new concept that an early embryo–uterine interaction during implantation sets up the subsequent developmental programming. It has a major clinical significance, as implantation in humans beyond the normal ‘window’ of uterine receptivity (8–10 days postovulation) is associated with higher risk of early pregnancy losses [30].

5. COX-2 deficiency leads multiple female reproductive failures Vasoactive, mitogenic and differentiating properties of COX-derived PGs [16] have been implicated in various female reproductive functions. For example, PGs are involved in follicular rupture during ovulation [31], and also implicated as important mediators of increased endometrial vascular permeability during implantation and decidualization [22]. However, direct evidence of differential roles of COX-1 and COX-2 in these reproductive events was lacking until the generation of Cox-1−/− and Cox-2−/− mice [32,33]. With the availability of these gene targeted mice, we explored in detail their reproductive phenotypes. Consistent with our previous report of uterine induction of COX-2 at the site of blastocyst implantation [22], mice with null mutation for COX-2 show multiple female reproductive deficiencies that span ovulation, fertilization, implantation and decidualization [34]. By contrast, female mice with null mutation for COX-1 are fertile with limited parturition defects [32,35]. Studies on Cox-2−/− mice further demonstrate that while prostacyclin (PGI2) plays a major role in implantation, PGE2 plays a complementary role in this process [36]. 5.1. Ovulation and fertilization are defective in Cox-2−/− mice Previous studies provided clear evidence that COX-2 deficiency is the major cause of ovulation and fertilization failures, primarily contributing to infertility in Cox-2−/− females [34,37]. This is consistent with gonadotropin-mediated induction of COX-2 in ovarian follicles preceding ovulation [31,38]. Apparently normal follicular development with severely compromised ovulation after gonadotropin stimulation in Cox-2−/− mice suggests that ovulation failure in these females is independent of gonadotropin deficiency or defective responsiveness of the null ovary to these hormones [34,37]. Fertilization failure in COX-2-deficient mice is presumably the result of defective oocyte maturation. This is consistent with observations of peri-nuclear accumulation of COX-2, but not COX-1, in the cumulus cells surrounding the ovulated eggs (Fig. 4). It is considered that lack of COX-2 leads to impaired cumulus cell–oocyte interactions, which is critical for the production of fertilization-competent eggs [39]. Indeed, defective cumulus expansion and decreased expression of a COX-2 target gene TSG-6 are evident the absence of COX-2 [40]. Of many of PGs, PGE2 has been shown to be the major contributor of follicular rupture during ovulation. It is assumed that defective ovulation and fertilization in Cox-2−/− mice result from PGE2 deficiency. Defective ovulation and fertilization in Cox-2−/− null females is

H. Wang, S.K. Dey / Prostaglandins & other Lipid Mediators 77 (2005) 84–102

93

Fig. 4. Immunostaining of COX-1 and COX-2 in the cumulus cell-enclosed ovulated eggs. Images depict antigen in green, nuclei in red, and the merge in yellow. Arrow indicates perinuclear localization of COX-2 in cumulus cells (scale bar, 50 ␮m).

consistent with this assumption. Collectively, differential roles of COX-1 and COX-2 in the preovulatory follicle rupture and oocyte maturation during ovulation and fertilization indicate that differential subcellular sites of PG production by COX-1 and COX-2 accounts for distinct roles of these two isoforms in various cellular functions. 5.2. Embryo implantation is impaired in the absence COX-2 Since COX-2 is expressed uniquely in the mouse uterus surrounding the implanting blastocyst [22], the critical role of COX-2 in embryo implantation was speculated. To address this issue, we employed blastocyst transfer experiments in wild-type and Cox-2−/− mice to examine whether uterine COX-2 deficiency impedes the implantation process. Indeed, majority of transferred wild-type blastocysts failed to implant in Cox-2−/− uteri. In rodents, the establishment of a differentiated uterus for supporting blastocyst development and implantation primarily depends on the coordinated effects of ovarian steroid hormones, estrogen and progesterone [2]. Thus, to exclude the potential effects of COX-2 deficient ovaries with a reduced rate of ovulation on the uterine receptivity for implantation, a physiologically relevant delayed implantation model was adopted. In this model, the ovarian source of estrogen and progesterone were eliminated by ovariectomy and then supplied with an appropriate regimen of progesterone and estrogen conducive to implantation. We observed that wild-type blastocysts transferred into the uteri of COX-2 deficient mice under such condition again failed to implant, suggesting that inadequacy of ovarian steroids was

94

H. Wang, S.K. Dey / Prostaglandins & other Lipid Mediators 77 (2005) 84–102

not the cause of implantation failure and attesting again to the critical role of COX-2 in implantation. Pharmacological interruption of COX-2 activity and implantation by a selective COX-2 inhibitor also corroborated our genetic evidence for a critical role of COX-2 in implantation [34,41]. Among various PGs, PGI2 is the most abundant PG in the early pregnant mouse uterus and is higher at implantation sites than in inter-implantation sites [36]. Expression studies demonstrated that Cox-2, Prostacyclin synthase and PPARδ are coexpressed at the implantation site, suggesting that PGI2 –PPAR␦ signaling is operative in the uterus during implantation. This suggestion was consistent with the finding of rescue of implantation defects in Cox-2−/− mice by a prostacyclin analogue or a combination of PPAR␦ and retinoid X receptor (RXR, a copartner of PPARs) agonists [36]. Collectively, several lines of evidence suggest that timely and cell-specific expression of COX-2 is essential for embryo implantation in mice. 5.3. Decidualization is defective in Cox-2−/− mice The attachment reaction is followed by extensive stromal cell proliferation and differentiation into decidual cells. In pregnant mice, the stimulus for decidualization is the implanting blastocyst. This process, however, can be induced artificially in pseudopregnant uterus by intraluminal infusion of oil as a deciduogenic stimulus [42]. This model has been used widely to study the mechanism(s) of decidualization. Although the blastocyst attachment fails to occur in the absence of COX-2, it was argued whether this isozyme is also critical for decidualization. Using this experimentally induced decidualization model, we showed that pseudopregnant Cox-2−/− uteri fail to response to the oil stimuli, indicating that both the initial attachment reaction and the subsequent decidualization process are impaired in the absence of COX-2. To unraveling the underlining cause for the decidualization failure in Cox-2−/− uteri, we examined the expression status of Cox-1, Cox-2 and PPARδ genes in the wild-type uterus after intraluminal infusion of oil on day 4 of pseudopregnancy. No changes in Cox-1 mRNA was noted after oil infusion. In contrast, high levels of Cox-2 mRNA rapidly accumulated in the luminal epithelium of the oil-infused horn within 2 h followed by precipitous decline by 8 h. However, with the initiation of extensive stromal cell proliferation by 24 h after oil infusion, Cox-2 expression was again evident, but this time, in the uterine stroma in a focal fashion, similar to the pattern observed during normal implantation [36,43]. Under similar conditions, PPARδ was coordinately expressed with Cox-2 in the oil-infused uterine horn [36]. These results again point toward the role of COX-2–PGI2 –PPAR␦ signaling in the initiation of decidualization, which is further confirmed by observations of partial rescue of defective decidualization in the Cox-2 deficient mice by administration of a prostacyclin analogue [34,36]. 5.4. Compensatory up-regulation of COX-1 rescues female infertility from the loss of COX-2: a function of genetic makeup There is now increasing evidence that mutation of a gene often results in substantially altered phenotypes depending on the genetic background of mice in which the mutation is maintained [44,45]. Association of aspirin (a COX inhibitor) resistance and genetic polymorphisms has also been noted in humans [46]. Thus, we explored whether genetic disparity

H. Wang, S.K. Dey / Prostaglandins & other Lipid Mediators 77 (2005) 84–102

95

plays any role in altering COX-2 functions. We generated Cox-2−/− mice on a CD1 background by back-crossing C57BL/6/129 Cox-2−/− mice with CD1 for several generations and compared potential changes in reproductive phenotypes between these two lines [47]. To our surprise, normal ovulation with improved fertilization; implantation and decidualization was noted in CD1 Cox-2−/− mice. This led us to explore the underlying molecular mechanisms for the improved female fertility in these null mice. COX-2, but not COX-1, is widely considered as a major contributor of normal ovulation [31,38], which is consistent with our previous observation of defective ovulation in C57Bl/6/129 Cox-2−/− mice [34,37]. We observed that gonadotropin stimulation can dramatically induce Cox-1 expression in mural granulosa layers, in addition to Cox-2 accumulation in both mural granulosa and cumulus layers in preovulatory follicles [47]. Furthermore, this induction of Cox-1 occurs rapidly (4 h) in CD1 mice when compared with that in C57Bl/6 mice. This observation highlights the contribution of COX-1 to normal ovulation and accounts for the normal follicular rupture and ovulation in CD1 Cox-2−/− females. However, lack of COX-1 expression in cumulus cells coincident with the lower fertilization rate in CD1 Cox-2−/− mice reinforces the essential role of COX-2 of cumulus cell origin in promoting oocyte maturation and competence to fertilization. This is consistent with the consensus that cumulus cell–oocyte interactions are important for the generation of fertilization-competent eggs [39]. These improved ovulation and fertilization in CD1 Cox-2−/− mice by differential expression of COX isozymes led us to propose that a compensatory COX-1 function might offset the deficiency of COX-2 required for implantation in CD1 mice. Indeed, a unique up-regulated COX-1 expression in the uterus at the site of the blastocyst was observed in CD1 mice lacking COX-2 (Fig. 5), and improved implantation in CD1 mice compared with complete failure of implantation in C57BL/6J/129 mice missing COX-2. This provides strong evidence that COX-1 is also inducible in a fashion similar to that of COX-2 within a specific genetic environment and can compensate for the loss of COX-2. However, a shifting of the normal window of implantation occurred for certain blastocysts in CD1 Cox-2−/− mice with subsequent developmental anomalies and small litter sizes, suggesting that COX-2 is still an important factor for the full restoration of female reproductive functions. As described earlier, similar findings were observed in cPLA2␣ −/− mice. These results constitute a new concept that a short delay in the initial attachment reaction propagates detrimental effects during the later course of pregnancy. Furthermore, our study provides direct in vivo evidence that a compensatory mechanism via COX-1 up-regulation substantially rescues COX-2-deficient female infertility in a genetic background-dependent manner. Considering the worldwide use of nonsteroidal anti-inflammatory drugs including selective COX-2 inhibitors and genetic disposition to drug responses [46], these results raise a cautionary note against therapeutic use and efficacy of COX-2 inhibitors in inflammatory diseases among human populations without regard for genetic and ethnic diversities, and their consumption during pregnancy [48].

6. Endocannabinoid signaling: a biphasic sensor in embryo implantation An emerging concept in implantation is the role of endocannabinoids, a group of endogenously generated lipid mediators that bind to and activate cannabinoid re-

96

H. Wang, S.K. Dey / Prostaglandins & other Lipid Mediators 77 (2005) 84–102

ceptors [7]. The identification of two classical G protein (Gi/o)-coupled cell surface cannabinoid receptors CB1 and CB2, and two major endocannabinoids anandamide (Narachidonoylethanolamine) and 2-arachidonoyl glycerol as ligands for these receptors, has demonstrated endocannabinoid signaling in many central and peripheral systems [12,49]. Anandamide, as a most widely studied ligand for CB receptors, is an ethanolamine amide of arachidonic acid, which is also a major precursor of prostaglandin biosynthesis (Fig. 1). Although synthesis of anandamide by condensation arachidonic acid with ethanolamine has been shown in cell-free systems, the existence of this pathway has not yet been documented in living cells. Recently, evidence for another pathway for anandamide synthesis has been reported [50]. This pathway involves hydrolysis of a phospholipid precursor, N-arachidonylphosphatidyl ethanolamine (NAPE) in the cell membrane by its selective phospholipase D (NAPE-PLD) (Fig. 1). Once synthesized, anandamide is released from the cell into the extra cellular space where it can act in an autocrine or paracrine manner. The effectiveness of anandamide depends on its concentration in the extracellular space, which is controlled by a two-step process: (a) cellular uptake by a specific anandamide transporter, and (b) intracellular degradation by fatty acid amide hydrolase (FAAH). Once internalized by the transporter, anandamide is metabolized to arachidonic acid and ethanolamine by FAAH (Fig. 1). With respect to the role of endocannabinoid system in female reproduction, we provided evidence in mice for the presence of functional CB1 in preimplantation embryos [51] and of anandamide in the oviduct and uterus [52,53], suggesting that endocannabinoid signaling is operative very early in pregnancy. This is further supported by the findings of biphasic effects of anandamide on embryo development and implantation [54,55]. We have shown that anandamide within a very narrow range regulates blastocyst function and implantation by differentially modulating mitogen-activated protein kinase (MAPK) signaling and Ca2+ channel activity via CB1 receptors. For example, anandamide at a low concentration (7 nM) induces extracellular regulated kinase (ERK) phosphorylation and nuclear translocation in trophectoderm cells without influencing Ca2+ channels, and renders the blastocyst competent for implantation in the receptive uterus. In contrast, anandamide at a higher concentration (28 nM) inhibits Ca channel activity and blastocyst competency for implantation without influencing MAPK signaling (Fig. 6). This finding provides for the first time a potential “cannabinoid sensor” mechanism for influencing crucial steps during early pregnancy. Recently, an association of spontaneous pregnancy losses with elevated anandamide levels in women has been demonstrated [56,57], reinforcing that endocannabinoid signaling is at least one of the pathways that determine the fate of embryo

Fig. 5. Compensatory uterine expression of COX-1 during implantation in CD1 mice missing COX-2. (A) Expression of Cox-1 mRNA in a representative implantation site of a Cox-2−/− mouse on day 5 of pregnancy. With the initiation of implantation, Cox-1 is expressed in the uterine luminal epithelium and subepithelial stromal cells at the site of the implanting blastocyst (blue band) on day 5 of pregnancy in CD1 Cox-2−/− mice in a pattern resembling the native expression of Cox-2 in wild-type (WT) mice. This compensatory Cox-1 expression was not observed in day 5 C57BL/6/129 Cox-2−/− recipient uteri after transfer of day 4 wild-type blastocysts. Arrows indicate the location of blastocysts. Bar, 200 ␮m. (B) Up-regulated COX-1 protein levels at the implantation sites (IS) in CD1 Cox-2−/− mice on day 5 of pregnancy as determined by Western blotting. Reprinted with permission from Ref. [47].

H. Wang, S.K. Dey / Prostaglandins & other Lipid Mediators 77 (2005) 84–102

Fig. 5. (Continued).

97

98

H. Wang, S.K. Dey / Prostaglandins & other Lipid Mediators 77 (2005) 84–102

Fig. 6. A scheme depicting biphasic mode of endocannabinoid signaling in blastocyst activation and implantation. The previous and present investigations provide evidence that cannabinoid or endocannabinoid signaling influences blastocyst functions relevant to implantation by using different signal transduction pathways that are dependent on the levels of the ligands and/or receptors. Reprinted with permission from Ref. [55].

implantation. We propose that critical levels of endocannabinoids of uterine origin interact with appropriately expressed blastocyst CB1 in synchronizing blastocyst activation with uterine receptivity for implantation, whereas aberrant levels of uterine endocannabinoids and/or blastocyst CB1 interfere with these processes, resulting in pregnancy termination (Fig. 6).

7. Perspective PGs possess vasoactive, mitogenic and differentiating properties and are implicated in various female reproductive functions. Expression studies and genetic studies in mice provide strong evidence that cPLA2 –COX-2 derived PGs are major players in female reproductive functions. Recent studies have also demonstrated that Cox-2 is expressed either in the uterus, blastocyst or both during implantation in a variety of species with different mode of implantation including sub-human primates and humans [58,59], suggesting a conserved function of COX-2 derived PG signaling in implantation in various species. This information has added meaningful insights into the molecular basis of embryo–uterine cross-talk during implantation. Besides unraveling the nature of these lipid mediators, our new findings in cPLA2 α and Cox-2 deficient mice also put forward a new concept that a short delay in on-time implantation creates a progressive adverse effect throughout the gestation. We have also shown that COX-1, widely considered as a constitutive house-keeping gene, can also function as an inducible molecule in a fashion similar to COX-2 and can compensate for the loss of COX-2 within a specific genetic environment. It is hoped that

H. Wang, S.K. Dey / Prostaglandins & other Lipid Mediators 77 (2005) 84–102

99

these observations will help to correct implantation failure and improve pregnancy rates in women. Recently, a new function has been ascribed to COX-2. There is evidence that COX-2 can metabolize anandamide into prostaglandin-ethanolamide [60,61], suggesting a close link between endocannabinoids and eicosanoids. The potential function and signaling pathways of these metabolic products in embryo implantation remain to be determined. Since lower levels of anandamide at the implantation sites are associated with accumulation of FAAH in the implanting blastocysts [53,62], it appears that heightened expression of COX-2 at these sites [22] contributes to lower levels of anandamide and higher levels of PGs. It is conceivable that both embryonic FAAH and uterine COX-2 at the site of implantation maintain the optimal levels of anandamide favorable to the initiation and progression of implantation. COX-2 may also compete for the substrate arachidonic acid, the precursor for the synthesis of both PGs and anandamide, at the implantation sites. Thus, COX-2 could be the ‘lock-key’ to switching the balance between these two lipid mediators favorable to embryo implantation.

Acknowledgements Studies by the authors incorporated in this review were supported by NIH grants HD29968, HD12304, HD33994 and DA06668. S.K. Dey is a recipient of an NICHD and an NIDA MERIT Awards. H. Wang is Lalor Foundation fellow.

References [1] Psychoyos A. Endocrine control of egg implantation. In: Greep RO, Astwood EG, Geiger SR, editors. Handbook of physiology. Washington, DC: American Physiology Society; 1973. p. 187–215. [2] Paria BC, Huet-Hudson YM, Dey SK. Blastocyst’s state of activity determines the “window” of implantation in the receptive mouse uterus. Proc Natl Acad Sci USA 1993;90:10159–62. [3] Enders AC, Schlafke S. A morphological analysis of the early implantation stages in the rat. Am J Anat 1967;120:185–226. [4] Das SK, Wang XN, Paria BC, et al. Heparin-binding EGF-like growth factor gene is induced in the mouse uterus temporally by the blastocyst solely at the site of its apposition: a possible ligand for interaction with blastocyst EGF-receptor in implantation. Development 1994;120:1071–83. [5] Parr EL, Tung HN, Parr MB. Apoptosis as the mode of uterine epithelial cell death during embryo implantation in mice and rates. Biol Reprod 1987;36:211–25. [6] McMaster MT, Dey SK, Andrews GK. Association of monocytes and neutrophils with early events of blastocyst implantation in mice. J Reprod Fertil 1993;99:561–9. [7] Paria BC, Dey SK. Ligand-receptor signaling with endocannabinoids in preimplantation embryo development and implantation. Chem Phys Lipids 2000;108:211–20. [8] Valentin E, Lambeau G. Increasing molecular diversity of secreted phospholipases A(2) and their receptors and binding proteins. Biochim Biophys Acta 2000;1488:59–70. [9] Gijon MA, Leslie CC. Regulation of arachidonic acid release and cytosolic phospholipase A2 activation. J Leukoc Biol 1999;65:330–6. [10] Murakami M, Nakatani Y, Kuwata H, Kudo I. Cellular components that functionally interact with signaling phospholipase A(2)s. Biochim Biophys Acta 2000;1488:159–66.

100

H. Wang, S.K. Dey / Prostaglandins & other Lipid Mediators 77 (2005) 84–102

[11] Clark JD, Schievella AR, Nalefski EA, Lin LL. Cytosolic phospholipase A2. J Lipid Mediat Cell Signal 1995;12:83–117. [12] Mechoulam R, Fride E, Di Marzo V. Endocannabinoids. Eur J Pharmacol 1998;359:1–18. [13] Serhan CN, Oliw E. Unorthodox routes to prostanoid formation: new twists in cyclooxygenase-initiated pathways. J Clin Invest 2001;107:1481–9. [14] Song C, Chang XJ, Bean KM, Proia MS, Knopf JL, Kriz RW. Molecular characterization of cytosolic phospholipase A2-beta. J Biol Chem 1999;274:17063–7. [15] Underwood KW, Song C, Kriz RW, Chang XJ, Knopf JL, Lin LL. A novel calcium-independent phospholipase A2, cPLA2-gamma, that is prenylated and contains homology to cPLA2. J Biol Chem 1998;273:21926– 32. [16] Smith WL, DeWitt DL, Garavito RM. Cyclooxygenases: structural, cellular, and molecular biology. Annu Rev Biochem 2000;69:145–82. [17] Kurumbail RG, Stevens AM, Gierse JK, et al. Structural basis for selective inhibition of cyclooxygenase-2 by anti-inflammatory agents. Nature 1996;384:644–8. [18] Ohno K, Fujiwara M, Fukushima M, Narumiya S. Metabolic dehydration of prostaglandin E2 and cellular uptake of the dehydration product: correlation with prostaglandin E2-induced growth inhibition. Biochem Biophys Res Commun 1986;139:808–15. [19] Fukushima M. Biological activities and mechanisms of action of PGJ2 and related compounds: an update. Prostaglandins Leukot Essent Fatty Acids 1992;47:1–12. [20] Forman BM, Tontonoz P, Chen J, Brun RP, Spiegelman BM, Evans RM. 15-Deoxy-delta 12,14-prostaglandin J2 is a ligand for the adipocyte determination factor PPARgamma. Cell 1995;83:803–12. [21] Forman BM, Chen J, Evans RM. Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors alpha and delta. Proc Natl Acad Sci USA 1997;94:4312–7. [22] Chakraborty I, Das SK, Wang J, Dey SK. Developmental expression of the cyclo-oxygenase-1 and cyclooxygenase-2 genes in the peri-implantation mouse uterus and their differential regulation by the blastocyst and ovarian steroids. J Mol Endocrinol 1996;16:107–22. [23] Song H, Lim H, Paria BC, et al. Cytosolic phospholipase A2alpha is crucial [correction of A2alpha deficiency is crucial] for ‘on-time’ embryo implantation that directs subsequent development. Development 2002;129:2879–89. [24] Reddy ST, Herschman HR. Prostaglandin synthase-1 and prostaglandin synthase-2 are coupled to distinct phospholipases for the generation of prostaglandin D2 in activated mast cells. J Biol Chem 1997;272: 3231–7. [25] Takano T, Panesar M, Papillon J, Cybulsky AV. Cyclooxygenases-1 and -2 couple to cytosolic but not group IIA phospholipase A2 in COS-1 cells. Prostaglandins Other Lipid Mediat 2000;60:15–26. [26] Bonventre JV, Huang Z, Taheri MR, et al. Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2. Nature 1997;390:622–5. [27] Uozumi N, Kume K, Nagase T, et al. Role of cytosolic phospholipase A2 in allergic response and parturition. Nature 1997;390:618–22. [28] McLaren A, Michie D. The spacing of implantations in the mouse uterus. Mem Soc Endocrinol 1959;6:65–74. [29] Well stead JR, Bruce NW, Rahima A. Effects of indomethacin on spacing of conceptuses within the uterine horn and on fetal and placental growth in the rat. Anat Rec 1989;225:101–5. [30] Wilcox AJ, Baird DD, Weinberg CR. Time of implantation of the conceptus and loss of pregnancy. N Engl J Med 1999;340:1796–9. [31] Sirois J, Simmons DL, Richards JS. Hormonal regulation of messenger ribonucleic acid encoding a novel isoform of prostaglandin endoperoxide H synthase in rat preovulatory follicles. Induction in vivo and in vitro. J Biol Chem 1992;267:11586–92. [32] Langenbach R, Morham SG, Tiano HF, et al. Prostaglandin synthase 1 gene disruption in mice reduces arachidonic acid-induced inflammation and indomethacin-induced gastric ulceration. Cell 1995;83:483– 92. [33] Dinchuk JE, Car BD, Focht RJ, et al. Renal abnormalities and an altered inflammatory response in mice lacking cyclooxygenase II. Nature 1995;378:406–9. [34] Lim H, Paria BC, Das SK, et al. Multiple female reproductive failures in cyclooxygenase 2-deficient mice. Cell 1997;91:197–208.

H. Wang, S.K. Dey / Prostaglandins & other Lipid Mediators 77 (2005) 84–102

101

[35] Reese J, Paria BC, Brown N, Zhao X, Morrow JD, Dey SK. Coordinated regulation of fetal and maternal prostaglandins directs successful birth and postnatal adaptation in the mouse. Proc Natl Acad Sci USA 2000;97:9759–64. [36] Lim H, Gupta RA, Ma WG, et al. Cyclo-oxygenase-2-derived prostacyclin mediates embryo implantation in the mouse via PPARdelta. Genes Dev 1999;13:1561–74. [37] Matsumoto H, Ma W, Smalley W, Trzaskos J, Breyer RM, Dey SK. Diversification of cyclooxygenase-2derived prostaglandins in ovulation and implantation. Biol Reprod 2001;64:1557–65. [38] Joyce IM, Pendola FL, O’Brien M, Eppig JJ. Regulation of prostaglandin-endoperoxide synthase 2 messenger ribonucleic acid expression in mouse granulosa cells during ovulation. Endocrinology 2001;142:3187–97. [39] Matzuk MM, Burns KH, Viveiros MM, Eppig JJ. Intercellular communication in the mammalian ovary: oocytes carry the conversation. Science 2002;296:2178–80. [40] Ochsner SA, Russell DL, Day AJ, Breyer RM, Richards JS. Decreased expression of tumor necrosis factoralpha-stimulated gene 6 in cumulus cells of the cyclooxygenase-2 and EP2 null mice. Endocrinology 2003;144:1008–19. [41] Reese J, Zhao X, Ma WG, Brown N, Maziasz TJ, Dey SK. Comparative analysis of pharmacologic and/or genetic disruption of cyclooxygenase-1 and cyclooxygenase-2 function in female reproduction in mice. Endocrinology 2001;142:3198–206. [42] Rankin JC, Ledford BE, Baggett B. Early involvement of cyclic nucleotides in the artificially stimulated decidual cell reaction in the mouse uterus. Biol Reprod 1977;17:549–54. [43] Scherle PA, Ma W, Lim H, Dey SK, Trzaskos JM. Regulation of cyclooxygenase-2 induction in the mouse uterus during decidualization. An event of early pregnancy. J Biol Chem 2000;275:37086–92. [44] Threadgill DW, Dlugosz AA, Hansen LA, et al. Targeted disruption of mouse EGF receptor: effect of genetic background on mutant phenotype. Science 1995;269:230–4. [45] Bonyadi M, Rusholme SA, Cousins FM, et al. Mapping of a major genetic modifier of embryonic lethality in TGF beta 1 knockout mice. Nat Genet 1997;15:207–11. [46] Cambria-Kiely JA, Gandhi PJ. Aspirin resistance and genetic polymorphisms. J Thromb Thrombolysis 2002;14:51–8. [47] Wang H, Ma WG, Tejada L, et al. Rescue of female infertility from the loss of cyclooxygenase-2 by compensatory up-regulation of cyclooxygenase-1 is a function of genetic makeup. J Biol Chem 2004;279:10649–58. [48] Li DK, Liu L, Odouli R. Exposure to non-steroidal anti-inflammatory drugs during pregnancy and risk of miscarriage: population based cohort study. BMJ 2003;327:368. [49] Maccarrone M, Finazzi-Agro A. Endocannabinoids and their actions. Vitam Horm 2002;65:225–55. [50] Okamoto Y, Morishita J, Tsuboi K, Tonai T, Ueda N. Molecular characterization of a phospholipase D generating anandamide and its congeners. J Biol Chem 2004;279:5298–305. [51] Paria BC, Das SK, Dey SK. The preimplantation mouse embryo is a target for cannabinoid ligand-receptor signaling. Proc Natl Acad Sci USA 1995;92:9460–4. [52] Das SK, Paria BC, Chakraborty I, Dey SK. Cannabinoid ligand-receptor signaling in the mouse uterus. Proc Natl Acad Sci USA 1995;92:4332–6. [53] Schmid PC, Paria BC, Krebsbach RJ, Schmid HH, Dey SK. Changes in anandamide levels in mouse uterus are associated with uterine receptivity for embryo implantation. Proc Natl Acad Sci USA 1997;94:4188–92. [54] Wang J, Paria BC, Dey SK, Armant DR. Stage-specific excitation of cannabinoid receptor exhibits differential effects on mouse embryonic development. Biol Reprod 1999;60:839–44. [55] Wang H, Matsumoto H, Guo Y, Paria BC, Roberts RL, Dey SK. Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation. Proc Natl Acad Sci USA 2003;100:14914–9. [56] Maccarrone M, Valensise H, Bari M, Lazzarin N, Romanini C, Finazzi-Agro A. Relation between decreased anandamide hydrolase concentrations in human lymphocytes and miscarriage. Lancet 2000;355:1326–9. [57] Maccarron M, Bisogno T, Valensise H, et al. Low fatty acid amide hydrolase and high anandamide levels are associated with failure to achieve an ongoing pregnancy after IVF and embryo transfer. Mol Hum Reprod 2002;8:188–95. [58] Kim JJ, Wang J, Bambra C, Das SK, Dey SK, Fazleabas AT. Expression of cyclooxygenase-1 and -2 in the baboon endometrium during the menstrual cycle and pregnancy. Endocrinology 1999;140:2672–8. [59] Marions L, Danielsson KG. Expression of cyclo-oxygenase in human endometrium during the implantation period. Mol Hum Reprod 1999;5:961–5.

102

H. Wang, S.K. Dey / Prostaglandins & other Lipid Mediators 77 (2005) 84–102

[60] Kozak KR, Crews BC, Morrow JD, et al. Metabolism of the endocannabinoids, 2-arachidonylglycerol and anandamide, into prostaglandin, thromboxane, and prostacyclin glycerol esters and ethanolamides. J Biol Chem 2002;277:44877–85. [61] Yu M, Ives D, Ramesha CS. Synthesis of prostaglandin E2 ethanolamide from anandamide by cyclooxygenase-2. J Biol Chem 1997;272:21181–6. [62] Paria BC, Zhao X, Wang J, Das SK, Dey SK. Fatty-acid amide hydrolase is expressed in the mouse uterus and embryo during the periimplantation period. Biol Reprod 1999;60:1151–7.