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Vincent Center for Reproductive Biology,4 Vincent Obstetrics and Gynecology Service, Massachusetts General Hospital, and Department of Obstetrics, Gynecology, and Reproductive Biology, Harvard Medical School, Boston, Massachusetts 02114
Department of Biomedical Sciences,5 Colorado State University, Fort Collins, Colorado 80523
ABSTRACT
During early gestation in invasively implanting species, the uterine stromal compartment undergoes dramatic remodeling, defined by the differentiation of stromal fibroblast cells into decidual cells. Lipid signaling molecules from a number of pathways are well-established functional components of this decidualization reaction. Because of a correlation in the events that transpire in the uterus during early implantation with known functions of bioactive sphingolipid metabolites established from studies in other organ systems, we hypothesized that uterine sphingolipid metabolism would change during implantation. By a combination of Northern blot, Western blot, and immunohistochemical analyses, we establish that enzymes at each of the major catalytic steps in the sphingolipid cascade become transcriptionally up-regulated in the uterus during decidualization. Each of the enzymes analyzed was up-regulated from Days of Pregnancy (DOP) 4.5–7.5. When comparing embryo-induced decidualization (decidual) with mechanically induced decidualization (deciduomal), sphingomyelin phosphodiesterase 1 (Smpd1) mRNA and sphingosine kinase 1 (SPHK1) protein were shown to be dually regulated in the endometrium by both maternal and embryonic factors. As measured by the diacyl glycerol kinase assay, ceramide levels rose in parallel with Smpd1 gene expression, suggesting that elevated transcription of sphingolipid enzymes results in heightened catalytic activity of the pathway. Altogether, these findings place sphingolipids on a growing list of lipid signaling molecules that become increasingly present at the maternal-embryonic interface.
ceramide,, decidua, decidualization, implantation, pregnancy, sphingolipid, sphingosine-1-phosphate, uterus
Unexplained pregnancy failure has been attributed to a variety of factors, including poor oocyte or embryo quality [1, 2]. Others suggest that luteal insufficiency [3] or endocrine disruption [4] results in habitual pregnancy loss. However, it is becoming increasingly clear from epidemiological studies in humans [5], as well as genetic studies in rodents [6], that failed pregnancy occurs in large part because of faulty uterine function or miscommunication between the embryo and mother prior to placentation. In invasively implanting species such as humans and rodents, the endometrial stromal compartment undergoes a dramatic change during early gestation involving proliferation, growth, and differentiation of resident stromal cells into large polyploidy decidual cells [7, 8]. This differentiated stromal tissue, called the deciduum, serves a variety of functions that are critical for pregnancy. For instance, the deciduum is by definition a secretory tissue producing a variety of important endocrine and paracrine signaling molecules, such as prolactin-related proteins [9], interleukins [10], cytokines [10], and prostanoids [11]. Additional known functions of the deciduum include its immunosuppressive actions [12, 13] and control of trophoblast growth and cell migration [14], providing a source of nutrients for the expanding trophectoderm [7, 8], and it also provides a rudimentary vascular network for nutrient/gas exchange for the embryo prior to development of the placenta [15]. Early decidualization is controlled primarily by maternal factors, because the hormonally primed uterus can be stimulated by mechanical means (e.g., infusion of sesame oil) to undergo decidualization [8]. The resultant endometrial structure (i.e., deciduoma) is similar to the embryo-induced deciduas in many respects, and yet it lacks substantial mesometrial vasculature. In addition to nutrient/gas exchange, the vascular component of the deciduum serves as a conduit for invading trophoblast cells that utilize the maternal blood vessels to establish a fetoplacental vascular connection with the maternal vascular system. In mice, decidualization begins shortly after the blastocyst adheres to the uterine epithelial lining on Day of Pregnancy (DOP) 4.5. The decidualized uterus must then provide a suitable environment for the developing embryo until the placenta becomes functionally competent. The deciduum therefore serves a complementary function to that of the placenta for 5–6 days, or approximately one third of the duration of pregnancy. This time is somewhat reduced in humans to approximately 20% of the pregnancy timeline.
Published reports focusing on lipid signaling in the uterus are plentiful, the most obvious and well studied being the steroid hormones estradiol and progesterone, which primarily regulate gene transcription through activation of nuclear receptors. More recently, however, other lipid signaling pathways have also proven to be essential for uterine function during implantation [16]. These pathways include signaling by various prostanoid [17] or arachidonic acid [18] derivatives, endocannabinoids (reviewed in Lim et al. [17]), and, most recently, lysophosphatidic acid [19]. Because of a correlation in the events that transpire in the uterus during early implantation with known functions of sphingolipid metabolites established from studies in other organ systems, we hypothesized that sphingolipid metabolism changes in the uterus as decidualization progresses. Sphingolipids play functional roles in angiogenesis, cell migration, apoptosis, regulation of secretion of extracellular materials, signal transduction, immunomodulation, and cellular differentiation [20, 21]. In the present study, we describe uterine expression and regulation of key enzymes coordinating the interconversion of the most well-studied sphingolipid metabolites (i.e., ceramide, sphingosine, and sphingosine-1-phosphate [S1P]) during the implantation phase of early gestation in mice.
Sexually mature female (6–10 wk of age) and male ICR mice (Charles River Laboratories, Wilmington, MA) were paired under an alternating 12L:12D cycle to establish pregnancies. For analysis of sphingomyelin phosphodiesterase 1 (Smpd1) expression, female mice were bred with intact or vasectomized males. Female mice were considered Day 0.5 of pregnancy or pseudopregnancy upon observation of a vaginal seminal plug. Uterine tissue (all days of pseudopregnancy and DOP 3.5) and whole implantation sites (DOP 4.5, 7.5, and 9.5) were collected and prepared for RNA isolation. Implantation sites were identified on DOP 4.5, 10 min after i.v. injection of Evans blue dye. For the remaining expression experiments, tissue was collected from implantation sites on DOP 4.5, and uterine decidual tissue was microdissected free of the embryo/fetus, extraembryonic membranes, and myometrium on DOP 7.5, 9.5, 11.5, 14.5, and 17.5. For measurements of ceramide concentrations in the uterus during early pregnancy, implantation sites were collected on DOP 4.5, 7.5, and 9.5 and microdissected free of embryo and associated membranes (DOP 7.5 and 9.5). To demonstrate embryonic regulation of select sphingolipid-metabolizing enzymes, female mice were placed with vasectomized male mice to induce pseudopregnancy. Sesame oil (10 µl) was then injected intraluminally on one side of the uterus immediately below the uterotubal junction to induce decidualization on Day 4 of pseudopregnancy. The contralateral uterine side was left unstimulated to avoid stromal decidualization. Uterine tissue was collected 72 h later, a time corresponding to Day 7.5 of pregnancy. All animal protocols were reviewed and approved by the Massachusetts General Hospital Institutional Care and Use Committee.
RNA Isolation and Northern Blot Analysis
Uterine tissues were collected for RNA isolation on Days 3.5, 4.5, 7.5, and 9.5 of pseudopregnancy and DOP 3.5, 4.5, 7.5, and 9.5. Embryos and extraembryonic membranes were removed by microdissection after DOP 4.5. Total cellular RNA was isolated from uterine tissue with Tri-reagent as described by the manufacturer (Sigma Chemical Co., St. Louis, MO). On DOP 4.5, implantation sites were identified by the blue dye method. The expression of Smpd1, N-acylsphingosine amidohydrolase 1 (Asah1), sphingosine-1-phosphate phosphohydrolase (S1pph), and sphingosine kinase 2 (Sphk2) was then evaluated by Northern blotting with riboprobes. Each cDNA was cloned by PCR with primer sets provided in Table 1. Figure 1 shows a schematic of the sphingomyelin pathway and highlights enzymes identified in this study as being transcriptionally regulated in the uterus during pregnancy. PCR-amplified cDNAs were then subcloned into the pGEM-T Easy cloning vector (Promega, Madison, WI), amplified in bacteria, and sequenced to confirm primer specificity. Northern blot analysis, as described in detail elsewhere [22, 23], was then used to assess mRNA expression of one representative sphingolipid-metabolizing enzyme at each step in the pathway. Briefly, 10 µg of total RNA was separated by electrophoresis on formaldehyde denaturing agarose gels. The RNA was transferred to nylon membrane (Amersham Biosciences, Piscataway, NJ) by capillary action and then probed with radiolabeled (Perkin Elmer, Boston, MA) Smpd1, Asah1, S1pph, and Sphk2 riboprobes. Blots were then reprobed with a radiolabeled 18S ribosomal RNA (Ambion, Austin, TX) cDNA, which served as an internal control.
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Dissected tissues were fixed in buffered 4% paraformaldehyde and then embedded in paraffin. Medial sections (6 µm) of implantation sites were deparaffinized with xylenes followed by graded rehydration in ethanol (100%, 95%, 80%, and 70%) and distilled water. Following peroxidase quenching (5 min in methanol containing 3% hydrogen peroxide), sphingosine kinase 1 (SPHK1) antigen was unmasked by high-temperature antigen retrieval (10 min of boiling in microwave) in which sections were immersed in 10 mM sodium citrate buffer (pH 6.5) and allowed to cool slowly to room temperature [24]. After equilibration in PBS and blocking (2% BSA and 1% normal donkey serum), sections were incubated overnight at 4°C with the primary antibody (rabbit anti-SPHK1 antiserum [25]) diluted (0.74 µg/ml) in TNK buffer (0.1 M Tris, pH 7.6; 0.55 M NaCl; 0.01 M KCl). For colorimetric detection, sections were then washed (PBS, 3x for 10 min each) and incubated for 1 h at room temperature with a 1:200 dilution of biotinylated secondary antibody (1:200; Santa Cruz Biotechnologies, Santa Cruz, CA); they were then washed again and incubated a final time with horseradish peroxidase (HRP)-conjugated streptavidin for 45 min at room temperature (Vector Laboratories, Burlingame, CA). After washing as before, sections were exposed to 3,3'-diaminobenzidine substrate (diluted to manufacturer's specifications; Sigma) for 2–10 min, counterstained with hematoxylin, dehydrated in ethanol and xylenes, and mounted for light microscopy. For confocal microscopy (Zeiss LSM 5 Pascal; Carl Zeiss Advanced Imaging Microscopy, Jena, Germany), Alexa 546 or Alexa 488 conjugated streptavidin (1:300; Invitrogen, Carlsbad, CA) was used in place of HRP-conjugated streptavidin as described for colorimetric staining. Sections were counterstained with To-Pro-3 (1:1000 dilution in PBS; Invitrogen) to visualize nuclei. Specificity of antigen detection was confirmed in control experiments in which tissue sections were 1) prepared as described above but with omission of the primary antibody and 2) incubated with rabbit immunoglobulin G (IgG, 0.74 µg/ml; Santa Cruz Biotechnologies) in place of the primary antibody.
Protein lysates were collected for Western blot analysis as described in detail [26]. Following separation by SDS-PAGE by the NuPage System (Invitrogen), proteins (20 µg/lane) were transferred (100 V, 1 h) to polyvinylidene difluoride membranes. Nonspecific binding was blocked with 5% fat-free milk in TBST buffer (50 mM Tris-HCl [pH 7.5], 0.15 NaCl, 0.05% Tween-20) for 1 h at room temperature. The SPHK1 protein was detected with a polyclonal antibody [25] diluted to 250 ng/ml in TBST. Membranes were then washed (3x for 10 min each) in TBST buffer and incubated with anti-rabbit IgG HRP conjugate (1:2500; Cell Signaling, Danvers, MA) for 1 h at room temperature. The membranes were washed with TBST as before, and bound antibody was detected with enhanced chemiluminescent reagents based on the manufacturer's recommendations (Amersham). To verify equal protein loading, membranes were then stripped (1 M glycine, pH 2.5, 1 h, 37°C) and reprobed with pan-AKT antibody (1:1000; Cell Signaling). Specificity of antigen detection was confirmed by negative controls in which blots were 1) prepared as described above but with omission of the primary antibody and 2) incubated with rabbit IgG (250 ng/ml; Santa Cruz Biotechnologies) in place of the primary antibody.
The amount of ceramide generated in the uterus during early gestation was determined by the diacyl glycerol kinase assay [27]. Membrane lipids were extracted (1 ml of methanol, 10 min, –80°C) from uterine tissues at implantation sites microdissected free of embryos and extraembryonic membranes (10 mg) on DOP 4.5, 7.5, and 9.5 and dried under nitrogen gas. Lipids were subjected to mild alkaline hydrolysis (500 µl of 0.1 N KOH for 1 h at 37°C); reextracted with 500 µl of chloroform, 270 µl of saline, and 30 µl of EDTA (1.5 mM); and dried under nitrogen gas. A reaction mixture containing 6 µl of cardiolipin (25 mg/ml; Avanti Polar Lipids, Alabaster, AL) and 20 µl of diethylenetriaminepentaacetic acid (DETAPAC, 1 mM) was vortexed vigorously, followed by the addition of 6.2 µl of octyl-ß-D-glucopyranoside, 50 µl of 2x reaction buffer (100 mM NaCl, 100 mM imidazole, 2 mM EDTA, and 25 mM MgCl2), 8 µl of imidazole:DETAPAC (10 mM:1 mM), 2 µl of dithiothreitol (100 mM), 1 µl of AMP (100 mM), 8 µl of water, and 3.5 µl of diacylglycerol kinase (1 mg/ml; Calbiochem). The mixture was vortexed briefly and preincubated for 30 min at 22°C. After the addition of 1 µl of [
-32P]ATP (3000 Ci/mmol; Amersham Pharmacia Biotech, Arlington Heights, IL), samples were incubated for 30 min at 22°C. The reaction was stopped with chloroform (500 µl) to extract lipids. The samples were vortexed and centrifuged briefly to separate the phases, and the organic phase was dried under nitrogen gas. The final residue of lipids was reconstituted in 50 µl of chloroform, of which 40 µl was loaded onto thin-layer chromatography plates (LK6D silica gel; Fisher Scientific, Pittsburgh, PA). The resulting product of the kinase reaction, ceramide-1-phosphate (C1P), was resolved in 65% chloroform-15% methanol-5% acetic acid. Plates were air dried and exposed to film for 2 h. The resolved band in each lane corresponding to C1P was cut from the chromatography plate and counted in a scintillation counter. Changes in uterine ceramide production in response to pregnancy are presented as nanomoles per milligram protein values.
Experimental Replication and Statistical Analysis
Each experiment was independently replicated at least three times, with different mice being used in each experiment. All data presented graphically are the mean ± SEM from replicated experiments. Assignment of mice to each experiment was made randomly. Raw data were analyzed with GraphPad PRISM software (version 4.0, GraphPad, San Diego, CA) by one- or two-way analysis of variance followed by the Tukey multiple comparison test to identify significant differences (P < 0.05) in mean values.
Smpd1 Is Up-Regulated in Whole Implantation Sites During Early Gestation
We first studied expression of Smpd1 mRNA throughout early gestation by Northern blot analysis. The SMPD1 enzyme catalyzes the hydrolysis of sphingomyelin, resulting in the formation of the bioactive compound ceramide (Fig. 1 [28–30]). As shown in Figure 2A, uterine expression of Smpd1 remained unchanged through all days of pseudopregnancy evaluated. However, a dramatic increase (P < 0.001) in Smpd1 was observed during the invasion phase of implantation (i.e., DOP 7.5; Fig. 2B), compared with the corresponding day of pseudopregnancy, and remained elevated through DOP 9.5.
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Elevated Expression of Multiple Sphingolipid Enzymes as Pregnancy Progresses
The SMPD1 enzyme is one of nearly two dozen known enzymes that regulate sphingolipid metabolism (reviewed in Futerman and Hannun [29]). Northern blotting was used to study the expression of representative genes from other enzyme families that regulate sphingolipid metabolism. As shown in Figure 3, Asah1, S1pph, and Sphk2 were significantly (P < 0.05) up-regulated from DOP 4.5 to DOP 7.5. Expression of Asah1 and S1pph remained elevated through DOP 9.5, whereas Sphk2 expression was reduced from DOP 7.5 to DOP 9.5 (Fig. 3B).
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SPHK1 Protein Expression Correlates with Uterine Stromal Cell Decidualization
We previously demonstrated the expression of S1P receptors at the maternal-embryonic interface and hypothesized that the enzymatic machinery for generating S1P was expressed at the implantation site [23]. In this next set of experiments, we characterized the uterine expression of the SPHK1, an enzyme that, in addition to SPHK2, catalyzes the conversion of sphingosine to its phosphorylated derivative S1P (Fig. 1). SPHK1 protein was minimally expressed in the uterus on DOP 4.5, but it then became up-regulated (P < 0.05) by DOP 7.5 (Fig. 4, A and B). Unlike Sphk2, SPHK1 peaked on DOP 9.5 and then declined steadily through the remainder of pregnancy. The antibody used in this experiment detected two isoforms (see arrows in Fig. 4A) of the SPHK1 enzyme. Each isoform appeared as a doublet on Western blots. The additional bands could be the encoded products of previously unreported mRNA splice variants, or they may represent posttranslationally modified forms of the enzyme, such as is often observed following protein phosphorylation [31]. No bands were detected in control experiments when the primary antibody was omitted or when the IgG antibody was applied in place of anti-SPHK1 antibodies (Fig. 4A). The representative immunoblot in Figure 4C emphasizes the elevated expression of SPHK1 protein in the uterus during pregnancy by comparison with the nondecidualized uterus of pseudopregnancy, as well as the lung, liver, spleen, and intestine.
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SPHK1 is expressed predominantly in the luminal epithelium on DOP 4.5, with little or no detectable expression in underlying stromal cells (Fig. 5A). By DOP 7.5, SPHK1 was observed throughout the decidualized endometrium, particularly at the antimesometrial pole (Fig. 5, C and G), with limited expression in the mesometrium. SPHK1 remained abundantly expressed in decidual stromal cells, encasing the embryonic chamber on DOP 9.5 (Fig. 5E), and was also easily detected in decidual tissue at the mesometrial pole by DOP 11.5 (Fig. 5H). As shown in Figure 5I, SPHK1 expression remained elevated at the mesometrial pole of the implantation site on DOP 14.5, where it was observed in cells immediately surrounding large maternal blood vessels. Little-to-no signal was observed at any stage of pregnancy when the primary antibody was omitted (data not shown) or when IgG was used in place of the SPHK1 antibody (Fig. 5, B, D, and F). Although the data in Figure 5 clearly demonstrate that SPHK1 is expressed by decidual cells, we cannot rule out the possibility that at least some SPHK1 protein within the deciduum, as shown by Western blotting (Fig. 4), derives from blood.
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Changes in Sphingolipid Content During Decidualization
To demonstrate that the sphingolipid pathway was not only transcriptionally and translationally up-regulated during implantation but that a concomitant rise in sphingolipid content was also evident, we measured ceramide in uterine tissue from implantation sites microdissected free of embryo and extraembryonic membranes by the diacyl glycerol kinase method [27]. Ceramide content increased (P < 0.05, Fig. 6) 2.8-fold from DOP 4.5 to DOP 7.5 and remained elevated through DOP 9.5.
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Maternal and Embryonic Factors Dually Regulate Expression of Sphingolipid-Metabolizing Enzymes During Uterine Decidualization
To begin to understand mechanisms regulating sphingolipid metabolism in the uterus during early gestation, we employed a comparative decidualization model in which stromal cell differentiation was initiated either by the embryo or mechanically by intrauterine infusion of sesame oil. As shown in Figure 7A, Smpd1 was minimally expressed in unstimulated uterine tissue on Day 7.5 of pseudopregnancy. By comparison, Smpd1 increased significantly in oil-induced uterine tissue on Day 7.5 of pseudopregnancy (deciduoma) but then became elevated further in embryo-induced decidual tissue on DOP 7.5 (Fig. 7B). Results from this experiment suggest that Smpd1 is regulated initially as part of the maternally controlled developmental program of decidualization, but then becomes up-regulated further by an embryonic factor(s). Similar findings were observed for SPHK1 in which expression of this enzyme correlated with decidualization, and the presence of the embryo further increased (P < 0.05) SPHK1 expression (Fig. 7, C and D).
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In the present study, it was established that several enzymes catalyzing the interconversion of sphingolipids are transcriptionally up-regulated in the uterus during early gestation. We measured uterine ceramide at three different time points during early pregnancy and found ceramide to increase significantly during decidualization. Another interesting finding is that while sphingolipid-metabolizing enzymes are up-regulated as part of the maternally controlled decidualization paradigm (i.e., deciduoma of pseudopregnancy), expression is elevated further during embryo-induced decidualization (i.e., decidua of pregnancy). Although it is possible that endometrial expression of these enzymes is simply delayed in the deciduomal model, a more likely explanation for why expression of the enzymes is increased in decidual tissue is that the embryo contributes to the positive regulation of genes encoding these enzymes. In support of this, a recent expression profiling study demonstrated that human trophoblast conditioned medium up-regulated expression of SPHK1 in decidualized uterine stromal cells [32]. Genetic studies in mice have clearly outlined a need for homeostatic sphingolipid metabolism and signaling during development (reviewed in Anliker and Chun [33] and Hla [34]). Evidence that sphingolipids actively participate in pregnancy is derived from three sources. Kharel et al. [35] published a recent study demonstrating that when compared with heterozygous or wild-type female mice, Sphk2-null dams were less apt to carry a pregnancy to term and gave birth to litters with fewer pups. Likewise, female mice lacking both S1P receptors S1P2 and S1P3 deliver fewer pups than wild-type littermates [36]. Studies from our own laboratory show that Smpd1-null female mice give birth to fewer pups and have an increased incidence of embryonic and fetal resorption during gestation, regardless of paternal genotype (Pru, unpublished results). The exact role or roles played by sphingolipid-metabolizing enzymes and S1P receptors during implantation remain to be established.
The terminal bioactive product in the sphingolipid pathway, S1P, is highly angiogenic. S1P receptors, of which there are five known (S1P1–5), are formerly called endothelial differentiation genes. S1P1, S1P2, and S1P3 are required for vascular development both during embryogenesis [37] and wound repair [38]. Perhaps uterine-derived S1P facilitates blood vessel formation at the mesometrial pole of the implantation site. Indeed, the temporal expression of SPHK1, which peaks on DOP 9.5, correlates well with uterine angiogenesis. We established previously that S1P1 and S1P2 are expressed on endothelial cells of the mesometrium [23]. Interestingly, detectable expression of these S1P receptors was diminished in deciduomal tissues (i.e., oil-induced decidual tissue), suggesting embryonic regulation of these genes [23]. The possibility that uterine-derived S1P coordinates mesometrial angiogenesis is at least plausible, because both the pathway that synthesizes S1P (present study) and the genes that promote angiogenesis [32] are regulated in decidualized stromal cells by the embryo.
Alternative to the potential function of S1P in uterine angiogenesis, S1P may affect some aspect of embryonic development. S1P1 and S1P2 have been localized to specific cells within the placenta by immunohistochemistry, as well as several tissues in the embryo and fetus [23, 37, 39]. This raises the possibility that the uterine deciduum, through synthesis of sphingolipid metabolites, regulates the invasive capacity of the embryo. We noted that the spatial expression pattern of SPHK1 was consistent with a barrier. Indeed, expression of this enzyme was predominantly antimesometrial and adjacent to the embryo in fully differentiated decidual cells during embryo invasion (DOP 7.5), but it then completely encircled the embryo by DOP 11.5. Perhaps uterine-derived sphingolipids attenuate trophoblast advancement into the uterus or regulate differentiation of the trophoblast lineage. Johnstone et al. [40] recently demonstrated that S1P inhibited differentiation of primary human cytotrophoblasts into syncytiotrophoblasts through a Gi-coupled receptor response, a pathway activated by S1P receptors. Sphingosine kinase 1 was also richly expressed in perivascular tissue surrounding larger maternal vascular canals. As suggested by Hemmings et al. [41], the possibility exists that faulty S1P signaling contributes to diseases of pregnancy such as intrauterine growth restriction or preeclampsia. Sphingosine-1-phosphate may also control local decidual lymphocyte trafficking or differentiation. A pivotal role has been established for S1P as a potent regulator of lymphocyte egress, blocking the release of lymphocytes from secondary lymphoid tissues [42]. Uterine-derived S1P may modify adaptive immune responses during pregnancy by regulating lymphocyte numbers or their activities within the deciduum or systemically by inhibiting lymphocyte egress from secondary lymphoid organs.
It is interesting that SphK1/SphK2 and S1PPH1, with opposing metabolic actions on S1P availability, were all up-regulated. Measuring other sphingolipids and the activities of enzymes that generate these bioactive molecules should shed light on functional roles of the sphingolipid pathway during pregnancy. Uterine-derived S1P levels, for example, may not change in association with expression of the sphingosine kinase enzymes. Because of the highly vascular nature of the deciduum, S1P may function systemically in endocrine fashion rather than locally as a paracrine/autocrine signaling molecule. A potential target of uterine-derived but systemically acting S1P would be the immune system.
Ceramide is situated at a critical branch point in the sphingomyelin pathway, utilized both as a backbone for the synthesis of more complex sphingolipids and for the production of other bioactive lipids, such as C1P, sphingosine, and S1P. Local sphingolipid synthesis in the uterine compartment may enhance the innate immune response or uterine mesometrial angiogenesis. Glycosphingolipids, such as those derived from the ceramide backbone, are now well-established activators of natural killer (NK) cells, and they also regulate cytokine secretion from NK cell [43]. In the deciduum of pregnancy, immune cells universally promote blood vessel formation at the implantation site in a number of species [44, 45]. Local production of sphingolipids, and glycoceramide derivatives in particular, may coordinate NK cell function at the implantation site. Although the present study adds to the likelihood that yet another lipid signaling pathway functions at the implantation site, many key questions remain to be resolved. For example, we established that multiple sphingolipid enzyme families with opposing actions are transcriptionally up-regulated. Interestingly, while an increase in ceramide content was noted from DOP 4.5 to DOP 7.5, a concomitant increase in Asah1 expression, which encodes an enzyme that metabolizes ceramide to sphingosine, was also observed. Why ceramide levels increase despite elevated Asah1 expression remains to be determined. However, it may be that while gene expression is up-regulated, ASAH1 enzyme activity may not be optimal or at least reduced in comparison to SMPD1 activity. This suggests that sphingolipid enzymes are posttranslational modified to regulate enzyme activity. In support of this, both SMPD1 and SPHK1 activities have been shown to be dependent on protein phosphorylation [31, 46]. What is clear from these studies is that the several components of the sphingolipid pathway are up-regulated in the decidualizing uterus during pregnancy and that ceramide content rises accordingly. One other explanation that could account for a global rise in sphingolipid content in the deciduum of early pregnancy is that uterine-derived sphingolipids may serve as a nutrient source for the developing embryo in the absence of a functional placenta. It will be necessary in future studies to measure decidual content for each of the sphingolipid molecules as well as the activity of each of the enzymes. Experiments such as this will be helpful in delineating which sphingolipid signaling molecules are most critical for pregnancy.
ACKNOWLEDGMENTS
We are grateful to Dr. Yoshiko Banno for kindly providing the SPHK1 antibodies.
FOOTNOTES
3These authors contributed equally to this study. 
1Supported in part by Vincent Memorial Research Funds and National Institutes of Health grants HD 032475 (T.R.H.) and ES012070 (J.K.P.).
Correspondence: 2James K. Pru, Vincent Center for Reproductive Biology, Vincent Obstetrics and Gynecology Service, Massachusetts General Hospital, Harvard Medical School, Thier Research Building, Room 931, 55 Fruit St., Boston, MA 02114. FAX: 617 724 9935; e-mail: jpru{at}partners.org
Received: 20 February 2007.
First decision: 18 March 2007.
Accepted: 18 June 2007.
REFERENCES
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