Maternal disturbance in activated sphingolipid metabolism causes pregnancy loss in mice (original) (raw)
Sphk1–/–Sphk2+/– female mutants are infertile. Sphk1_–/–_Sphk2+/– females were phenotypically normal. Unexpectedly, however, when they were mated, they were found to be infertile. Mating of Sphk1_–/–_Sphk2+/– females with fertile males of any genotype did not produce offspring. In contrast, male Sphk1_–/–_Sphk2+/– mice were normally fertile. Normal mating behavior was observed in the mutant females when they were caged with males. To determine the causes of impaired fertility in Sphk1_–/–_Sphk2+/– females, we examined in detail the reproductive phenotypes of these mice during pregnancy. The appearance and histology of the ovaries were normal in Sphk1_–/–_Sphk2+/– females, with corpora lutea, various stages of follicular development, and lesser amounts of interstitial tissue, which are all compatible with wild-type mice (Figure 1A).
Normal ovarian functions and implantation in Sphk1_–/–_Sphk2+/– female mice. (A) Representative photographs of H&E staining of ovaries (n = 3). (B) Serum P4 levels on day 6.5 pc, day 7.5 pc, day 8.5 pc, and day 11.5 pc (n = 3). (C–E) Measurement of Sphk activity in ovariectomized wild-type females. The mice were given a single injection of E2 (100 ng/mouse), a single injection of P4 (2 mg/mouse), or E2 plus P4, and sacrificed 6 hours or 12 hours later (C and D). Another group of mice received a regimen designed to mimic the P4 and E2 levels during the estrous cycle and early pregnancy described in Methods (E). The assay was performed in the presence of Triton X-100 (C and E) or of BSA complexes without Triton X-100 (D and E). The data represent mean values ± SE (n = 3, *P < 0.01, paired Student’s t test). (F) Ovulation and fertilization rates on day 1.5 pc. Results of ovulation are mean values ± SE. n = 4 (wild-type), n = 3 (Sphk1_–/–_Sphk2+/–), unpaired Student’s t test. (G) Representative photographs of uteri with implantation sites (blue bands) on day 5.5 pc. Arrows indicate implantation sites. (H) The number and weight of implantation sites were examined on day 5.5 pc by the blue dye method. The data represent mean values ± SE. n = 3 (wild-type); n = 5 (Sphk1_–/–_Sphk2+/–); unpaired Student’s t test. Scale bars: 200 μm (A); 1 mm (G).
Circulating serum progesterone (P4) levels in Sphk1_–/–_Sphk2+/– females were not significantly different from those in wild-type females on days 6.5 pc, 7.5 pc, 8.5 pc, and 11.5 pc (Figure 1B), suggesting that luteal P4 secretion is normal during pregnancy in the mutant females. Although the P4 levels showed a downward trend as developmental stages advanced, it was not statistically significant. To determine whether Sphk activity is regulated by ovarian steroid hormones, ovariectomized mice received a subcutaneous injection of estrogen (E2; 100 ng/mouse), P4 (2 mg/mouse), or both (E2 + P4), and were sacrificed at 6 and 12 hours. Mice injected with oil (vehicle) served as controls. Total Sphk activity was measured in homogenates of whole uteri in the presence of Triton X-100 (Figure 1C), which stimulates Sphk1 and inhibits Sphk2. The Sphk activity was not significantly induced by any injections (Figure 1C). Similar results were obtained when the assay was run in the absence of Triton X-100 and with sphingosine added as a complex with BSA, conditions in which both Sphk1 and Sphk2 are normally active (Figure 1D). When ovariectomized mice were treated with a regimen that mimics the estradiol and P4 levels of the estrous cycle and early pregnancy described in Methods, Sphk activity was slightly increased (1.5-fold) in the presence of Triton X-100, suggesting that the Sphk1 activity could be weakly induced by P4 with estrogen priming (Figure 1E).
Ovulation and fertilization were examined by counting the number of ovulated eggs and fertilized 2-cell embryos on day 1.5 pc. There were no significant differences in the ovulated eggs and fertilization rate between wild-type and Sphk1_–/–_Sphk2+/– females (Figure 1F). Implantation was assessed by using a blue dye reaction and gross histological analysis. The number, size, weight and intensity of implantation sites in day 5.5 pc wild-type and Sphk1_–/–_Sphk2+/– females were similar (Figure 1, G and H), indicating that normal implantation had occurred in the mutant females. There were no spacing abnormalities (Figure 1G). These results suggest that the infertility observed in Sphk1_–/–_Sphk2+/– females is unlikely to be caused by defective steroid hormone secretion, ovulation, fertilization, or implantation.
Defective uterine functions in Sphk1–/–Sphk2+/– female mutants lead to pregnancy loss. To elucidate the cause of the infertility in Sphk1_–/–_Sphk2+/– mice, pregnant uteri and embryos were inspected at various developmental stages. In this study, all females were mated with wild-type males. On day 6.5 pc, embryos in Sphk1_–/–_Sphk2+/– uteri did not differ significantly from those in wild-type uteri (Figure 2, A–D). On day 7.5 pc, however, 27% of embryos examined (n = 243) in Sphk1_–/–_Sphk2+/– uteri were considerably smaller than those found in wild-type uteri (Figure 2, E–H and Q). The embryos were frequently found absorbed even at this stage (52%) (Figure 2Q). Some embryos (21%) still looked normal at this stage. On day 8.5 pc in Sphk1_–/–_Sphk2+/– uteri, most embryos had been absorbed (79%, n = 86), just leaving traces of yolk sac and no placenta formations (Figure 2, I–L and Q). On days 9.5 pc and 10.5 pc, almost all Sphk1_–/–_Sphk2+/– uteri looked dark red, reminiscent of hemorrhage, in sharp contrast with the normally developed embryos and placentas in wild-type uteri (Figure 2, M–P).
Defective uterine function leading to early pregnancy loss in Sphk1_–/–_Sphk2+/– female mice. (A–P) Photographs of whole uteri (including embryos) and embryos on day 6.5 pc (A–D), day 7.5 pc (E–H), day 8.5 pc (I–L), day 9.5 pc (M and N), and day 10.5 pc (O and P). (A, E, I, M, and O) Whole uteri from wild-type female mice. (B, F, J, N, and P) Whole uteri from Sphk1_–/–_Sphk2+/– female mice. (C, G, and K) Embryos from uteri of wild-type female mice. (D, H, and L) Embryos from uteri of Sphk1_–/–_Sphk2+/– female mice. Scale bars: 200 μm (C, D, G, H, K, and L); 1 mm (A, B, E, F, I, J, and M–P). (Q) Summary of the embryonic phenotype from day 7.5 pc (wild-type, n = 256; Sphk1_–/–_Sphk2+/+, n = 101; Sphk1+/+_Sphk2_–/–, n = 69; and Sphk1_–/–_Sphk2+/–, n = 243), day 8.5 pc (wild-type, n = 40; Sphk1_–/–_Sphk2+/–, n = 86), and day 9.5 pc (wild-type, n = 7; Sphk1_–/–_Sphk2+/–, n = 49) uteri. Embryos were counted according to this classification: normal, developmental delay (the length of long axis: < 1.0 mm, day 7.5 pc; < 2.0 mm, day 8.5 pc; < 2.5 mm, day 9.5 pc), or absorbed, and the percentages were calculated for inclusion in the figure.
To exclude embryonic effects, day 3.5 pc embryos were collected from wild-type females that had been mated with wild-type males and transferred to the uteri of recipient pseudopregnant (day 2.5 pc) wild-type and Sphk1_–/–_Sphk2+/– female mice. Examination of the uteri at day 8.5 pc indicated that wild-type embryos were unable to survive in the uteri of Sphk1_–/–_Sphk2+/– mice (0%, n = 26) while most of the embryos survived in the uteri of wild-type mice (71%, n = 28). Furthermore, there was no difference in the number of pups derived from wild-type (8.8, n = 29) and Sphk1_–/–_Sphk2+/– (9.0, n = 27) females on day 7.5 pc. Taken together, these results demonstrated that the infertility observed in Sphk1_–/–_Sphk2+/– females is attributable to maternal effects.
Uterine decidualization is impaired in pregnant Sphk1–/–Sphk2+/– mice. To explore the pathological changes that underlie the pregnancy loss observed in Sphk1_–/–_Sphk2+/– mice, pregnant uteri in wild-type and Sphk1_–/–_Sphk2+/– female mice between days 4.5 pc and 7.5 pc were examined histologically. On day 4.5 and day 5.5 pc, the appearance of Sphk1_–/–_Sphk2+/– uteri did not significantly differ from that of uteri from wild-type mice. The decidual cells and embryos in the mutant uteri were morphologically indistinguishable from those in wild-type, suggesting that the decidualization had begun normally in the mutant mice (Figure 3, A and B, and data not shown). However, day 6.5 pc uteri from Sphk1_–/–_Sphk2+/– mice revealed hemorrhage and multinucleated neutrophil infiltration in the area surrounding the embryo, although the embryos were still viable (Figure 3, C–F). On day 7.5 pc, the hemorrhage and neutrophil infiltration in the decidual region were more noticeable (Figure 3, G–K), with necrotic embryos often observed (Figure 3K). The neutrophils were identified by immunostaining with an anti-neutrophil antibody (Figure 3L).
Defective decidualization in Sphk1_–/–_Sphk2+/– uteri. (A–K) H&E staining of longitudinal sections from wild-type and Sphk1_–/–_Sphk2+/– uteri on day 5.5 pc (A and B), day 6.5 pc (C–F), and day 7.5 pc (G–K). (A, C, G, and H) Wild-type uteri. (B, D–F, and I–K) Sphk1_–/–_Sphk2+/– uteri. Arrows in D, F, and I–K indicate hemorrhage, and arrowheads in E and K indicate neutrophil infiltration in the decidua of Sphk1_–/–_Sphk2+/– uteri. (L) Immunostaining with anti-neutrophil antibody on day 7.5 pc Sphk1_–/–_Sphk2+/– uteri. Arrowheads indicate positive neutrophils. De, decidua; Em, embryo; He, hemorrhage; Np, neutrophil; Ne; necrotic embryo. (M and N) In situ hybridization for Sphk1 in wild-type uteri using antisense (M) or sense (N) probes. Arrows in M indicate Sphk1 expression. Scale bars: 100 μm (A–D, G–J, and L); 50 μm (E, F, and K); and 1 mm (M and N). In all experiments, more than 3 animals were examined. (O) Artificial decidualization. Wild-type or Sphk1_–/–_Sphk2+/– mice were given intraluminal oil infusion on day 3.5 pc of pseudopregnancy. On day 7.5 pc, the uteri were weighed. Fold increases denote comparison of weights between infused and noninfused uterine horns. The numbers above the bars indicate the number of responding mice/total number of mice. Results are expressed as mean ± SE. *P < 0.01, unpaired Student’s t test.
Sphk1 mRNA expression was examined in day 7.5 pc wild-type uteri by in situ hybridization. The Sphk1 mRNA was localized in the antimesometrial portion of the decidua, with more abundant expression in the secondary decidual zone (Figure 3, M and N), consistent with a role of Sphk1 in decidualization.
Decidualization can be induced experimentally in pseudopregnant or steroid hormonally treated uteri by intraluminal oil infusion. Thus, we examined decidualization in Sphk1_–/–_Sphk2+/– mice by intraluminal oil infusion on day 3.5 pc. Sphk1_–/–_Sphk2+/– mice showed a significantly lower decidual response than wild-type mice with respect to increased uterine weight (4.3 ± 3.1 versus 18.4 ± 4.2) (Figure 3O). While 100% (10/10) of the wild-type mice responded to the stimuli, only 56% (5/9) of Sphk1_–/–_Sphk2+/– mice showed this response. Taken together, these results suggest a crucial role of Sphk in decidualization.
We assessed cell mitosis by anti-phosphohistone H3 immunostaining on day 5.5 pc (Figure 4, A–D) and day 6.5 pc (Figure 4, E–H) uteri. The number of mitotic cells was significantly decreased in the Sphk1_–/–_Sphk2+/– uteri compared with wild-type uteri at both stages (Figure 4I). We further studied cell survival of decidual cells in pregnant uteri. Apoptosis was assessed in wild-type and Sphk1_–/–_Sphk2+/– uteri on day 5.5 pc and day 7.5 pc by the TUNEL assay. A mild increase in cell death was already observed in the decidual zone of day 5.5 pc Sphk1_–/–_Sphk2+/– uteri immediately surrounding the embryo, although they grossly looked normal (Figure 4, J and K). The day 7.5 pc Sphk1_–/–_Sphk2+/– uteri revealed remarkably increased apoptosis in the area surrounding the embryo, particularly in the primary decidual zone (Figure 4, L–O). To determine what cell types were undergoing increased cell death, immunostaining was performed using anti-cytokeratin and anti-desmin antibodies to label trophoblast cells and decidual cells, respectively (23–26). Desmin staining was observed throughout the decidual tissues, where shrunken, apparently dying cells were stained in the primary decidual zone (Figure 4, R and S). On the other hand, excessive trophoblast giant cell invasion was not observed in the decidual tissues, as denoted by the cytokeratin staining (Figure 4, P and Q). These results suggest that decidual cells are specifically undergoing increased cell death, which is not caused by increased trophoblast giant cell invasion.
Decreased cell mitosis and increased cell death in Sphk1–/–Sphk2+/– decidua. (A–H) Immunostaining with anti-phospho-histone H3 on wild-type (A, B, E, and F) and Sphk1_–/–_Sphk2+/– (C, D, G, and H) uteri on day 5.5 pc (A–D) and day 6.5 pc (E–H). (I) Percentage of phospho-histone H3–positive cells. n = 6 matched pairs. *P < 0.05; **P < 0.01, paired Student’s t test. (J and K) TUNEL assay on wild-type (J) and Sphk1_–/–_Sphk2+/– (K) uteri on day 5.5 pc. Arrows in K indicate the increased cell death in Sphk1_–/–_Sphk2+/– uteri. (L–O) TUNEL assay on wild-type (L and M) and Sphk1_–/–_Sphk2+/– (N and O) uteri on day 7.5 pc. M and O represent high-power views of the boxed areas in L and N, respectively. Arrows in N indicate the increased cell death in Sphk1_–/–_Sphk2+/– decidua. (P and Q) Immunostaining with anti-cytokeratin to label trophoblast cells on day 7.5 pc Sphk1_–/–_Sphk2+/– uteri. Q represents a high-power view of the boxed area in P. Arrows in P correspond to those in N. Note no positive staining in the area indicated by arrows. Arrowheads indicate positive trophoblast cells. (R and S) Immunostaining with anti-desmin to label decidual cells on day 7.5 pc Sphk1_–/–_Sphk2+/– uteri. S shows a high-power view of the boxed area in R. Arrows in R correspond to those in N. Note shrunken decidual cells and normal decidual cells indicated by arrows and asterisks, respectively, in S. Scale bars: 100 μm (A–H and L–S); 50 μm (J and K). In all experiments, more than 3 animals were examined.
Defects in decidual cells and decidual blood vessels in Sphk1–/–Sphk2+/– uteri. Day 7.5 pc uteri of pregnant mice were further analyzed by electron microscopy. In Sphk1_–/–_Sphk2+/– uteri, all decidual blood vessels immediately surrounding embryos revealed severe endothelial cell breakage. Cellular organelles, such as the nucleus, mitochondria, ribosomes, and endoplastic reticulum as well as collagen fibers, were freely floating in the lumen of the blood vessels, and intact basal lamina and tight junctions were rarely detectable (Figure 5, D and E). Blood cell components from broken blood vessels were sometimes in contact with decidual cells directly (Figure 5G). Surprisingly, multilayered membranous cytoplasmic bodies, reminiscent of those found in sphingolipid storage diseases, were observed in the cytoplasm of decidual cells (Figure 5, H and I). The endothelial cells also included these cytoplasmic bodies, which were often found to be floating in the lumen of blood vessels (Figure 5F). Interestingly, Sphk1_–/–_Sphk2+/+ uteri also showed defects, although milder, in decidual blood vessels and decidual cells, in spite of the fact that they exhibited normal fertility. Blood vessels from Sphk1_–/–_Sphk2+/+ decidua contained a mixture of nearly normal and aberrant endothelial cells with light and coarse cytoplasm (Figure 5J). The endothelial cells in Sphk1_–/–_Sphk2+/+ decidua were occasionally disrupted similarly to those in Sphk1_–/–_Sphk2+/– decidua (Figure 5K). Furthermore, Sphk1_–/–_Sphk2+/+ decidua also included multilayered membranous cytoplasmic bodies (Figure 5L) although they were less prominent than those observed in Sphk1_–/–_Sphk2+/– decidua. Neither defective endothelial cells nor membranous cytoplasmic bodies were observed in wild-type decidua (Figure 5, A and C), and all wild-type blood vessels showed clear tight junctions and basal lamina (Figure 5B). The membranous cytoplasmic bodies were not found in any cells of brain, liver, and spleen from day 7.5 pc Sphk1_–/–_Sphk2+/– females; these are target organs of many sphingolipid storage diseases (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI30674DS1). Blood vessels were also intact in these organs (Supplemental Figure 1). These data suggest that the cellular defects are specific to pregnant uteri.
Transmission electron microscopic analysis of decidual cells and decidual blood vessels from wild-type (A–C), Sphk1_–/–_Sphk2+/– (D–I), and Sphk1_–/–_Sphk2+/+ (J–L) uteri. aEC, aberrant endothelial cell with light and coarse cytoplasm; bEC, broken endothelial cell; BL, basal lamina; CB; multilayered membranous cytoplasmic body; DC, decidual cell; EC, endothelial cell; EJ, endothelial cell-cell junction; RBC, red blood cell; WBC, white blood cell. Arrows in D–F and K indicate broken endothelial cells. Arrows in H, I, and L indicate multilayered membranous cytoplasmic bodies. Original magnification, ×2,000 (A, C, D, G, H, and K); ×16,000 (B, F, and I); ×6,300 (E and L); and ×3,150 (J).
We further examined the expression of factors implicated in angiogenesis, vascular endothelial growth factor (Vegf), angiopoietin 1 (Angpt1), angiopoietin 2 (Angpt2), and endothelial-specific receptor tyrosine kinase (Tie2) on day 6.5 pc and day 7.5 pc decidua from wild-type and Sphk1_–/–_Sphk2+/– females (Figure 6, A–D). There was no difference in the expression levels of these factors in wild-type and Sphk1_–/–_Sphk2+/– decidua either on day 6.5 pc or day 7.5 pc, indicating that these factors would not be the cause of disturbance in Sphk1_–/–_Sphk2+/– uteri.
Vegf, Angpt1, Angpt2, and Tie2 mRNA expression. Vegf (A), Angpt1 (B), Angpt2 (C), and Tie2 (D) mRNA expression on day 6.5 pc and day 7.5 pc decidua from wild-type and Sphk1_–/–_Sphk2+/– females as determined by real-time PCR. The expression levels are shown relative to those in wild-type decidua. Data represent mean values ± SE. n = 3.
The sphingolipid metabolic pathway is highly activated in the decidua during pregnancy. Total Sphk activity was measured in homogenates of nonpregnant whole uteri, day 7.5 pc decidua of pregnant mice, and day 7.5 pc interimplantation tissues (uterine tissues lacking implantation sites) from wild-type, Sphk1_–/–_Sphk2+/+, Sphk1+/+_Sphk2_–/–, and Sphk1_–/–_Sphk2+/– female mice, from which the embryos had been removed. Strikingly, Sphk activity was increased 9.8-fold in day 7.5 pc wild-type decidua of pregnant mice compared with that in day 7.5 pc wild-type interimplantation tissues in pregnant mice in the presence of Triton X-100 (Figure 7, A and B), which stimulates Sphk1 and inhibits Sphk2. Sphk activity was also increased in Sphk1+/+_Sphk2_–/– decidua (12.0-fold) compared with that in Sphk1+/+_Sphk2_–/– interimplantation tissues while Sphk activity was barely detectable either in Sphk1_–/–_Sphk2+/+ or Sphk1_–/–_Sphk2+/– uteri, irrespective of whether they were decidua or interimplantation tissues (Figure 7, A and B). Similar results were obtained in the absence of Triton X-100, a condition in which both Sphk1 and Sphk2 are normally active, although Sphk activity was detectable to some extent in Sphk1_–/–_Sphk2+/+ and Sphk1_–/–_Sphk2+/– uteri in this assay system (Figure 7, C and D). Nonpregnant whole uteri showed a trend of Sphk activity similar to that of interimplantation tissues in all genotypes of mice (Figure 7, A–D). These results suggest that Sphk activity was dramatically increased during pregnancy in wild-type and Sphk1+/+_Sphk2_–/– decidua whereas it was not inducible during pregnancy in either Sphk1_–/–_Sphk2+/+ or Sphk1_–/–_Sphk2+/– decidua.
Measurement of Sphk activity. Sphk enzymatic activity was determined in nonpregnant whole uteri, day 7.5 pc decidua, and day 7.5 pc interimplantation tissues from wild-type, Sphk1_–/–_Sphk2+/+, Sphk1+/+Sphk2–/–, and Sphk1_–/–_Sphk2+/– females. The assay was performed in the presence of Triton X-100 (A and B) or of BSA complexes without Triton X-100 (C and D). (A and C) TLC analysis of [32P] S1P formed by Sphk in uterine samples. The bands for S1P and origin are shown. (B and D) Sphk activity. The data represent mean values ± SE and were compared between day 7.5 pc decidua and day 7.5 pc interimplantation tissues. n = 3. *P < 0.05, paired Student’s t test.
To investigate sphingolipid metabolism in normal pregnancy, we examined the mRNA levels for major genes involved in the sphingolipid metabolic pathway (Figure 8) in nonpregnant whole uteri, day 7.5 pc decidua of pregnant mice, and day 7.5 pc interimplantation tissues from wild-type and Sphk1_–/–_Sphk2+/– female mice, from which the embryos had been removed. In line with Sphk activity, the Sphk1 mRNA levels were 9.3-fold higher in day 7.5 pc wild-type decidua of pregnant mice than in day 7.5 pc wild-type interimplantation tissues while the _Sphk_2 mRNA levels were not significantly altered (Figure 9A). The expression of other key genes (Figure 8) was also upregulated in pregnant wild-type decidua compared with pregnant wild-type interimplantation tissues: serine palmitoyl transferase 1 and 2 (Sptlc1, 8.7-fold; Sptlc2, 6.3-fold), S1P lyase (Sgpl1; 11.3-fold), S1P phosphatase 1 (Sgpp1; 7.7-fold), and sphingomyelinase 1 (Smpd1; 5.3-fold) (Figure 9A). The mRNA levels of the genes involved in the sphingolipid pathway, other than Sphk1 and Sphk2, were also elevated in Sphk1_–/–_Sphk2+/– decidua compared with Sphk1_–/–_Sphk2+/– interimplantation tissues and did not significantly differ from those in wild-type decidua (Figure 9A). Interestingly, the Lass genes, which encode dihydroceramide synthases (27), were not highly elevated in the wild-type decidua during pregnancy compared with interimplantation tissues although the expression levels of the Lass 5 and Lass 6 were slightly increased (1.4-fold and 2.8-fold, respectively) (Figure 9B). The expression profile of nonpregnant whole uteri was similar to that of interimplantation tissues in both wild-type and Sphk1_–/–_Sphk2+/– mice (Figure 9, A and B). Furthermore, day 7.5 pc uteri from Sphk1_–/–_Sphk2+/+ and Sphk1+/–_Sphk2_–/– pregnant females, both of which are fertile, exhibited a similar expression profile to the wild-type uteri, except for the lack of Sphk1 and Sphk2 expression, respectively (Supplemental Figure 2, A and B). Taken together, these results suggest that the de novo synthesis pathway for sphingolipids, in particular the portion leading to the production and degradation of S1P, is highly activated at the transcription level in the decidua during pregnancy and that the reproductive phenotype observed in Sphk1_–/–_Sphk2+/– females can be mainly attributed to the Sphk1 expression, but with a dosage effect of Sphk2.
Sphingolipid and related glycerophospholipid biosynthetic pathways. Enzymes, expression levels of which are increased during pregnancy, are indicated in red color. X, genetic disruption of Sphk genes.
The expression levels of enzymes involved in sphingolipid metabolism during pregnancy. Relative mRNA expression of Sphk1, Sphk2, Sptlc1, Sptlc2, Sgpl1, Sgpp1, Smpd1, Smpd2, Smpd3, Asah1, Asah2, Lass1, Lass2, Lass4, Lass5, Lass6, and Degs1 in nonpregnant whole uteri, day 7.5 pc decidua of pregnant mice, and day 7.5 pc interimplantation tissues from wild-type and Sphk1_–/–_Sphk2+/– females as determined by real-time PCR. Expression levels are shown relative to those in nonpregnant wild-type uteri. Data represent mean values ± SE and were compared between day 7.5 pc decidua and day 7.5 pc interimplantation tissues. n = 3. *P < 0.05; **P < 0.01, paired Student’s t test.
Sphingoid bases abnormally accumulate in pregnant Sphk1–/–Sphk2+/– decidua. Sphingolipid levels were measured by mass spectrometry in homogenates of nonpregnant whole uteri (wild-type and Sphk1_–/–_Sphk2+/–) and day 7.5 pc decidua of pregnant mice (wild-type, Sphk1_–/–_Sphk2+/+, Sphk1+/+_Sphk2_–/–, and Sphk1_–/–_Sphk2+/–). In day 7.5 pc Sphk1_–/–_Sphk2+/+ and Sphk1_–/–_Sphk2+/– decidua, dihydroS1P levels were slightly higher than in day 7.5 pc wild-type decidua (1.6-fold and 1.9-fold, respectively) (Figure 10A). S1P levels were not significantly altered in all types of pregnant decidua examined (Figure 10B). In sharp contrast, both dihydrosphingosine (sphinganine) and sphingosine levels were substantially increased (70.5-fold and 18.0-fold, respectively) in day 7.5 pc Sphk1_–/–_Sphk2+/– decidua as compared with those in day 7.5 pc wild-type decidua (Figure 10, C and D). Higher levels of sphingoid bases were also observed in Sphk1_–/–_Sphk2+/+ decidua, although less than in Sphk1_–/–_Sphk2+/– decidua (Figure 10, C and D). Ceramide and sphingomyelin levels were not significantly different in all types of day 7.5 pc decidua examined (Figure 10, E and F). In summary, pregnant Sphk1_–/–_Sphk2+/+ and Sphk1_–/–_Sphk2+/– decidua exhibited an abnormal accumulation of dihydrosphingosine and sphingosine, which was more pronounced in the Sphk1_–/–_Sphk2+/– uteri. The sphingoid base accumulation can be attributed to the defective Sphk activity in the pregnant uteri. From these results, it is possible that the membranous cytoplasmic bodies observed in the electron microscopic analysis resulted from sphingosine and dihydrosphingosine accumulation. These results obtained by mass spectrometry were confirmed by alternative methods described previously (data not shown) (19, 28).
Measurement of sphingolipid levels. DihydroS1P (A), S1P (B), dihydrosphingosine (C), sphingosine (D), ceramide (E), and sphingomyelin (F) levels were determined in nonpregnant whole uteri (wild type and Sphk1_–/–_Sphk2+/–) and day 7.5 pc decidua (wild type, Sphk1_–/–_Sphk2+/+, Sphk1+/+_Sphk2_–/–, and Sphk1_–/–_Sphk2+/–) by mass spectrometry. Data represent mean values ± SE. n = 3. *P < 0.05; **P < 0.01, paired Student’s t test.
Phosphatidylethanolamine levels are reduced in Sphk1–/–Sphk2+/– decidua. Next, we measured levels of the glycerophospholipids cardiolipin (CL), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) in nonpregnant whole uteri (wild-type and Sphk1_–/–_Sphk2+/–) and day 7.5 pc decidua of pregnant mice (wild-type and Sphk1_–/–_Sphk2+/–), from which the embryos had been removed. In nonpregnant Sphk1_–/–_Sphk2+/– uteri, PE levels were reduced by about 37% compared with nonpregnant wild-type uteri (Figure 11C). A similar level of reduction was also observed in pregnant Sphk1_–/–_Sphk2+/– decidua compared with pregnant wild-type decidua (Figure 11C). S1P and dihydroS1P were degraded by S1P lyase to produce fatty aldehyde and phosphoethanolamine, which were then reused for biosynthesis of PE (Figure 8). Thus, the decreased PE levels in Sphk1_–/–_Sphk2+/– uteri would be reasonable to expect if products derived from the sphingolipid metabolic pathway were being utilized for PE biosynthesis. The levels of CL, PC, and PS were not significantly different in the 4 types of uteri examined (Figure 11, A, B, and D). There were no significant differences in the levels of CL, PC, PE, and PS between day 6.5 pc wild-type and Sphk1_–/–_Sphk2+/– decidua (Supplemental Figure 3, A–D). These results indicate that the diminution of PE levels observed in day 7.5 pc Sphk1_–/–_Sphk2+/– decidua is present when defective decidualization is apparent. Thus, the PE deficiency may accelerate the defect leading to the pregnancy loss, rather than causing the primary defect.
Measurement of glycerophospholipid levels. CL (A), PC (B), PE (C), and PS (D) levels were determined in nonpregnant whole uteri and day 7.5 pc decidua from wild-type and Sphk1_–/–_Sphk2+/– pregnant female mice. Data represent mean values ± SE. n = 3. **P < 0.05, *P = 0.05, paired Student’s t test.
The PG pathway is not involved in the pathogenesis of Sphk1–/–Sphk2+/– uteri. The PG pathway plays crucial roles in female reproduction. PG-endoperoxidase synthase-2 (Ptgs2), also known as COX2, converts arachidonic acid to PG H2 (PGH2) and is a key enzyme in the biosynthesis of PGs. The PGH2 is then converted to various PGs by specific enzymes. Deficiency of Ptgs2 causes multiple female reproductive failures, including defective attachment reaction and defective decidualization in mice (29). Thus, we examined the Ptgs2 expression and prostaglandin E2 (PGE2) levels in day 6.5 pc and day 7.5 pc (wild-type and Sphk1_–/–_Sphk2+/–) decidua of pregnant mice. The Ptgs2 expression in Sphk1_–/–_Sphk2+/– decidua did not significantly differ from that of wild-type decidua, either on day 6.5 pc or day 7.5 pc (Figure 12A). The parallel result was obtained with PGE2 levels, where there was no difference between wild-type and Sphk1_–/–_Sphk2+/– decidua (Figure 12B). These results suggest that PG biosynthesis is unlikely to be involved in the pathogenesis of Sphk1_–/–_Sphk2+/– uteri.
Ptgs2 mRNA expression and PGE2 levels. (A) Ptgs2 mRNA expression on day 6.5 pc and day 7.5 pc decidua from wild-type and Sphk1_–/–_Sphk2+/– females as determined by real-time PCR. The expression levels are shown relative to those in wild-type decidua. (B) PGE2 levels in day 7.5 pc decidua from wild-type and Sphk1_–/–_Sphk2+/– pregnant female mice. The data represent mean values ± SE. n = 3.