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Background: Prostacyclin (PGI(2)) plays an important role in mouse embryo development and implantation. However, it is unclear whether its action is mediated via the I prostaglandin receptor (IP)., Methods: We compared the preimplantation development of IP deleted (IP-/-) embryos and wild-type (WT) embryos. We also evaluated the effect of iloprost, a stable PGI(2) analog, and L-165041, a peroxisome proliferator activated receptor delta (PPARdelta) ligand, on IP-/- versus WT embryos. Finally, we compared the development of heterozygous IP deficient embryos carrying a normal maternal IP allele versus paternal IP allele., Results: Development of IP-/- embryos lagged behind WT embryos and was not enhanced by either the PGI(2) analog or the PPARdelta ligand. WT embryos had slightly higher, although statistically not significant, implantation rates than IP-/- embryos. Heterozygous IP deficient embryos carrying a normal maternal IP allele showed better development and responded to the PGI(2) analog, unlike those carrying the normal paternal IP allele., Conclusions: IP receptors play an important role in preimplantation embryo development and mediate the embryo's response to exogenous PGI(2). Early embryo development depends on the oocyte IP receptor.

Little is known about prostaglandin synthesis and function in embryonic stem cells. We postulated that mouse embryonic stem (mES) cells possess enzymes to synthesize protective prostaglandins. Compared with differentiated adult cells, mES cells were less susceptible to H(2)O(2)-induced apoptosis. However, their apoptosis was enhanced by indomethacin or SC-236, a selective inhibitor of cyclooxygenase (COX)-2. Analysis of COX pathway enzymes by Western blotting revealed expression of COX-2 and cytosolic and microsomal prostaglandin E(2) (PGE(2)) synthases. COX-1 and prostacyclin (PGI(2)) synthases were undetectable. mES cells produced PGE(2) but not PGI(2). Importantly, PGE(2) rescued mES cells from apoptosis. To elucidate the signaling mechanism by which PGE(2) inhibits apoptosis, we analyzed E-type prostaglandin (EP) receptors by Western blots. All EP isoforms were detected except EP4. Butaprost, a specific EP2 agonist, rescued mES cells from apoptosis, whereas sulprostone, an EP1/EP3 agonist, had no effect, suggesting selective interaction of PGE(2) with EP2. The antiapoptotic effect of PGE(2) was abrogated by Ly-294002 or wortmannin but not H-89 or a specific inhibitor of protein kinase A, suggesting signaling via phosphatidylinositol-3 kinase (PI-3K). Akt was constitutively active in mES cells, which were inhibited by indomethacin and rescued by PGE(2). The rescuing effect of PGE(2) was abrogated by Ly-294002. These results indicate that mES cells constitutively express COX-2 and PGE synthases and produce PGE(2), which confers resistance to apoptosis via EP2-mediated activation of PI-3K to the Akt pathway. Disclosure of potential conflicts of interest is found at the end of this article.

Background: Successful IVF depends in part on quality embryos. Recent work suggests that prostaglandin I(2) (PGI(2) or prostacyclin) promotes the development of embryos in vitro and enhances their implantation potential. The mechanism underlying the effects of PGI(2) is unclear. It has been reported that peroxisome proliferator-activated receptor delta (PPARdelta) mediates the effects of PGI(2) at the implantation sites., Methods: The expression of PPARdelta in the preimplantation embryos was examined by RT-PCR, western blot analysis and immunohistochemistry. Synthetic PPARdelta ligand (L-165041) and PPARdelta targeted (PPARdelta(-/-)) embryos were used to reveal the roles of PPARdelta in PGI(2)-stimulated and spontaneous embryo development., Results: Preimplantation embryos express PPARdelta, which is essential for the enhancing effect of PGI(2) and the spontaneous progression of preimplantation embryos. Enhanced blastocyst hatching by PGI(2) (P < 0.05) was abrogated by PPARdelta deletion. Blastocyst formation and embryo hatching were impaired in PPARdelta(-/-) embryos. PPARdelta deletion significantly reduced embryo cell proliferation (P < 0.01); PPARdelta activation increased embryo cell proliferation (P < 0.05). PPARdelta activation enhanced the implantation of wild-type (WT) embryos (P < 0.05); PPARdelta deletion reduced embryo implantation (P < 0.05)., Conclusions: PPARdelta is essential for spontaneous and PGI(2)-stimulated embryo development and blastocyst hatching. The implantation of cultured embryos is enhanced by PPARdelta activation. PPARdelta represents a novel therapeutic target to improve IVF outcome.

Background: Recently we discovered that human oviducts produce a significant amount of prostacyclin (prostaglandin I2, PGI2) and that PGI2 enhances the potentials of live birth of mouse embryos. However, the eicosanoid profile of mouse oviducts remains unknown., Methods: The metabolites of [14C]arachidonic acid by mouse oviducts were analysed by high-performance liquid chromatography. The expression of cyclooxygenase (COX)-1, COX-2 and PGI2 synthase (PGIS) was analysed by western blot analysis and immunohistochemistry. The PGI2 synthetic capacities and the COX transcripts during the preimplantation period were compared. The effects of COX-2 inhibitor on PGI2 production were ascertained., Results: Mouse oviducts produced, in order of abundance, PGI2, PGD2 and PGE2. Western blot analysis confirmed the expression of COX-1, -2 and PGIS which were expressed by luminal epithelia and smooth muscle cells. Day 2-3 post-coitus (p.c.) oviducts produced PGI2 10-fold higher than day 4 p.c. oviducts (P = 0.0087); day 1 p.c. oviducts expressed COX-2 transcript 5-fold higher than day 3 p.c. oviducts (P = 0.0004). The PGI2 production was markedly reduced by a selective COX-2 inhibitor., Conclusions: Mouse oviducts synthesized maximal PGI2 during day 2-3 p.c., coinciding with the transformation of 2-cell embryos to morulae. The results suggest that oviduct-derived PGI2 may enhance embryo development in a paracrine fashion.

Introduction: The role of prostaglandins (PGs) in embryo hatching remains controversial. In addition, there is no direct evidence that mouse embryos synthesize PGs., Methods: The effects of endogenous PG on mouse embryo hatching were evaluated by blocking endogenous PG synthesis with indomethacin. Specific cyclooxygenase (COX) inhibitors were used to identify the role of COX-1- and COX-2-derived PGs. An eicosanoid profile was generated by incubating blastocysts with [3H]arachidonic acid and analysing the metabolites by high performance liquid chromatography. The expression and the localization of COX-1, COX-2 and prostacyclin synthase (PGIS) were examined by western blot analysis and immunohistochemistry., Results: The hatching of embryos cultured in 30 microl of protein-free medium was blocked by indomethacin (P = 0.007) or a selective COX-2 inhibitor (P = 0.004). Adding back iloprost, a prostacyclin analogue, abolished the effects of the COX-2 inhibitor. Prostacyclin was the most abundant PG produced by mouse blastocysts, which expressed COX-1, COX-2 and PGIS. COX-1, COX-2 and PGIS were expressed in 4-cell stage embryos and beyond; they were present in the inner cell mass and the trophectoderm of the blastocysts., Conclusion: Mouse embryos express COX-1, COX-2 and PGIS which catalyse the formation of PGI2; COX-2-derived PGI2 plays a critical role in embryo hatching.

Background: Recently we discovered that the human oviduct synthesizes abundant prostacyclin (PGI(2)). Gene knock-out studies suggest that PGI(2) is essential to endometrial decidualization, but the effects of PGI(2) on sperm and embryos have not been reported., Methods: The effects of PGI(2) on human sperm were analysed by a computer-assisted semen analysis system. The effects of PGI(2) on mouse embryos were examined based on the rates of complete hatching. The expression of PGI(2) receptor (IP) was evaluated by Western blot analysis and immunohistochemistry. The binding of PGI(2) to embryos was confirmed by radioligand binding assay. Finally, cAMP levels were assessed in PGI(2)-challenged embryos., Results: Iloprost (a stable PGI(2) analogue) did not affect the motility or the overnight survivability of human sperm. Western blot analysis did not detect IP in the sperm plasma membrane. In contrast, the hatching of mouse embryos was enhanced by iloprost (ED(50) 6.7 nmol/l). Exposure to iloprost during 8-cell to morulae or morulae to early blastocyst stages was critical to enhanced hatching. This coincided with the developmental stage-specific expression of IP. Although iloprost bound to blastocysts, it did not significantly increase cAMP., Conclusion: PGI(2) enhanced the hatching of mouse embryos but not the motility of human sperm.

Animal studies unequivocally support the indispensable role of prostaglandin (PG) and cyclooxygenase (COX) in ovulation and implantation. Available data also suggest that PG and COX may be important in the transport of embryos. The effects of PGE(2) and PGF(2alpha) on the contractility of human tubal muscle have been studied extensively; the expression of COX in human fallopian tubes was also reported. Despite all these, two fundamentally important questions remained to be answered: 1) which PGs are produced by human fallopian tubes; and 2) which COX isoform(s) is expressed by the fallopian tubes. We used reverse-phase HPLC to study the metabolism of [1-(14)C] arachidonic acid by the fallopian tubes. We found that 6 keto-PGF(1alpha), a stable metabolite of prostacyclin (PGI), and PGE(2) constituted 56% +/- 10% and 35% +/- 10% (mean +/- SEM, four samples), respectively, of total eicosanoids synthesized. Western blot analysis revealed the expression of both COX isoforms. Immunohistochemistry study showed that both COX-1 and -2 were localized to nonciliated epithelia and tubal smooth muscle. In addition, COX-2 was also expressed in ciliated epithelial cells. Western blot analysis revealed the expression of PGI synthase (PGIS) and PGI receptor by fallopian tubes. Immunohistochemistry confirmed the expression of PGIS by luminal epithelia, tubal smooth muscle, vascular endothelial cells, and vascular smooth muscle cells. Iloprost, a PGI analog, inhibited the activities of circular and longitudinal muscles of the fallopian tube. Thus, the fallopian tube expresses both COX isoforms and PGIS. Furthermore, it is a source and a target of PGI. PGI and COX may be important to gamete function, embryo transport, and embryo development.

The complex polyploid genomes of three Saccharum species have been aligned with the compact diploid genome of Sorghum (2n = 2x = 20). A set of 428 DNA probes from different Poaceae (grasses) detected 2460 loci in F1 progeny of the crosses Saccharum officinarum Green German x S. spontaneum IND 81-146, and S. spontaneum PIN 84-1 x S. officinarum Muntok Java. Thirty-one DNA probes detected 226 loci in S. officinarum LA Purple x S. robustum Molokai 5829. Genetic maps of the six Saccharum genotypes, including up to 72 linkage groups, were assembled into "homologous groups" based on parallel arrangements of duplicated loci. About 84% of the loci mapped by 242 common probes were homologous between Saccharum and Sorghum. Only one interchromosomal and two intrachromosomal rearrangements differentiated both S. officinarum and S. spontaneum from Sorghum, but 11 additional cases of chromosome structural polymorphism were found within Saccharum. Diploidization was advanced in S. robustum, incipient in S. officinarum, and absent in S. spontaneum, consistent with biogeographic data suggesting that S. robustum is the ancestor of S. officinarum, but raising new questions about the antiquity of S. spontaneum. The densely mapped Sorghum genome will be a valuable tool in ongoing molecular analysis of the complex Saccharum genome.

Increasing evidence indicates that PGs may play an obligatory role in blastocyst implantation. Cyclooxygenase (also known as PGH synthase) isozymes 1 and 2 catalyze the rate limiting steps in the biosynthesis of PGs. The ubiquitous cyclooxygenase-1 (COX-1) subserves housekeeping functions, whereas the inducible cyclooxygenase-2 (COX-2) is expressed by limited cell types and tightly controlled. Here we report the induction of COX-2 gene expression by interleukin-1 beta (IL-1 beta) in cultured human endometrial stromal cells. COX-2 activity was induced by IL-1 beta (1 ng/mL); conversion of exogenous arachidonic acid to PGF2 alpha increased from 2.6 +/- 0.6 ng/well (mean +/- SEM; n = 6) to 22.2 +/- 5.6 ng, but was completely blocked (2.8 +/- 0.7 ng/well) by NS-398, a specific COX-2 inhibitor. Undetectable in quiescent stromal cells, messenger ribonucleic acid for COX-2 was induced 30 min after IL-1 beta treatment, reached a maximum at 4 h, and decreased after 15 h. Protein synthesis was not required for induction of the COX-2 gene, as it was blocked by actinomycin D but not by cycloheximide. The 70-kDa COX-2 protein was not detected in quiescent cells, became detectable 6 h after IL-1 beta treatment, and remained detectable even after 15 h. IL-1 beta (0.1-100 ng/mL) increased the luciferase activity in promoterless luciferase reporter containing the 900-bp 5'-flanking sequence (-891 to +9) of the COX-2 gene in a dose-dependent manner, with an ED50 of 0.1-1 ng/mL.

A 3-D model of human thromboxane A2 synthase (TXAS) was constructed using a homology modeling approach based on information from the 2.0 A crystal structure of the hemoprotein domains of cytochrome P450BM-3 and P450cam. P450BM-3 is a bacterial fatty acid monooxygenase resembling eukaryotic microsomal cytochrome P450s in primary structure and function. TXAS shares 26.4% residue identity and 48.4% residue similarity with the P450BM-3 hemoprotein domain. The homology score between TXAS and P450BM-3 is much higher than that between TXAS and P450cam. Alignment between TXAS and the P450BM-3 hemoprotein domain or P450cam was determined through sequence searches. The P450BM-3 or P450cam main-chain coordinates were applied to the TXAS main chain in those segments where the two sequences were well aligned. These segments were linked to one another using a fragment search method, and the side chains were added to produce a 3-D model for TXAS. A TXAS substrate, prostaglandin H2 (PGH2) was docked into the TXAS cavity corresponding to the arachidonic acid binding pocket in P450BM-3 or camphor binding site in P450cam. Regions of the heme and putative PGH2 binding cavities in the TXAS model were identified and analyzed. The segments and residues involved in the active-site pocket of the TXAS model provide reasonable candidates for TXAS protein engineering and inhibitor design. Comparison of the TXAS model based on P450BM-3 with another TXAS model based on the P450cam structure indicated that P450BM-3 is a more suitable template for homology modeling of TXAS.

Mortality rates of coronary heart disease are much lower in Japan than in the United States. The authors' previous report on coagulation factors showed that population levels of plasma fibrinogen and factor VII activity parallel this mortality difference. To investigate other hemostatic variables, the authors assessed indicators of fibrinolytic activity (tissue plasminogen activator antigen) and coagulation inhibition (antithrombin III activity and protein C) in 136 men aged 34-55 years in four different samples: rural Japanese, urban Japanese, Japanese Americans, and Caucasian Americans. Mean tissue plasminogen activator antigen was higher in Caucasians and Japanese Americans than in rural and urban Japanese (p less than 0.01), while a contrasting trend in mean antithrombin III activity was suggested (p = 0.10). No significant differences were observed in mean levels of protein C. After controlling for known coronary risk factors, mean levels of tissue plasminogen activator antigen remained significantly different across the four samples (p less than 0.01); mean antithrombin III activity was not different (p = 0.23). Population differences in tissue plasminogen activator antigen parallel the coronary heart disease mortality difference between Japan and the United States. Although no definite evidence is available showing that tissue plasminogen activator antigen is a risk factor for coronary heart disease, the present study suggests a positive ecologic association between this hemostatic factor and coronary heart disease mortality.

Mortality rates of coronary heart disease are much lower and hemorrhagic stroke rates are higher in Japanese than in Caucasians. To investigate whether population differences in plasma concentrations of coagulation factors are consistent with these mortality differences, the authors examined, in 1987, a total of 136 men aged 34-55 years in four different samples: rural Japanese living in Akita, Japan; urban Japanese living in Osaka, Japan; and Japanese Americans and Caucasian Americans living in Minneapolis-St. Paul, Minnesota. The mean plasma fibrinogen level in Caucasians was 290 mg/dl, which was significantly higher than that in each of the Japanese samples (223-250 mg/dl; test for difference: p less than 0.001). The mean coagulation activities of factor VII and factor VIII (factor VIIc and factor VIIIc) were higher in Caucasian and Japanese Americans than in rural and urban Japanese (p less than 0.01 for factor VIIc and p = 0.03 for factor VIIIc). von Willebrand factor did not differ significantly across the populations. The relations of these coagulation factors with other cardiovascular risk factors (age, body mass index (weight (kg)/height (m)2), blood pressure, serum total cholesterol, serum triglyceride, cigarette smoking, and alcohol intake) were also examined. Mean plasma fibrinogen was consistently higher in current smokers than in nonsmokers within each sample. Factor VIIc and factor VIIIc levels were positively associated with serum total cholesterol and serum triglyceride. No consistent associations were seen between von Willebrand factor and cardiovascular risk factors. After the authors controlled for these covariates, mean fibrinogen and factor VIIc levels remained significantly different, but factor VIIIc levels did not. Different levels of coagulation factors across these samples are probably attributable to differences in environmental factors, especially diet, as well as genetic differences between Caucasians and Japanese. Furthermore, the differences in plasma fibrinogen and factor VIIc levels may explain part of the difference in mortality from cardiovascular disease across these populations.