Piotr Kaczynski

Autocrine and Paracrine Mechanisms of Prostaglandin E2 Action on Trophoblast/Conceptus Cells through the Prostaglandin E2 Receptor (PTGER2) during Implantation

The conceptus and endometrium secrete large amounts of prostaglandin E₂ (PGE₂) into the porcine uterine lumen during the periimplantation period. We hypothesized that PGE₂ acts on conceptus/trophoblast cells through auto- and paracrine mechanisms. Real-time RT-PCR analysis revealed that PGE₂ receptor (PTGER)2 mRNA was 14-fold greater in conceptuses/trophoblasts on days 14-25 (implantation and early placentation period) vs preimplantation day 10-13 conceptuses (P < .05). Similarly, expression of PTGER2 protein increased during implantation. Conceptus expression of PTGER4 mRNA and protein did not differ on days 10-19. PGE₂ stimulated PTGER2 mRNA expression in day 15 trophoblast cells through PTGER2 receptor signaling. PGE₂ elevated aromatase expression and estradiol-17β secretion by trophoblast cells. Moreover, PGE₂ and the PTGER2 agonist, butaprost, increased the adhesive capacity of both human HTR-8/SVneo trophoblast and primary porcine trophoblast cells to extracellular matrix. This PGE₂-induced alteration in trophoblast cell adhesion to extracellular matrix was abolished by incubation of these cells with AH6809 (PTGER2 antagonist), ITGAVB3-directed tetrapeptide arg-gly-asp-ser or integrin ITGAVB3 antibody. PGE₂ stimulated adhesion of porcine trophoblast cells via the estrogen receptor and MEK/MAPK signaling pathway. PGE₂ induced phosphorylation of MAPK1/MAPK3 through PTGER2 and up-regulated expression of cell adhesion proteins such as focal adhesion kinase and intercellular adhesion molecule-1. Our study indicates that elevated PGE₂ in the periimplantation uterine lumen stimulates conceptus PTGER2 expression, which in turn promotes trophoblast adhesion via integrins, and synthesis and secretion of the porcine embryonic signal estradiol-17β. Moreover, the mechanism through which PGE₂ increases trophoblast adhesion is not species specific because it is PTGER2- and integrin-dependent in both porcine and human trophoblast cells.

Prostaglandin F2α promotes angiogenesis and embryo–maternal interactions during implantation

Implantation in humans and other mammals is a critical period during which high embryonic mortality rates occur. Prostaglandins (PGs) are key mediators regulating interactions between the reproductive tract and the conceptus (embryo with extraembryonic membranes). Although the significance of PGF2α as a regulator of corpus luteum regression is well established, the role of its high amounts in the uterine lumen in most mammals, regardless of placentation type, during the implantation period remains unresolved. We hypothesized that PGF2α acting as an embryonic signal mediator contributes to pregnancy establishment. Using a porcine model, we demonstrated that the conceptus and its signal (estradiol-17β) elevated endometrial expression of PGF2α receptor (PTGFR) in vivo and in vitro PTGFR protein was expressed mainly in luminal epithelial (LE) and glandular epithelial cells and blood vessels in the endometrium. PGF2α stimulated the MAPK1/3 pathway in endometrial LE cells that coincided with elevated gene expression and secretion of endometrial vascular endothelial growth factor A (VEGFA) protein. PGF2α-PTGFR and adenylyl cyclase signaling were involved in this process. PGF2α-induced VEGFA acting through its receptors stimulated proliferation of endometrial endothelial cells. Moreover, PGF2α elevated gene expression of biglycan, matrix metalloproteinase 9, transforming growth factor β3, and interleukin 1α in the endometrium. In summary, our study indicates that PGF2α participates in pregnancy establishment by promoting angiogenesis and expression of genes involved in tissue remodeling and conceptus-maternal interactions in porcine endometrium during early pregnancy.

Steroid hormones, prostanoids, and angiogenic systems during rescue of the corpus luteum in pigs.

In order to characterize the transition of the corpora lutea (CL) from acquisition of luteolytic sensitivity to rescue of luteal function: i) the expression of 38 factors associated with steroids, prostanoids, and angiogenic systems and ii) concentrations of the main hormones responsible for maintenance of CL function in cyclic and pregnant pigs were examined. Additionally, the effect of prostaglandin (PG) E2 and F2 α on luteal function during the estrous cycle and pregnancy was evaluated in vitro. Significantly up-regulated gene expression was revealed in CL collected on day 14 of the estrous cycle (CYP19A1, ESR2, PTGS2, HIF1A, and EDN1) and on days 12-14 of pregnancy (SCARB1, PGRMC1, STAR, HSD3B1, NR5A1, PTGFR, PTGER4, and VEGFA). Elevated concentrations of estradiol-17β and PGE2 occurred in CL on days 12 and 14 of pregnancy respectively, while an increased intraluteal PGF2 α content was noted on day 14 of the estrous cycle. Both PGs increased the synthesis of progesterone by cultured luteal slices obtained on day 14 of pregnancy, in contrast to the action of PGF2 α on the corresponding day of the estrous cycle. PGE2 stimulated cAMP production via PTGER2 and PTGER4, while PGF2 α elevated the content of CREB in cultured luteal slices from CL of pregnant pigs. In silico analysis showed that infiltration of lymphocytes and apoptosis of microvascular endothelium were activated in CL on day 12 of the estrous cycle vs pregnancy. Summarizing, an abundance of E2 and PGE2 during pregnancy regulates specific pathways responsible for steroidogenesis, the prostanoid signaling system and angiogenesis during rescue from luteolysis in porcine CL.

Embryo-maternal dialogue during pregnancy establishment and implantation in the pig

Porcine conceptuses secrete pregnancy‐recognition signals (estrogens, including estradiol‐17β) that inhibit luteolysis, thereby prolonging progesterone production by corpora lutea. The supportive mechanism by which the conceptus also inhibits luteolysis is by shifting endometrial prostaglandin (PG) synthesis to luteoprotective PGE2. Progesterone stimulates endometrial production of factors that are essential for conceptus development. Priming the uterus by progesterone and loss of progesterone receptors from the uterine epithelium by D1ay 10–12 after estrus are key for achieving endometrial receptivity for implantation. Conceptus implantation involves a series of events, many resembling the inflammatory reaction, that are greatly influenced by cytokines, growth factors, and prostaglandins. We herein present a novel, dual role for PGF2α in corpora lutea that depends on the acquisition of luteolytic sensitivity, based on the knowledge that PGF2α triggers pathways involved in luteolysis during the estrous cycle or/and may have an alternative function in maintaining progesterone synthesis during pregnancy. We also point out a new role for PGF2α that, together with PGE2, can act as embryonic signal mediators. PGF2α, which until recently was considered undesirable for promoting pregnancy, is now known to stimulate conceptus‐maternal interactions and angiogenesis in the endometrium. This function is in line with other important prostaglandin functions, such as stimulating adhesion of trophoblasts (PGE2, PGI2) as well as endometrial vascular functions and trophoblast cell proliferation (PGI2). Finally, microRNAs have emerged as important post‐transcriptional regulators of gene function, adding a new area of investigation that may enhance understanding of conceptus‐endometrial interactions.

Effect of conceptus on expression of prostaglandin F2α receptor in the porcine endometrium

Increased synthesis of prostaglandin F(2α) (PGF(2α)) in the endometrium and conceptus during the implantation period results in elevated concentration of PGF(2α) in the uterine lumen in pregnant gilts. PGF(2α) exerts its effects through PGF(2α) receptor (PTGFR), a G-protein-coupled receptor. However, besides studies concerning the function of PTGFR in endometrial abnormalities, the role of PTGFR in the endometrium during early pregnancy has not been elucidated. Therefore, the aim of this study was: (1) to evaluate the profile of PTGFR gene and protein expression in the porcine endometrium during early pregnancy and the estrous cycle; (2) to determine if the effect of conceptus on PTGFR expression is dependent on type of endometrial cells-luminal epithelial (LE) or stromal (ST) cells; and (3) to elucidate if the putative effect of conceptus on endometrial PTGFR expression is mediated by estrogen receptor. We evaluated the expression pattern of PTGFR gene and protein in the endometrium during day 9, 11, 12, 15, and 18 of the estrous cycle and pregnancy (N = 4-6 per group). The expression of PTGFR mRNA was greater on day 18 of pregnancy and the estrous cycle (vs. days 9-15 of the estrous cycle and pregnancy, P < 0.05). Expression of PTGFR protein was approximately 10-fold upregulated in the endometrium on day 15 of pregnancy when compared with day 15 of the estrous cycle (P < 0.01). Endometrial expression of PTGFR protein increased from day 12 to 18 of pregnancy (P < 0.05). PTGFR mRNA was expressed in LE and ST cells. In a subsequent experiment, we used a coculture model in which LE cells were cultured on collagen-coated inserts placed in wells plated with ST cells. Day 11 or 15 conceptus-exposed medium (CEM) elevated expression of PTGFR mRNA (2- and 1.5-fold, respectively, P < 0.05) in LE cells cocultured with ST cells. CEM did not have an effect on PTGFR mRNA expression in ST cells. The 11-day CEM-induced increase of PTGFR mRNA was abolished by incubation of LE cells in the presence of the estrogen receptor antagonist (ICI-182,780; P < 0.01). Summarizing, the conceptus upregulated expression of PTGFR in the endometrium during the implantation period. Moreover, this study indicates that expression of PTGFR gene was elevated in LE cells of endometrium by embryonic signal of estradiol. Our results suggest para- and autocrine effects of PGF(2α) through its receptor PTGFR in the porcine endometrium, especially in luminal epithelium which is in direct contact with the conceptus during the implantation period.

Leakage flow reduction in different configuration of labyrinth seal on a turbine blade tip

This paper contains experimental and numerical investigation of the leakage flow over the blade tip in turbine stage. Experiments were conducted in non-rotating linear channel. Test rig was intended to model the geometry of the labyrinth seal of the blades of the lower pressure turbine stage. Investigated model contained two different geometries of labyrinth fins. Moreover smooth and honeycomb stator landing were tested. Experimental measurements have been supported by CFD simulation which gives valuable information of flow structure in labyrinth seal and show complex flow physics in the investigated model.

Transition Effect on Shock Wave Boundary Layer Interaction on Compressor Blade

Intensive research on the laminar flow is carried out nowadays. The main objective is to keep the laminar boundary layer as long as possible for the drag or losses reduction, depending on the application. Nevertheless, in some applications, the laminar layer interaction with the shock wave may lead to a strong flow separation. The process of separation usually becomes unsteady and causes buffeting at airfoils and shock oscillations in the internal flows. Shock oscillation causes pulsation of pressure, and consequently a change of the blade load. The main objective of this research is to study the effect of transition on the flow structure of the shock wave boundary layer interaction. This paper focuses on the influence of boundary layer transition on the flow pattern in the blade passage of a compressor cascade. The main question is whether the induced transition upstream of the shock can improve the pressure unsteadiness and the flow downstream of the interaction. The results presented here concern experimental investigations and the used combination of various methods allowed gaining an insightful analysis of the application of transition control devices into a complex flow structure in a compressor cascade passage.