Agnieszka Blitek

Estradiol-17β, Prostaglandin E2 (PGE2), and the PGE2 Receptor Are Involved in PGE2 Positive Feedback Loop in the Porcine Endometrium

Before implantation, the porcine endometrium and trophoblast synthesize elevated amounts of luteoprotective prostaglandin estradiol-17beta (E(2)) (PGE(2)). We hypothesized that embryo signal, E(2), and PGE(2) modulate expression of key enzymes in PG synthesis: PG-endoperoxide synthase-2 (PTGS2), microsomal PGE synthase (mPGES-1), PGF synthase (PGFS), and PG 9-ketoreductase (CBR1) as well as PGE(2) receptor (PTGER2 and -4) expression and signaling within the endometrium. We determined the site of action of PGE(2) in endometrium during the estrous cycle and pregnancy. Endometrial tissue explants obtained from gilts (n = 6) on d 11-12 of the estrous cycle were treated with vehicle (control), PGE(2) (100 nM), E(2) (1-100 nm), or phorbol 12-myristate 13-acetate (100 nm, positive control). E(2) increased PGE(2) secretion through elevating expression of mPGES-1 mRNA and PTGS2 and mPGES-1 protein in endometrial explants. By contrast, E(2) decreased PGFS and CBR1 protein expression. E(2) also stimulated PTGER2 but not PTGER4 protein content. PGE(2) enhanced mPGES-1 and PTGER2 mRNA as well as PTGS2, mPGES-1, and PTGER2 protein expression. PGE(2) had no effect on PGFS, CBR1, and PTGER4 expression and PGF(2alpha) release. Treatment of endometrial tissue with PGE(2) increased cAMP production. Cotreatment with PTGER2 antagonist (AH6809) but not PTGER4 antagonist (GW 627368X) inhibited significantly PGE(2)-mediated cAMP production. PTGER2 protein was localized in luminal and glandular epithelium and blood vessels of endometrium and was significantly up-regulated on d 11-12 of pregnancy. Our results suggest that E(2) prevents luteolysis through enzymatic modification of PG synthesis and that E(2), PGE(2), and endometrial PTGER2 are involved in a PGE(2) positive feedback loop in porcine endometrium.

Mechanisms for the Establishment of Pregnancy in the Pig

Establishment of pregnancy in pigs requires continuous function of corpora lutea and endometrial preparation for embryo implantation. Progesterone regulates expression of many proteins necessary for endometrial remodelling and embryo-maternal communications. Attaining the uterine receptivity involves progesterone priming and loss of progesterone receptors in the uterine epithelium before days 10-12 after oestrus. Spermatozoa and oocytes in oviduct alter secretion of specific proteins that exert beneficial effect on gametes and embryos. Moreover, an appropriate leucocyte activation and maintenance of delicate cytokine balance within the oviduct and uterus are important for early pregnancy. This early local immune response is rather mediated by seminal plasma components. These components also influence prostaglandin (PG) synthesis in the oviduct that is important for gamete and embryo transport. Pregnancy establishment requires the biphasic pattern of oestrogen secretion by conceptuses on days 11-12 and 15-30. Conceptus affects lipid signalling system consisting of prostaglandins and lysophosphatic acid. PG synthesis is changed by conceptus signals in favour of luteoprotective PGE(2) . Additionally, existence of PGE(2) positive feedback loop in the endometrium contributes to increased PGE(2) /PGF(2α) ratio during the peri-implantation period. PGE(2) through endometrial PGE(2) receptor (PTGER2) elevates the expression of enzymes involved in PGE(2) synthesis. Higher PGE(2) secretion in uterine lumen coincides with the elevated expression of HOXA10 transcription factor critical for implantation. A stable adhesion between conceptus and endometrium requires reduction in mucin-1 on the apical surface of epithelium and integrin activation by extracellular matrix proteins. Furthermore, growth factors, cytokines and its receptors are involved in embryo-maternal interactions.

Novel biological and possible applicable roles of LH/hCG receptor

Luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptors are widely expressed in gonadal cells, however, the presence of these receptors has also been demonstrated in several other non-gonadal female and male tissues. The expression level of non-gonadal LH/hCG receptors is much lower than in gonads, although their expression is regulated by similar mechanisms and they also exert biological effects using similar signaling pathways. Hormonally regulated LH/hCG receptor expression in the oviduct suggests that LH could be involved in the regulation of its contraction, gametes/embryos transport and synchronization of the fertilization. One of the major roles of the myometrial LH/hCG receptors may also be the stimulation of growth and maintenance of the uterine relaxation during pregnancy. In pigs, LH seems to be one of the pleiotropic factors which influence the endometrial prostaglandin F(2alpha) synthesis and initiation of the luteolysis. The LH/hCG receptor expression in several cancer cells provides new possibilities for developing new strategies for targeted cancer therapy based on lytic LH/hCG conjugates.

Assessment of VEGF-receptor system expression in the porcine endometrial stromal cells in response to insulin-like growth factor-I, relaxin, oxytocin and prostaglandin E2

Several factors participate in regulation of growth and development as well as angiogenesis of the uterus during pregnancy, and hence little is known about the role of hormonal regulation of vascular endothelial growth factor (VEGF)-receptor system expression. This study has examined the effect of insulin-like growth factor-I (IGF-I), relaxin (RLX), oxytocin (OT) and prostaglandin (PG) E(2), on VEGF secretion and VEGF-receptor system mRNA expression in the porcine endometrial stromal cells. IGF-I and RLX were identified as the most effective inducers of VEGF secretion and mRNA expression. Although PGE(2) stimulated VEGF secretion and VEGF164 mRNA expression, OT inhibited both secretion and mRNA expression of VEGF. When tested for VEGF receptors (R), all factors failed to affect their mRNA expression. Media conditioned by stromal cells collected after IGF-I and RLX treatment significantly increased endothelial cell proliferation and this effect was blocked by soluble VEGFR-1. These data suggest that during early pregnancy IGF-I, RLX and PGE(2) can affect VEGF expression in the endometrium and therefore may support uterine and embryo development, implantation and pregnancy.

Regulation of expression and role of leukemia inhibitory factor and interleukin-6 in the uterus of early pregnant pigs

Cytokines, which are generally involved in the process of inflammation, may also play a critical role in conceptus implantation. We examined: (1) the expression profiles of leukemia inhibitory factor (LIF) and interleukin (IL)-6 mRNA and their protein content in the endometrium of cyclic and pregnant gilts on Days 10 to 18 after estrus; (2) the effect of conceptus-exposed medium on LIF and IL-6 synthesis in the endometrium; (3) the profiles of IL6R and LIFR mRNA expression in pig conceptuses collected on Days 10 to 18 of pregnancy; and (4) the effect of LIF and IL-6 on the attachment and proliferation of porcine trophoblast cells. The expression of LIF mRNA in the endometrium increased between Days 10 and 12 in both cyclic and pregnant gilts, and tended to be higher in Day 12 pregnant animals compared with nonpregnant ones. The LIF protein content in the uterine lumen peaked on Day 12 of pregnancy, and was higher than on Day 12 of the estrous cycle. Endometrial IL-6 mRNA expression was upregulated on Day 12 in pregnant gilts compared with nonpregnant animals. Moreover, a higher content of IL-6 protein was observed in pregnant than in cyclic gilts. The addition of conceptus-exposed medium resulted in up-regulation of LIF and IL6 mRNA expression, and increased IL-6 content in endometrial slices. In conceptuses, increased mRNA expression was detected on Days 10 to 14 for IL6R and on Day 14 for LIFR, when compared with other days studied. LIF and IL-6 stimulated the attachment and proliferation of trophoblast cells in vitro. In summary, LIF and IL-6 are important components of embryo-uterine interactions during early pregnancy in the pig, and may contribute to successful conceptus implantation.

Oxytocin and tumor necrosis factor α stimulate expression of prostaglandin E2 synthase and secretion of prostaglandin E2 by luminal epithelial cells of the porcine endometrium during early pregnancy

Oxytocin (OXT) and tumor necrosis factor α (TNF) have been implicated in the control of luteolysis by stimulating endometrial secretion of luteolytic prostaglandin F(2α) (PGF(2α)). Nevertheless, OXT concentration in porcine uterine lumen increases markedly on days 11-12 of pregnancy, and TNF is expressed in endometrium during pregnancy. The objective of the study was to determine the effect of OXT and TNF on expression of the enzymes involved in PG synthesis: PG-endoperoxide synthase 2 (PTGS2), PGE(2) synthase (mPGES-1) and PGF synthase, and PGE(2) receptor (PTGER2), as well as on PG secretion by endometrial luminal epithelial cells (LECs) on days 11-12 of the estrous cycle and pregnancy. LECs isolated from gilts on days 11-12 of the estrous cycle (n=8) and pregnancy (n=7) were treated with OXT (100  nmol/l) and TNF (0.6  nmol/l) for 24  h. OXT increased PTGS2 mRNA and mPGES-1 protein contents, as well as PGE(2) secretion but only on days 11-12 of pregnancy. TNF stimulated PTGS2 and mPGES-1 mRNA, as well as mPGES-1 protein expression and PGE(2) release on days 11-12 of pregnancy and the estrous cycle. In addition, expressions of PTGER2 and PTGER4 were determined in corpus luteum (CL). Abundance of PTGER2 mRNA and PTGER4 protein in CL was upregulated on day 14 of pregnancy versus day 14 of the estrous cycle. This study indicates that TNF and OXT regulate PGE(2) synthesis in LECs during early pregnancy. PGE(2) secreted by LECs, after reaching ovaries, could have a luteoprotective effect through luteal PTGER2 and PTGER4, or may directly promote uterine function and conceptus development.

Endometrial and conceptus expression of HoxA10, transforming growth factor β1, leukemia inhibitory factor, and prostaglandin H synthase-2 in early pregnant pigs with gonadotropin-induced estrus

This study was conducted to evaluate the effect of estrus induction with gonadotropins on endometrial and conceptus expression of HoxA10, transforming growth factor (TGF) beta1, leukemia inhibitory factor (LIF), and prostaglandin H synthase-2 (PGHS-2) during early pregnancy in pigs. Twenty-four prepubertal gilts received 750 IU of pregnant mare serum gonadotropin (PMSG) and 500 IU of human chorionic gonadotropin (hCG) 72h later. Gilts in the control group (n=23) were observed daily for estrus behavior. Endometrial tissue samples, conceptuses, blood serum, and uterine luminal flushings (ULFs) were collected on days 10, 11, 12, and 15 after insemination. There was no effect of estrus induction on estradiol content in ULFs, or on ovulation and fertilization rates in studied gilts. However, the content of progesterone in the blood serum was greater in naturally ovulated gilts in comparison to gonadotropin-treated animals on day 12 of pregnancy (P<0.05). HoxA10 expression was up-regulated in the endometrium of pregnant gilts, with natural ovulation on days 12 (P<0.05) and 15 (P<0.001) in comparison to days 10 and 11. When compared to control gilts, administration of PMSG/hCG resulted in decreased expression of endometrial HoxA10, TGFbeta, LIF, and PGHS-2 on day 12 of pregnancy (P<0.05). Conceptus expression of studied factors was not affected by gonadotropin treatment. Overall, these results suggest improper endometrial preparation for implantation in prepubertal gilts induced to ovulate with PMSG/hCG.

Seminal plasma affects prostaglandin synthesis in the porcine oviduct

Seminal fluids introduced to the female reproductive tract at mating can affect subsequent events, such as ovulation, fertilization, conception, and pregnancy. Bioactive molecules present in seminal plasma can modify the cellular composition, structure, and function of local tissues and of tissues distal to the tract. The oviduct plays a decisive role in reproduction providing a beneficial milieu for gamete maturation, fertilization, and early embryonic development. Therefore we have investigated whether intrauterine infusion of seminal plasma can modulate prostaglandin (PG) synthesis in the porcine oviduct through regulation of gene and protein expression of enzymes of prostaglandin synthesis pathway. Among several enzymes involved in the prostaglandin synthesis pathway tested in the present study PGF(2α) synthase (PTGFS) and prostaglandin 9-ketoreductase (CBR1), which convert PGE(2) to PGF(2α), expression were significantly down-regulated in the oviducts on Day 1 after seminal plasma infusion into the uterine horns. The effects of the treatment were transient and by Day 5 levels of PTGFS and CBR1 were comparable in seminal plasma-treated and control animals. Additionally, increased PGE(2) to PGF(2α) and PGFM to PGF(2α) ratios in the oviductal tissues were indicated. Our results clearly demonstrate that seminal plasma affects prostaglandin synthesis in the porcine oviduct. Altered PTGFS and CBR1 expression in consequence changed PGE(2) to PGF(2α) and PGFM to PGF(2α) ratios in the porcine oviduct.

Characterization of bovine immortalized luteal endothelial cells: action of cytokines on production and content of arachidonic acid metabolites

BackgroundThe interactions between luteal, vascular endothelial, immune cells and its products: steroids, peptide hormones, prostaglandins (PGs), growth factors and cytokines play a pivotal role in the regulation of corpus luteum (CL) function. Luteal endothelial cells undergo many dynamic morphological changes and their action is regulated by cytokines. The aims are: (1) to establish in vitro model for bovine luteal endothelial cells examination; (2) to study the effect of cytokines: tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) on cell viability, leukotrienes (LTs) and PG synthases, and endothelin-1 (EDN-1) mRNA, protein expression and their secretion in bovine immortalized luteal endothelial (EnCL-1) cells.MethodsThe primary cultures of bovine luteal endothelial cells were immortalized by transfection with vector carrying the Simian virus 40 T-antigen (SV40 T-ag) sequence. Expression of SV40 T-ag gene in EnCL-1 cells was confirmed by RT-PCR and immunofluorescence staining showed the presence of endothelial cell markers: VE-cadherin and von Willebrand factor. EnCL-1 cells were stimulated by TNFalpha with IFNgamma (50 ng/ml each) for 24 h. Cell viability, mRNA expression (real time RT-PCR), protein expression (western blotting) for LTC4 synthase (LTC4S), LTA4 hydrolase (LTA4H), PGE2 and PGF2alpha synthases and endothelin-1 (EDN-1), and levels of LTs (B4 and C4) and PGs (E2 and F2alpha) and EDN-1 in the medium (EIA) were evaluated.ResultsWe received immortalized luteal endothelial cell line (EnCL-1). Cytokines did not change EnCL-1 cell viability but increased mRNA expression of LTC4S, LTA4H, PGE2 and PGF2alpha synthases and EDN-1. EDN-1/2/3, LTC4 and PGF2alpha synthases protein expression were elevated in the presence of TNFalpha/IFNgamma, and accompanied by increased EDN-1, LTC4 and PGF2alpha secretion. Cytokines had no effect on PGES and LTA4H protein expression, and PGE2 and LTB4 release.ConclusionsTNFalpha and IFNgamma modulate EnCL-1 cell function. Moreover, established EnCL-1 cell line appears to be a good model for investigating the molecular mechanisms related to cytokines action and aa metabolites production in cattle.

Effect of estrus induction on prostaglandin content and prostaglandin synthesis enzyme expression in the uterus of early pregnant pigs.

Prostaglandins (PGs) play a pivotal role in maternal recognition of pregnancy and implantation in pigs. In the present study, PGE(2), PGF(2alpha), and PGFM (PGF(2alpha) metabolite) content, as well as PGE(2) synthase (mPGES-1) and PGF(2alpha) synthase (PGFS) expression was investigated in early pregnant gilts with natural (n=21) and PMSG/hCG-stimulated (n=19) estrus. Endometrial tissue samples, uterine luminal flushings (ULFs), and blood serum were collected on days 10-11, 12, and 15 after insemination. Additionally, day 15 conceptuses were collected for mPGES-1 and PGFS protein expression. Effect of estrus induction was observed on day 15 of pregnancy, when the content of PGE(2) in the uterine lumen was fourfold lower in gonadotropin-stimulated gilts in comparison to controls (P<0.001). Decreased PGE(2) content in ULFs of gonadotropin-treated pigs was preceded by lower endometrial mPGES-1 gene expression in hormonally-stimulated animals in comparison to control gilts (P<0.01). On the other hand, estrus induction with PMSG/hCG resulted in higher PGE(2) accumulation in the endometrial tissue on day 15 of pregnancy (P<0.01). Furthermore, PGF(2alpha) content in the endometrium and PGFM levels in blood serum were lower in gonadotropin-treated gilts, especially on day 12 after insemination when compared to control gilts (P<0.01). Finally, PGFS expression in day 15 conceptuses was decreased in animals with hormonally-induced estrus. We conclude that PMSG/hCG stimulation of prepubertal gilts to induce estrus results in changes of PG production and secretion during early pregnancy, which, in turn, may affect conceptus development, implantation, and the course of pregnancy.