Prostacyclin synthesis and prostacyclin receptor expression in the porcine corpus luteum: evidence for a luteotropic role in vitro.

The prostacyclin (prostaglandin I2) signaling system is an essential regulator of vascular homeostasis. Since the corpus luteum is a highly vascularized gland, prostacyclin seems to be crucial for luteal development and function. Although progress has been made in understanding the luteotropic action of prostacyclin in mammals, its role in the porcine corpus luteum remains to be determined. Therefore, studies were conducted to (1) determine profiles of prostacyclin synthase expression and prostacyclin metabolite concentration, as well as prostacyclin G-protein-coupled receptor expression in porcine luteal tissue on days 2 to 16 of the estrous cycle and days 10 to 30 of pregnancy using real-time PCR, western blot, or enzyme immunoassay; and (2) examine the effect of prostacyclin on progesterone synthesis in vitro. To accomplish the second aim, luteal cells were treated with prostacyclin analogs, iloprost and carbaprostacyclin, in the presence or absence of prostacyclin receptor antagonists. The mRNA expression of cytochrome P450 family 11 subfamily A member 1 and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 was analyzed using real-time PCR, while progesterone concentration in culture medium was assessed by radioimmunoassay.Dynamic changes of prostacyclin synthase and prostacyclin receptor expression were observed in porcine luteal tissue during the estrous cycle and early pregnancy. Moreover, prostacyclin stimulated progesterone production and this effect was abolished by the addition of prostacyclin receptor antagonists. Our findings provide strong evidence that prostacyclin and its signaling system are present in corpus luteum of the pig and may directly promote luteotropic activity through upregulation of progesterone synthesis.

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

Porcine conceptuses secrete pregnancy-recognition signals (estrogens, including estradiol-17ß) 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 transforming growth factor (TGF) ß1 mRNA expression and protein concentration in the porcine endometrium--in vivo and in vitro studies.

Transforming growth factor (TGF) ß and its receptors are expressed at the conceptus-maternal interface during early pregnancy in the pig. The present studies were conducted to examine: (1) the effect of conceptus products on TGFß1 mRNA expression and protein concentration in the porcine endometrium using in vivo and in vitro models, and (2) the effect of TGFß1 on proliferation of porcine trophoblast cells in vitro. During in vivo experiments, gilts with one surgically detached uterine horn were slaughtered on days 11 or 14 of the estrous cycle and pregnancy. For in vitro studies, endometrial explants and luminal epithelial (LE) cells co-cultured with stromal (ST) cells were treated with conceptus-exposed medium (CEM). Moreover, porcine trophoblast cells were treated with TGFß1, and the number of viable cells was measured. On day 11, the presence of conceptuses had no effect on TGFß1 mRNA expression, but decreased the TGFß1 protein concentration in the connected uterine horn compared with the detached uterine horn. In contrast to day 11, on day 14 after estrus, TGFß1 mRNA expression and protein content in the endometrium collected from the gravid uterine horn were greater when compared with the contralateral uterine horn. The treatment of endometrial slices with CEM resulted in greater TGFß1 mRNA expression and protein secretion. LE cells responded to CEM with an increased TGFß1 mRNA level. Moreover, TGFß1 stimulated the proliferation of day 14 trophoblast cells. In summary, porcine conceptuses may regulate TGFß1 synthesis in the endometrium at the time of implantation. TGFß1, in turn, may promote conceptus development by increasing the proliferation of trophoblast cells.

Diverse effects of prostacyclin on angiogenesis-related processes in the porcine endometrium.

Angiogenesis is important for endometrial remodeling in mature females. The endometrium synthesizes high amounts of prostacyclin (PGI2) but the role of PGI2 in angiogenesis-related events in this tissue was not fully described. In the present study, porcine endometrial endothelial (pEETH) cells and/or a swine umbilical vein endothelial cell line (G1410 cells) were used to determine the regulation of PGI2 synthesis and PGI2 receptor (PTGIR) expression by cytokines and to evaluate the effect of PGI2 on pro-angiogenic gene expression, intracellular signaling activation, cell proliferation and migration, cell cycle distribution, and capillary-like structure formation. We found that IL1ß, IFNγ, and/or TNFα increased PGI2 secretion and PTGIR expression in pEETH cells. Iloprost (a PGI2 analogue) acting through PTGIR enhanced the transcript abundance of KDR, FGFR2, and ANGPT2 and increased proliferation of pEETH cells. This latter was mediated by PI3K and mTOR activation. In support, transfection of G1410 cells with siRNA targeting PGI2 synthase decreased pro-angiogenic gene expression and cell proliferation. Furthermore, iloprost accelerated the gap closure and promoted cell cycle progression. Intriguingly, the formation of capillary-like structures was inhibited but not completely blocked by iloprost. These findings point to a complex pleiotropic role of PGI2 in angiogenesis-related events in the porcine uterus.

Prostacyclin Synthesis and Prostacyclin Receptor Expression in the Porcine Myometrium: Prostacyclin Potential to Regulate Fatty Acid Transporters, Cytokines and Contractility-Related Factors.

Although prostacyclin (PGI2) has been well described as a regulator of smooth muscle activity, limited data are available concerning its role in the myometrium of pigs. The present research aimed to examine profiles of PGI2 synthase (PTGIS) and PGI2 receptor (PTGIR) expression and 6-keto PGF1α (a PGI2 metabolite) concentrations in the myometrium of gilts throughout the estrous cycle and during early pregnancy using qPCR, Western blot, and/or ELISA methods. Furthermore, myometrial explants were exposed to iloprost (a stable PGI2 analog) to investigate the effect of PGI2 on the mRNA expression of factors engaged in smooth muscle contraction, nutrient transport, prostaglandin synthesis and action, and inflammatory response. PTGIS mRNA expression was greater in cyclic than in pregnant gilts on days 11-12 after estrus and was accompanied by greater concentrations of 6-keto PGF1α detected in cyclic than in pregnant animals on days 11-20. Iloprost stimulated fatty acid transporters and contractility-related calponin 1 and caldesmon 1 mRNA expression and decreased interleukin 1ß and tumor necrosis factor transcript abundance. The obtained results indicate a physiologically relevant role of PGI2 during the estrous cycle in the porcine myometrium with its importance for regulating the expression of contractility-, nutrient transport- and inflammatory response-related factors.

Immortalization of swine umbilical vein endothelial cells (SUVECs) with the simian virus 40 large-T antigen.

Implementation of the swine umbilical vein endothelial cells (SUVECs) model in vitro can be instrumental in determining the biology of endothelial cells. We have generated an immortalized endothelial cell line, G-1410, using Simian virus 40 T-antigen (SV40 T-ag) primarily to overcome the short life span before the onset of senescence and high variability among enzymatically isolated cells of primary cultures. Fast proliferating cells were selected from cultures and, after a fifth passage, examined for the presence of the SV40 T-ag by PCR and immunocytochemistry. Phase contrast and transmission electron microscopy revealed that G-1410 cells did not differ morphologically from SUVECs. The G-1410 cells exhibited positive staining for vascular endothelial (VE)-cadherin and von Willebrand factor (vWF), and formed capillary-like tube structures on Matrigel. Despite the strong oncogenic signal provided by SV40 T-ag, these transformed G-1410 cells have remained karyotypically normal and non-tumorigenic. G-1410 cells also responded to stimulation with VEGF, FGF-2, and newborn calf serum. Moreover, G-1410 cells showed elevated expression of VEGF120, VEGF164 (VEGF-A), and FGF-2 at both mRNA and protein levels. In conclusion, based on the cytological and functional evaluation of the newly obtained immortalized cell line, it can be concluded that G-1410 cells provide a useful tool for studying the effects of VEGF and FGF systems, and other signal transduction pathways related to angiogenesis.

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

BACKGROUND: The 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. METHODS: The 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. RESULTS: We 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. CONCLUSIONS: TNFalpha 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.

Oxytocin and tumor necrosis factor alpha 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.

Effect of steroids on HOXA10 mRNA and protein expression and prostaglandin production in the porcine endometrium.

The homeobox A (HOXA) family of genes is responsible for segmental development of the female reproductive tract during embryogenesis. However, HOXA10 has been shown to be essential not only for uterus development, but also for implantation. Persistent expression and steroid-dependent regulation of this gene has been demonstrated in adult human, primate, murine and canine uteri. Moreover, HOXA10-dependent expression of prostaglandin H synthase-2 (PGHS-2), a key enzyme in prostaglandin production, has been previously detected. The role of the HOXA10 gene in the porcine uterus is not well established. Therefore, the present studies were undertaken to 1) examine the effect of E(2) and P(4) on HOXA10 mRNA and protein content in the endometrium collected on day 9 of the estrous cycle and 2) determine the PGHS-2 protein expression and PGE(2) and PGF(2α) secretion from endometrial tissue in response to steroid treatment. Endometrial explants collected from mature gilts on day 9 of the estrous cycle were incubated with E(2) (1-100 nM), P(4) (10-1000 nM) or E(2) (10 nM) and P(4) (100 nM) for 24 h. E(2) alone or E(2) in the presence of P(4) increased HOXA10 mRNA expression in the endometrium (P<0.05). The HOXA10 protein level was upregulated in response to E(2), P(4) and both steroids administered simultaneously (P<0.05). Moreover, E(2) and P(4) stimulated PGHS-2 protein expression in cultured endometrial explants. PGE(2), but not PGF(2α), secretion increased in the presence of E(2) (P<0.05). However, the release of both prostaglandins was decreased after treatment of endometrial explants with the highest dose of P(4) (P<0.01). These results demonstrate that E(2) and P(4) are important regulators of HOXA10 gene expression in the adult porcine endometrium during the mid-luteal phase of the estrous cycle. Additionally, the similar profiles of endometrial HOXA10 and PGHS-2 expression in the presence of E(2) and P(4) indicate that both genes are simultaneously regulated by steroids in the porcine uterus.

Peroxisome proliferator-activated receptor ß/δ and γ agonists differentially affect prostaglandin E2 and cytokine synthesis and nutrient transporter expression in porcine trophoblast cells during implantation.

Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor family of ligand-dependent transcription factors. PPARs have been shown to be important regulators of female reproductive functions, including conceptus development and placenta formation. This study examines the effect of PPARß/δ and PPARγ agonists and antagonists on (1) the synthesis of prostaglandin (PG) E2, interleukin (IL) 6, interferon (IFN) γ, and tumor necrosis factor (TNF) α and (2) the mRNA expression of genes encoding nutrient transporters and/or binding proteins in Day 15 conceptus trophoblast cells. The study also examines whether PPAR agonist-modulated IL6, IFNγ, and TNFα secretion is mediated via mitogen-activated protein kinase (MAPK) pathways. Trophoblast cells were exposed to L-165,041 (a PPARß/δ agonist) or rosiglitazone (a PPARγ agonist) in the presence or absence of GSK3787 (a PPARß/δ antagonist) or GW9662 (a PPARγ antagonist) or in the presence or absence of U0126 (a MAPK inhibitor). Rosiglitazone stimulated PGE synthase and IFNG mRNA expression in trophoblast cells and enhanced PGE2 concentrations in the incubation medium. Moreover, cells treated with rosiglitazone exhibited increased abundance of the solute carrier organic anion transporter family member 2A1 (SLCO2A1, a PG transporter) and of fatty acid binding protein (FABP) 5 transcripts. All these effects were abolished by the addition of GW9662, which indicates that the action of rosiglitazone is PPARγ-dependent in the studied cells. L-165,041 inhibited TNFα synthesis and decreased the mRNA expression of FABP3 and IL6 in trophoblast cells. However, this effect was not abolished by the addition of GSK3787 into the incubation medium, suggesting that L-165,041 action is independent of PPARß/δ. The inhibitory effect of L-165,041 on TNFα concentration and the stimulatory effect of rosiglitazone on IFNγ accumulation in the medium were not observed in the presence of the MAPK inhibitor, suggesting that the action of both agonists may be mediated by MAPKs. In conclusion, PPARß/δ and PPARγ agonists are differentially involved in the trophoblast expression of genes related to conceptus development and implantation in pigs. Furthermore, L-165,041 and rosiglitazone may have PPAR-dependent and -independent effects in conceptus trophoblast cells.

In vivo response of the corpus luteum to progesterone treatment of gilts during early gestation.

Supplementation of progesterone (P4) in pregnant gilts increases concentrations of circulating P4 and stimulates the secretory activity of the endometrium. In this study, there was examination of the consequences of exogenous P4 administration on luteal P4 content and the expression of genes related to the corpus luteum (CL) function. Gilts with gonadotropin-induced estrus were administered daily injections of corn oil (n = 8) or P4 (n = 8) on days 3 through 10 after insemination. The animals were slaughtered on day 12 of pregnancy to obtain corpora lutea for real-time polymerase chain reaction analyses of selected genes and for enzyme immunoassay of P4. Injections with P4 had no effect on the concentration of P4 and the relative abundance of mRNA transcripts of cholesterol transport-related proteins, steroidogenic enzymes, and receptors for luteotropic factors in the luteal tissue. The abundance of prostaglandin (PG) endoperoxide synthase 2, PGI2 synthase, PGI2 receptor, fibroblast growth factor 2, peroxisome proliferator-activated receptor γ, and tumor necrosis factor α receptor type I transcripts increased after P4 treatment. In contrast, the relative abundance of angiopoietin 2 mRNA decreased in response to P4 administration. In summary, P4 supplementation in pregnant gilts does not affect luteal steroidogenesis but modulates the abundance of factors related to vascular function. Given that the endometrium is the main target tissue for P4, an indirect uterine-mediated effect of exogenous P4 on CL function is likely.

Regulation of expression and role of peroxisome proliferator-activated receptors (PPARs) in luminal epithelial and stromal cells of the porcine endometrium.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor family of ligand-dependent transcription factors. PPARs are important regulators of glucose and fatty acid metabolism, apoptosis, angiogenesis, cell proliferation and differentiation, and immune response. Their possible role in the female reproductive tract was demonstrated. In the present study, cultured luminal epithelial (LE) and stromal (ST) cells of the porcine endometrium were used to examine (1) the effect of conceptus exposed medium (CEM) on mRNA and protein expression and DNA binding activity of PPARA, PPARD, and PPARG isoforms, and (2) the effect of PPARA, PPARD, and PPARG agonists on the expression of selected genes, apoptosis, and cell proliferation. The addition of CEM stimulated PPARA expression and DNA binding activity of this isoform in LE and ST cells (P < 0.05). Increased expression of PPARD mRNA in the presence of CEM was detected in ST cells (P < 0.05), while the concentration of PPARG transcripts decreased in response to CEM in both cell types (P < 0.05). LE and ST cells of the pig endometrium possess PPARA, PPARD, and PPARG proteins, with clear nuclear staining visible predominately in ST cells. In LE cells, activation of PPARG with 15-deoxy-Δ12,14-prostaglandin(PG)J2 down-regulated the expression of genes encoding amino acid transporter 1 (SLC38A1), leukemia inhibitory factor (LIF) and enzymes involved in PG synthesis (P < 0.05). In ST cells, activation of PPARD isoform with both agonists used (L-165,041 and cPGI2) and PPARG isoform with 15-deoxy-Δ12,14-PGJ2 increased vascular endothelial growth factor A (VEGFA) mRNA expression (P < 0.05). Moreover, GW9578 (PPARA agonist) and 15-deoxy-Δ12,14-PGJ2 stimulated glucose transporter 1 (SLC2A1) gene expression in ST cells. 15-deoxy-Δ12,14-PGJ2 was also effective in up-regulation of the ratio of BAX/BCL2 mRNA expression and active caspase-3 concentration in ST cells (P < 0.05). Finally, GW9578 stimulated LE and ST cell proliferation, while rosiglitazone (PPARG agonist) increased the number of viable ST but not LE cells. In conclusion, this study demonstrated that conceptus products differentially modulate PPARs expression and activity in the porcine endometrium. Activation of PPARs may in turn affect nutrient transport, PG synthesis, angiogenesis, apoptosis, or cell proliferation in this tissue. Therefore, PPAR isoforms seem to play an important role in development and function of the porcine uterus.

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.

The effect of insulin-like growth factor-I, relaxin and luteinizing hormone on vascular endothelial growth factor secretion by cultured endometrial stromal cells on different days of early pregnancy in pigs.

The effect of insulin-like growth factor-I (IGF-I), relaxin (RLX) and luteinizing hormone (LH) on vascular endothelial growth factor (VEGF) in vitro secretion by endometrial stromal cells in pigs was investigated on days 10-12 and 20-22 of gestation. LH-stimulated stromal cell secretion of VEGF did not differ among tested days of early pregnancy. However, IGF-I- and RLX-mediated release of VEGF depended on the day of pregnancy. It seems that IGF-I and RLX may be considered as potent activators of VEGF-mediated angiogenesis in porcine endometrium, and their action may be more pronounced during maternal recognition of pregnancy.

Development of real-time PCR assays in the study of gonadotropin subunits, follistatin and prolactin genes expression in the porcine anterior pituitary during the preovulatory period.

OBJECTIVES: Neural control of the anterior pituitary function consists of the interplay of neuropeptides action, gonadal steroid hormones and many other factors. The physiological effect of this regulatory action is the release and synthesis of protein hormones in the precise time and quantity. The main factor responsible for the gonadotropins release and synthesis is the gonadotropin-releasing hormone (GnRH). We must still study the modulation of the synthesis of the gonadotropins subunits - LHbeta, FSHbeta and alpha subunit by different forms of GnRH and by its analogs, in order to better understand the regulation of gonadotropin release and synthesis. THE AIM of this study was to develop real-time PCR assays of five candidate reference genes for normalization purposes in order to quantify target transcripts in anterior pituitary cells during the preovulatory period. Moreover, we focused on the influence of GnRH receptor antagonist (antide) treatment on mRNA expression levels of GPalpha, LHbeta, FSHbeta, FST(follistatin) and PRL(prolactin) genes in these cells. MATERIAL AND METHODS: Anterior pituitary cells were obtained from pituitary glands of four mature pigs at the preovulatory phase. Cells were incubated with or without antide and relative mRNA level of target genes was measured using the Applied Biosystems 7500 Real Time System. For an exact comparison of mRNA quantity, the stability of five reference genes, ACTB, B2M, GAPDH, RPL1, and TOP2B was evaluated to choose the most appropriate reference gene for qRT-PCR normalization in the pituitary cells. Expression stability of reference genes was calculated using the geNorm application. The developed method of PCR assay was applied to study gene expression in pig pituitary cells in short culture. RESULTS: The most stably expressed genes in the pituitary cells were GAPDH and TOP2B. The expression of ACTB, B2M and RPL1 appeared to be highly unstable. After normalization to the GAPDH/TOP2B, results showed that the mRNA expression of the FSHbeta gene was highest in comparison with LHbeta, GPalpha, FST and PRL genes (p<0.005). Pre-treatment of cells by the antide resulted in lower mRNA expression of these genes, while FSHbeta mRNA had a significantly lower expression (p<0.05) in comparison with control. CONCLUSIONS: Real-time PCR analysis of the expression of LHbeta, FSHbeta, alpha subunit, follistatin and prolactin genes in porcine anterior pituitary cells during the preovulatory period is suitable for the study of modulatory action of metal complexes with GnRH on the expression of these genes.

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.

Effect of steroids on basal and LH-stimulated prostaglandins F(2alpha) and E(2) release and cyclooxygenase-2 expression in cultured porcine endometrial stromal cells.

Ovarian steroids modulate uterine receptivity in domestic species. Luteinizing hormone (LH) stimulates prostaglandin (PG)F(2alpha) release from the porcine endometrium. However, the combined action of LH and steroids on PGs secretion has not yet been studied in pigs. The aim of the present study was to examine the effect of estradiol (E(2)) and progesterone (P(4)) on basal and LH-stimulated PGF(2alpha) and PGE(2) secretion and cyclooxygenase-2 (COX-2) protein expression in porcine endometrial stromal cells obtained on days 12-13 of the estrous cycle. Cells were cultured for 48 h in a medium containing charcoal-stripped newborn calf serum alone or supplemented with 10 nM E(2) and/or 50 nM P(4). Then, the cells were incubated for 6 h in the presence or absence of LH (20 ng/ml). Long exposure of stromal cells to steroids had no effect on PGF(2alpha) secretion, but PGE(2) release increased in the presence of E(2) plus P(4) (p<0.05). Pre-incubation of cells with E(2) plus P(4) resulted in enhanced PGF(2alpha) (p<0.05) and PGE(2) (p<0.001) secretion. Moreover, LH increased PG(2alpha) secretion in control (p<0.05) and E(2)-treated stromal cells (p<0.01). LH tended (p=0.07) to elevate PGE(2) release only in cells pre-exposed to E(2) plus P(4). The expression of COX-2 protein was increased by LH (p<0.05), but not by steroids. These results confirm the stimulatory effect of LH on PGF(2alpha) secretion and COX-2 expression in porcine stromal cells before luteolysis. PG release from porcine endometrium seems to be controlled by ovarian steroids, however only E(2)-treated-treated cells responded to LH.

Peroxisome proliferator-activated receptor (PPAR) isoforms are differentially expressed in peri-implantation porcine conceptuses.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor family of ligand-dependent transcription factors. PPARs are critical regulators of glucose homeostasis and lipid metabolism, and affect cell proliferation and differentiation. In the current study, we examined (1) the profiles of PPARA, PPARD, and PPARG mRNA expression and DNA binding activity in porcine conceptuses collected on Days 10-11 (spherical and tubular conceptuses), 11-12 (filamentous conceptuses), 13-14, and 15-16 (elongated conceptuses) of pregnancy, (2) the presence of PPARA, PPARD, and PPARG proteins in Days 10, 12, and 15 conceptuses. Moreover, we analyzed the abundance of retinoid X receptor (RXR; PPARs heterodimer partner) transcripts as well as the correlation between PPARs mRNA expression and the expression of genes important for and/or associated with elongation of porcine conceptuses: aromatase (CYP19A1), prostaglandin endoperoxide synthase 2 (PTGS2), glucose transporter 1 (SLC2A1), and interleukin 1B (IL1B). PPARA mRNA expression in conceptuses did not change during Days 10-14 of gestation, but was greater on Days 15-16 compared to Days 10-11 (P < 0.05). A considerable increase in PPARD and PPARG mRNA expression was observed in filamentous conceptuses from Days 11-12 compared to spherical and tubular conceptuses from Days 10-11 (P < 0.01), followed by a decrease on Days 13-14 and 15-16 (P < 0.05). PPARA, PPARD, and PPARG proteins were present in conceptus tissue demonstrating nuclear localization clearly visible on Days 12 and 15 of pregnancy. DNA binding activity of the PPARD isoform was greater in filamentous conceptuses from Days 11-12 than in spherical and tubular conceptuses from Days 10-11 (P < 0.01). Moreover, concentrations of active PPARD and PPARG proteins in nuclear fractions of conceptus tissue were greater on Days 11-12 compared to Days 13-14 and 15-16 of pregnancy (P < 0.05). RXRA, RXRD, and RXRG mRNA expression in conceptuses increased on Days 11-12 compared to Days 10-11 (P < 0.05). PPARD and PPARG mRNA expression showed strong positive correlations with PTGS2 mRNA expression (P < 0.0001). Additionally, PPARD gene expression correlated with SLC2A1 and IL1B mRNA expression (P < 0.01). Collectively, these results indicate that among all three PPARs expressed in peri-implantation porcine conceptuses, PPARD and PPARG may be involved in conceptus elongation before implantation.

Molecular cloning and spatiotemporal expression of prostaglandin F synthase and microsomal prostaglandin E synthase-1 in porcine endometrium.

Endometrial prostaglandins (PGs) and the PGE2/PGF2alpha ratio play an important role in regulating the estrous cycle and establishment of pregnancy. The enzymes downstream of cyclooxygenase-2 may determine the PGE2/PGF2alpha ratio in the porcine uterus. Thus, we have cloned porcine PGF synthase (PGFS) and microsomal PGE synthase-1 (mPGES-1) and characterized their expression in porcine endometrium during the estrous cycle and early pregnancy. PGFS and mPGES-1 amino acid sequences possessed a high degree (>67% and >77%, respectively) of identity with the other mammalian homologs. There was little modulation of mPGES-1 throughout the estrous cycle; however, PGFS expression was highly up-regulated in endometrium around the time of luteolysis. During early pregnancy, PGFS at the protein level showed a time-dependent increase (low on d 10-13, intermediate on d 14-23, and high on d 24-25). In pregnancy, expression of mPGES-1 was intermediate on d 10-11 and low on d 14-17 and then increased after d 22, reaching the maximum on d 24-25. Immunohistochemistry showed localization of PGFS and mPGES-1 proteins mainly in luminal and glandular epithelium. Concluding, the spatiotemporal expression of PGFS throughout the estrous cycle indicates an involvement of PGFS in regulating luteolysis in the pig. The comparison of endometrial PGFS and mPGES-1 expression on d 10-13 of the estrous cycle and pregnancy suggest a supportive role of these enzymes in determining the increase of uterine PGE2/PGF2alpha ratio during maternal recognition of pregnancy. Moreover, high expression of both PG synthases after initiation of implantation may indicate their significant role in placentation.

Mechanisms ensuring optimal conditions of implantation and embryo development in the pig.

The paper summarizes results of a series of studies concerning luteolysis and early pregnancy in pigs. The involvement of the oxytocin (OT)/OT receptor system in the mechanism of corpus luteum (CL) protection during early pregnancy as well as the implication of luteinizing hormone (LH) in the endometrial prostaglandin (PG) release and synthesis are described. In addition, the role of leptin in the regulation of ovarian steroidogenesis and the expression of leptin and its receptor (OB-Rb) genes in hypothalamus, pituitary and reproductive tissues are reported. Moreover, a strong emphasis was placed on the mechanism of PGE2 participation in the local endocrine regulations of reproductive processes occurring in the utero-ovarian area as well as on the vascular endothelial growth factor (VEGF) ligand-receptor system in the ovary and uterus.