The regulation of the expression of prokineticin 1 and its receptors and its mechanism of action in the porcine corpus luteum.

Prokineticin 1 (PROK1) is an important factor in pregnancy establishment in pigs, acting at the embryo-maternal interface and the corpus luteum (CL). Estradiol-17ß (E2) is the primary pregnancy recognition signal in pigs, and its effects are augmented by luteotropic prostaglandin E2 (PGE2). On the contrary, prostaglandin F2α (PGF2α) exerts mainly a luteolytic effect. The present study aimed to elucidate whether E2, PGE2, and PGF2α regulate the expression of PROK1 and its receptors in the porcine CL and to determine the PROK1 effect on luteal endothelial cells and pathways that may be involved in this regulation. The effects of E2, PGE2, and PGF2α on the expressions of PROK1 and its receptors in the CL were studied using an in vitro model of ultrathin luteal tissue explants model. Additionally, the effects of E2 and PGE2 on the PROK1 system were determined using an in vivo approach, in which the hormones were administered into the uterine lumen to imitate their secretion by embryos. Endothelial cell proliferation was measured using the colorimetric method. E2 acting via estrogen receptors simulated the mRNA and protein expressions of PROK1 and PROKR1 in CL explants in vitro (p < 0.05). The simultaneous action of E2 with PGE2 enhanced the expression of luteal PROK1 mRNA in vitro (p < 0.05). Estradiol-17ß acting alone significantly increased PROK1 mRNA levels in vivo, whereas E2 simultaneously administered with PGE2 significantly elevated the PROK1 mRNA expression and PROKR1 mRNA and protein contents in CLs adjacent to uterine horns receiving hormonal infusion compared with CLs adjacent to placebo-treated uterine horns (p < 0.01). The PROK1 protein expression was significantly higher in the CLs of pigs treated with E2, PGE2, and E2 together with PGE2 than in the control group. PGF2α increased the PROK1 mRNA content in CLs on days 12 and 14 of the estrous cycle (p < 0.05). The expression of PROKR2 at the mRNA and protein levels remained unchanged in response to in vitro and in vivo treatments. PROK1 stimulated the proliferation of luteal endothelial cells by activating the MAPK, AKT, and mTOR pathways (p < 0.05). In summary, the luteal expressions of PROK1 and PROKR1 in early pregnancy are regulated by E2 and PGE2. PROK1 stimulates luteal angiogenesis by activating the MAPK, AKT, and mTOR pathways. The regulation of luteal PROK1 expression by PGF2α indicates PROK1's putative role during luteolysis. We conclude that PROK1-PROKR1 signaling supports luteal function during CL rescue in pregnancy in pigs.

Prokineticin 1 is a novel factor regulating porcine corpus luteum function.

Prokineticin 1 (PROK1) is a pleiotropic factor secreted by endocrine glands; however, its role has not been studied in the corpus luteum (CL) during pregnancy in any species. The present study aimed to investigate the contribution of PROK1 in regulating processes related to porcine CL function and regression: steroidogenesis, luteal cell apoptosis and viability, and angiogenesis. The luteal expression of PROK1 was greater on Days 12 and 14 of pregnancy compared to Day 9. PROK1 protein expression during pregnancy increased gradually and peaked on Day 14, when it was also significantly higher than that on Day 14 of the estrous cycle. Prokineticin receptor 1 (PROKR1) mRNA abundance increased on Days 12 and 14 of pregnancy, whereas PROKR2 elevated on Day 14 of the estrous cycle. PROK1, acting via PROKR1, stimulated the expression of genes involved in progesterone synthesis, as well as progesterone secretion by luteal tissue. PROK1-PROKR1 signaling reduced apoptosis and increased the viability of luteal cells. PROK1 acting through PROKR1 stimulated angiogenesis by increasing capillary-like structure formation by luteal endothelial cells and elevating angiogenin gene expression and VEGFA secretion by luteal tissue. Our results indicate that PROK1 regulates processes vital for maintaining luteal function during early pregnancy and the mid-luteal phase.

Novel insights into conceptus-maternal signaling during pregnancy establishment in pigs.

Pregnancy establishment in mammals, including pigs, requires coordinated communication between developing conceptuses (embryos with associated membranes) and the maternal organism. Porcine conceptuses signalize their presence by secreting multiple factors, of which estradiol-17ß (E2) is considered the major embryonic signal initiating the maternal recognition of pregnancy. During this time, a limited supply of prostaglandin (PGF2α) to the corpora lutea and an increased secretion of luteoprotective factors (e.g., E2 and prostaglandin E2 [PGE2]) lead to the corpus luteum's maintained function of secreting progesterone, which in turn primes the uterus for implantation. Further, embryo implantation is related to establishing an appropriate proinflammatory environment coordinated by the secretion of proinflammatory mediators including cytokines, growth factors, and lipid mediators of both endometrial and conceptus origin. The novel, dual role of PGF2α has been underlined. Recent studies involving high-throughput technologies and sophisticated experimental models identified a number of novel factors and revealed complex relationships between these factors and those already established. Hence, it seems that early pregnancy should be regarded as a sequence of processes orchestrated by pleiotropic factors that are involved in redundancy and compensatory mechanisms that preserve the essential functions critical for implantation and placenta formation. Therefore, establishing the hierarchy between all molecules present at the embryo-maternal interface is now even more challenging.

Novel role for conceptus signals in mRNA expression regulation by DNA methylation in porcine endometrium during early pregnancy†.

During early pregnancy, porcine conceptuses (the embryos with associated membranes) secrete estradiol-17ß (E2)-their major signal for maternal recognition of pregnancy-and prostaglandin E2 (PGE2). Both hormones induce prominent changes of the endometrial transcriptome in vivo. Studies on endometrial pathologies have shown that E2 affects gene expression by epigenetic mechanisms related to DNA methylation. Herein, we determined the effects of E2 and PGE2 alone, and a combined E2 + PGE2 treatment administered into the uterine lumen in vivo on the expression and activity of DNA-methyltransferases (DNMTs) and on CpG methylation patterns of selected genes in porcine endometrium. To compare the effect of treatment with the physiological effect of pregnancy, endometria from day 12 pregnant/cyclic gilts were included. Both E2 and PGE2 significantly reduced the expression of DNMTs. Likewise, the expressions of DNMT1 and DNMT3A were decreased on day 12 of pregnancy compared to the estrous cycle. DNMT activity increased in endometrial samples following E2 treatment and in gilts on day 12 of pregnancy. Treatment with E2 alone and/or simultaneously with PGE2 altered endometrial DNA methylation of CpG sites of ADAMTS20, ADH1C, BGN, PSAT1, and WNT5A. Different CpG methylation patterns of ADAMTS20, BGN, DMBT1, RASSF1, and WNT5A were found in the endometrium on day 12 of pregnancy compared to day 12 of the estrous cycle. Significant correlations were detected between CpG methylation and gene expression for ADAMTS20, ADH1C, BGN, DMBT1, PSAT1, and WNT5A. Our results indicate that CpG methylation induced by embryonic signals may contribute to regulating endometrial gene expression during pregnancy establishment.

Prokineticin 1-prokineticin receptor 1 signaling in trophoblast promotes embryo implantation and placenta development.

Successful pregnancy establishment in mammals depends on proper embryo-maternal communication. Prokineticin 1 (PROK1) is a secretory protein that exerts pleiotropic functions in various tissues. Despite the studies that have primarily been performed with human cell lines and mice, the function of PROK1 in trophoblasts has still not been fully elucidated. Hence, the aim of this study was to establish the role of PROK1 in trophoblasts during implantation and placentation. Prokineticin 1 mRNA was elevated in porcine trophoblasts during implantation and the early placentation period. Furthermore, we reveal that PROK1-PROKR1 signaling induces the expression of genes involved in the regulation of angiogenesis, immunological response, trophoblast cell adhesion, invasion, and proliferation, as well as stimulating phosphorylation of MAPK and PTK2. Ingenuity Pathway Analysis identified the aforementioned and also other functions associated with PROK1-regulated genes/proteins, such as cell-to-cell contact, epithelial tissue differentiation, Ca2+ release, lipid synthesis, and chemotaxis. We also showed evidence that PROK1 acting via PROKR1 increased trophoblast cell proliferation and adhesion. The PROK1-stimulated cell proliferation was mediated by PI3K/AKT/mTOR, MAPK, and cAMP, whereas adhesion was mediated by MAPK and/or PI3K/AKT signaling pathways. Concluding, our study suggests that PROK1 plays a pleiotropic role in trophoblast function during implantation and early placentation.

Estradiol-17ß Regulates Expression of Luteal DNA Methyltransferases and Genes Involved in the Porcine Corpus Luteum Function In Vivo.

The corpus luteum (CL) is a temporary endocrine gland vital for pregnancy establishment and maintenance. Estradiol-17ß (E2) is the major embryonic signal in pigs supporting the CL's function. The mechanisms of the luteoprotective action of E2 are still unclear. The present study aimed to determine the effect of E2 on luteal expression of factors involved in CL function. An in vivo model of intrauterine E2 infusions was applied. Gilts on day 12 of pregnancy and the estrous cycle were used as referential groups. Concentrations of E2 and progesterone were elevated in CLs of gilts receiving E2 infusions, compared to placebo-treated gilts. Estradiol-17ß stimulated luteal expression of DNA-methyltransferase 1 (DNMT1), but decreased expression of DNMT3B gene and protein, as well as DNMT3A protein. Similar results for DNMT3A and 3B were observed in CLs on day 12 of pregnancy compared to day 12 of the estrous cycle. Intrauterine infusions of E2 altered luteal expression of the genes involved in CL function: PTGFR, PTGES, STAR, HSD17B1, CYP19A1, and PGRMC1. Our findings indicate a role for E2 in expression regulation of factors related to CL function and a novel potential for E2 to regulate DNA methylation as putative physiological mechanisms controlling luteal gene expression.

Synergistic action of estradiol and PGE2 on endometrial transcriptome in vivo resembles pregnancy effects better than estradiol alone†.

Successful pregnancy establishment in mammals depends on numerous interactions between embryos and the maternal organism. Estradiol-17ß (E2) is the primary embryonic signal in the pig, and its importance has been questioned recently. However, E2 is not the only molecule of embryonic origin. In pigs, prostaglandin E2 (PGE2) is abundantly synthesized and secreted by conceptuses and endometrium. The present study aimed to determine the role of PGE2 and its simultaneous action with E2 in changes in porcine endometrial transcriptome during pregnancy establishment. The effects of PGE2 and PGE2 acting with E2 were studied using an in vivo model of intrauterine hormone infusions, and were compared to the effects of E2 alone and conceptuses' presence on day 12 of pregnancy. The endometrial transcriptome was profiled using gene expression microarrays followed by statistical analyses. Downstream analyses were performed using bioinformatics tools. Differential expression of selected genes was verified by quantitative polymerase chain reaction. Microarray analysis revealed 2413 differentially expressed genes (DEGs) in the endometrium treated simultaneously with PGE2 and E2 (P < 0.01). No significant effect of PGE2 administered alone on endometrial transcriptome was detected. Gene ontology annotations enriched for DEGs were related to multiple processes such as: focal adhesion, vascularization, cell migration and proliferation, glucose metabolism, tissue remodeling, and activation of immune response. Simultaneous administration of E2 and PGE2 induced more changes within endometrial transcriptome characteristic to pregnancy than infusion of E2 alone. The present findings suggest that synergistic action of estradiol-17ß and PGE2 resembles the effects of pregnancy on endometrial transcriptome better than E2 alone.

Pleiotropic role of prokineticin 1 in the porcine endometrium during pregnancy establishment and embryo implantation †.

Acquisition of endometrial receptivity for embryo implantation is one of the crucial processes during pregnancy and is induced mainly by progesterone and enhanced by conceptus signals. Prokineticin 1 (PROK1) is characterized as a secretory protein with diverse functions in various tissues, including the reproductive tract. PROK1, with its receptor PROKR1, are up-regulated in the porcine endometrium during implantation and in women's receptive endometrium and decidua. However, the function of PROK1 in embryo-maternal communication has still not been fully elucidated. Hence, we hypothesize that PROK1 is involved in endometrial receptivity development and implantation in pigs. In this study, using the porcine in vivo model of intrauterine infusions of estradiol-17ß (E2) and prostaglandin E2 (PGE2), we revealed that these hormones elevated endometrial expression of PROK1 and PROKR1 mRNA, respectively. Moreover, E2, acting synergistically with PGE2, increased PROKR1 protein expression. We also evidenced that PROK1-PROKR1 signaling induced expression of following genes and/or proteins CCN2, CDH13, FGF2, NFATC2, ANGPT1, ANGPT2, CDH1, MUC4, SPP1, IFNG, IL6, LIF, LIFR, TNF, TGFB3, and FGF9, as well as phosphorylation of PTK2 and secretion of IL6 and IL11 by endometrial explants in vitro. Ingenuity pathway analysis revealed that functions associated with the PROK1-regulated genes/proteins include cell-to-cell contact, cell attachment, migration and viability, differentiation of epithelial tissue, leukocyte migration, inflammatory response, angiogenesis, and vasculogenesis. Summarizing, our study suggests that PROK1 acts pleiotropically as an embryonic signal mediator that regulates endometrial receptivity by increasing the expression of the genes and proteins involved in implantation and pregnancy establishment in pigs.

Prokineticin 1-prokineticin receptor 1 signaling promotes angiogenesis in the porcine endometrium during pregnancy†.

Pregnancy establishment in mammals, including pigs, requires proper communication between embryos and the maternal reproductive tract. Prokineticin 1 (PROK1) has been described as a secretory protein with pleiotropic functions and as a novel tissue-specific angiogenic factor. However, despite the studies performed mainly on human cell lines and in mice, the function of PROK1 in the endometrium during early pregnancy is still not fully elucidated. We hypothesized that PROK1 contributes to pregnancy establishment in pigs. The present study is the first to report that the expression of PROK1 and its receptor (PROKR1) is elevated in the porcine endometrium during the implantation and early placentation period. PROK1 protein was detected mainly in luminal epithelial cells, glandular epithelial cells, and blood vessels in the endometrium. Using the porcine in vivo model of unilateral pregnancy, we revealed that conceptuses induced the endometrial expression of PROK1 and PROKR1. Moreover, the embryonic signal, estradiol-17ß, as well as progesterone, stimulated the endometrial expression of PROK1 and PROKR1. We also evidenced that PROK1-PROKR1 signaling supports endometrial angiogenesis in pigs. The PROK1-stimulated proliferation of primary porcine endometrial endothelial (PEE) cells involved PI3K/AKT/mTOR, MAPK, cAMP, and NFKB signaling pathways. Furthermore, PROK1 via PROKR1 promoted the formation of capillary-like structures by PEE cells. PROK1 also stimulated VEGFA and PGF2α secretion, which in turn may indirectly support angiogenic changes within endometrial tissue. In summary, our study suggests that PROK1 acts as an embryonic signal mediator that regulates endometrial angiogenesis and secretory function during the implantation and early placentation period in pigs.

Estradiol-17ß-Induced Changes in the Porcine Endometrial Transcriptome In Vivo.

Estradiol-17ß (E2) is a key hormone regulating reproductive functions in females. In pigs, E2, as the main conceptus signal, initiates processes resulting in prolonged corpus luteum function, embryo development, and implantation. During early pregnancy the endometrium undergoes morphological and physiological transitions that are tightly related to transcriptome changes. Recently, however, the importance of E2 as a primary conceptus signal in the pig has been questionable. Thus, the aim of the present study was to determine the effects of E2 on the porcine endometrial transcriptome in vivo and to compare these effects with transcriptome profiles on day 12 of pregnancy. Microarray analysis revealed differentially expressed genes (DEGs) in response to E2 with overrepresented functional terms related to secretive functions, extracellular vesicles, cell adhesion, proliferation and differentiation, tissue rearrangements, immune response, lipid metabolism, and many others. Numerous common DEGs and processes for the endometrium on day 12 of pregnancy and E2-treated endometrium were identified. In summary, the present study is the first evidence for the effect of E2 on transcriptome profiles in porcine endometrium in vivo in the period corresponding to the maternal recognition of pregnancy. The presented results provide a valuable resource for further targeted studies considering genes and pathways regulated by conceptus-derived estrogens and their role in pregnancy establishment.

Prostaglandin F2α stimulates angiogenesis at the embryo-maternal interface during early pregnancy in the pig.

Blood vessel formation is a critical process for successful pregnancy establishment and placenta formation. Angiogenic factors such as vascular endothelial growth factor (VEGF), angiopoietins (ANGPTs) or fibroblast growth factor 2 (FGF2) are known to be involved in angiogenesis. However, the mechanism regulating their expression in the porcine endometrium and trophoblast has not been described during early pregnancy establishment. Recently, we reported an important role for prostaglandin F2α (PGF2α) in supporting processes accompanying the peri-implantation period in the pig. The aim of the present study was to determine the effect of PGF2α on angiogenic factor gene and protein expression at the embryo-maternal interface and on capillary-like structure formation by endometrial endothelial cells. In the present study, we used various in vitro models involving endometrial tissue explants, primary porcine trophoblast and endometrial endothelial cells, as well as a swine umbilical vein endothelial cell line (G1410). ANGPT1, ANGPT2 and FGF2 gene expression was analyzed in porcine endometrial explants and in primary trophoblast cells incubated with PGF2α (100 nM, 1 µM). VEGFA gene expression and protein secretion by porcine primary trophoblast cells were studied in vitro using primary trophoblast cells. A network formation assay using the G1410 cell line and primary endothelial cells of endometrial origin was performed to assess the effect of PGF2α on capillary-like structure formation. We found that PGF2α stimulated VEGFA gene expression (1 µM) and secretion of this protein (100 nM) by porcine trophoblast cells (P < 0.05). In endometrial explants, PGF2α increased the expression of the ANGPT1, ANGPT2 and FGF2 genes (P < 0.05). PGF2α stimulated the formation of capillary-like structures acting on porcine endometrial endothelial cells on days 15 and 20 of pregnancy and in the G1410 cell line (P < 0.05). PGF2α-stimulated endothelial cell network formation was diminished by using a MEK kinase inhibitor in G1410 cells. Our results indicate an important role for PGF2α in the regulation of angiogenesis at the embryo-maternal interface. PGF2α promotes angiogenesis in the porcine endometrium by activating the MAPK signaling pathway. The stimulating effect of PGF2α on the formation of capillary-like structures by endothelial cells, together with our previous findings, supports the hypothesis that PGF2α is an important factor promoting the development of the placenta during early pregnancy in the pig.

Prostaglandin F2α stimulates adhesion, migration, invasion and proliferation of the human trophoblast cell line HTR-8/SVneo.

INTRODUCTION: The amount of prostaglandin F2α (PGF2α) in the uterine lumen increases during the window of implantation in many mammals, including humans. We hypothesized that PGF2α regulates processes related to human embryo implantation. METHODS: The effect of PGF2α was studied using an in vitro model of human extravillous trophoblast (EVT) cell line (HTR-8/SVneo). Adhesion, proliferation, invasion and migration assays, zymography for metalloproteinases (MMP) activity, and gene/protein expression analyses were applied. Doses of 100 nM and/or 1 µM of PGF2α and fluprostenol were used. PGF2α receptor (PTGFR), MMP9 and MMP2 proteins in the human first trimester placenta were localized by immunohistochemistry and immunofluorescence. RESULTS: This study is the first reporting the expression of PTGFR protein in the first trimester placenta, as well as in HTR-8/SVneo cells. PGF2α and fluprostenol increased HTR-8/SVneo cell proliferation and adhesion to extracellular matrix protein (P < 0.05). This effect was abolished by mitogen activated protein kinases (MAPK) inhibitor. PGF2α induced phosphorylation of focal adhesion kinase and MAPK1/3 (P < 0.05). PGF2α increased mRNA content and protein activity of MMP9, and gene and protein expression of interleukin-6 (P < 0.05). EVT cell migration and invasiveness were stimulated by PGF2α (P < 0.05). The PGF2α effect on cell invasion was reduced by inhibitors of MMP2, MMP9 and mTOR. In all experiments, the stimulatory effects of PGF2α were diminished by using a PTGFR antagonist. DISCUSSION: Our findings suggest a significant role for PGF2α in mechanisms associated with implantation. PGF2α acting by PTGFR in HTR-8/SVneo cells stimulates their adhesion and proliferation through the MAPK signaling pathway and increases invasiveness inducing MMP proteolytic activity and mTOR signaling.

Prostaglandin F2α promotes embryo implantation and development in the pig.

Successful establishment and development of pregnancy requires proper communication between developing conceptuses and the maternal reproductive tract. Prostaglandins are key players involved in the regulation of reproductive processes in mammals including pigs. Due to its luteolytic action, prostaglandin F2-alpha (PGF2α) is mainly considered as an undesirable factor during early pregnancy. However, its content in the uterine lumen is elevated in pigs and other mammals. Recently, we reported an important role of PGF2α in the endometrium during early pregnancy in the pig. Thus, the aim of the present study was to determine whether PGF2α can act on porcine trophoblast and if so, to elucidate what effect it could exert. We detected increased expression of PGF2α receptor during the implantation period (from day 14 until day 19 of pregnancy). Global gene expression profiling using microarrays and quantitative PCR studies revealed that PGF2α acting on porcine trophoblast cells in vitro alters expression of genes potentially involved in processes related to implantation, such as: cell proliferation, focal adhesion, extracellular matrix binding, cell migration, cytoskeleton organization, immune interactions, ion homeostasis, and lipid metabolism. Using primary porcine trophoblast cells, we demonstrated that PGF2α stimulated trophoblast cell proliferation and adhesion to extracellular matrix protein. This was likely mediated by mitogen-activated protein kinases (MAPK1/3) and focal adhesion kinase (FAK) since we observed increased phosphorylation of MAPK1/3 and FAK in trophoblast cells treated with PGF2α. To conclude, the present report indicates a novel role for PGF2α in the porcine conceptus as a para- and autocrine factor supporting pregnancy establishment.

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.

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-17ß) 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 ß3, 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.

Functional consequences of knocking down porcine prostaglandin synthases in SK-6 swine kidney cell line.

We hypothesized that in vitro knock-down of the previously cloned genes of prostaglandin synthases will result in a reduction of synthesis, and thus secretion of their respective products, i.e., prostaglandin (PG) E2 or PGF2α. For this purpose, we designed short hairpin RNA (shRNA)-encoding constructs to knock down porcine mPGES-1 and PGFS (also named as AKR1CL1 in GenBank) and used them to transfect swine kidney SK-6 cells. Knocking down PGFS or mPGES-1 transcripts resulted in at least 50% inhibition of protein expression of each respective enzyme, as well as a reduction in the production of their respective prostaglandin, PGF(2α) or PGE(2). These results confirmed the identities of PGFS and mPGES-1. Moreover, they illustrate a unique opportunity to use the gene knock-down constructs in primary endometrial cells in order to study their biological roles in the porcine endometrium, particularly during the establishment of pregnancy.

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.

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-17ß 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-17ß. 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.

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.

Novel insights into the mechanisms of pregnancy establishment: regulation of prostaglandin synthesis and signaling in the pig.

Ovarian progesterone induces essential changes leading to a temporary state of uterine receptivity for conceptus implantation. Estrogens secreted by the porcine conceptus on days 11 and 12 of pregnancy provide the initial signal for maternal recognition of pregnancy and maintenance of a functional corpus luteum (CL) for continued production of progesterone. As prostaglandins F(2)(α) (PGF(2)(α)) and E(2) (PGE(2)) exert opposing actions on the CL, a tight control over their synthesis and secretion is critical either for the initiation of luteolysis or maintenance of pregnancy. One of the supportive mechanisms by which conceptus inhibits luteolysis is changing PG synthesis in favor of luteoprotective PGE(2). Conceptus PGE(2) could be amplified by PGE(2) feedback loop in the endometrium. In pigs, as in other species, implantation and establishment of pregnancy is associated with upregulation of expression of proinflammatory factors, which include cytokines, growth factors, and lipid mediators. The conceptus produces inflammatory mediators: interferon γ and interferon δ, interleukins IL1B and IL6, and PGs, which probably activate inflammatory pathways in the endometrium. The endometrium responds to these embryonic signals by enhancing further progesterone-induced uterine receptivity. Understanding the mechanisms of pregnancy establishment is required for translational research to increase reproductive efficiencies and fertility in humans and animals.